Accepted Manuscript

Association of first-trimester angiogenic factors with placental histological findings in

late-onset preeclampsia

Stefania Triunfo, MD PhD, Francesca Crovetto, MD, Fatima Crispi, MD PhD, Victor
Rodriguez-Sureda, PhD, Carmen Dominguez, PhD, Alfons Nadal, MD PhD, Anna
Peguero, MD, Eduard Gratacos, MD PhD, Francesc Figueras, MD PhD
PII: S0143-4004(16)30056-X
DOI: 10.1016/j.placenta.2016.04.005
Reference: YPLAC 3393

To appear in: Placenta

Received Date: 8 February 2016

Revised Date: 4 April 2016
Accepted Date: 5 April 2016

Please cite this article as: Triunfo S, Crovetto F, Crispi F, Rodriguez-Sureda V, Dominguez C, Nadal
A, Peguero A, Gratacos E, Figueras F, Association of first-trimester angiogenic factors with placental
histological findings in late-onset preeclampsia, Placenta (2016), doi: 10.1016/j.placenta.2016.04.005.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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 ACCEPTED MANUSCRIPT
1 Association of first-trimester angiogenic factors with placental histological findings in late-
2 onset preeclampsia
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5 Stefania Triunfo1, MD, PhD; Francesca Crovetto1,2, MD; Fatima Crispi1, MD, PhD; Victor Rodriguez-
6 Sureda3, PhD, Carmen Dominguez3, PhD; Alfons Nadal4, MD, PhD; Anna Peguero1, MD; Eduard
7 Gratacos1, MD, PhD; Francesc Figueras1, MD, PhD.
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9 BCNatal - Barcelona Center for Maternal-Fetal and Neonatal Medicine (Hospital Clínic and Hospital

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10 Sant Joan de Deu), IDIBAPS, University of Barcelona, and Centre for Biomedical Research on Rare
11 Diseases (CIBER-ER), Barcelona, Spain.
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12 Ca’ Granda, Ospedale Maggiore Policlinico, Dipartimento Ostetricia e Ginecologia; Università degli

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13 Studi di Milano, Milan, Italy.
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14 Biochemistry and Molecular Biology Research Centre for Nanomedicine, Hospital Univeritari Vall d-
15 Hebron, Barcelona, Spain; Centre for Biomedical Research on rare Disease (CIBERER), Instituto de

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16 Salud Carlos III, Spain.
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17 Department of Pathology, Hospital Clinic-IDIBAPS, University of Barcelona, Barcelona, Spain.
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23 Correspondence and reprint request to

24 Stefania Triunfo
25 Maternal-Fetal Medicine Department
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26 Hospital Clinic, University of Barcelona

27 Sabino de Arana 1, 08028
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28 Barcelona
29 Spain
30 Telephone: +34 93 227 5600
31 Fax: +34 (0) 93 227 5605
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32 E-mail: TRIUNFO@clinic.ub.es
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40 SHORT TITLE
41 Angiogenic factors and placental findings in late-onset preeclampsia with placental insufficiency
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1 ABSTRACT

2 Objective: To explore in women with late-onset preeclampsia (PE) the association between maternal
3 levels of angiogenic/antiangiogenic factors in the first trimester of pregnancy and histological
4 findings attributable to placental underperfusion (PUP).

5 Methods: A nested case-control cohort study was conducted in 73 women with pregnancies

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6 complicated by late-onset PE (>34 weeks at delivery) matched with controls. First trimester uterine
7 artery Doppler (UtA); maternal levels of placental growth factor (PlGF) and soluble fms-like tyrosine
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kinase-1 (sFlt-1) were retrieved. Placentas were histologically evaluated using a hierarchical and
9 standardized classification system. One-way ANOVA with linear polynomial contrast or linear-by-
10 linear association test was performed to test the hypothesis of a linear association across study

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11 groups (controls, PE without PUP and PE with PUP).

