prediction of preeclampsia by placental protein 13 and background risk factors and its prevention by...

DOI 10.1515/jpm-2013-0298 J. Perinat. Med. 2014; 42(5): 591–601

Hamutal Meiri*, Marei Sammar, Ayelet Herzog, Yael-Inna Grimpel, Galina Fihaman, Aliza Cohen, Vered Kivity, Adi Sharabi-Nov and Ron Gonen

Prediction of preeclampsia by placental protein 13 and background risk factors and its prevention by aspirin

Abstract

Aim: Evaluation of placental protein 13 (PP13) and risk factors (RFs) as markers for predicting preeclampsia (PE) and use of aspirin for PE prevention.Materials and methods: First-trimester pregnancy screen-ing was based on having PP13 level ≤ 0.4 multiple of the median (MoM) and/or at least one major risk factor (RF) for PE. Management was by routine care or combined with daily treatment with 75 mg aspirin between 14 and 35 weeks of gestation.Results: Of 820 deliveries, 63 women developed PE (7.7%). Median PP13 levels was 0.2MoM in the PE group com-pared with 0.83MoM among unaffected and 1.0MoM in unaffected not treated with aspirin (P < 0.0001). Low PP13 was a better predictor for PE versus major RFs, particu-larly for young nuliparous. Combining low PP13 with RFs increased prediction accuracy. Mean arterial pressure (not included in the initial prediction), could add to prediction accuracy when combined with low PP13 and RFs. PE pre-vention by aspirin was most effective when the risk was determined by low PP13 alone, less effective for combin-ing low PP13 with RFs, and ineffective when determined by RFs alone.Conclusion: When PE risk is determined by low first tri-mester PP13 or by combined low PP13 and RFs, prevention with aspirin is warranted.

Keywords: Aspirin prevention; in-vitro fertilization; mean arterial pressure; multiple of the medians; placenta; pla-cental protein 13; preeclampsia; risk factors; risk predic-tion; serum biomarkers.

*Corresponding author: Dr. Hamutal Meiri, TeleMarpe Ltd., 12 Barasani Moshe St., Tel Aviv 69121, Israel, Tel.: +972-54-7774762, E-mail: [email protected] Sammar: Department of Biotechnology Engineering, ORT Braude College, Karmiel, IsraelAyelet Herzog: Hy-Laboratories Ltd., Rehovot, IsraelYael-Inna Grimpel, Galina Fihaman, Aliza Cohen and Vered Kivity: TeleMarpe Ltd., Tel Aviv, IsraelAdi Sharabi-Nov: Ziv Medical Center, Safed, Israel

Ron Gonen: Bnai Zion Medical Center, Rappaport Faculty of Medicine, Technion, Haifa, Israel

IntroductionPreeclampsia (PE) is a pregnancy disorder that affects about 2%–8% of all pregnancies around the globe. It is also a major reason for the mortality and morbidity of mothers, fetuses, and neonates. The disorder com-prises new-onset hypertension coupled with damage to the kidney, occasionally to the liver, and to the cardiovascular system [13, 29, 34]. Although the eti-ology of PE remains unclear, it is attributed to multi-factorial causes associated with impaired placentation [23, 25, 26]. In the last decade, a handful of markers were developed to predict PE [6, 9]. The development of a multi-marker screening coupled to logistic regression algorithms have shown that the accuracy of the detec-tion rate (DR) could reach 80%–93% for 5%–10% false positive rate (FPR) [1, 7].

Placental protein 13 (PP13) is a galectin, which is gen-erated by the syncytiotrophoblast and released into the maternal blood [12, 27, 32]. A recent meta-analysis per-formed by members of our group, which included 15,585 patients in 19 studies, showed that at 10% FPR, the DR for first trimester PE prediction by PP13 ranged from 47% for all cases of PE (n = 1109) to 76% for preterm PE (n = 532) [15]. The prediction improved when PP13 was combined with placental growth factor (PIGF) [35], or when it was evaluated as a component of a multiple marker panel with prior risk factors (RFs), mean arterial pressure (MAP), and Doppler pulsatility index (Doppler PI) for the blood flow through the uterine arteries [1].

A kit for determining PP13 obtained a CE Mark for in-vitro diagnosis in 2006 for PP13 ELISA kits as an aiding tool for predicting pregnancy disorders. Israel’s Ministry of Health (MOH) approved the kit inclusion for aiding PE prediction in 2007. The current study was a part of a Post-marketing approval report to the Israel MOH on the

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592 Meiri et al., Aspirin prevention of preeclampsia predicted with PP13

PP13 Kit performance in the perinatal clinical, as was per-formed in Israel between 2007 and 2011.

