Lack of an association between first‐trimester concentration of mid‐regional pro‐atrial natriuretic peptide and risk of early‐onset preeclampsia <34 weeks' gestation

We examined the heart failure biomarker mid‐regional pro‐atrial natriuretic peptide during the first trimester of pregnancy in relation to early‐onset preeclampsia <34 weeks.


INTRODUCTION
Preeclampsia affects 3%-8% of all pregnancies worldwide and is a major cause of maternal and fetal morbidity and mortality. 1 The pathology of preeclampsia involves impaired placentation and has been investigated for several years but is still not fully understood and is considered multifactorial. The current understanding is that failure in the remodeling of the uterine spiral arteries and inadequate trophoblast invasion and migration play a major part. On a molecular level, proangiogenic and antiangiogenic factors, like placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1), have been found to be involved, but the molecular mechanisms are not fully elucidated. [2][3][4] Due to the immediate maternal and fetal adverse consequences of preeclampsia, and the possibly future increased risk of cardiovascular disease for both mother and child, it is important to find preventive methods for preeclampsia. Especially early identification of high-risk patients is of great value, because low-dose aspirin treatment initiated before 16 weeks of gestation has proven to be effective as a prophylactic treatment for preterm preeclampsia. 1,5 In uncomplicated pregnancies, cardiac output (CO) is increased as early as 5 weeks of gestation, 6 and the increase in CO is accompanied by a decrease in mean arterial pressure and reduction in systemic vascular resistance to avoid high blood pressure and pathological strain to the heart. 7 The preclinical phase of preeclampsia is characterized by a hyperdynamic highoutput-low-resistance state, whereas the clinical phase is characterized by a low-output-high-resistance state. 6,8 Correspondingly, maternal CO at 11+0 to 13 +6 weeks of gestation has been found to be higher in women destined to develop preeclampsia compared to women, who did not develop preeclampsia. 6 Natriuretic peptides, like brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP), are excreted primarily from the ventricular and atrial myocytes, respectively, in the respond to stretching of the ventricular or atrial wall due to an overload of the heart. The function of natriuretic peptides is to decrease the workload of the heart, and they do so by increasing natriuresis and diuresis, enhancing capillary permeability and relaxing the peripheral vasculature, 9 thus, decreasing afterload. ANP is stored as ProANP in the atrial myocytes and is cleaved in equimolar amounts into mid-regional pro-atrial natriuretic peptide (MRproANP) and the biologically active ANP. MRproANP is more stable and has a longer half-life than ANP and is, correspondingly, often used as a surrogate biomarker for plasma ANP. [10][11][12] MRproANP is a well-known diagnostic biomarker for the presence and severity of hemodynamic cardiac stress and heart failure, 13 and it is a useful biomarker for prediction of major adverse cardiovascular events in patients with diabetes. 14,15 Also, MRproANP has been found to be inversely associated with incident type 2 diabetes. 16 In uncomplicated pregnancies MRproANP plasma concentrations are similar or slightly higher than in the nonpregnant state, and MRproANP concentrations have been shown to be elevated in the clinical phase of preeclampsia, likely due to the strain on the heart that preeclampsia exerts. 9,[17][18][19] MRproANP levels in the first trimester have not been investigated extensively, and most of the published studies focus on the MRproANP in the second and third trimester. Inconsistent results have been found, but some studies have shown elevated concentrations of MRproANP from 22 weeks of gestation in the preclinical phase of preeclampsia. One study has indicated higher levels of MRproANP in the first trimester in pregnancies destined to develop preeclampsia, but results were not statistically significant. 20 The aim of this study was to further investigate the association between first-trimester MRproANP and early-onset preeclampsia, that is, severe preeclampsia requiring delivery before 34 weeks of gestation.