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12 Results: In 54 (74%) placentas, 89 placental histological findings qualifying for PUP were found.
13 Across study groups, significant values were observed in maternal levels of decreased PlGF (MoM
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14 values: 1.53, 1.41 and 1.37; p<0.001), increased sFlt-1 (MoM values: 3.11, 3.11 and 3.22; p=0.002),
15 increased sFlt-1/PlGF ratio (MoM values: 2.3, 2.3 and 2.44; p<0.001), abnormal UtA Doppler (MoM
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16 values: 1, 1.26 and 1.32; p<0.001), and worse perinatal outcomes in terms of gestational age at
17 delivery, cesarean section for not reassuring fetal status, birth weight and neonatal acidosis.
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18 Discussion: In late-onset PE an imbalance of circulating angiogenic and anti-angiogenic factors

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19 already present at 8-10 weeks of pregnancy was associated with histological findings reflecting
20 placental insufficiency. An early first trimester screening by angiogenic factors might help to identify
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patients with placental involvement among late-onset PE cases.

22 Conclusion: In late-onset preeclampsia, first-trimester uterine Doppler and circulating levels of

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23 angiogenic/antiangiogenic factors are associated with placental underperfusion.

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28 KEYWORDS

29 Preeclampsia; First trimester screening; Placental growth factor; Soluble Fms-like tyrosine kinase-1;
30 Histological Findings; Placental underperfusion.
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1 HIGHLIGHTS (separate file, 85 characters totally).
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Late-onset preeclampsia is associated with adverse perinatal outcomes.
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First study to investigate the relationship between first trimester PlGF-sFlt-1 and placental
4 histology.
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First trimester PlGF and sFlt-1 levels are associated with histological placental insufficiency.
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14 ABBREVIATIONS

15 PE: preeclampsia;
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16 FGR: fetal growth restriction;
17 GA: gestational age;
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18 PlGF: placental growth factor;

19 sFlt-1: soluble fms-like tyrosine kinase-1;
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20 MoM: multiples of normal median;

21 EFW: estimated fetal weight;
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22 UtA: uterine artery;

23 UA: umbilical artery;
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24 MCA: middle cerebral artery;

25 CPR: cerebroplacental ratio;
26 PI: Doppler pulsatility index;
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27 BW: birth weight;

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28 PUP: placental underperfusion

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1 INTRODUCTION

2 Preeclampsia (PE) is a major contributor to maternal and perinatal morbidity and mortality [1,2].
3 Accumulating evidence indicates that a central feature in the pathophysiology of PE is a failure of the
4 physiologic transformation of spiral arteries [3-6]. Although the primary insults for these
5 abnormalities remain elusive [7,8], it is postulated that the resulting poor placentation and reduced
6 blood supply to the placenta in early pregnancy lead to the release of factors into the maternal

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7 circulation causing the systemic manifestations of the syndrome.

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While this pathophysiology is behind the vast majority of cases of early-onset PE, in late-onset PE
9 there is a broader spectrum of involved mechanisms [9]. This has a major clinical impact because
10 whilst a combination of maternal characteristics and some biomarkers (such as blood pressure (BP)

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11 and uterine Doppler evaluation) at first-early second trimesters predict most cases that develop
12 early-onset PE, this same predictive strategy fails to detect the majority of instances of late-onset PE

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13 [10]. This poor predictive performance could be overcome by a better phenotyping of the late PE
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14 syndrome, similarly what has been proposed in other obstetric syndromes (i.e. prematurity) [11].

15 A proportion of women with late-onset PE had signs of placental insufficiency at histological

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16 evaluation [12], and they exhibited an imbalance in their angiogenic profile [13]. Increased risks of
17 adverse perinatal outcome have been described in presence of simultaneous late-onset PE and
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18 placental insufficiency [14]. It follows from this evidence that for an efficient prevention of late-
19 onset PE, targeting those instances that exhibit placental insufficiency is key. However, it remains
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20 unclear whether late-onset PE cases with true placental under perfusion already show an imbalance
21 in their angiogenic profile in the first trimester. This could help to define predictive and preventive
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22 strategies.

23 The aim of this study was to explore the association between maternal plasma concentrations of
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24 angiogenic factors in the first trimester of pregnancy and histological findings attributable to PUP in
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25 late-onset PE.

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1 PATIENTS AND METHODS

2 Participants
3 A nested case-control study nested in a cohort of unselected singleton pregnancies referred for
4 routine first-trimester screening for aneuploidies (11+0-13+6 weeks of gestation) was conducted.
5 Eligible cases were women without chronic hypertension subsequently developing late-onset PE

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6 (delivering above 34 weeks of gestation). Cases were matched with controls, defined as pregnancies
7 not complicated by PE delivering immediately after each case. Exclusion criteria were congenital
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malformations and suspected chorioamnionitis.
9 Plasmatic levels of angiogenic factors were determined in stored samples. Gestational age (GA) in all
10 pregnancies was calculated based on the crown-rump length (CRL) at first-trimester ultrasound [15].