In 2010, Bujold et al. [4] published a meta-analysis, which indicated that daily treatment with 75 mg aspirin starting before 16 weeks of gestation was associated with a significantly lower frequency of risk of PE, particularly early PE. His study was cited by the World Health Organi-zation (WHO) in a white paper published in 2011, which recommended the use of aspirin to prevent PE in women with high risk of developing the disorder [34]. A position paper published by the Israel Society for Maternal Fetal Medicine during that time proposed the same recommen-dation; however, the guidelines left the decision to use aspirin to the individual physician [3] . The guidelines remained silent about using aspirin when the patient’s risk was established by any biomarker, including PP13. This was because Israel was the first to include a predict-ing test in pregnancy management, and no randomized study had yet to show the relation between risk predicted by biomarkers and aspirin benefits. The current study describes not only the screening efficacy but also the results of using aspirin to prevent PE. Unlike our former publication [30], this study covered both aspirin treated and untreated patients, but only deal with singleton pregnancies.

Materials and methodsThis post-approval study was conducted under informed consent according to the Israeli MOH with permit #11720000. Pregnant women who attended doctors’ offices or community clinics for first trimester evaluation of pregnancy from July 2007 to February 2011 were enrolled in the study. About 3 to 50 patients were enrolled each week by 153 physicians across Israel. Women < 18 years of age were not enrolled as were those with non-viable fetuses identified by the absence of fetal heart beat. In the end, a total of 947 women were enrolled. All twin pregnancies (n = 34) were excluded from the anal-ysis of this study and their results were described by Svirsky et al. [30]. Twin pregnancies, in which one fetus vanished (n = 7) were also excluded. In the final analysis, 93 patients were excluded due to preg-nancy loss before 22 weeks (n = 36); pregnancy termination due to fetal malformation (n = 6); loss to follow-up due to address change or due to moving to other countries (n = 15); and multiple gestation preg-nancies (n = 36). In vitro fertilization (IVF) patients (n = 66), including those with oocyte donation (n = 17), were enrolled but only after dis-continuing the treatment for hormonal support of placentation. The analysis was performed on 820 pregnant women.

Blood was drawn from pregnant women between 8 and 14 weeks of gestation. Blood was left to coagulate at room temperature for 30–90 min and then centrifuged for 15 min at 3000 × g, after which the serum was collected and stored at –20°C for up to four days until it was transferred to a central laboratory in cool boxes kept at this temperature. PP13 test was performed within 2 to 12 days of blood

drawing. Sample stability was standardized and showed consistency within this time frame. PP13 levels were prospectively determined using enzyme-linked immunosorbent assay (ELISA), according to the sandwich method as previously published [4, 5, 7]. A different kit lot (12 altogether) was used every three to four months throughout the testing period. To determine variations within a kit (7.9%), between kits (13%) and between lots (13%), aliquots from a pool of serum from pregnant women were placed in several wells on each plate. Each sample was determined as the mean of four to eight repeats on the same plate, and when coefficient of variance exceeded 7.9%, the sample was retested on an additional plate. The laboratory techni-cian used barcode-marked samples and was blinded to any patient information.

Gestational age (GA) was determined by the last menstrual period and confirmed by first trimester crown rump length (CRL) measurements [17]. Demographics, medical, and pregnancy his-tory were obtained at enrolment. PP13 analysis was performed by Zer HiTech Central Laboratory and by American Medical Laborato-ries, both in Tel Aviv, Israel. Pregnancy outcome after delivery was obtained from the patients’ medical records and hospital discharge form (85%). Otherwise, a detailed telephone interview was con-ducted with a) patients who had delivered at home (13 cases), b) delivered abroad (when contact information was available), and 3) patients who did not keep their hospital discharge form as well as with the patient’s physician.

PE was diagnosed according to the criteria of the International Society for the Study of Hypertension in Pregnancy (ISSHP) as updated by Lindheimer [19], and according to those issued by Sibai for the Society of Maternal and Fetal Medicine (SMFM) [29]. The cri-teria were as follows: systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg on at least two occasions 4 h apart developed after 20 weeks of gestation in previously normoten-sive women combined with proteinuria of ≥ 300 mg in 24 h, or two readings of at least 2+ on dipstick analysis of midstream or catheter urine specimens, if no 24 h collection was available. Severe PE was defined according to systolic blood pressure ≥ 160 mmHg or diastolic blood pressure ≥ 110 mmHg, proteinuria ≥ 3 g in 24-h urine specimen or ≥ 3+ on deep stick as above [12, 21]. Preterm PE cases and Early PE were all delivered before 37 and 34 weeks, respectively [6].

