A longitudinal analysis of angiotensin II type 1 receptor antibody and angiogenic markers in pregnancy

Accepted Manuscript A longitudinal analysis of Angiotensin II type 1 receptor antibody and angiogenic markers in pregnancy Dr Shikha Aggarwal, MBBS(HONS), FRACP, Dr Neroli Sunderland, Ms Charlene Thornton, Dr Bei Xu, Professor Annemarie Hennessy, MBBS, FRACP, PhD, Dr Angela Makris, MBBS, FRACP, PhD PII:

S0002-9378(16)30923-1

DOI:

10.1016/j.ajog.2016.10.028

Reference:

YMOB 11358

To appear in:

American Journal of Obstetrics and Gynecology

Received Date: 27 July 2016 Revised Date:

28 September 2016

Accepted Date: 18 October 2016

Please cite this article as: Aggarwal S, Sunderland N, Thornton C, Xu B, Hennessy A, Makris A, A longitudinal analysis of Angiotensin II type 1 receptor antibody and angiogenic markers in pregnancy, American Journal of Obstetrics and Gynecology (2016), doi: 10.1016/j.ajog.2016.10.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT TITLE PAGE Title A longitudinal analysis of Angiotensin II type 1 receptor antibody and angiogenic markers in

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pregnancy

Authors Dr Shikha AGGARWAL1,2, MBBS(HONS),FRACP

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Dr Neroli SUNDERLAND 3, Ms Charlene THORNTON 4,

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Dr Bei XU 2,

Professor Annemarie HENNESSY 1,2,4 , MBBS,FRACP,PhD Dr Angela MAKRIS1,2,4, MBBS,FRACP,PhD

School of Medicine, Western Sydney University, Sydney, Australia

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Heart Research Institute, Sydney, Australia

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Royal Prince Alfred Hospital, Sydney, Australia

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Department of Renal Medicine, South Western Sydney Local Health District, Sydney,

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Australia

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Disclosure Statement

The authors have nothing to disclose and no conflicts of interest.

Funding Kidney Health Australia: provided a research grant which was utilised to purchase research assays and equipment.

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ACCEPTED MANUSCRIPT Preeclampsia associated research laboratories at Heart Research Institute (PEARLS): provided a research grant which was utilised to purchase research assays and equipment.

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Corresponding Author Shikha Aggarwal (MBBS, FRACP) Vascular Immunology research group

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Heart Research Institute, 7 Eliza St Newtown, NSW, Australia, 2042

e-mail: [email protected]

Word Count: Abstract: 309

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Manuscript: 3198

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Phone +61-413030995

Number of figures and tables: 5

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Condensation and short version of title

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Condensation: This longitudinal prospective cohort observational study assesses the changes in Angiotensin II type 1 receptor antibodies and other angiogenic markers throughout pregnancy.

Short title: AT1AA and angiogenic markers in pregnancy

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ACCEPTED MANUSCRIPT ABSTRACT

Key words

Preeclampsia, Angiotensin II type 1 receptor antibodies (AT1AA), soluble fms-like tyrosine

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kinase like receptor 1 (sFlt-1), soluble endogolin (sEng), placental growth factor (PlGF).

Background

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Preeclampsia can be caused by shallow trophoblast invasion and results in endothelial dysfunction. Angiotensin II type 1 receptor antibodies (AT1AA) may have a role in

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both processes. Other angiogenic markers (placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and endoglin (sEng) alter before clinically evident preeclampsia.

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Objectives

The aim of this study is to assess the longitudinal changes and utility of biomarkers AT1AA and angiogenic markers in hypertensive disorders of pregnancy; gestational

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Study Design

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hypertension (GHT) and preeclampsia (PE).

A longitudinal prospective cohort observational study of angiogenic markers and a secondary retrospective case-control study of AT1AAchanges were conducted. The studies were conducted in a large tertiary metropolitan teaching hospital (Sydney, Australia). Sequential recruitment of women with a singleton pregnancy (N= 351) was undertaken. Plasma concentrations of AT1AA, PlGF, sFlt-1 and sEng were measured using validated enzyme-linked immunosorbent assays at 12, 18, 28, 36, 40 weeks

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ACCEPTED MANUSCRIPT gestation and 6 weeks post-partum. Clinical, demographic and pregnancy data were prospectively collected. Pregnancy outcomes were classified as normotensive, GHT or PE. Analyses were carried out using SPSS and significance set at p<0.05.

