ORIGINAL ARTICLE

Predictive Role of First-trimester ELABELA Levels for Late-onset Preeclampsia Gulten Ozgen1, Gultekin Adanas Aydin1 and Habibe Ayvaci2 1Department of Obstetrics and Gynecology, Bursa Yüksek İhtisas Training and Research Hospital, Bursa, Turkey 2Department of Obstetrics and Gynecology, Zeynep Kamil Women and Children Disease Training and Research Hospital, Istanbul, Turkey

ABSTRACT Objective: To determine the predictive role of first trimester maternal serum ELABELA (ELA) levels in late-onset preeclampsia (PE). Study Design: Comparative descriptive study. Place and Duration of Study: Department of Obstetrics and Gynecology, Bursa Yüksek İhtisas Training and Research Hospital, Bursa, Turkey between January and September 2019. Methodology: A total of 600 pregnant women, whose maternal serum samples were collected through the first trimester screening test, were recruited. Twenty-three patients with late-onset PE group and 47 without PE group were included. All parti- cipants in whom maternal serum samples were collected between 11th and 14th weeks of were followed until delivery. Results: Median ELA level was 654.53 [217.67-870.20] pg/mL in the PE group and 645.80 [367.25-1833.17] pg/mL in the control group, indicating no significant difference between the groups (p=0.408). There was a statistically significant difference between the two groups in terms of frequency of history of hypertension, intrauterine growth restriction (IUGR) and acute fetal distress (AFD), (p=0.003, p=0.016, and p=0.005, respectively). Three patients had preterm delivery in the PE group which was non-significant, while seven patients had gestational diabetes mellitus in PE group that was significantly higher compared to controls (p<0.001). Conclusion: Only ELA level in the first trimester of pregnancy may not be of much value in predicting late-onset PE.

Key Words: ELABELA, Preeclampsia, Late-onset, Predictive marker.

How to cite this article: Ozgen G, Aydin GA, Ayvaci H. Predictive Role of First-trimester ELABELA Levels for Late-onset Preeclampsia. J Coll Physicians Surg Pak 2021; 31(08):916-920.

INTRODUCTION The placental hypoxia contributes to the formation of inade- quate syncytiotrophoblasts and results in downregulation of A successful pregnancy is defined as a healthy embryonic devel- syncytin-1 found in early-onset preeclampsia (PE) and intrau- opment and viable pregnancy with favourable maternal and terine growth restriction (IUGR). 2,3 fetal outcomes; and appropriate placental development is essential for a successful pregnancy.1 Placental perfusion is Preeclampsia is a pregnancy condition which accounts for 5-8% achieved by adequate remodelling of the spiral arteries of of all and is associated with increased fetal and 4 uterus in early pregnancy, resulting in invasion of the extra-vil- maternal morbidity and mortality, if left untreated. Its aeti- loustrophoblasts.1 ology is multifactorial and environmental factors, health and disease status, maternal or fetal malnutrition, have been Reperfusion accompanied by placental hypoperfusion and mostly blamed.5 ischemic injury leads to the release of circulating antiangio- genic factors, which are associated with maternal vascular ELABELA (ELA) is a novel which encodes an endogenous dysfunction. ligand for the apelin/AJP receptor, a member of Class A G-pro- tein coupled receptor family, which shares common features Correspondence to: Gulten Ozgen, Department of Obstet- with angiotensin receptor type 1 including regulation of blood rics and Gynecology, Bursa Yüksek Ihtisas Training and pressure, electrolyte balance, energy metabolism, inflamma- Research Hospital, Bursa, Turkey tory process, oxidative stress, vasodilation, vasoconstriction, E-mail: [email protected] cardiac contractility, and angiogenesis.6 It is expressed in ...... human embryonic stem cells and has involvement in self-re- Received: December 23, 2021; Revised: April 07, 2021; newal and embryonic apoptosis.7 Although early studies Accepted: June 28, 2021 showed that ELA was involved in the , kidney and pros- DOI: https://doi.org/10.29271/jcpsp.2021.08.916 tate tissue; however, recent studies have demonstrated that it