A multiple regression analysis was performed to analyze covari-ates that could affect marker values, including gestational age, body mass index (BMI), smoking, maternal age, parity and conception through IVF. The correlation of each confounder with PP13 level was assessed beyond the step-wise regression. Covariates found to be significant from step-wise regression were selected for adjust-ing PP13. For the current study, maternal serum PP13 levels were expressed as gestational week specific multiple of the median (MoM) [7, 11, 32]. MoM was further adjusted to BMI, method of conception, smoking status, parity, and maternal age. Although patients reported their racial origin, we were unable to use this information. This was because, as was frequently reported by others studies for Israel, a high rate of intermarriage among individuals of different ethnic ori-gins yielded a majority of a mixed Sephardic and Ashkenazi origin (28). MoM calculations were updated every 9 months during the study to better account for a potential change in patient characteris-tics during the course of the testing period.

Test Report form informed the patient of having the following: 1) PP13 MoM above or below the cutoff of 0.4 MoM or 2) major RFs for PE. The definition of being at risk to PP13 was based on having PP13 ≤ 0.4 MoM or having major RFs or both. The list of major RFs



Meiri et al., Aspirin prevention of preeclampsia predicted with PP13 593

Table 1A Patient characteristics at enrolment and at delivery.

Parameter

Unaffected singletonn = 757

PEn = 63

P-value

Characteristics at enrollment Maternal age (years) 30 (18–54) 32 (21–47) NS Gestational age at enrollment (weeks) 12 (6–14) 11 (7–13) NS Nullipara 405 (54) 24 (38) < 0.001 Caucasian 717 (95) 61 (97) NS Smoking 27 (3.6) 2 (3.2) NS BMI (kg/m2) 22.3 (17–47) 24.5 (18–40) NS MAP at enrollment (mmHg) 77 (57–113) 89 (66–103) < 0.001 In vitro fertilization 55 (7.3) 11 (17.5) < 0.001 All Risk factors 262 (34.6) 33 (52.4) < 0.001 Placental protein 13 (multiple of the medians) 0.83 (0.08–2.5) 0.27 (0.0–1.5) < 0.0001Characteristics at delivery Gestational age at delivery (weeks) 39.3 (37–42) 38 (27–42) NS Cesarian section 209 (27.6) 38 (60.3) < 0.001 MAP at delivery (mmHg) 80 (65.7–95) 119 (109–168) < 0.001 Urine protein (g/dL) 0 (0–0.4) 1.050 (0.4–9.0) < 0.001 Maternal hospitalization (days) 3 (0–7) 5 (2–14) < 0.005 Newborn birth weight (g) 3220 (2220–4200) 2775 (1120–4000) < 0.005 Newborn hospitalization (days) 3 (0–28) 5 (2–54) < 0.001

Continuous values are presented as medians (range) and categorical values are presented as n (%); NS = Not Significant.

included the following: 1) maternal diseases (chronic hypertension or diabetes or kidney disorders) or cardiovascular diseases, phos-pholipid syndrome, thrombophilia, and lupus erythromatosus; 2) a history of PE; 3) a BMI > 35 or maternal age > 40; and 4) concep-tion by assisted fertility [IVF or Intra-cytoplasmic sperm injection (ICSI)]. Patient prior risk was semi quantified according to the arithmetic sum of RF categories (e.g., between zero to four). MAP was calculated as [(diastolic blood pressure × 2)+systolic BP]/3 (6), and converted to MoM according to the mean value for non-PE patients.

Patient management followed the guidelines of the Israel Soci-ety for Maternal Fetal Medicine Bar [3]. It remained the physician’s decision to prescribe aspirin based on major RFs alone, based on PP13 test results alone, or based on having both. Aspirin was pre-scribed for daily use at 75 mg/day between gestational weeks 14–35; otherwise, the high risk were managed by close surveillance.

Statistical analysisPP13 MoMs were compared using a two-tailed non-parametric Wil-coxon Rank Sum test. Given that PP13 levels follow an approximately log Gaussian distribution [20, 22], standard deviations of log10 MoM were estimated from the 10th to 90th percentile range divided by 2.563. DR and FPR were assessed retrospectively according to test perfor-mance or by receiver operating characteristics (ROC) analysis using MoM values. Odds Ratio (OR) was determined by [Sensitivity/(1–Sen-sitivity)]/[(1–Specificity)/Specificity)]. Baseline and delivery vari-ables were compared among singletons using Fisher’s exact test for categorical variables and the Wilcoxon Rank Sum test for continuous variables. Analysis was performed by the statistical laboratory at Ziv Medical Center, Galil Faculty of Medicine, Bar Ilan University, Safed, Israel.