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11 The local Ethics Committee approved the study protocol and each patient provided written informed
12 consent.

13 Data collection
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14 Baseline maternal characteristics, including age, ethnicity, body mass index (BMI), parity, maternal
15 and paternal smoking, known chronic disease (i.e., diabetes mellitus, renal disease, and autoimmune
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16 disorders), and obstetric history were recorded in the hospital database upon study admittance. Data
17 on postpartum follow-up or subsequent complications of pregnancy, ultrasound evaluations, and
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18 perinatal outcomes were also collected prospectively.

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19 Outcome measures
20 PE was defined according to the guidelines of the International Society for the Study of Hypertension
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in Pregnancy. This requires two recordings of systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg at
22 least 4 h apart in previously normotensive women after 20 weeks of gestation, and proteinuria of
23 300 mg or more in 24 h [16].
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24 Small for gestational age (SGA) newborns were defined with birth weight below the 10th centile
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25 according to local standards [17].

26 Intrauterine growth restriction (IUGR) fetus was defined with an estimated fetal weight (EFW) below
27 the 10th centile according to local standards [17], with abnormalities in Doppler evaluation
28 (cerebroplacental ratio (CPR) < 5th centile or uterine artery (UtA) Doppler pulsatility index (PI) > 95th
29 centile) [18,19].

30 Biophysical parameters

31 In each patient transvaginal UtA Doppler assessment was performed at the time of first-trimester

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1 scan (11+0- 13+6 weeks of gestation) in order to measure the PI in the left and right uterine arteries,
2 as previously described [18]. Maternal BP was recorded at time of ultrasound with automated
3 devices OMRON M6 Comfort (OMRON Corporation, Kyoto, Japan). Mean arterial pressure (MAP) was
4 measured in one arm (right or left) while women were seated and after a 5-minute rest. MAP was
5 calculated as: diastolic BP + (systolic BP – diastolic BP)/3.

6 Blood sampling and testing procedures

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7 Venous blood samples were drawn from each woman between 8+0 and 11+6 weeks’ gestation
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(mean 10.6, SD 3.85). They were collected in an EDTA-containing tube and processed within one
9 hour. Plasma was separated by centrifugation at 3000 rpm for 10 minutes at 4°C, and samples were
10 immediately stored at -80°C until assayed.

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11 Levels of PlGF and sFlt-1 in maternal plasma were measured by the corresponding ELISA kits
12 according to the manufacturer's instructions (R&D Systems Europe Ltd, Abingdon, UK). The assay kits

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13 used detect only free analytics, not the bound fractions. All samples were collected, handled, and
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14 stored under the same conditions and tested in duplicate. Plate-to-plate variability was controlled by
15 running an internal control along the samples. Test sera and matched controls were simultaneously
16 and blindly run on the same plates. Intra-assay (<5%) and inter-assay (10%) precision of all kits were
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17 consistent. Linear regression coefficients of standard curves were never <0.99.

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18 Clinical management
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19 Deliveries were attended by a staff obstetrician blinded to angiogenic factor determinations.

20 Continuous fetal heart monitoring was performed during labor, graded (normal, suspicious, or
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abnormal) in terms of presence, type, and length of decelerations; bradycardia; tachycardia; and
22 assessment of variability [20,21]. In instances of two or more suspicious variables or with at least one
23 abnormal parameter unresponsive to digital fetal scalp stimulation, fetal scalp blood pH was tested
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24 [22]. Any pH value <7.15 or <7.20 on two attempts 30 min apart were regarded as abnormal [22].
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25 Cesarean section was done for non-reassuring fetal status, based on intrapartum fetal abnormalities
26 in heart rate and scalp blood pH [22]. Cesarean delivery for non-reassuring fetal status was indicated
27 for a persistently abnormal heart tracing after pessary withdrawal, oxytocin suspension, and a 10-
28 minute intravenous infusion of ritrodine (200 μg/min). All cases with adverse outcome were formally
29 assessed to ensure that the management protocol had been followed correctly.