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Results

351 women were recruited, 17 developed GHT and 18 PE. Women with PE at

baseline were heavier (p=0.015), taller (p=0.046) had higher systolic (p=0.029) and

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diastolic (p=0.006) blood pressure. The PE group had higher sFlt-1 from 28 weeks

onwards (p= 0.003) and lower PlGF from 18 weeks (p= 0.004). sEng and AT1AA did

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not vary over time or between groups. AT1AA (12 weeks) was positively correlated with serum PAPP-A (p= 0.008) and BHCG (p= 0.04).

Conclusion

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Angiogenic markers vary longitudinally during pregnancy and PlGF and sFlt-1 have a role for predicting and diagnosing PE later in disease. Our data shows that AT1AA are not sensitive for disease and hence not useful as a biomarker. Larger studies are

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required to describe the role and functionality of AT1AA in PE.

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ACCEPTED MANUSCRIPT INTRODUCTION

Preeclampsia is a hypertensive disorder of pregnancy that continues to affect 3-5% of all pregnancies worldwide resulting in maternal and neonatal morbidity and

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mortality.1 The disease is characterised by the onset of hypertension and proteinuria after 20 weeks gestation and in severe cases progression to renal failure, elevated liver enzymes with low platelets (HELLP syndrome) and cerebral oedema with seizures.2

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The disease is a disorder of the maternal endothelium and is made up of two stages;3 inadequate placental implantation resulting in placental ischaemia4 followed by a

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maternal inflammatory syndrome.5 The release of inflammatory molecules such as cytokines and anti-angiogenic factors alter the function of the maternal endothelial cell and contribute to the clinical features of preeclampsia.6 In preeclampsia there is an imbalance between pro-angiogenic factors such as vascular endothelial growth

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factor (VEGF), placental growth factor (PlGF) and anti-angiogenic factors such as soluble fms-like tyrosine kinase 1 receptor (sFlt-1) and serum endogolin (sEng). The mechanism for this imbalance remains unclear although placental ischaemia7,8 and

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implicated.9,10

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more recently angiotensin II type 1 receptor autoantibodies (AT1AA) have been

AT1AA were first described in preeclampsia by Wallukat et al.11 Administration of the antibodies to animals can induce the syndrome of preeclampsia12 and in vitro the antibodies cause vasoconstriction13 and endothelial cell damage.14 AT1AA are detectable as early as 16 weeks gestation15 and hence may prove a useful biomarker for predicting preeclampsia. However there is no data in the current literature evaluating AT1AA in early gestation (11-14 weeks), at the critical time of trophoblast

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invasion.16 It is unclear whether AT1AA precede disease onset or develop as a consequence of placental ischaemia as demonstrated in animal models.17 Although they have been shown to present in disease, there is little data about their change

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during pregnancy.15

We performed a longitudinal cohort study of angiogenic biomarkers; sFlt-1, PlGF, sEng to assess their role in identifying women early in pregnancy who will develop

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hypertensive disorders of pregnancy (gestational hypertension [GHT] and preeclampsia [PE]). A longitudinal case control study assessing the changes in

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AT1AA in women who develop PE, GHT compared to age matched controls was also performed with the secondary aim of examining the relationship between AT1AA and angiogenesis.

Subjects

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MATERIALS AND METHODS

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Women were consecutively recruited from antenatal and ultrasound clinics

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(performing nuchal translucency scans) at Royal Prince Alfred Hospital, Sydney Australia. At this time, nuchal translucency scans were offered to all women who consented. All women less than 14 weeks gestation (measured by ultrasound) with a singleton pregnancy were included in the study. Written informed consent was obtained from all participants. The women were prospectively followed longitudinally throughout their pregnancy.

Women were categorised into 3 groups at time of delivery, Group 1 had preeclampsia

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ACCEPTED MANUSCRIPT (PE), Group 2 had gestational hypertension (GHT) and Group 3 were normotensive and acted as the control group (CT). Women were considered to have PE or GHT as defined by the International Society for the study of hypertension in pregnancy guideline 2001.18 Briefly preeclampsia was defined as blood pressure ≥140mmHg

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systolic and/or or ≥90mmHg diastolic after 20 weeks gestation as on two occasions at least four hours apart. This was associated with evidence of fetal growth restriction and end organ dysfunction, including proteinuria (greater than or equal to 2+ on

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dipstick or 300mg/24hr) or renal insufficiency (serum creatinine greater than

0.09mmol/L), liver disease (raised serum transaminases or severe epigastric pain),

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neurological problems (including convulsions) and haematological disturbances (thrombocytopenia, disseminated intravascular coagulation or hemolysis), all of which resolved within three months post partum. GHT was defined as women with blood pressure ≥140mmHg systolic and/or or ≥90mmHg diastolic after 20 weeks

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gestation.