Results

Development of PE and patient characteristics

Data were available for 820 singleton gestations deliv-ered beyond 22 weeks. PE occurred in 63 women (7.7%), of which six had early PE (delivery < 34 weeks), 21 had preterm PE (delivery at 340–36+6 weeks), and 36 had PE at term (delivery ≥ 370 weeks). PE was combined with intra-uterine growth restriction (IUGR) in 12 subjects and was complicated by the hypertension elevated liver enzyme and low platelets (HELLP) syndrome in four subjects.

At enrollment, the PE group median MAP was signifi-cantly higher by 12 ± 3 mmHg compared with the unaffected group (P < 0.0001) (Table 1A). In addition, a larger propor-tion of women in the PE group conceived by IVF compared with those who were unaffected (17.5% vs. 7.3%, P < 0.0001). The proportion of unaffected patients with any RF was higher in the current study compared with other pregnancy cohorts in Israel (34.6% of the unaffected group compared with 11.1% in the previously used cohorts) [11, 28]. Regard-less of the above, the proportion of women who had any RF was higher in the PE group compared with that of the unaffected group (52.4% vs. 34.6%, P < 0.001). The pro-portions of patients with 1) nephropathy; 2) previous PE, gestational hypertension or IUGR; and 3) those who con-ceived by assisted fertility, were significantly higher in the




PE group (P < 0.001 for all parameters). No differences were observed in terms of maternal age, BMI, and parity between the PE and the unaffected group (Table 1B). Unlike other studies [19, 29], there were significantly less nullipara in the PE group compared with the unaffected group (P < 0.001). At delivery, the PE group had higher proteinuria and MAP compared with the unaffected group (P < 0.001 for each parameter; Table 1A). Among women with PE, more cesar-ian sections were performed (60% vs. 27.6%, P < 0.001), their newborns were smaller by 445 ± 108 g on average, and were kept longer at intensive care units (Table 1A).

MoM adjustment during the study

The correlation between PP13 level to the various con-founders and the significance of PP13 distribution accord-ing to each parameter is detailed in Table 2. Similar to previous studies [11, 30], PP13 levels moderately increased with GA and decreased with BMI (r > 0.9, P < 0.0001, for each). PP13 levels significantly increased with age among women > 40 years (11.9%) but moderately increased in younger women as previously described [11]. Meanwhile, the correlation between PP13 levels and smoking or parity remained low [11, 12] and was even lower for those who conceived by IVF; however, all the above co-variates were utilized to adjust PP13 to MoM (Table 2).

Table 1B Frequency of risk factors.

Risk factors Unaffected singleton

n = 757

PEn = 63

P-value

Maternal disease in this pregnancy Chronic hypertension in current pregnancy 3.2% 0 0.09 Pre-gestational diabetes 0.6% 0 0.17 Nephropathy 0.1% 7.9% < 0.001 Antiphospholipid antibodies (APLA) syndrome 0.6% 0 0.13 Thrombophilia 1.7% 0 0.11 Lupus 0.1% 0 0.19 Others 0.4% 0 0.15Total with maternal disease in this pregnancy 10.3% 11.1% 0.17Maternal Demography Maternal age > 40 years 6.2% 3.2% 0.07 BMI > 35 years 3.8% 7.9% 0.09Total maternal demography risk factors 9.0% 10.2% 0.13PE or GH or intra-uterine growth restriction in previous pregnancy 20.5% 34.9% < 0.001In vitro fertilization 7.3% 17.5% < 0.001Total with significant risk factor/s (%) 34.6% 52.4% < 0.005

Women often had more than only 1 maternal disease (e.g., chronic hypertension and pre gestational diabetes or thrombophilia with nephropathy). Thus, the total frequency of women with maternal disease in this pregnancy is smaller than the arithmetic sum of the differ-ent frequency of each maternal disease. This is also true in the case of maternal demography where some women are both obese and at age > 40 years. Similarly, women who conceived through IVF may also have maternal diseases or at advanced age. Thus, the total frequency of women with any risk factor appearing in the last line is smaller from the arithmetic sum of all the individual frequencies.

Table 2 Correlation between PP13 and confounders.

Parameter Correlation P-value by t-test

Gestation week r = 0.974 < 0.0001Maternal body mass index r = 0.916 < 0.0001Smoking r = 0.78 < 0.01Maternal age r = 0.99 < 0.01Parity r = 0.74 < 0.05In vitro fertilization r = 0.67 < 0.05

A multiple regression analysis was performed to analyze covari-ates that could affect marker values including GA, BMI, smoking, maternal age, parity, and conception through IVF. Covariates found to be significantly different from step wise regression were selected for adjusting PP13.

