Original Investigation

Comparison of maternal serum NRG-4 levels in healthy and preeclamptic pregnancies


  • Kadriye Yakut
  • Filiz Halıcı Öztürk
  • Doğa Fatma Öcal
  • Betül Yakıştıran
  • Fatma Didem Yücel Yetişkin
  • Turhan Çağlar

Received Date: 28.11.2020 Accepted Date: 08.09.2021 J Turk Ger Gynecol Assoc 2022;23(1):8-13 PMID: 35263833


The new adipokine, neuregulin-4 (NRG-4), acts as a signaling protein and plays a role in lipogenesis, inflammatory events and atherosclerosis. The aim was to investigate maternal levels of NRG-4 in preeclampsia (PE) disease.

Material and Methods:

Pregnant women with PE, divided into severe and mild PE, and gestational age-matched healthy pregnant women, as a control group, were recruited. NRG-4 levels were measured using an ELISA. NRG-4 levels in the groups and the relation between NRG-4 and clinical and laboratory parameters were analyzed.


There were 41 women in the PE group, 11 (26.8%) in the severe and 30 (73.2%) in the mild subgroups and 41 controls. There were no significant differences between the groups in terms of maternal age, gravidity, parity, abortion, gestational week at the time of blood sampling, levels of hemoglobin, platelet count, alanine and aspartate transaminases (p=0.067, p=0.819, p=0.957, p=0.503, p=0.054, p=0.217, p=0.306, and p=0.270 respectively). The PE group had higher body mass index, nitrogen urea and creatinine values, and diastolic and systolic blood pressure (p=0.005, p<0.001, p<0.001, p<0.001, and p<0.001 respectively). In addition, earlier gestational week at delivery, lower birth weight and Apgar scores at 1 and 5 minutes and the occurrence of non-reassuring fetal heart rate tracing were found in the PE group (p=0.010, p=0.004, p=0.005, p=0.005, and p=0.026 respectively). There were no significant differences between the groups in terms of NRG-4 (p=0.611). No correlation was identified between clinical parameters examined and NRG-4 levels (p=0.722).


No association was found between NRG-4 concentrations and PE patients, regardless of severity of PE, compared to healthy pregnancies. Future longitudinal studies are needed to confirm this lack of association in PE.

Keywords: NRG-4, neuregulin, preeclampsia, perinatal outcome


Preeclampsia (PE) is a serious cardiovascular disorder of pregnancy, which is characterized by hypertension in addition to proteinuria or hypertension and end-organ dysfunction in the absence of proteinuria (1). The course of the disorder is unpredictable, and associated with neonatal and maternal life-threatening complications (1,2,3). The pathogenesis of PE is multifactorial and involves abnormal development of placenta, endothelial dysfunction, and immunological and genetic factors (4,5,6). Changes in the balance of angiogenic and antiangiogenic factors, and the presence of insulin resistance and/or obesity contribute to the pathogenesis of the PE and clinical symptoms (7).

Neuregulins are members of the endothelial growth factor-like growth factor family. Four subtypes of neureglins have been identified, one of which is neuregulin-4 (NRG-4) (8). NRG-4 is mainly produced by brown adipose tissue and plays a role as signaling protein during cell-to-cell interaction (9). In vivo studies have suggested that NRG-4 levels change during the process of lipogenesis, inflammatory events and as a result of changes in energy metabolism (10,11). It has been suggested that NRG-4 positively correlates with the development of obesity related disorders, such as type 2 diabetes mellitus (DM) and non-alcoholic fatty liver disease (NAFLD) and plays a role in the development of coronary artery disease (CAD) by promoting atherosclerosis (12,13,14,15,16,17).

The relationship between adipokines and metabolic disorders is complex and is not fully understood. In addition to adipose tissue, placental tissue is thought to be a source of adipokines. The relationship between metabolically active proteins, such as leptin, resistin, adiponectin and tumor necrosis factor-a has been investigated in the pathogenesis of PE. However, no clear relationship between adipokine levels and PE has been found (18,19,20,21). Therefore, the aim of this study was to investigate if there was an association between PE and NRG-4 levels for the first time. We also aimed to understand whether NRG-4 levels are associated with the severity of PE and neonatal and maternal clinical parameters.