Study population
In this retrospective case-control study, the population comprised pregnant women attending the routine firsttrimester ultrasound scan at 11-13+6 weeks of gestation between August 2010 and October 2015 at the Department of Obstetrics at Rigshospitalet, Copenhagen, Denmark. Women had a routine blood sample taken at 8-13+6 weeks of gestation, which was analyzed for pregnancy-associated plasma protein A (PAPP-A) and free beta human chorionic gonadotrophin (free β-hCG). Furthermore, the women had a first-trimester scan including measurement of crown-rump length and nuchal translucency thickness. These markers were used in the risk calculation for aneuploidies and diagnosis of major fetal abnormalities, and the crown-rump length was measured to determine the gestational age. All blood samples were stored in the hospital biobank freezer at À80 C.
We included singleton pregnant women with earlyonset preeclampsia defined as a preeclampsia diagnosis and delivery before 34 weeks of gestation. 1 For each blood sample from a preeclampsia case, two to three control samples were randomly selected from the local biobank by matching on the analysis day of the firsttrimester blood sample. Through information in the local obstetric database, control samples were excluded if the pregnancy was complicated by preterm delivery or preeclampsia. All multiple pregnancies were excluded.
The study was approved by the Danish Data Protection Agency (H-2016-52, I-Suite no.: 04448) and the local ethics committee approved the study, with dispensation from consent (H-16002032).
The study population comprised 34 singleton pregnancies with early-onset preeclampsia and 91 singleton control pregnancies matched by time of routine firsttrimester blood sampling where MRproANP had subsequently been measured in the biobank samples.

Maternal characteristics and history
Maternal characteristics and obstetric and medical history were retrieved from the local Astraia database (Astraia GmbH, Munich, Germany). The maternal characteristics comprised age, height, and weight at the time of the routine first-trimester ultrasound scan, ethnicity (Caucasian or non-Caucasian), and smoking during pregnancy. Maternal obstetric history included parity (nulliparous or multiparous) and medical history, method of conception (spontaneous or nonspontaneous) and diabetes mellitus.

Biochemical analyses
The first-trimester blood samples were collected between 8-13+6 weeks of gestation and were centrifuged at 2000g for 10 min within 6 h after collection. The samples were analyzed routinely for free βhCG and PAPP-A as part of the first-trimester screening for chromosomal anomalies. Excess serum was stored at À80 C, and samples were thawed and analyzed for PlGF, sFlt-1, and MRproANP. All analyses were performed using the BRAHMS KRYPTOR compact PLUS platform, which is based on TRACE (Time-Resolved Amplified Cryptate Emission) Technology. The detection limit was 3.6 pg/mL, 22 pg/ mL, and 2.1 pmol/L for PlGF, sFlt-1 and MRproANP, respectively. The functional assay sensitivity, detected by inter-assay precision of 20% of coefficient variability (CV), has been assessed as being lower than 10 pmol/L for MRproANP and 6.72 pg/mL and 29 pg/mL for PlGF and sFlt-1, respectively.

Definition of preeclampsia
Preeclampsia was defined according to the Danish guideline from 2018 (www.dsog.dk/obstetrik) and comprised the development of de novo hypertension and proteinuria after 20 weeks of gestation and/or one of the following signs of organ failure: thrombocytopenia, renal insufficiency, liver insufficiency, pulmonary edema, cerebral or visual symptoms, or intrauterine growth restriction. In patients with pregestational hypertension (hypertension before pregnancy or before 20 weeks of gestation) preeclampsia was defined as an additional increase in blood pressure and proteinuria and/or one of the abovementioned symptoms or signs of organ failure after 20 weeks of gestation. Hypertension was defined as systolic blood pressure ≥140 mmHg and/or a diastolic blood pressure ≥90 mmHg measured at two different occasions at least 4 h apart. If blood pressure was ≥160 mmHg systolic and/or ≥110 mmHg diastolic, hypertension could be diagnosed within a shorter time interval (min) to ensure that treatment be initiated in a timely manner. Proteinuria was defined as ≥1 + (30 mg/dL) on a urine dipstick, or a spot urine analysis with protein/ creatinine ratio ≥30 mg/mmol (0.3 mg/mg) and/or albumin/creatinine ratio ≥8 mg/mmol (70 mg/g) or protein ≥0.3 g in a 24-h urine specimen.