30 Placental evaluation

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1 Placental examinations adhered to standard laboratory protocol. Fresh and trimmed (after removal
2 of the membranes, cord, and any blood clots, the placenta) placental weight were recorded.
3 Trimmed placental weight centiles were assigned based on GA-specific placental weight charts [23].
4 The feto-placental weight ratio (birth weight: fresh placental weight) was also expressed as a
5 percentile, drawn from GA-specific ranges [24].
6 Placentas were fixed in 10% buffered formalin. After gross examination, routine samples of each

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7 specimen were taken for routine processing: one transverse section of cord, one rolled strip of
8 membranes, and three blocks of villous parenchyma. All macroscopic lesions were sampled as well.
9 Finished slides were hematoxylin and eosin-stained. A single senior pathologist (AN) supervised all

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10 examinations. For purposes of this study PUP-related histologic manifestations were further
11 designated as maternal or fetal in origin [25,26].

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12 Among maternal vascular supply disruptions, specific vascular alterations qualifying for maternal
13 vascular maldevelopment were: superficial implantation/decidual arteriopathy (acute atherosis and

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14 mural hypertrophy [mean wall diameter>30% of overall vessel diameter of arterioles in the decidua
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15 parietalis]), undergrowth/distal villous hypoplasia (decrease in the number and modal diameter of
16 distal villi at the center of the lobule after adjustment for plane of section and gestational age, in the
17 lower 75% of a full-thickness section), excessive intervillous fibrin (basal layer of fibrinoid material
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18 involving > 30% of the placental maternal surface) and migration disorders. Specific vascular
19 alterations qualifying for maternal vascular obstruction were: syncytial knots involving terminal villi
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20 (affecting >50% of the terminal villi), villous agglutination (>50%), villous agglutination (eccentric
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21 aggregates on intervillous fibrin on proximal and distal villi affecting >50% of the villi in a single slide)
22 and villous infarcts (>30% of villous loss). Specific vascular alterations qualifying for maternal vascular
23 loss of integrity were: arterial rupture (abruption placenta), venous rupture (acute chronic marginal
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24 abruption).
25 Among fetal vascular supply disruptions, lesions qualifying for maldevelopment were:
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26 chorioangioma, chorioangiosis and distal villous immaturity. Lesions qualifying for obstruction were
27 considered those secondary to vascular thrombo-occlusive disease (thrombosis of chorionic plate
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28 and stem villous channels with distal villous avascularity affecting large groups).
29 When any of these lesions above described were present, the placental was considered to qualify for
30 PUP.

31 Statistical analysis

32 Normal distributions were assured by the Shapiro-Wilk test. One-way ANOVA with linear polynomial
33 orthogonal contrast or linear-by-linear association test X2 were used to test the hypothesis of linear

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1 trend across the study groups (controls, PE without PUP and PE with PUP) of quantitative and
2 qualitative variables, respectively.
3 All statistical analyses were performed with standard software (SPSS v. 20.0, SPSS Inc, Chicago, IL,
4 USA), with statistical significance set at p<0.05. Graphs were constructed with R version 3.2.2 (The R
5 Foundation for Statistical Computing).
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1 RESULTS

2 A total of 76 late-onset PE pregnancies were recruited for study. Of these, 3 were excluded because
3 of abnormalities detected in late pregnancy [(mild ventriculomegaly, (n=1); moderate pyelectasis
4 (n=1)] and suspected chorioamnionitis (n=1), leaving 73 cases for analysis matched with 73 controls.

5 In a total of 54 placentas (74%), 89 instances of PUP-related findings were identified (Table 1).

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6 Placentas with signs of underperfusion had non-significant lower mean values of weight (386.9 [SD
7 135.8] vs 400.7 [SD 120.6]; p=0.696) and fetoplacental weight ratio (6.7 [SD 3.6] vs 6.4 [SD
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3.2];p=0.733).

9 Table 2 details baseline and clinical characteristics of the study population, segregated by the

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10 presence of PUP-related changes. Caucasian ethnicity, BMI at beginning, smoking status and
11 obstetric history, including previous PE, FGR and stillbirth, were statistically significant among the

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12 study groups. AN
13 Maternal BP, first trimester scan, uterine Doppler, and levels of angiogenic factor are itemized in
14 Table 3. Both biophysical (MAP, UtA) and biochemical (PlGF, sFlt-1) parameters significantly differed
15 in women that developed late-onset PE with signs of PUP. Figure 1 shows PlGF, sFlt-1 and ratio
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16 distribution by the presence of signs of PUP.