Material and Methods

Study participants

This was a case-control study. Pregnant women with PE, divided into two subgroups as severe PE and mild PE, and gestational age-matched healthy pregnant women as a control group were recruited. All data were collected between September 2018 and March 2019 at the Department of Perinatology of Zekai Tahir Burak Women’s Health Training and Research Hospital in Ankara, Turkey. The study design was approved by the institutional research ethics committee (approval number: 28/2019) and written informed consent was obtained from all participants. The study was performed according to the universal principles expressed in the Declaration of Helsinki. All participants were in the third trimester of pregnancy. Exclusion criteria included any patient having: a chronic systemic disease; an autoimmune disease; chronic drug use; or presence of multiple gestation; presence of fetal congenital abnormality; and presence of complication of pregnancy including gestational DM, chorioamnionitis, and premature preterm rupture of pregnancy. Body mass index (BMI) was calculated as body weight (in kilograms) divided by squared height (in metres). According to the ACOG guideline (1), the criteria for the diagnosis of PE and in which cases it is called PE with severe features (the group called severe PE according to the old nomenclature) are given below.

Diagnostic criteria for preeclampsia:

- New onset of hypertension, defined as systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg in at least two measurements over four hours or six hours apart, or systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥110 mmHg confirmed within a short interval (minutes).


- Proteinuria defined as protein/creatinine ratio ≥0.3 mg/dL, dipstick reading of 2+ or ≥300 mg in a 24-hour urine collection after 20 weeks of gestation,


- In the absence of proteinuria - new onset of hypertension with signs of end-organ dysfunction, such as platelet count <100,000/mL, increased liver transaminases on at least two occasions, pulmonary edema, serum creatinine >1.1 mg/dL or two-fold elevation of basal creatinine level or new-onset headache, unresponsive to medication, or visual symptoms

The presence of any of the following criteria was grouped as “severe PE” (according to the old nomenclature, and “severe PE with severe feature” according to the new nomenclature).

PE with severe features as following:

- Systolic blood pressure ≥160 mmHg or diastolic blood pressure ≥110 mmHg on two occasions at least 4 hours apart (unless antihypertensive therapy was initiated),

- Platelet count <100,000/mL,

- At least two episodes of increased liver transaminases,

- Pulmonary edema,

- Serum creatinine >1.1 mg/dL or there was a two-fold elevation of basal creatinine level,

- New-onset headache unresponsive to medication,

- Visual symptoms,

- Severe persistent right upper quadrant or epigastric pain unresponsive to medications (Gestational Hypertension and Preeclampsia: ACOG Practice Bulletin, Number 222 Obstet Gynecol 2020; 135: e237-60. doi: 10.1097/AOG.0000000000003891).

Based on the ACOG guideline (1), patients who met the above-mentioned PE diagnostic criteria but did not have the criteria of PE with severe features; were classified as mild PE group (mild PE with the old nomenclature, PE without severe features with the new nomenclature).

Blood samples

All blood samples were taken from the antecubital vein. Samples for the measurement of NRG-4 levels were taken into tubes containing ethylene diamine tetra-acetic acid blood samples of 5 mL volume were centrifuged for 10 minutes at 1,000×g at 2-8 °C within 30 minutes of collection. Then plasma was stored at -80 °C until analysis. Concentrations of alanine transaminases, aspartate transaminases, creatinine, urea, hemoglobin and platelet count were measured as routine laboratory parameters. NRG-4 levels were measured using an ELISA (Human NRG-4 ELISA Kit, Cloud-Clone Corp., Katy, TX 77494, USA) following manufacturer’s instructions. The intra- and inter-assay coefficients of variation were <10% and <12%, respectively. The detection range was 0.156-10 ng/mL. The minimum detectable dose of NRG-4 is typically less than 0.056 ng/mL.

Statistical analysis

IBM SPSS Statistics, version 21.0 (IBM Corp. Armonk, NY, USA) was used to analyze the collected data. Kolmogorov-Smirnov test was used to evaluate the closeness of data sets to normal distribution. Descriptive statistics were expressed as mean ± standard deviation and median (minimum-maximum). The parametric Sample t-test and non-parametric Mann-Whitney U test were used to determine statistically significance between two independent groups, as appropriate. Comparison of two qualitative variables was done with the chi-square test, according to the expected value levels. Spearman and Pearson correlation tests were used to examine the association between two variables. Statistical significance was assumed when p<0.05.


Eighty-two women participated in this case-control study, equally divided between the PE group (n=41) and control group (n=41). Clinical and demographic parameters of study groups are shown in Table 1. There were no significant differences between the PE and control groups in terms of maternal age, gravidity, parity, abortion and gestational week at the time of blood sampling. BMI was significantly higher in the PE group compared to the control group. Similarly, there was no difference between the PE and control groups in terms of hemoglobin concentration, platelet count, and alanine and aspartate transaminases levels. However, renal function tests such as blood nitrogen urea and creatinine were significantly elevated in the PE group in comparison with the control group (Table 1).