Statistical analysis
Descriptive statistical analyses were performed for maternal characteristics and obstetric and medical history for early-onset preeclampsia cases compared to the control group, reported as the mean and SD, median and interquartile range (IQR) or frequencies. For comparison of the characteristics between preeclampsia cases and the control group Students t-test, Mann-Whitney U test or chi-squared test were applied, as appropriate.
The distribution of the measured concentration of all five biomarkers was compared between early-onset preeclampsia cases and the control group using the Mann-Whitney U test. For PAPP-A and free β-hCG multiple of the medians (MoMs) were calculated routinely in Astraia with adjustment for maternal weight, ethnicity, smoking status, mode of conception, parity, and diabetes mellitus. For PlGF, sFlt-1, and MRproANP univariable linear regression analysis was done on data for the women in the control group to determine which factors among the maternal characteristics and obstetric and medical history were significant contributors to the respective biomarker log-transformed concentrations. Multivariable linear regression analysis with backward elimination was performed based on p values < 0.1 in the univariable linear regression analysis. Variables with p values > 0.05 were excluded in the backward elimination. However, for all three biomarkers, results were adjusted for gestational age. The resulting equation was used to calculate the expected concentration for each biomarker and the data was converted into MoM values.
Level of significance was set at p < 0.05. With the sample size of 34 cases and 91 controls, a power of 0.80 with α = 0.05, we would be able to detect a difference in biomarkers levels of 0.05 MoMs for cases compared to controls. All statistical analyses were done using the R studio software package (R version 3.5.1) and Stata (Stata version 15.1).

RESULTS
Data on maternal characteristics and obstetric and medical history are presented in Table 1. Women with early-onset preeclampsia were significantly older ( p = 0.01) and their pregnancies were less likely to be spontaneously conceived ( p = 0.04) compared to the women in the control group. Median gestational age at the time of blood sampling was slightly lower among women with early-onset preeclampsia than in the control group ( p = 0.02).
The relation between the measured concentration and the gestational age at blood sample collection is shown as scatter plots in Figure 1 for MRproANP, PlGF, and sFlt-1. We found that gestational age was significantly associated with the measured concentration of PlGF ( p < 0.001) and sFlt-1 ( p = 0.02) in these first-trimester blood samples, whereas there was no evidence of a significant association between gestational age and the measured concentration of MRproANP ( p = 0.84).
In the univariable linear regression analyses performed on the women in the control group, we found no significant association with the measured biomarker concentration in relation to neither maternal characteristics, nor obstetric and medical history for MRproANP and sFlt-1 in these first-trimester samples. However, we found a significant association with the measured concentration of PlGF for parity (p = 0.004) with a lower concentration in nulliparous women compared to multiparous women.
Neither measured MRproANP concentrations nor MRproANP MoM levels were significantly different in women with early-onset preeclampsia compared to the control group ( p = 0.41 and p = 0.48, Table 2), whereas median PlGF and PAPP-A levels, as expected, were significantly lower (p < 0.001 and p = 0.03, respectively, for median MoM levels). We found no statistically significant T A B L E 1 Maternal characteristics and obstetric and medical history and comparison of gestational age at the time of blood sampling between women with early-onset preeclampsia <34 weeks and uncomplicated pregnancies (control group).

Maternal characteristics
Early-onset preeclampsia (n = 34) Control group (n = 91) p-Value differences in sFlt-1 levels or free β-hCG levels in women with early-onset preeclampsia compared to controls (p = 0.11 and p = 0.45, respectively, for MoM levels). As a higher body mass index is known to be associated with lower circulating concentrations of natriuretic peptides, 21 we performed sensitivity analyses after matching the early-onset preeclampsia cases with controls by body mass index. These analyses showed similar results to the nonmatched analyses (data not shown).

