DOI 10.1515/jpm-2013-0298J. Perinat. Med.

2014; 42(5): 591601

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

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 screening 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 75mg 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 compared 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, particularly 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 prevention by aspirin was most effective when the risk was
determined by low PP13 alone, less effective for combining low PP13 with RFs, and ineffective when determined
by RFs alone.
Conclusion: When PE risk is determined by low first trimester 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; placental protein 13; preeclampsia; risk factors; risk prediction; serum biomarkers.
*Corresponding author: Dr. Hamutal Meiri, TeleMarpe Ltd., 12
Barasani Moshe St., Tel Aviv 69121, Israel, Tel.: +972-54-7774762,
E-mail: hamutal.meiri@gmail.com
Marei Sammar: Department of Biotechnology Engineering, ORT
Braude College, Karmiel, Israel
Ayelet Herzog: Hy-Laboratories Ltd., Rehovot, Israel
Yael-Inna Grimpel, Galina Fihaman, Aliza Cohen and Vered Kivity:
TeleMarpe Ltd., Tel Aviv, Israel
Adi Sharabi-Nov: Ziv Medical Center, Safed, Israel

Introduction
Preeclampsia (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 comprises new-onset hypertension coupled with damage
to the kidney, occasionally to the liver, and to the
cardiovascular system [13, 29, 34]. Although the etiology of PE remains unclear, it is attributed to multifactorial 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 detection rate (DR) could reach 80%93% for 5%10% false
positive rate (FPR) [1, 7].
Placental protein 13 (PP13) is a galectin, which is generated by the syncytiotrophoblast and released into the
maternal blood [12, 27, 32]. A recent meta-analysis performed 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 invitro diagnosis in 2006 for PP13 ELISA kits as an aiding
tool for predicting pregnancy disorders. Israels 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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592Meiri etal., Aspirin prevention of preeclampsia predicted with PP13

PP13 Kit performance in the perinatal clinical, as was performed 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 Organization (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 recommendation; however, the guidelines left the decision to use
aspirin to the individual physician [3] . The guidelines
remained silent about using aspirin when the patients
risk was established by any biomarker, including PP13.
This was because Israel was the first to include a predicting 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 methods

This 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 <18years 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 analysis 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 pregnancy 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 pregnancies (n=36). In vitro fertilization (IVF) patients (n=66), including
those with oocyte donation (n=17), were enrolled but only after discontinuing 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 3090min and then centrifuged for 15min at 3000g, after which
the serum was collected and stored at 20C 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 technician 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 history were obtained at enrolment. PP13 analysis was performed by
Zer HiTech Central Laboratory and by American Medical Laboratories, 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 conducted 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 patients 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 criteria 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 normotensive women combined with proteinuria of 300mg in 24 h, or two
readings of at least 2+ on dipstick analysis of midstream or catheter
urine specimens, if no 24h 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 covariates 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 adjusting 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 origins 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 characteristics 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

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Meiri etal., Aspirin prevention of preeclampsia predicted with PP13593

included the following: 1) maternal diseases (chronic hypertension
or diabetes or kidney disorders) or cardiovascular diseases, phospholipid syndrome, thrombophilia, and lupus erythromatosus; 2)
a history of PE; 3) a BMI >35 or maternal age >40; and 4) conception 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 pressure2 )+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 Society for Maternal Fetal Medicine Bar [3]. It remained the physicians
decision to prescribe aspirin based on major RFs alone, based on
PP13 test results alone, or based on having both. Aspirin was prescribed for daily use at 75mg/day between gestational weeks 1435;
otherwise, the high risk were managed by close surveillance.

Statistical analysis
PP13 MoMs were compared using a two-tailed non-parametric Wilcoxon 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 performance or by receiver operating characteristics (ROC) analysis using
MoM values. Odds Ratio (OR) was determined by [Sensitivity/(1Sensitivity)]/[(1Specificity)/Specificity)]. Baseline and delivery variables were compared among singletons using Fishers 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 delivered 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 34036+6 weeks), and 36 had PE at
term (delivery 370 weeks). PE was combined with intrauterine 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 significantly higher by 123 mmHg compared with the unaffected
group (P<0.0001) (Table 1A). In addition, a larger proportion 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]. Regardless 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 proportions of patients with 1) nephropathy; 2) previous PE,
gestational hypertension or IUGR; and 3) those who conceived by assisted fertility, were significantly higher in the

Table 1APatient characteristics at enrolment and at delivery.