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17 Obstetric and perinatal outcomes of the study population are shown in Table 4. As expected, adverse
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18 perinatal outcomes, more frequent in cases with late-onset PE and signs of PUP, were statistically
19 significant.
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1 DISCUSSION

2 The present study shows that in late-onset PE, first trimester uterine Doppler and circulating levels of
3 angiogenic/antiangiogenic factors revealed a biological gradient between controls, PE without PUP
4 and PE with PUP.

5 The classification of PE into early- and late-onset has prognostic value, since early-onset disease is

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6 associated with higher perinatal and maternal morbidity and mortality rates than late-onset disease,
7 mainly related to gestational age at delivery [27,28]. Moreover, as late-onset PE is far more
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prevalent than early-onset PE, it accounts for a larger etiological proportion of the occurrence of
9 adverse perinatal outcomes [29,30]. However, further phenotyping of cases with late-onset PE may
10 permit tailored management. Our results show that late-onset PE cases with unbalanced angiogenic

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11 profiles are at higher risk of adverse outcomes.

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12 Several lines of evidence support the hypothesis that early- and late-onset PE may have different
13 pathophysiologic pathways. First, several recognized risk factors for PE such as maternal history of
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14 PE, chronic hypertension and use of drugs to induce ovulation seem to be more related to the early-
15 onset form of the disease [31]. Second, an increasing number of biochemical non-angiogenic [32-35]
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16 and angiogenic markers have been shown to be also differentially associated with early disease.
17 Among the latter, sFlt-1 [36-39] and PlGF [40,41] are more frequently abnormal in early than in late
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18 disease. Third, uterine artery Doppler velocimetry in the first [42,43] or second [44] trimester is also
19 more predictive of early than late-onset PE. As known, an inadequate remodeling of the spiral
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20 arteries may induce placental hypoxia and oxidative stress, and the subsequent association between
21 increased impedance to blood flow in the uterine arteries and early-onset PE suggests a greater
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22 contribution of PUP preceding early- than late-onset disease [45]. Fourth, the prevalence of placental
23 findings suggestive of maternal underperfusion in patients with PE decreased gradually with GA [46].
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24 In addition, in a placental morphometric study, Egbor et al observed a significant reduction in the

25 volume and surface of the terminal villi in the placentas of patients with early-onset PE delivering
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26 before 34 weeks compared to gestational age-matched controls [47]. Differences in the placental
27 pathologic findings between early- and late-onset PE were confirmed in a small case-control study
28 showing higher rates of placental infarctions, decidual arteriopathy and villous hypermaturation in
29 early-onset than in late-onset PE [26].

30 Evidence of a relationship between maternal levels of angiogenic/anti-angiogenic factors in the first

31 trimester and placental findings in late-onset PE is scant, being described in only one previous study;
32 however, the authors evaluated it in the third trimester and reported low plasma concentrations of

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1 PlGF and higher sFlt-1 in the presence of placental lesions [46]. Our study adds to the growing body
2 of evidence by maintaining that an imbalance of angiogenic factors and PE are associated, and shows
3 that, to some extent, this link could be traced back to the first trimester.

4 At present, potential interventions exist to ameliorate this imbalance, such as early administration of
5 low-dose aspirin (LDA) and, hence, might provide opportunities to improve perinatal outcomes [48].
6 While LDA clearly reduces early-onset PE when started before 16 weeks, this strategy fails to reduce

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7 the incidence of term PE [8]. It could be speculated that, as late-onset PE is a heterogeneous
8 condition in which maternal predisposition and defective trophoblastic invasion contribute to

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9 variably in its pathophysiology, cases with evidence of an angiogenic imbalance are candidates for
10 such prophylaxis. We provide evidence that this imbalance could already be detected at first

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11 trimester, a period when LDA is most likely to be effective [7].