Comparison of PP13 levels between unaffected and PE pregnancies

The median MoM PP13 level in the 63 PE cases was lower than in the 757 unaffected pregnancies (0.28 median MoM vs. 0.83 median MoM, respectively, P < 0.0001). As described in a previous work [30] after excluding 115 cases treated with aspirin, the median MoM PP13 level was 0.45 in PE cases (n = 43) compared with 1.0 in unaffected ones (n = 656) (P < 0.01, Figure 1 and Table 3). Given that PP13 was determined prior to aspirin treatment, this difference is attributed to PE prevention by aspirin. The median PP13




MoM did not vary significantly among term, preterm, or early PE or if PE was combined with IUGR. PP13 levels in patients who had gestational diabetes, constituent preterm delivery, or gestational hypertension were also similar to those in the unaffected group (data not shown).

Prospective prediction of PE-PP13 alone

Screening for PE by PP13 alone predicted 80% of cases at 20% FPR, and at 13% FPR when aspirin-treated

patients were excluded. For RFs alone, screening for PE by any RF identified 55% of cases at 45% FPR (OR = 1.49, Table 4). Selecting for RFs, nephropathy, previous PE, or IVF increased the OR to 2.9 (data not shown). Fpr PP13+RFs, pre-diction by combining PP13 with all of the RFs yielded a DR of 85% at 15% FPR (OR = 32.1, Table 4). Using only nephropa-thy, previous PE or IVF increased the DR to 87% at 12% FPR (OR = 41.51) (data not shown). MAP was not included in the prospective panel to determine the risk for PE but was col-lected throughout the study. The DR for MAP alone was 43% at 15% FPR (OR = 40.43). Exclusion of the aspirin-treated

2.5

2.0

1.5

PP13

MoM

1.0

0.5

0

Unaffected Unaffected(ASP+)

Unaffected(ASP-)

All PE Preterm PE Early PE PE+IUGR

Figure 1 Distribution of PP13 MoM.Box plot of Median MoM of PP13 showing the unaffected pregnancy group and various PE subgroups; PE = preeclampsia, IUGR = intra-uterine growth restriction, ASP = aspirin, ASP+ = treated with aspirin, ASP– = untreated.

Table 3 PP13 multiple of the medians (MOM).

Group n Median MoM

(95% confidence interval)

P-value (compared with unaffected w/p aspirin

Unaffected without aspirin 656 1.00 (0.95–1.13) All unaffected 757 0.83 (0.72–1.03) < 0.01Unaffected treated with aspirin after test 101 0.37 (0.48–0.73) < 0.001All PE 63 0.28 (0.09–0.52) < 0.0001PE without aspirin 45 0.44 (0.28–0.69) < 0.0001a

PE treated with aspirin after test 18 0.24 (0.08–0.49) < 0.0001a

PE subtypes 25 0.27 (0.21–0.34) < 0.0001 Early PE 4 0.28 (0.24–0.31) < 0.05 PE+IUGR 13 0.24 (0.15–0.25) < 0.001 Preterm PE 25 0.27 (0.21–0.34) < 0.0001Assisted fertility Unaffected – spontaneous 701 0.96 (0.93–1.00) Unaffected+IVF - 55 0.72 (0.63–0.78) < 0.001 PE-spontaneous 52 0.34 (0.19–0.53) PE + IVF 11 0.26 (0.09–0.38)

MoM = multiple of the medians, IVF = in vitro fertilization, PE = preeclampsia, IUGR = intra-uterine growth restriction.




pregnancies yielded a DR of 49% and a higher OR. However, as was previously described by Poon et al. and by Akolekar et al. [1, 24], combining MAP with PP13 and all RFs increased the DR to 93% at FPR = 10% (OR = 119.6, Table 4), and to 94% at 9% FPR when those treated by aspirin were omitted.

ROC analysis

ROC curve analysis (Figure 2 and Table 5) showed that the area under the curve (AUC) was 0.84 and the DR was 52%

Sens

itivi

ty

1.0

PP13 PP13+RF PP13+RF+MAPRF

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

00 0.1 0.2 0.3 0.4

1-Specificity

0.5 0.6 0.7 0.8 0.9 1.0

Figure 2 Receiver operation characteristic (ROC) curve analysis.Placental protein 13 (PP13)-blue line, Major Risk Factors for PE-green line, PP13+RF-red line, PP13+RF+MAP-black line

Table 5 Sensitivity and specificity from ROC analysis.

Marker Area under the curveMean (95%CI)

Sensitivity% (95% CI)

10% FPR 15% FPR 20% FPR

PP13 0.84 (0.78–0.90) 52 (44–55) 76 (72–81) 81 (76–57)RF 0.63 (0.55–0.78) 18 (16–20) 55 (49–61) 5 (49–61)PP13+RF 0.89 (0.81–0.93) 59 (89–93) 91 (89–93) 100PP13+RF+MAP 0.93 (0.87–0.95) 93 (87–100) 100

CI = confidence interval, FPR = false positive rate, PP13 = placental protein 13, RF = risk factors, MAP = mean arterial pressure.