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This study was approved by the South Western Sydney Human Ethics committee.

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Data collection

Baseline clinical data was collected for all women recruited into the study at the first antenatal visit. Baseline data included maternal age at time of delivery, height (measured in meters), weight (measured in kilograms), BMI (calculated as weight/height squared). Detailed personal, family medical and obstetric history (in particular chronic infections, hypertension, preeclampsia, miscarriages, termination) along with results of routine infectious serology (Hepatitis B, C antibody testing, syphilis and human immunodeficiency virus (HIV)), blood group, smoking history,

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ACCEPTED MANUSCRIPT medications taken prior to and during pregnancy, illicit drug usage and previous delivery methods was documented.

Clinical data was collected throughout the pregnancy at time points; 11-14 weeks

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gestation (T1), 16-20 weeks (T2), 26-28 weeks (T3), 34-36 weeks (T4), term (T5), 6 weeks postnatal (T6). Data collected included blood pressure (measured by midwife or obstetrician in antenatal clinic, sitting down after 5 minute rest) and results of

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antenatal investigations such serum PAPP-A, BHCG levels, glucose tolerance test

results and nuchal translucency test. At the time of delivery the following data was

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collected: gestation at delivery, weight (g at birth), infant length (cm), infant sex, infant Apgar scores at one and five minutes, requirement of any neonatal supportive care (such as intensive care, or high dependency monitoring), delivery method and any complications that occurred during the delivery (for example post partum

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haemorrhage). In the postpartum period the data collected included: blood pressure, breast feeding status and post delivery complications. (Table 1)

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Sample collection and processing

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Maternal blood was collected at six time points; 11-14 weeks gestation, 16-20 weeks, 26-28 weeks, 34-36 weeks, term, 6 weeks postnatal. Venous blood was collected into 2 tubes; a polyethylenetherephthalate (PET) tube containing a clot activator (silica particles) for serum samples (sFlt-1, PlGF, sEng) and a PET tube containing 7.2mg potassium ethylenediaminetetraacetic acid (K2EDTA) (BD, NSW, Australia) for plasma samples (AT1AA). The blood was centrifuged, within 4 hours of venipuncture, for 10 minutes at 3000 revolutions per minute (rpm) (GS-6 Centrifuge, Beckman, California, USA). The serum was then aliquoted (250µL) and stored at –

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ACCEPTED MANUSCRIPT 80°C until required.

Enzyme Linked Immunsorbent Assay Concentrations of sFlt-1, PlGF, sEng and AT1AA were measured using commercial

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enzyme linked immunosorbent assay (ELISA) kits (sFlt-1 Bender Medsystems,

Vienna, Austria; PlGF R & D Systems, Minneapolis, USA; sEng R&D Systems, Minneapolis, USA ; AT1AA One Lambda, California, USA). All ELISAs were

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performed according to the manufacturers instructions, in duplicate. If the CV was

greater than or equal to 10%, the results of the ELISA were discarded and the entire

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ELISA plate repeated. The inter-assay and intra-assay coefficients of variation were 5.8 and 9.9 percent respectively for sFlt-1; 2.3 and 7.7 percent respectively for PlGF; 3.8 and 10.8 percent for sEng; 3.29 and 11.41 percent respectively for AT1AA. As reported by the manufacturers the intra-assay precision (CV) for ten identical samples

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was 3.9% and the inter-assay precision (CV) for twenty identical samples was 5.1%. The lower limit of detection for the AT1AA assay was 2.5U/ml. In the current study the ELISA we utilised has undergone robust scientific validation by Dragun et al19 who first used

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the kit in the renal transplant population however the kit has not been validated in pregnancy.

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Statistical Methods

Statistical analysis was carried out using SPSSTM v 23 (Illinois, USA) software. The distribution of the data was analysed using the Kolmogorov-Smirnov test and where applicable the data were logarithmically transformed. The data is presented as mean+/-SEM. In cases where the data could not be normalised, non-parametric tests such as Kruskal-Wallis or Mann Whitney U tests were applied. In this case the data is presented as median (IQR). Although the overall significance level was set at 0.05,

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the Sharpened Bonferroni method was used to adjust the individual alpha level when multiple comparisons were performed. A sFlt-1 to PlGF ratio was calculated by dividing the sFlt-1 concentrations by the PlGF concentrations at various time points for each individual patient. The sample size was powered on a 50% difference in

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protein concentrations at 12 weeks specifically designed to look at mechanisms for

predicting preeclampsia. The AT1AA assay was subsequently available and a nested

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case control study was conducted based on sample availability.