916 Journal of the College of Physicians and Surgeons Pakistan 2021, Vol. 31(08): 916-920 Gulten Ozgen, Gultekin Adanas Aydin and Habibe Ayvaci is additionally released in lungs and endothelia of the common uteroplacental dysfunction (IUGR and impaired Doppler velocit- vessels.8-10 ometry).4 Late-onset PE was defined as the presence of PE after 34 weeks of gestation. 14,15 Furthermore, apelin and ELA have been recently investigated as predictors of PE in placenta and maternal circula- Maternal venous blood samples were obtained between 11th tion samples of PE patients and healthy controls. 11 In an animal and 14 thweeks of gestation and centrifuged at 4,000 rpm for 10 model, Ho et al. reported that decreased placental ELA level was minutes. Then, they were stored at °C-80 until analysis. a predictor of PE and maternal systemic vascular injury.12 The serum levels of ELA in duplicate were analysed using the However, Pritchardet al. measured placental messenger human ELA (ELA32 isoform) ELISA kit (Sunred Biological Tech- ribonucleic acid (mRNA) and circulating ELA in PE patients and nology, Shanghai, China). The serum ELA concentration of this healthy controls and found no significant correlation between assay ranges between 0.01 to 100 ng/mL. PE and ELA levels. 13 Statistical analysis was performed using the SPSS version 22.0 There are controversial results regarding the role of ELA, apelin (IBM Corp., Armonk, NY, USA). Shapiro-wilk test was used to or APJ in the literature. The aim of the present study was to determine the normality of distribution of variables. Descriptive investigate the predictive value of the first trimester maternal data were presented as mean ± standard deviation for normally th th serum ELA levels in late-onset PE. distributed variables and as median [IQR]: (25 -75 percentile)] for non-normally distributed ones. Categorical variables were METHODOLOGY shown in number and percentage. Student’s t-test was used for This prospective, case-control study was approved by the local variables showing normal distribution between the two groups Ethics Committee vide decision No. 2011-KAEK-25-2019/12-10 and the Mann-Whitney U-test was performed to compare non- and performed between January and September 2019. Anormally distributed variables between the two groups. Cate- written informed consent was obtained from each patient. The gorical variables were compared between the groups using the study was conducted in accordance with the principles of the Pearson Chi-square and Fisher’s Exact test. A p value of <0.05 Declaration of Helsinki. was considered statistically significant. A total of 600 singleton pregnant women with an age range of 18 RESULTS to 40 years, whose serum samples were collected for the first Late-onset PE developed in 23 (3.8%) of 600 patients. A total of th th trimester screening test (11 to 14 weeks), were screened.- 70 participants including 23 (32.9%) late-onset PE patients Women with multiple pregnancy, morbid obesity, malignancy, and 47 (67.1%) healthy pregnancy were included in this study. active labour, chorioamnionitis, systemic inflammatory condi- The mean age was 31.4 ± 6.6years in the PE group and 28.1 ± tions and non-steroidal anti-inflammatory drug use, severe 4.4 years in the control group. The median BMI was 30.12 liver and kidney failure were excluded from the study. Among [27.34-35.11] kg/m2 in the PE group and 28.51 [27.18-31.4] the patients with PE, only those with late-onset PE werekg/m2. A statistically significant difference was found between included. The control group consisted of healthy age- and body the two groups in terms of age (p=0.037). There was no statisti- mass index (BMI)-matched individuals. Finally, 23 patients with cally significant difference in the BMI values between the late-onset PE (PE group) and 47 without PE (control group) were groups (p=0.277). Demographic characteristics of the study included in the study. population are shown in Table I. Demographic and clinical characteristics of the participants The mean birth weight was 2910 [2680-3360] g in the PE group and perinatal and postnatal outcomes including age, height, and 3395 [3012.5-3600] g in the control group (p=0.002). The BMI, gravidity and parity, maternal height and weight, gesta- median ELA level was 654.53 [217.67-870.20] pg/mL in the PE tional age, birth weight, delivery mode, APGAR (Appearance, group and 645.80 [367.25-1833.17] pg/mL in control group, pulse, grimace, activity and respiration) score, adverse indicating no significant difference between the groups obstetric outcomes such as PE, gestational diabetes mellitus (p=0.408). Clinical characteristics of the study population are (GDM), preterm premature rupture of membranes (PPROM), also presented in Table I. There was a statistically significant IUGR, and preterm birth obtained using electronic medical difference between the two groups in terms of the frequency of records. hypertension, IUGR, and acute fetal distress (AFD), (p=0.003, The PE was diagnosed with the presence of the above, accom- p=0.016, and p=0.005, respectively). Three patients had panied by one of the followings; (1) new onset hypertension [at preterm delivery in the PE group, which was non-significant, least two high systolic (≥140mmHg) or diastolic (≥90 mmHg) while seven patients had GDM in the PE group that was signifi- measurements at 4-hour intervals], (2) protein- cantly higher compared to the controls (p<0.001). A total of uria (≥300 mg/24 h, ≥30 mg/moL protein: creatinine ratio; or 87% cases in the PE group and 50% cases in the control group adipstick-test result ≥ 2+); (3) other maternal organ dysfunc- underwent caesarean-section delivery, indicating a statisti- tion (neurological complications such as , stroke, cally significant difference between the groups (p=0.003, headaches, blindness, kidney or liver impairments); and (4) Table II).