Table 4 First trimester prediction of PE.

Marker Case detected

Detection rate

False positive rate

Odds ratio

PP13 50/63 80% 21.5% 14.6RFs 35/63 55% 45% 1.49PP13+RF 53/63 85% 15% 32.1PP13+RF+MAP 59/63 93% 10% 119.6

PP13 = placental protein 13, RF = risk factors, MAP = mean arterial pressure.

for PP13 alone at 10% FPR. The AUC = 0.63 and DR = 18%, for RFs alone, and AUC = 0.89 and DR = 59% for PP13+RFs. Combining PP13 with RFs and MAP increased the AUC and the DR to 0.93 and 93%, respectively (Table 5).

Effect of treatment with aspirin on PE incidence

The women’s compliance with the doctor’s decision to treat them with aspirin was 95%. As described in Figure 3A–D, there were 377 women with PP13 levels within normal range who did not have any prior RFs, and none was treated by aspirin. Only 2 (0.53%) women developed PE. Meanwhile, the group of first trimester PP13 ≤ 0.4MoM and no RFs included 127 women. Of this group, there were 31 women treated with aspirin and only one (3.2%) developed PE compared with 26 PE cases among the 96 women (27.08%) who were not treated with aspirin. Next, in the group of 219 women who had only RFs (but PP13 levels > 0.4MoM), there were 39 who were treated by aspirin and only five developed PE (12.8%) com-pared with five PE cases out of 180 (2.77%) of women who were untreated. Finally, of the 97 women who had both low PP13 levels and RFs, eight out of 45 (17.78%) women who were treated with aspirin developed PE compared with 16 out of 52 untreated women (30.77%).

Discussion

Major findings

Performed in a clinical setting, we found that RFs were weak predictors of PE compared with PP13 and MAP; however, combining these three parameters increased their prediction power to good clinical performance by WHO criteria [34]. Second, we also found that the efficacy of aspirin use before 16 weeks in PE prevention appeared to be related to the way the risk was determined, thus




emphasizing the importance of low PP13 for estimating the effectiveness of aspirin in PE prevention.

RFs

RFs were found to be low predictors, either individually or combined; however, nephropathy, assisted fertility, and previous PE seemed to be better predictors of PE compared with other known prior RFs, such as diabe-tes mellitus, maternal age, or high BMI. Nephropathy is

35A30

25

20

15

Pree

clam

psia

(%

)

10

5 3.2%

Low PP13 Low PP13+Risk factorsRisk factors

12.8%

2.7%

17.7%

30.7%

27%

Treated Untreated

0

Low PP13 and RFOnly RFOnly Low PP13Aspirin effect

B

1.730.218.43Relative ratio

PP13 = placental protein 13, RF = risk factors

D

No RiskN= 377

PP13 + RFN=96

Only RFN=219

Only low PP13N=127

Group

2 (3.2%)24 (38.1%)10 (15.9%)27 (42.8%)Preeclampsia

PP13=placental protein 13, RF=risk factors

C

Patie

nt (

#)

400

350

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250

200

150

100

50

0

27

100

10

209

24

72

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375

Low PP13 Low PP13+Riskfactors

No RiskRisk factors

UnaffectedPE

Figure 3 (A) The effect of aspirin on PE by risk group. (B) Treatment effectiveness by risk group. (C) Frequency of PE by risk group. (D) PE frequency by risk group.

rare among women at fertility age, and women with the disorder form a very high risk group. Previous PE cases accounted for a significant proportion and are known to be at high risk to PE. The proportion of women who con-ceive by IVF is constantly increasing. Thus, these three RFs should be gaining greater attention when evaluating the risk to develop PE in various algorithms and compared with the current WHO [34] or NICE [19] guidelines

Screening for PE by PP13

Overall, the use of PP13 for screening of PE appeared to be effective, particularly for young nuliparous with unknown risk. PP13 alone predicted 80% of PE cases at 20% FPR, as reported previously by Gonen et al. [11], but only predicted 54% of cases at 10% FPR, which can be mainly attributed to the relatively low proportion of early onset cases. In a recent meta-analysis, Huppertz et al. [15] found that for 15,585 patients in 19 studies, the DR for reduced levels of maternal blood PP13, by itself as a first trimester marker of PE assessed at 10%, FPR ranged from 47% for all cases of PE (n = 1109) to 76% for combined preterm and early onset PE (n = 532) [15]. These results correspond with the clinical performance reported here.