DISCUSSION
In this study, we examined the first-trimester plasma concentration of MRproANP in a case-control study with 34 women with preeclampsia and delivery before 34 weeks of gestation and 91 women without preeclampsia including the more well-described biomarkers, free β-hCG, PAPP-A, PlGF, and sFlt-1.
The first-trimester plasma concentration of MRproANP was not significantly different in the group of women who subsequently developed early-onset preeclampsia compared to the control group, but as described in the literature, we found that concentrations of both PlGF and PAPP-A were significantly lower in women with early-onset preeclampsia compared to the control group. As expected, we did not find an association with sFlt-1 and free β-hCG levels.
Only one other study has investigated the first-trimester levels of MRproANP in relation to preeclampsia. 20 In line with our results, they also found no significant difference of MRproANP levels between 11 women with early-onset preeclampsia and 100 women in the control group. The fact that MRproANP concentrations were not found elevated in the first trimester in pregnancies destined to develop early-onset preeclampsia could be explained by the increased afterload strain on the heart not being high enough to give measurably elevated concentrations of MRproANP in the first trimester, most likely because the body's adaptive system is still sufficient at this early point in pregnancy. This corresponds to the observed mechanisms in uncomplicated pregnancies, where CO is higher in the first trimester compared to nonpregnant women.
In recent years, more studies about preeclampsia and maternal cardiovascular function have been published, where hemodynamics, biomarkers, fetal development (measured as small for gestational age [SGA] and fetal growth restriction [FGR]), and gestational age at delivery have been examined. De Paco et al. 6 found that CO was higher in the first trimester in women with pregnancies destined to develop preeclampsia, but lower in pregnancies with SGA, compared to unaffected pregnancies. They further subdivided the women with preeclampsia into cases with and without SGA and found no difference in CO in women with preeclampsia with SGA compared to unaffected pregnancies. Another study, by Giannubilo et al., 7 examined pregnant women in the clinical phase of preeclampsia and also found a difference in maternal hemodynamics when they subdivided preeclampsia into preeclampsia with and without FGR. They confirmed that plasma BNP was elevated in preeclampsia pregnancies compared to unaffected pregnancies, but more interestingly, they found that the lower the CO the higher the plasma BNP levels, and the higher the plasma BNP levels, the lower the birth weight, but this was only found in the preeclampsia group with FGR. So, in the clinical phase of preeclampsia, the subgroup with FGR has lower CO, higher vascular resistance and higher plasma BNP levels compared to the group without FGR. These results support the current understanding that preeclampsia is a multifactorial disease with different phenotypes and pathology, which could explain the different results in the different trimesters.
With respect to handling, storage, and analysis of the MRproANP samples, studies have shown that the F I G U R E 1 Measured concentration according to gestational age at blood sampling in women with early-onset preeclampsia (black dots) and uncomplicated pregnancies (gray dots) for: (a) Mid-regional proatrial natriuretic peptide (MRproANP), (b) Placental growth factor (PlGF), (c) soluble Fms-like tyrosine kinase-1 (sFlt-1). proANP molecule, containing the MRproANP site, is well suited as a surrogate analyte instead of the biologically active ANP, because it has been proven to be more stable. Furthermore, studies have shown that the relatively new assay targeting the MRproANP site on proANP, which was used in this study, is equally good or better than older assays targeting the N-terminal site of proANP. 10,12 A study by Goode et al 11 showed no significant degradation of MRproANP, when blood was stored for up to 24 h prior to centrifugation. Therefore, the handling, storage, and analysis of our MRproANP samples is not likely to have affected our results.
The lower first-trimester PAPP-A and PlGF concentration in pregnancies destined to develop early-onset preeclampsia in our study is very much consistent with current literature and supports the well-established hypothesis that imbalances in angiogenic factors play an important role in the development of preeclampsia. 1,[22][23][24][25][26][27][28] The fact that PAPP-A and PlGF show consistent results with current literature, strengthens the results obtained for MRproANP, which has not been investigated as well as the others.