Parameter

Characteristics at enrollment
Maternal age (years)

Gestational age at enrollment (weeks)

Nullipara

Caucasian

Smoking

BMI (kg/m2)

MAP at enrollment (mmHg)

In vitro fertilization

All Risk factors

Placental protein 13 (multiple of the medians)

Characteristics at delivery
Gestational age at delivery (weeks)

Cesarian section

MAP at delivery (mmHg)

Urine protein (g/dL)

Maternal hospitalization (days)

Newborn birth weight (g)

Newborn hospitalization (days)

Unaffected singleton
n=757

PE
n=63

30 (1854)
12 (614)
405 (54)
717 (95)
27 (3.6)
22.3 (1747)
77 (57113)
55 (7.3)
262 (34.6)
0.83 (0.082.5)

32 (2147)
11 (713)
24 (38)
61 (97)
2 (3.2)
24.5 (1840)
89 (66103)
11 (17.5)
33 (52.4)
0.27 (0.01.5)

39.3 (3742)
209 (27.6)
80 (65.795)
0 (00.4)
3 (07)
3220 (22204200)
3 (028)

38 (2742)

38 (60.3)

119 (109168)

1.050 (0.49.0)

5 (214)
2775 (11204000)

5 (254)

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

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P-value

NS

NS
<0.001

NS

NS

NS
<0.001
<0.001
<0.001
<0.0001

NS
<0.001
<0.001
<0.001
<0.005
<0.005
<0.001

594Meiri etal., Aspirin prevention of preeclampsia predicted with PP13

Table 1BFrequency of risk factors.
Risk factors

Unaffected
singleton
n=757

Maternal disease in this pregnancy

Chronic hypertension in current pregnancy

Pre-gestational diabetes

Nephropathy

Antiphospholipid antibodies (APLA) syndrome

Thrombophilia

Lupus

Others

Total with maternal disease in this pregnancy

Maternal Demography

Maternal age >40 years

BMI >35 years

Total maternal demography risk factors

PE or GH or intra-uterine growth restriction in previous pregnancy

In vitro fertilization

Total with significant risk factor/s (%)

PE
n=63

P-value

0
7.9%

0
11.1%

3.2%
7.9%
10.2%
34.9%
17.5%
52.4%

3.2%
0.6%
0.1%
0.6%
1.7%
0.1%
0.4%
10.3%
6.2%
3.8%
9.0%
20.5%
7.3%
34.6%

0.09
0.17
<0.001
0.13
0.11
0.19
0.15
0.17
0.07
0.09
0.13
<0.001
<0.001
<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 different 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.

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 cesarian sections were performed (60% vs. 27.6%, P<0.001),
their newborns were smaller by 445108 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 confounders and the significance of PP13 distribution according 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).

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

Table 2Correlation between PP13 and confounders.

Parameter

Correlation

Gestation week

Maternal body mass index

Smoking

Maternal age

Parity

In vitro fertilization

r=0.974
r=0.916
r=0.78
r=0.99
r=0.74
r=0.67

P-value by t-test

<0.0001
<0.0001
<0.01
<0.01
<0.05
<0.05

A multiple regression analysis was performed to analyze covariates 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.

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Meiri etal., Aspirin prevention of preeclampsia predicted with PP13595

2.5

PP13 MoM

2.0

1.5

1.0

0.5

0
Unaffected

All PE

Preterm PE Early PE

PE+IUGR Unaffected Unaffected

(ASP+)
(ASP-)

Figure 1Distribution 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.

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, prediction by combining PP13 with all of the RFs yielded a DR
of 85% at 15% FPR (OR=32.1, Table 4). Using only nephropathy, 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 collected throughout the study. The DR for MAP alone was 43%
at 15% FPR (OR=40.43). Exclusion of the aspirin-treated

Table 3PP13 multiple of the medians (MOM).

Group

Median
MoM

(95% confidence
interval)

P-value (compared with

unaffected w/p aspirin

Unaffected without aspirin

All unaffected

Unaffected treated with aspirin after test

All PE

PE without aspirin

PE treated with aspirin after test

PE subtypes

Early PE

PE+IUGR

Preterm PE

Assisted fertility
Unaffected spontaneous

Unaffected+IVF
PE-spontaneous

PE + IVF

656
757
101
63
45
18
25
4
13
25

1.00
0.83
0.37
0.28
0.44
0.24
0.27
0.28
0.24
0.27

(0.951.13)
(0.721.03)
(0.480.73)
(0.090.52)
(0.280.69)
(0.080.49)
(0.210.34)
(0.240.31)
(0.150.25)
(0.210.34)

701
55
52
11

0.96
0.72
0.34
0.26

(0.931.00)
(0.630.78)
(0.190.53)
(0.090.38)

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

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<0.01
<0.001
<0.0001
<0.0001a
<0.0001a
<0.0001
<0.05
<0.001
<0.0001

<0.001

596Meiri etal., Aspirin prevention of preeclampsia predicted with PP13

Table 4First trimester prediction of PE.
Marker

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).