Recently, Gizurarson et al. [10] demonstrated in exper-imental pregnant rats that PP13 reduced blood pressure and increased angiogenesis of the utero-placental artery, causing artery expansion that, in turn led to increased size of the placenta and increased newborn weight. Other studies showed a correlation between low PP13 levels and a smaller placenta [14], less elaboration of the utero-pla-cental vasculator [12], calcium flux [2], interaction with phospholipid regulation and the lipid raft system [2, 33], and genetic factors [8, 31]. A new theory by Malchiore and Thilaganathan [18] connects between the risk to develop PE to the maternal heart function and its capability to adjust to the burden of pregnancy. Therefore, it is impor-tant to investigate the major causes for reduced PP13. As discussed later, it appears that identifying patients with low PP13 level improves the prediction of the efficacy of a subsequent aspirin treatment.

Combining low PP13 with RFs

PE prediction was more effective for this combination com-pared with each marker alone. As specified above, in the combined analysis when the RFs included only previous PE, conception by IVF and having nephropathy yielded better results. The improvement in this combination can be due




to several reasons. First, nephropathy probably enhanced PP13 removal from the body through the kidney, thereby reducing the circulating blood PP13 levels, and contribut-ing to higher prediction accuracy. Second, previous PE was 10% more frequent in the PE group (30.4%) compared with the unaffected group (20.5%, P < 0.001). In addition, there was an inverse correlation (R2 = 0.79 by ANOVA) between low PP13 in the current pregnancy and PE in the previ-ous pregnancy, especially among women who developed preterm and early PE and among those with PE compli-cated by IUGR. Thus, previous PE may indicate a tendency for reduced PP13 expression and for impaired placentation, which could be derived by the ovum and maternal body “memory” or other intrinsic factors. Third, IVF frequency was 27% in PE cases compared with 7.7% in the unaffected ones. The correlation between low PP13 and conceiving by IVF has R2 = 0.91 (ANOVA). This might be related to the underlying metabolic or hormonal impairment, which can lead to increased PE in IVF patients or for the need of these patients to undergo IVF in the first place [5].

Screening by MAP

MAP by itself appears to be a reasonable predictor for PE, as was previously demonstrated by Poon et al. [21]. It had a very large impact when added to a panel consisting of PP13 and RFs. Given that all three parameters are very simple and can be used in most clinical set-ups, their com-bination may be a good fit for the majority of the health-care systems in both developing and developed countries.

Aspirin

Enroling women who were aware of their condition and of the need to modify pregnancy management was probably associated with the 95% compliance of the women with aspirin treatment. In this study, aspirin was administered prior to GA of 16 weeks as recomended by Bujold et al. [4]. The generalization from this study was limited by the fact that the decision to treat with aspirin in the clinical setting was not randomized, but based on how the attending phy-sician perceived the risk of the women to develop PE, his/her trust with the PP13 test [15], and his/her convincing of the anticipated efficacy of aspirin [4].

Perceived aspirin efficacy

The study showed that only 17.8% of the women were treated with aspirin when the risk was determined on the

basis of RFs alone. These results indicate that currently, the professional community is not convinced that there is benefit to be gained in the use of aspirin in preventing PE in cases of having RFs. In comparison, when the risk was determined by low PP13 levels alone, 24.4% of the women were treated with aspirin, and when the risk was deter-mined by having both RFs and low PP13, almost half of the patients (46.4%) were treated with aspirin. This analysis indicates that physicians trust the PP13 test results on their own, but only a little better than they do when the risk is defined based on RFs alone. Under such circumstances, they may undertreat nuliparous women whose risk was determined by PP13 alone. However, having indication that the risk for developing PE was derived from two inde-pendent sources of information encouraged the physicians to use aspirin for prevention. These results showed that doctors actually looked for a comprehensive risk assess-ment before opting to have the aspirin treatment.

The model of aspirin work

Grimpel et al. [12] tested placental explants obtained from first trimester pregnancy termination and cultured them for 24 h with increasing doses of aspirin or without it. They have found that aspirin increases the release of PP13 from the placental explants in a dose-dependent manner, with maximum effect obtained with 150 mg per day. These results derived from in-vitro models may account for the higher effectiveness of aspirin when used in the context of risk predicted based on low PP13. A new model by Wright et al. [36] for PE prediction has estimated that PE frequency could have reached 100% if pregnancy lasted 80 instead of 40 weeks. Using the same rationale, aspirin is proposed to shift to the right (later time) the fre-quency of PE. Support for this model may be offered from PP13 MoM assessment for all unaffected cases compared with those untreated with aspirin (MoM = 0.85 compared with MoM = 1.0, respectively). This can also be compared with median MoM = 0.28 in the entire PE group compared with MoM = 0.45 among those who were not subsequently treated with aspirin.