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An analysis of repeated measures was employed to assess the changes of the markers over time and to assess between-subject effects. Cross-sectional analysis at a single time point was performed by Kruskal-Wallis tests. Bivariate correlation analysis using Spearman tests were conducted to assess relationships between the markers and

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demographic risk factors. A univariate analysis was conducted to assess whether individual demographic or serum factors were predictive of disease. Diagnosis was

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PE).

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grouped for this analysis to include all hypertensive disorders of pregnancy (GHT and

RESULTS

Baseline characteristics Three hundred and fifty one women were recruited into the study. Eighteen women developed preeclampsia and seventeen developed gestational hypertension. Of the remaining three hundred and thirty three, a total of sixteen women were excluded; nine had early fetal demise and seven were lost to follow up (Figure 1).

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The maternal age was similar for all the groups (PE, CT, GHT). There was a statistically significant difference in maternal weight (p=0.015), height (p=0.046) and BMI (p=0.041) between the groups (Kruskal-Wallis test). On multiple comparisons

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only the PE group 1.6[1.54-1.65] m was statistically shorter than the GHT group

1.67[1.65-1.68] m and the GHT group 70[60-88] kg was significantly heavier that the CT group 62[57-70] kg. Baseline systolic and diastolic blood pressure was

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significantly different across groups with p values of 0.029 and 0.006 respectively

(Kruskal-Wallis). However on multiple comparisons only the baseline diastolic blood

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pressure in the PE group 75.5 [60-81.25] mmHg was statistically significant compared to the control group 65[60-70] mmHg. The PE group had significantly higher rates of PE history 27.78% compared to 1.33% in CT p=<0.001 (KruskalWallis Analysis). There was no significant difference in the rate of smoking,

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primiparity and gestational diabetes mellitus (GDM) (Table 2). As indicated in Table 1 all patients had their final blood collection taken at term; there were no preterm

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deliveries in this study.

Longitudinal changes sFlt-1 Serum sFlt-1 concentrations significantly increased progressively during the pregnancy. From 12 weeks 0.25[0.03-0.55] ng/ml to 18 weeks 0.14[0-0.32] ng/ml sFlt-1 increased significantly (p 0.006), as was the case from 28 weeks 0.2[0.06-0.4] ng/ml to 36 weeks 0.64[0.28-1.02] ng/ml (p <0.001) and from 36 weeks 0.64[0.281.02] ng/ml to term 0.84[0.57-1.22] ng/ml (p <0.001) on repeated measures analysis with Bonferroni correction. (Figure 2.1)

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There was also a statistically significant difference between the three diagnostic groups from 28 to 36 weeks (p<0.001) and 36 weeks to term (p=0.002) on repeated measures analysis. The sFlt-1 concentration in the PE group (28:36 weeks; 0.37[0.19-

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0.72]: 1.12[0.66-1.99] ng/ml) increased to a greater extent compared to the CT group (28:36 weeks; 0.2[0.58-0.4]: 0.63[0.26-0.98] ng/ml (Tukey’s T Test; p<0.001) but was not statistically different to the GHT group (28:36 weeks; 0.15[0.01-0.38]:

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0.79[0.22-0.99] ng/ml) (Tukey’s T test p 0.08). From 36 weeks to term the PE group (36weeks:term; 1.12[0.66-1.99]: 1.61[0.89-2.77] ng/ml) continued to significantly

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differ from the CT group (36weeks:term; 0.63[0.26-0.98]: 0.8[0.51-1.13] ng/ml) (Tukey’s T test p=0.005) but not the GHT group (36 weeks: term; 0.79[0.22-0.99]: 1.13[0.84-1.82] ng/ml) (Tukey’s T test 0.069).

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The sFlt-1 concentration between the three groups did not differ significantly in the post partum period (K-W: p=0.245).

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Longitudinal changes PlGF

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Plasma PlGF concentrations increased until it reached a peak at 28 weeks gestation 267.5[174.25-389.64] pg/ml and then progressively decreased as the pregnancy advanced (Figure 2.2). The PLG concentration were significantly higher from 12 14.21[4.69-27.19] ng/ml to 18 weeks 85.28[57.74-132.62] ng/ml (p <0.001) and from 18 weeks 85.28[57.74-132.62] ng/ml to 28 weeks 267.5[174.25-389.64] pg/ml (p <0.001) (repeated measures with Bonferroni correction). The concentration at 28 weeks 267.5[174.25-389.64] pg/ml was significantly higher than 36 weeks 104.29[39.90-237.25] pg/ml (p <0.001) as was the case from 36 weeks 104.29[39.90-

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ACCEPTED MANUSCRIPT 237.25] pg/ml to term 8.1[1.74-18.36] pg/ml (p=0.025) (repeated measures with Bonferroni correction).