Journal of the College of Physicians and Surgeons Pakistan 2021, Vol. 31(08): 916-920 917 Predictive role of first-trimester ELABELA levels for late-onset preeclampsia

Table I: Demographic and clinical characteristics of study population. Control group (n=47) PE group (n=23) p-value Age, year 28.1±4.4 31.4±6.6 0.037b Height, cm 161.5± 3.5 160.1±4.1 0.145b Body weight, kg 75 [70-82] 77 [70-85] 0.487a BMI, kg/m2 28.51 [27.18-31.24] 30.12 [27.34-35.11] 0.277a First trimester screening test, week 12 [12-12.5] 12 [12-13] 0.399a Birth weight, g 3395 [3012.5-3600] 2910 [2680-3360] 0.002a APGAR score, 1 min 9 [9-9] 9 [8-9] 0.004a APGAR score, 5 min 10 [10-10] 10 [9-10] 0.004a ELA level, pg/mL 645.80 [367.25-1833.17] 654.53 [217.67-870.20 ] 0.408a Data are given in mean ± SD or median [IQR]: (25th-75th percentile. aMann-Whitney U-test. bIndependent samples t-test. PE: Preeclampsia. APGAR: Appearance, pulse, grimace, activity, and respiration; ELA: ELABELA.

Table II: Distribution of categorical variables according to study groups. Control PE group Total p-value n % n % n %

No 46a 100.0 18b 78.3 64 92.8 History of HT 0.003a Yes 0a 0 5b 21.7 5 7.2

No 44a 100.0 16b 69.6 60 89.6 GDM <0.001a Yes 0a 0.0 7b 30.4 7 10.4

No 43a 97.7 18b 78.3. 61 91.0 IUGR 0.016a Yes 1a 2.3 5b 21.7 6 9.0

No 43a 97.7 17b 73.9 60 89.6 AFD 0.005a Yes 1a 2.3 6b 26.1 7 10.4

No 44a 100.0 19b 82.6. 63 94.0 Placental abruption 0.012a Yes 0a 0.0 4b 17.4 4 6.0

No 42a 95.5 20a 87.0 62 92.5 Preterm delivery 0.330a Yes 2a 4.5 3a 13.0 5 7.5

No 22a 50.0 3b 13.0 25 37.3 Caesarean-section delivery 0.003b Yes 22a 50.0 20b 87.0 42 62.7

Neonatal ICU No 39a 92.9 18a 81.8 57 89.1 0.220a Yes 3a 7.1 4a 18.2 7 10.9 Data are given in number and frequency, unless otherwise stated. aFisher’s Exact test. bPearson chi-square test. PE, Preeclampsia; HT: Hypertension; GDM: Gestational diabetes mellitus; OGTT: Oral glucose tolerance test; IUGR/ Intrauterine growth restriction.

DISCUSSION apoptosis, reduced proliferation, and delayed syncytiotro- phoblast differentiation.12 In this study, exogenous ELA Preeclampsia is one of the most common complications of administration could reverse the destructive effects by pregnancy and is the leading cause of fetal and maternal inhibiting angiotensin-converting enzyme system through its 16 morbidity. The underlying aetiopathologic mechanisms of protective effects. PE have not been fully understood yet, and there are many studies searching for the mechanisms, biomarkers and treat- In another animal model, Yang et al. reported that exoge- ment options in this area. Early-onset PE is primarily associ- nous ELA infusion exerted cardiovascular protective effects ated with abnormal remodelling of the uterine arteries, while and prevented pulmonary arterial hypertension and experi- late-onset PE is a maternal rather than a placental disease mental septic shock with reversal of the destruction in rats. 20 with a more diverse underlying pathophysiology than early- In addition, Huang et al. longitudinally examined, for the onset type. The latter form of PE is more common, first time, the ELA levels before pregnancy and at each 21 accounting for 90% of all cases.17-19 trimester. The authors concluded that maternal blood ELA levels were elevated in the first and second trimesters in It is of utmost importance to identify high-risk patients for PE patients with late in pregnancy; to initiate targeted therapies. The ELA is a novel peptide however, the ELA levels were not significantly correlated which has been recently discovered and acts as an indepen- with PE during any stage of pregnancy. The detection of ELA dent angiogenic factor in PE pathogenesis. In ELA-knockout in non- pregnant women was suggestive of the fact that ELA mice, Ho et al. found that the of the ELA-knockout was not a pregnancy-specific hormone.20 However, elevated mice had thin labyrinths with poor vascularization, increased levels of ELA in pregnant women than non-pregnant women