12 Strengths of this study are its prospective design, the application of validated and reproducible

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13 histological criteria for classifying placental lesions, and the adjusted PlGF and sFlt-1 levels according
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14 to gestational age. Unfortunately, we must also concede some limitations. First, we evaluated the
15 angiogenic factors early in the first trimester (most cases before 10 weeks). It is likely that the
16 angiogenic imbalance would be more pronounced later in the first trimester. However, these
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17 significant differences in pro and anti-angiogenic profile so early in pregnancies complicated by

18 placental under perfusion in late-onset PE could be valuable to placental literature. Secondly,
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19 longitudinal angiogenic factor determinations may have shed more light on their temporal
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20 relationship with placental damage. Thirdly, no placental examinations, in term of weight,

21 placental/BW ratio, and histologic findings have been performed in the control group. Finally, the
22 limited sampling size renders our study inadequately powered to assess the potential impact of
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23 angiogenic factors within each category of PUP.

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24 In conclusion, a subset of patients with late-onset PE already has an imbalance of angiogenic/anti-

25 angiogenic factors in maternal blood already present in the first trimester, and this finding is
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26 associated with a higher frequency of placental histological findings.

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1 ACKNOWLEDGEMENTS

2 This work was supported by grants from The Cerebra Foundation for the Brain Injured Child
3 (Carmarthen, Wales, UK), Obra Social “la Caixa” and AGAUR 2014 SGR grant nº 928. This publication
4 has been funded with support of the Erasmus + Programme of the European Union (Framework
5 Agreement number: 2013-0040). This publication reflects the views only of the author, and the
6 Commission cannot be held responsible for any use which may be made of the information
7 contained therein.

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8 The samples used in this study were provided by the Hospital Clínic-IDIBAPS Biobank with
9 appropriate ethical approval.
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15 FINANCIAL DISCLOSURE
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16 The authors have indicated that they have no financial relationships to disclose relevant to this
17 article.
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23 CONFLICTS OF INTEREST
24 The authors have no conflicts of interest relevant to this article to disclose.
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30 CONDENSATION
31 This study is the first to ascertain the association between angiogenic factors evaluated at first
32 trimester and placental under perfusion-related histopathology in pregnancies complicated by late-
33 onset preeclampsia.
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1 Table 1. Categories/subcategories of placental attributes (n=89) consistent with under perfusion in
2 study population.

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Categories of placental Subcategories
injury of placental injury
n (%) n (%)
Maldevelopment

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32 (42.1)
Maternal
Obstruction
vascular supply
39 (51.3)

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76 (85.4)
Loss of integrity
5 (6.6)

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Maldevelopment
4 (30.8)
Fetal
Obstruction
vascular supply
5 (38.4)

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13 (14.6)
Loss of integrity
4 (30.8)
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Table 2. Baseline characteristics in pregnancies complicated by late-onset preeclampsia, with
and without placental signs attributable to PUP.

Controls PUP – PUP + Linear

Characteristics
(n=73) (n=19) (n=54) Tendency P†
Age (yrs) 32.4 (5.1) 33.2 (6.3) 32.4 (5.9) 0.947
BMI (Kg/m2) 23.5 (3.1) 25.4 (4.5) 25.5 (4.8) 0.005
Ethnicity

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White 53 (72.6) 12 (63.2) 30 (55.6) 0.046
Black 1 (1.4) 1 (5.3) 0 (0) 0.570
Latin-American 14 (19.2) 5 (26.2) 18 (33.3) 0.070

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Others 5 (6.8) 1 (5.3) 6 (11.1) 0.405
Smoking status
No smokers 63 (86.3) 16 (84.2) 48 (88.9) 0.687

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<10 7 (9.6) 3 (15.8) 5 (9.3) 0.002
≥10 3 (4.1) 0 (0) 1 (1.8)
Low SES* 35 (50) 8 (42.1) 20 (37.0) <0.001
Medical history

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Chronic hypertension 1 (1.4) 2 (10.5) 4 (7.4) 0.102
0.806
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Diabetes mellitus 1 (1.4) 1 (5.3) 1 (1.8)
Renal disease 0 (0) 0 (0) 1(1.8) 0.842
Autoimmune disease 4 (5.5) 0 (0) 4 (7.4) 0.683
Coagulation disorders 1 (1.4) 0 (0) 2 (3.7) 0.381
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Obstetric history
Nulliparous 46 (63) 13 (68.4) 34 (63) 0.985
Previous PE 2 (2.7) 1 (5.3) 10 (18.5) 0.002
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Previous FGR 1 (1.4) 0 (0) 5 (9.3) 0.031

Previous stillbirth 0 (0) 0 (0) 3 (5.6) 0.033
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ART 5 (6.5) 2 (10.5) 3 (5.6) 0.805

PUP: Placental under perfusion; BMI: Body-mass index; SES: socio-economic status; PE:
Preeclampsia; FGR: Fetal growth restriction; ART: Assisted reproductive technologies.
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*Routine occupations, long-term unemployment or never worked.