Case Detection
False Odds ratio
detected
rate positive rate

PP13

RFs

PP13+RF

PP13+RF+MAP

50/63
35/63
53/63
59/63

80%
55%
85%
93%

21.5%
45%
15%
10%

14.6
1.49
32.1
119.6

Effect of treatment with aspirin on PE

incidence

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

pressure.

The womens compliance with the doctors decision

to treat them with aspirin was 95%. As described in
Figure 3AD, 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
PP130.4MoM 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%) compared 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%).

pregnancies yielded a DR of 49% and a higher OR. However,

as was previously described by Poon etal. and by Akolekar
etal. [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%
PP13

RF

PP13+RF

PP13+RF+MAP

1.0
0.9
0.8

Sensitivity

0.7
0.6

Discussion

0.5
0.4

Major findings

0.3
0.2

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

0.1
0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1-Specificity
Figure 2Receiver 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 5Sensitivity and specificity from ROC analysis.
Marker

PP13

RF

PP13+RF

PP13+RF+MAP

Area under the curve

Mean (95%CI)

Sensitivity% (95% CI)

10% FPR

0.84 (0.780.90)
0.63 (0.550.78)
0.89 (0.810.93)
0.93 (0.870.95)

52 (4455)
18 (1620)
59 (8993)
93 (87100)

15% FPR

76 (7281)
55 (4961)
91 (8993)

100

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

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20% FPR

81 (7657)
5 (4961)

100

Meiri etal., Aspirin prevention of preeclampsia predicted with PP13597

35

Treated

Preeclampsia (%)

30

Untreated

30.7%

27%

25
20

17.7%

15

12.8%

10
5

3.2%

2.7%

0
Low PP13

Risk factors

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 conceive 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

Low PP13+Risk factors

Screening for PE by PP13

B
Aspirin effect

Only Low PP13

Only RF

Low PP13 and RF

Relative ratio

8.43

0.21

1.73

emphasizing the importance of low PP13 for estimating

the effectiveness of aspirin in PE prevention.

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 etal. [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 etal. [10] demonstrated in experimental 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-placental 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 important 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.

RFs

Combining low PP13 with 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 diabetes mellitus, maternal age, or high BMI. Nephropathy is

PE prediction was more effective for this combination compared 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

PP13 = placental protein 13, RF = risk factors

C 400
PE

375

Unaffected

350

Patient (#)

300
250
209

200
150
100

100
50
0

72
27

24

10

Low PP13

Risk factors

Low PP13+Risk
factors

No Risk

D
Group
Preeclampsia

Only low PP13 Only RF

N=127
N=219
27 (42.8%)
10 (15.9%)

PP13 + RF
N=96
24 (38.1%)

No Risk
N= 377
2 (3.2%)

PP13=placental protein 13, RF=risk factors

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.

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598Meiri etal., Aspirin prevention of preeclampsia predicted with PP13

to several reasons. First, nephropathy probably enhanced
PP13 removal from the body through the kidney, thereby
reducing the circulating blood PP13 levels, and contributing 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 previous pregnancy, especially among women who developed
preterm and early PE and among those with PE complicated 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 etal. [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 combination may be a good fit for the majority of the healthcare 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 etal. [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 physician 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 determined 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 independent sources of information encouraged the physicians
to use aspirin for prevention. These results showed that
doctors actually looked for a comprehensive risk assessment 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 24h 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 etal. [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 frequency 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 attending 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

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Meiri etal., Aspirin prevention of preeclampsia predicted with PP13599

3.2% in the low PP13 alone compared with 17.78% in the

RFs combined with low PP13 group) (Figure 3). This indicates 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 physicians
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 subanalyses 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 etal. [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.

Conclusion
This study reported the screening potential of the PP13
biomarker in predicting PE in the first trimester of pregnancy. 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 doctors 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 evaluating 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 trimester 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, acquisition 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 algorithm 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. performed 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 HiTech 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
Authors 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 continuation 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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600Meiri etal., Aspirin prevention of preeclampsia predicted with PP13

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