There was a statistically significant difference between the three diagnostic groups

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from18 to 28 weeks (p 0.014), from 28 to 36 weeks (p 0.001) and from 36 weeks to term (p 0.014) on repeated measures analysis. The PlGF concentrations were

significantly lower in the PE group (18:28 weeks; 61[38.16-98.62]: 89.14[63.32-

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252.15] pg/ml) compared to CT (18:28 weeks; 88.64[60-133.95]: 280[185.34-407.63] pg/ml) (Tukey’s T test p=0.026) but were not statistically different to the GHT group

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(18:28 weeks; 62.8[41.39-108.27]: 209.29[157.35-319.35] pg/ml) (Tukey’s Test p=0.742).

From 28 to 36 weeks the PE group (28:36 weeks; 89.14[63.32-252.15]: 14.2[5.8479.52] pg/ml) continued to have lower PLGF concentrations compared to the CT

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group (28:36 weeks; 280[185.34-407.63]: 132.81[55.92-259.16] pg/ml (Tukey’s T test p=0.002) as was the case from 36 weeks to term (Tukey’s T test p=0.026).

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There was no significant difference between the three groups in the post partum

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period (Kruskal-Wallis p=0.28).

sFlt-1 : PlGF

The ratio between sFlt-1 and PlGF was statistically different between diagnostic groups at time point 28 weeks (p value 0.005), 36 weeks (p value <0.001) and term (p value <0.001) (Kruskal-Wallis analysis). (Figure 3)

Longitudinal changes in sEng

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The concentration of sEng (ng/ml) generally increased throughout gestation until term then decreased in the post-natal period (Figure 2.3). The sEng concentration was significantly higher from 18 weeks 27.53[24.14-31.35] ng/ml to 28 weeks 30.23[27.18-35.01] ng/ml (p<0.001) and from 28 weeks 30.23[27.18-35.01] ng/ml 36

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weeks 36.02[32.33-40.06] ng/ml (p<0.001) (repeated measures with Bonferroni

correction). The sEng concentration was significantly lower post partum 31.24[27.1636.21] ng/ml compared to term 37.82[32.41-42.96] ng/ml (p=0.03) (repeated

Longitudinal changes AT1AA

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three diagnostic groups at any time-point.

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measures with Bonferroni correction). There was no statistical difference between the

AT1AA measures were only conducted in a total of seventy six patients (seventeen with preeclampsia, fifteen with gestational hypertension and forty four age matched

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controls) due to lack of sample availability. Where available plasma samples were utilized for analysis, however in 10% of cases serum samples were used due to lack of

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plasma availability.

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In this case control study the concentration of AT1AA did not change throughout pregnancy the concentration at 12 weeks gestation was 15.44[12.77-17.86] u/ml and at term 11.53[5.75-17.78] u/ml (Figure 2.4) this was not statistically significant (p>0.05) (repeated measures with Bonferroni correction). More importantly there was also no difference between the diagnostic groups at any specific time point (Kruskal-Wallis test; 12 weeks p=0.234, 18 weeks p=0.326, 28 weeks p=0.862, 36 weeks p=0.975, term p=0.576 and post partum p=0.118)

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There was no significant correlation between AT1AA at time point 12 weeks and age, body mass index, previous history of preeclampsia, gestational diabetes, baseline blood pressure (at 20 weeks) and smoking (Table 2).

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There was a positive correlation between AT1AA at 12 weeks with serum PAPP-A and BHCG with correlation coefficient and p values of 0.33; 0.008 and 0.261; 0.04 respectively (Figure 4). When looking at the different diagnostic groups this

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correlation was only significant in the CT group (p= 0.012) and not in the GHT or PE

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groups.

Univariate analysis

On univariate analysis a combined diagnosis of preeclampsia or gestational hypertension was predicted by a history or previous preeclampsia (p<0.01), booking

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in diastolic blood pressure (p<0.05) and serum endogolin level at 12 weeks (p=0.04).

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COMMENT

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AT1AA at 12 weeks did not have predictive value on univariate analysis (p=0.25).