918 Journal of the College of Physicians and Surgeons Pakistan 2021, Vol. 31(08): 916-920 Gulten Ozgen, Gultekin Adanas Aydin and Habibe Ayvaci could be attributed to the ELA release by embryo and nancy, seems to be a response against hypoxia and oxida- . The authors also found that ELA levels tive stress resulting from the divergence between the increased in each trimester, although this increase was not maternal perfusion and placental/fetal metabolic demand. significant. Compared to the control group, ELA levels were also increased in the first and second trimester in onlyIn experimental animal models, ELA has been shown to 21 increase vasodilation, cardiac contractility and cardiac patients with gestational hypertension. In their study, Deniz output, and ejection fraction.20,21 In the late pregnancy, et al. compared maternal serum ELA, apelin, and NO levels elevated ELA levels exhibit a protective effect against renal between PE and healthy controls and also PE and severe PE compensation and/or endothelial stimulation in women with groups.22 They found that the mean maternal blood ELA gestational hypertension.21 Considering the fact that total levels were significantly lower in PE, compared to severe PE vascular resistance is lower and cardiac output is higher in cases. The decreased levels of ELA in that study could be late-onset PE than early-onset PE, elevated ELA levels seem related to the late collection week of serum samples at 36 to be a protective mechanism against increased cardiac weeks of gestation. output. These findings are consistent with the previous In previous studies, insufficient extratrophoblast proliferation report by Huang et al. showing that ELA levels were elevated in the maternal spiral arteries has been suggested to cause from the first trimesters with slight increases in further 21 impairment in placental perfusion.23 trimesters. In this study, however, there were relatively low ELA concentrations than those found by Panaitescuet al. In this study, maternal serum ELA concentrations from age- and Pritchard et al.’s studies.11,13 This discrepancy can be and BMI-matched healthy controls and from PE patients in attributed to the differences in the methodology of these the first trimester of pregnancy were investigated the predic- studies, such as the collection of serum samples at first, tive value of ELA for late-onset PE. The median maternal second or third gestational week. Therefore, further studies serum ELA concentration was 654.53pg/mL in the late-onset using similar study protocols are warranted. Unlike other PE group and 645.80pg/mL in the control group, indicating studies, these findings do not support the hypothesis that PE no statistically significant difference (p=0.408). In addition, is characterised by an early deficiency of circulating ELA. there was no significant difference in the ELA levels in the first trimester of pregnancy between the groups. Similarly, in CONCLUSION 24 a recent study, Villie et al. examined whether ELA acted Molecular biomarkers play a central role in research and clin- independently and earlier than sFlt-1 and was dysregulated ical practice. The discrepancy between the present results before the onset of PE. They found no significant difference and previous reports can be attributed to the use of different in the ELA levels between the control group and PE patients. ligand profiles in different tissues and sampling of maternal This finding suggests that human PE is not characterised by serum in first trimester. Based on the present results, ELA an early deficiency in ELA. Similarly, the discrepancy in the alone may not be a valuable biomarker in the first trimester ELA levels in late-onset PE versus the control group in the of pregnancy to predict late-onset PE. However, further 24 study of Villie et al. and this study can be explained by the large-scale, prospective studies are needed to establish a fact that serum ELA concentrations might have increased in definite conclusion. the second and third trimesters, but not in the first trimester, to compensate maternal perfusion in late-onset ETHICAL APPROVAL: PE. The trial protocol was approved by the local Ethics Committee No. 2011-KAEK-25-2019/12-10. Zhou et al. evaluated the maternal serum, urine and placental tissues ELA levels in PE and healthy pregnants.23 PATIENTS’ CONSENT: Written consent was obtained from each patient participating The ELA levels were found to significantly lower in late-onset in this study. PE compared to control ones. This finding indicates that ELA may play a major role in the pathogenesis of late-onset PE. CONFLICT OF INTEREST:

In another study, Para et al. examined whether a model The authors declared no conflict of interest. combining the measurement of the Fas/Fas ligand system (sFas) and ELA in the maternal circulation could be used as a AUTHORS’ CONTRIBUTION: clinical biomarker for early- and late-onset PE.25 The authors GO: Conception and design analysis. showed that late-onset PE patients, but not early-onset PE, GAA: Design, analysis. HA: Acquisition, analysis, interpretation of data. had elevated concentrations of ELA, compared to gestational age-matched controls and elevated ELA and sFas concentra- REFERENCES tions were more strongly correlated with late-onset form of

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Journal of the College of Physicians and Surgeons Pakistan 2021, Vol. 31(08): 916-920 919 Predictive role of first-trimester ELABELA levels for late-onset preeclampsia

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