Prediction and aspirin efficacy

Contrary to the perceived benefit of aspirin by the attend-ing physicians, this drug appeared to be more effective when the risk was determined by low PP13 levels alone compared with a risk predicted based on PP13 combined with RFs (PE frequnecy of the aspirin treated groups was




3.2% in the low PP13 alone compared with 17.78% in the RFs combined with low PP13 group) (Figure 3). This indi-cates that aspirin efficacy may be related to the cause underlying the risks for developing PE. Low PP13 indicates a placental derived risk, whereas RFs are mainly derived of maternal factors. Thus, aspirin may not be a sufficient preventing agent when the risk for PE is multifactorial.

Limitations

The main limitation of this study lies in the fact that it has been conducted within a clinical setting, in which the use of aspirin is not randomized but a result of the physician’s attitude to several factors, including the risks involved for the patients, PP13 test accuracy, and aspirin efficacy. This bias prohibits generalization of the study results on the effect of aspirin in PE prevention in the risk prediction setting we have earlier described. In addition, the small size of the PE group (n = 63) prevents meaningful sub-analyses of individual RFs using parametric regression, while the a-parametric analysis suffers due to its nature of eliciting overfitting. Thus, fine tuning is anticipated with a larger cohort. In addition, the study results may be affected by socio-economic bias due to the enrolment of patients who could pay for the tests, since the test is not yet included in routine public health services. There is also a disproportionate enrolment of patients with high prior risk compared with the national frequency of RFs in Israel [11]. While the frequency of PE is much higher than that of the general population of Israel [5], it has already been found by Khalil et al. [16] that the prediction of PE by PP13 in high-risk populations has similar efficacy compared with PP13 efficacy in predicting PE in the low-risk groups.

ConclusionThis study reported the screening potential of the PP13 biomarker in predicting PE in the first trimester of preg-nancy. It appeared that the use of RFs in combination with PP13 and MAP for predicting PE risk yielded better performance compared with each individual parameter alone. These results provide additional reasons for issuing guidelines related to PE prediction by first trimester screening. Women who were identified to be at high risk either by having low PP13, major RFs or both, were either managed by close surveillance or by daily use of aspirin at their doctor’s discretion. Even if the use of aspirin was not randomized, this study shows a very important result and a first hint to a treatment strategy of PE based on first

trimester screening. Members of our team are currently participating in a multi-national randomized study, the ASPRE project funded by the EC, which aims at evaluat-ing the use of aspirin versus placebo for PE prevention based on a multi-marker risk prediction. The study will recruit 33,000 women during the first trimester, and will likely provide sufficient evidence for correlating first tri-mester screening to the risk of developing PE as well as the benefit of using aspirin to prevent such condition.

Authorship contributions: All authors contributed to manuscript writing. R.G. supervised clinical definitions, service design, and held a help desk for low-dose aspirin. A.H. was in charge of collecting the delivery outcomes and supervised the completion of the combined enrolment and delivery database. Laboratory personnel training, acquisi-tion of patient data, and uploading them to the database were performed by G.F., V.K., A.C. and Y-I.G. V.K. was the clinical service manager. Y-I.G. was responsible for algo-rithm calculation and adjustment during the course of the study, and G.F. oversaw the making of PP13 kits and conducting quality analysis. M.S. produced the PP13 for the study, and assisted the analysis. H.M. was involved in all aspects of the study, including securing MOH approval, service design, data acquisition and analysis. A.S-N. per-formed the statistical analysis.

Acknowledgments: The authors wish to thank Dr. Ruti Cohen and the Hy-Laboratory team for their assistance in collecting the pregnancy outcomes. We also thank Zer Hi-Tech and American Medical Laboratories for performing the PP13 testing and improving test accuracy. The authors also wish to thank all former employees of Diagnostic Technologies for their valuable assistance in making the kits and all the support they provided in many ways. The preparation of this manuscript was sponsored in part by the EC FP7 project ASPRE (#601852.)

Conflict of interest statement

Author’s conflict of interest disclosure: A.H. used to be an employee of and H.M. is currently a consultant of Hy Laboratories, which acquired the rights to use the PP13 technology. H.M., G.F., M.S., V.K., A.C., and Y-I.G. were employees of the former owner of PP13 technologies until the company stopped its operation in September 2010. They then volunteered time and efforts to the continua-tion of the clinical service until February 2011.

Received November 3, 2013. Accepted February 7, 2014. Previously published online March 7, 2014.




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The authors stated that there are no conflicts of interest regarding the publication of this article.



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