We have described that AT1AA are detectable early in pregnancy however are not specific for disease and have limited predictive value. The results of our case control longitudinal study have shown that AT1AA were detectable in 93.4%, 100% and 95.2% of patients with PE, GHT and control respectively who deliver at term. AT1AA were detectable as early as 12 weeks, however were not specific for disease later in the pregnancy and the concentrations were similar between all three diagnostic groups throughout the pregnancy. AT1AA was not significantly correlated

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with sFlt-1, PlGF and sEng. Our study is the first to utilise a commercially available, validated ELISA kit for measuring AT1AA levels in a longitudinal analysis of pregnant women.

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Our results differ to those of Siddiqui et al20 who showed that AT1AA were present in greater than 95% of women with PE and in less than 30% of women without disease. The assay was qualitative and not quantitative as they used a luciferase assay to detect

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AT1AA. Sahay et al15 described higher AT1AA levels in women with the PE between 26-30 weeks compared to controls. However they utilised a different ELISA

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(MyBiosource) and detected lower concentrations in all women, many near the lower limit of the assay.

The detection of AT1AA as early as 12 weeks is a novel finding and challenges

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concepts surrounding the role of AT1AA and placental ischaemia shown in some animal models. La marca et al have shown that AT1AA can be induced by reducing placental perfusion surgically in rats.21 This supports the theory that like sFlt-1, AT1AA is a consequence of placental hypoxia/ischaemia. However the presence of

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AT1AA as early as 12 weeks in human gestation demonstrates the antibodies precede the placental development phase of trophoblast invasion and spiral artery remodelling

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and hence are not a result of placental hypoxia. In-vitro studies have shown that AT1AA can impair trophoblast invasion through activation of the AT1- receptor and PAI-1.22 Our data support the findings of this study and suggest that AT1AA may indeed be a mediator of preeclampsia through reduced trophoblast invasion and inadequate placentation. The correlation between AT1AA concentrations particularly in early pregnancy with PAPP-A and BHCG may indicate a relationship with placental

dysfunction. The usefulness of adding AT1AA to current combination screening models required further testing.

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Other angiogenic markers including sFlt-1, PLGF and sEng have been widely studied due to ease of measurement with readily available ELISA kits. In contrast studies on the role of AT1AA and longitudinal changes in concentration levels have been limited due to difficulty in measuring these antibodies. Previously AT1AA in preeclampsia

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have been measured using either degrees of increased contraction rate of neonatal rat cardiomyocyte23 or luciferase bioassays.24 This study is the first to quantitatively

measure the AT1AA concentrations using a validated ELISA19. Further studies using

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explaining the exact role of AT1AA in this disease.

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this ELISA kit in preeclampsia may provide accuracy of measurement and assist in

Although there was no change in the AT1AA longitudinally, there were significant changes of sFlt-1 and PlGF, which is consistent with published data.25 Although the value of sFlt-1 and PlGF for disease prediction in the first trimester had poor

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predictive utility26 they have been shown to correlate with diagnosis, adverse disease outcomes especially with premature disease onset (<34 weeks).27 The use of the sFlt1:PlGF ratio has resulted in improved prediction and disease detection compared to

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either marker alone, with differences between control and PE groups described as

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early as 17-20 weeks.28 In our study we found the ratio was significantly different between the three diagnostic groups from 28 weeks onwards. Newer assays have provided accurate diagnostic cut-offs for utilising this ratio as a predictive tool.29,30

In this study sEng concentrations were consistently increased throughout gestation with subsequent decline in the post-partum period in all groups. This differs from some published reports28 in that this study included baseline characteristics such as older population, multiparous and gestational diabetic cases and a wider range of

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ethnic groups. The study was an observational cohort study and others performed case control studies28. This has resulted in a much higher rate of PE and GHT than the average population, which may bias the results. Levine et al28 showed that sEng in addition to the sFlt-1:PlGF ratio may have predictive value, however recent studies

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have shown that when measured early in pregnancy sEng does not perform well on statistical tests for disease prediction.31

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We understand that our study has limitations. In particular the small event rate and

small sample size reduces the power of this study, specimens were stored for a period

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of time (especially prior to AT1AA measurement) which may affect detection, however all specimens were treated identically. The strengths of our study include the study design and recruitment process. All pregnancy data was collected prospectively and our recruitment method provided an un-differentiated group of women with no

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exclusions based on age or socioeconomic status. Further research in the role of angiogenic markers in particular AT1AA may provide insight into disease

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pathogenesis and therapeutic options.