†StaZsZcally significant p<0.05 using one-way ANOVA with linear polynomial contrast or linear-
by-linear association test, as appropriate.
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Data are expressed as median (SD) or n (%).

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Table 3. Biophysical and biochemical parameters in pregnancies complicated by late-onset
preeclampsia, with and without placental signs attributable to PUP.

Controls PUP – PUP + Linear

Characteristics
(n=73) (n=19) (n=54) Tendency P†

Biophysical parameters
MAP (MoM) 0.96 (0.09) 1.11 (0.22) 1.08 (0.12) <0.001
CRL (mm) 62.8 (7.4) 64.2 (10.8) 65.7 (9.9) 0.068

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NT (MoM) 0.9 (0.3) 1.11 (0.31) 1.06 (0.36) 0.251
Mean UtA (MoM) 1 (0.35) 1.26 (0.38) 1.32 (0.46) <0.001

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Biochemical parameters
PlGF (MoM) 1.53 (0.21) 1.41 (0.25) 1.37 (0.26) <0.001
sFlt-1 (MoM) 3.11 (0.19) 3.11 (0.16) 3.22 (0.19) 0.002

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sFlt-1/ PlGF (MoM) 2.3 (0.27) 2.3 (0.56) 2.44 (0.53) <0.001

PUP: Placental under perfusion; MAP: Mean arterial pressure; MoM: Multiple of the expected
median; NT: Nucal translucency; UtA: Uterine artery; PI: Pulsatility index; PlGF: Placental growth

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factor; sFlt-1: Soluble Fms-like tyrosine kinase-1.
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Data are expressed as median (SD).
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1 Table 4. Obstetrical and perinatal outcomes in pregnancies complicated by late-onset preeclampsia,
2 with and without PUP.

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Controls PUP – PUP +
Characteristics Tendency P†
(n=73) (n=19) (n=54)
GA at diagnosis of PE (wks) - 35.5 (4.5) 35.3 (3.8) 0.831‡
HELLP syndrome 0 (0) 4 (21.1) 3 (5.5) 0.089
Eclampsia 0 (0) 0 (0) 1 (1.9) 0.224

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Abruptio placentae 0 (0) 0 (0) 1 (1.9) 0.335
GA at delivery (wks) 39.6 (1.4) 36.0 (3.9) 35.6 (3.5) <0.001
CS for NRFS 5 (6.9) 5 (35.7) 12 (37.5) 0.014

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BW (g) 3394 (420) 2395 (1010) 2319.4 (948.6) <0.001
BW percentile 52.3 (20.1) 27.4 (32.7) 22.7 (30.6) <0.001
BW < 10th percentile 0 (0) 10 (52.6) 28 (51.9) <0.001
Male gender 36 (49.3) 7 (36.8) 23 (42.6) 0.429

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APGAR at 5 min <7 0 (0) 2 (1.1) 4 (1.9) 0.073
Neonatal acidosis (pH<7.10) 0 (0) 0 (0) 5 (9.3) 0.006
NICU admission 0 (0) 1 (5.3) 0 (0) 0.807

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Placental weight (g) - 400.7 (120.6) 386.9 (135.8) 0.696‡
BW/Placental weight ratio - 6.4 (3.2) 6.7 (3.6) 0.733‡
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PUP: placental under perfusion; GA: Gestational age; PE: Preeclampsia; HELLP: Hemolysis,
Elevated Liver enzymes and Low Platelets; CS: Cesarean section; NRFS: Non-Reassuring Fetal
Status; BW: birth weight; NICU: neonatal intensive care unit.
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†StaZsZcally significant p<0.05 using one-way ANOVA with linear polynomial contrast or
linear-by-linear association test, as appropriate.
‡ Only between cases (PUP+ vs. PUP -)
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1 Figure 1. Distribution of markers by study group.
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