The literature on first trimester sFlt-1 levels in pregnancies destined to develop preeclampsia is inconsistent. Both no difference, decreased as well as increased concentrations of sFlt-1 in the first trimester have been described, 1,22,26,[29][30][31] but, one of the most recent reviews from 2019 1 regarding first-trimester preeclampsia screening concluded that most of the included studies showed no significant evidence for using the firsttrimester sFlt-1 level as a screening marker for preeclampsia, as sFlt-1 levels only increased significantly after the first trimester.
Our study holds some limitations. Of the known risk factors for developing preeclampsia, 1,32-34 we included diabetes mellitus, height, weight, body mass index, maternal age, method of conception, and parity. Unfortunately, some other well-known risk factors of preeclampsia were not included in our study, because they were not routinely registered in the Astraia database at the time. Therefore, the population may include women with cardiac and renal disease, conditions that may affect the plasma MRproANP concentration. 35 Our study population mainly included Caucasians ($95%), which limits the results to not being applicable for other ethnicities. Preanalytical factors such as exercise and hydration status may affect plasma MRproANP concentrations. [36][37][38] In our study, the blood samples were collected in our blood test clinic, where the joint EFLM-COLABIOCLI recommendation for venous blood sampling 39 are followed including resting for 15 min in the waiting area, but we do not have information on preanalytical factors such as level of physical activity prior to blood sampling.
T A B L E 2 Comparison of biomarker levels between women with early-onset preeclampsia <34 weeks and uncomplicated pregnancies (control group). The relatively large number of early-onset preeclampsia cases, a rare form of preeclampsia associated with a high risk of neonatal complications, 40 is a strength to our study. Aspirin treatment to women at high risk at firsttrimester screening for preeclampsia has been shown to have the greatest effect for prevention of early-onset preeclampsia. The only previous study examining firsttrimester MRproANP 20 analyzed samples from only 11 women with early-onset preeclampsia, but they also included 24 women with preterm (i.e., delivery between 34 and 37 weeks) preeclampsia. It is also strength of our study that all cases were validated in the local hospital database as to whether the cases fulfilled the preeclampsia definition set in the study. This validation ensures that cases registered with faulty diagnosis codes are not included.
In conclusion, the maternal first-trimester concentration of MRproANP, a peptide with multiple biological functions including a relation to cardiovascular disease, was not significantly different in women destined to develop early-onset preeclampsia compared to controls. To date, PlGF and PAPP-A remain the most interesting first-trimester markers for prediction of early-onset preeclampsia. Future studies should try to further elucidate the potentially different phenotypes of preeclampsia 41 and enhance the understanding of the pathology behind preeclampsia and, thereby, improve first-trimester screening and treatment of both the preclinical phase and the clinical phase of preeclampsia.
AUTHOR CONTRIBUTIONS Signe Milling Mortensen: Protocol development, data management, data analysis, and manuscript preparation and editing; Charlotte Kvist Ekelund: Protocol development, data collection, and manuscript editing; Berit Woetmann Pedersen: Protocol development, data collection, and manuscript editing; Ann Tabor: Protocol development, data collection, and manuscript editing; Line Rode: Protocol development, data collection, data analysis, and manuscript preparation and editing. All authors made substantial contribution to the conception of the work and the interpretation of the data, revised it critically for important intellectual content and approved the final version.

CONFLICT OF INTEREST STATEMENT
All authors declared that they have no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. The Danish Patient Safety Authorities do not allow data sharing without individual approvals. It is therefore not possible to publicly share our data.
However, upon request we will be able to apply for approval for data sharing.