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Angiogenic markers vary longitudinally during pregnancy and PlGF and sFlt-1 have a role for predicting and diagnosing PE later in disease. Our data has shown that AT1AA are not specific for disease at term and whether they play a role in the pathophysiology of early or late PE remains uncertain.

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ACCEPTED MANUSCRIPT REFERENCES 1. 2.

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Espinoza J, Romero R, Mee Kim Y, et al. Normal and abnormal transformation of the spiral arteries during pregnancy. Journal Perinat Med. Vol 342006:447. LaMarca B, Parrish MR, Wallace K. Agonistic autoantibodies to the angiotensin II type I receptor cause pathophysiologic characteristics of preeclampsia. Gend Med. 2012;9(3):139-146. Brown MA, Lindheimer MD, de Swiet M, Van Assche A, Moutquin JM. The classification and diagnosis of the hypertensive disorders of pregnancy: statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy. 2001;20(1):Ix-xiv. Dragun D. The detection of antibodies to the Angiotensin II-type 1 receptor in transplantation. Methods Mol Biol. 2013;1034:331-333. Siddiqui AH, Irani RA, Blackwell SC, Ramin SM, Kellems RE, Xia Y. Angiotensin receptor agonistic autoantibody is highly prevalent in preeclampsia: correlation with disease severity. Hypertension. 2010;55(2):386-393. LaMarca B, Wallukat G, Llinas M, Herse F, Dechend R, Granger JP. Autoantibodies to the angiotensin type I receptor in response to placental ischemia and tumor necrosis factor alpha in pregnant rats. Hypertension. 2008;52(6):1168-1172. Xia Y, Wen H, Bobst S, Day MC, Kellems RE. Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig. 2003;10(2):82-93. Wallukat G, Nissen E, Neichel D, Harris J. Spontaneously beating neonatal rat heart myocyte culture-a model to characterize angiotensin II at(1) receptor autoantibodies in patients with preeclampsia. In Vitro Cell Dev Biol Anim. 2002;38(7):376-377. Dechend R, Homuth V, Wallukat G, et al. AT(1) receptor agonistic antibodies from preeclamptic patients cause vascular cells to express tissue factor. Circulation. 2000;101(20):2382-2387. Levine RJ, Maynard SE, Qian C, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med. 2004;350(7):672-683. Akolekar R, de Cruz J, Foidart JM, Munaut C, Nicolaides KH. Maternal plasma soluble fms-like tyrosine kinase-1 and free vascular endothelial growth factor at 11 to 13 weeks of gestation in preeclampsia. Prenat Diagn. 2010;30(3):191-197. Goel A, Rana S. Angiogenic factors in preeclampsia: potential for diagnosis and treatment. Curr Opin Nephrol Hypertens. 2013;22(6):643-650. Levine RJ, Lam C, Qian C, et al. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. N Engl J Med. 2006;355(10):9921005. Stepan H, Hund M, Gencay M, et al. A comparison of the diagnostic utility of the sFlt-1/PlGF ratio versus PlGF alone for the detection of preeclampsia/HELLP syndrome. Hypertension in pregnancy. 2016:1-11. Zeisler H, Llurba E, Chantraine F, et al. Predictive Value of the sFlt-1:PlGF Ratio in Women with Suspected Preeclampsia. N Engl J Med. 2016;374(1):13-22.

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Widmer M, Cuesta C, Khan KS, et al. Accuracy of angiogenic biomarkers at 20weeks' gestation in predicting the risk of pre-eclampsia: A WHO multicentre study. Pregnancy Hypertens. 2015;5(4):330-338.

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Table 1. Data Collection 12

18

28

Type of data

weeks *

weeks

weeks

Study Blood









Blood Pressure









Blood

Infectious

-

GTT

FBC

Investigations

Serology

Ultrasound

Nuchal

Ultrasound

Investigations

thickness

details of

Serum PAPP-

anomaly

A

scan

TE D

* Recruitment Data collected at this point

EP

Natal







-

-

-

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Term

SC

-

-

M AN U

FBC, Full blood count; GTT, Glucose tolerance test

AC C

36 weeks

FBC

Serum BHCG

- No data collected

Post

-

-

23

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Table 2. Baseline Characteristics Gestational Preeclampsia

Hypertension

Group

Group

Group

Characteristic

(n=307)

(n= 18)

Age (yrs)

33 [30-36]

32 [29.75-35.5]

Height (m)

1.65 [1.6-1.7]

1.6 [1.54-1.65] #

Weight (kg)

62 [57-70]

65 [58.25-67]

70 [60-88] *

0.015

BMI (kg/m2)

22.72 [20-97-25.0)]

24.02 [21.61-25.68]

26.3 [21.65-

0.041

(n=17)

P value

33 [30.5-35.5]

0.886

1.67 [1.65-1.68]

0.046

SC

M AN U

Blood Pressure

RI PT

Control

30.30]

110 [100-120]

120 [100-130]

110 [108-125]

0.029

Diastolic (mmHg)

65 [60-70]

75.5[60-81.25] *

70 [60-80]

0.006

20.67(62)

16.66(3)

5.88(1)

0.285

56 (168)

66.67 (12)

70.59 (12)

0.355

1.33 (4)

27.78(5)

0(0)

<0.001

76.67 (23)

11.11 (2)

11.76(2)

0.792

40 (39-40)

37.5 (35.75-40)*#

40 (38.5-40)

<0.001

Caesarean delivery % (n)

26.33 (79)

55.56 (10) *

41.18 (7)

0.015

Fetal weight centile

60.5 [34.25-80.75]

41.5 [7.5-79]

39 [23.5-71]

0.056

Smoking rate % (n) Primiparous rate % (n)

GDM % (n)

EP

History of PE % (n)

TE D

Systolic (mmHg)

AC C

Gestational age at delivery

Data are median [interquartile range] unless otherwise stated. % indicates rate of incidence. P value= Kruskal-Wallis analysis. * GHT or PE groups significantly different from control (Mann-Whitney U test and Bonferroni correction) #

PE group significantly different from GHT group (Mann-Whitney U test and Bonferroni correction)

24

ACCEPTED MANUSCRIPT FIGURE LEGENDS Figure 1: Study Recruitment A total of 351 women were recruited with 342 included in the study.

RI PT

PE; preeclampsia, GHT; gestational hypertension

Figure 2: Longitudinal changes of pregnancy markers (median with 25th-75th centiles)

SC

Longitudinal median changes of pregnancy markers over time for (A) soluble fms like

M AN U

tyrosine kinase 1 receptor [sFlt-1], (B) placental growth factor [PlGF], (C) soluble endogolin [sEng], (D) Angiotensin II type 1 receptor antibody [AT1AA]. PE; preeclampsia, GHT; gestational hypertension.

* statistically significant over time compared to previous time point (p<0.05).

TE D

# statistically significant between groups at that time point (p<0.05).

Figure 3: sFlt-1:PLGF ratio throughout gestation(median with 25th-75th centiles)

EP

Longitudinal changes of the ratio of soluble fms like tyrosine kinase 1 receptor [sFlt1] to placental growth factor [PLGF].

AC C

PE; preeclampsia, GHT; gestational hypertension. # statistically significant between groups at that time point (p<0.05).

Figure 4: Correlation scatter plots Serum Angiotensin II type 1 receptor antibody [AT1AA] measured at 12 weeks and (A) serum PAAP-A and (B) serum BHCG measured at 12 weeks. P <0.05 indicates statistical significance.

AC C

EP

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M AN U

SC

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Data 3 150

100

50

0 0

50

100

150

A GHT

SC

B PE

PE

Normal *

3.0

#

GHT

Normal

*

M AN U

450

#

400

2.5 *

350

#

2.0

PlGF pg/ml

sFlt-1 ng/ml

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1.5 1.0

* #

0.5

300

* #

250 200

*

*

#

150

#

100

*

50

TE D

0.0

m tu

rm

ar

te

36

28

18

12

m

EP

Time point (weeks)

AC C

C PE

GHT

45

po

GHT

Normal

40 35

AT1AA U/ml

*

30 25 20 15

30 25 20 15

m -p ar

po st

po

Time point (weeks)

tu

m te r

36

28

18

st -p

ar t

te

rm

um

0 36

0

28

5

18

5

12

10

10

12

sEng ng/ml

PE

Normal

*

35

Time point (weeks)

D

*

40

st

po st

-p

-p

ar tu

te rm

36

28

18

12

0

Time point (weeks)

GHT

TE D

m

#

tu

rm

ar st -p

Time point (weeks)

po

18

AC C

0.1

36

EP

0.2

#

28

0.3

12

sFlt-1:PLGF ratio

0.4

-0.1

#

te

0.5

0.0

Normal

M AN U

PE

SC

RI PT

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M AN U

SC

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A 8

TE D

PAPP-A

6 4

p = 0.003 r2 = 0.38

0 0

10

20

EP

2

30

40

50

AC C

AT1AA u/ml

B 5

BHCG

4 p = 0.004 r2 = 0.27

3 2 1 0 0

10

20

30

AT1AA U/ml

40

50