Perinatal outcomes of the study groups are shown in Table 2. Gestational week at the time of delivery was earlier in the PE group than in the control group. Babies born to mothers in the control group had significantly elevated birth weight compared to babies born to mothers with PE. Mothers with PE were more likely to deliver by cesarean section when compared with controls. Apgar scores at 1 minute and 5 minutes were much lower in babies from the PE group but there was no difference between the groups in terms of neonatal intensive care unit admission likelihood (Table 2).

The PE group was divided into severe (n=11, 26.8%) and mild (n=30, 73.2%). Statistically, there was no difference in terms of NRG-4 levels between the PE group as a whole and the control group (Table 1). In addition, there was no difference in NRG-4 levels between the severe and mild PE groups (p=0.72). As shown Table 3, no significant correlations were identified between NRG-4 levels and clinical, laboratory and demographic parameters.


NRG-4, a new brown adipose tissue-associated adipokine, has been reported to play an important role in the regulation of energy metabolism and in the development of obesity related diseases (12,13,14,15,16). Besides acting in paracrine and autocrine signal transduction, NRG-4 decreases hepatic lipogenesis and increases fatty acid beta-oxidation in an endocrine fashion (9). Thus, NRG-4 contributes to lipid and glucose homeostasis. It has been reported that NRG-4 promotes the development of obesity-related disorders, such as type-2 DM and NAFLD (12,13,14,15,16). In addition to the metabolic roles ascribed to NRG-4, Ma et al. (21) suggested that excessive production of NRG-4 may have anti-atherogenic and anti-inflammatory effects. Similarly, it has been suggested that decreased NRG-4 levels may induce the development of atherosclerosis (20). Sato and Minatsuki (17) reported that NRG-4 levels were lower in patients with CAD than in the control group. However, it has not been clearly established which metabolic pathways NRG-4 interacts with. Activating ERB B4, a member of the epidermal growth factor receptor and plays role in the diseases such as cancers, DM may lead to the progression of atherosclerosis by inhibiting the apoptosis of endothelial cells via NRG-4 (14). It has also been shown that reduced NRG-4 levels were related to increased carotid intima thickness and carotid plaque in a group with obesity when compared to a control group (22). In the light of this evidence, we investigated whether there was an alteration of NRG-4 levels in women with PE. This seemed a reasonable hypothesis, given that PE is known to be associated with obesity, endothelial dysfunction, inflammation and metabolic diseases. Considering the similarities between mechanisms and risk factors, it was expected that there would be a correlation between NRG-4 and PE. However, we did not find any such relationship. As has been previously reported, we found higher BMI in the PE group, but there were no difference in NRG-4 levels between the groups. Furthermore, there was no correlation between NRG-4 level and the severity of PE, nor with clinical and laboratory parameters. It is thought that NRG-4 is a marker of brown adipocytes in human adipose tissue and is also associated with obesity. No such relationship was evident in after analysis of the data from our study populations; we could not find any correlation between NRG-4 levels and BMI.

There may be a number of possible reasons why these findings emerged from our study. Firstly, the pathogenesis of PE involves a dynamic process, so there may be temporal changes in concentrations of circulating cytokines and adipokines as the pathogenic process progresses. These cytokine/adipokine concentrations may be normalized again by the time clinical symptoms and overt PE appears. However, it is known that obesity, especially with intensive visceral adiposity, can contribute to the pathogenesis of PE by increasing proinflammatory cytokines and adipokines. There have been contrasting reports of the utility of assessing concentrations of visceral mass-derived adipokines (such as resistin, visfatin) or adipokines reflecting general adiposity (adiponectin, leptin) for the prediction of the development of PE (18,20,23,24,25). In the study of Chandrasekaran et al. (19), it was demonstrated that PE was associated with elevated level of visceral fat mass-derived adipokines and leptin, but there was no relation between the groups for adiponectin levels. These analyses bring into question the importance of the effect of placentally derived adipokines in the pathogenesis of PE.

Secondly, although higher BMI is known to contribute to developing PE and there is correlation between BMI and increasing PE severity. BMI, which is especially affected by white adipose tissue, may not fully reflect the increased brown adipose tissue. Therefore, BMI may be an insufficient indicator to reflect body fat tissue distribution. Chandrasekaran et al. (19) showed that normal weight and obese women in the PE and control groups had similar levels of visceral mass-derived adipokines, cytokines and inflammatory markers. Kurek Eken et al. (26) demonstrated that serum NRG-4 levels were higher in patients diagnosed with gestational DM compared to healthy pregnant women. Moreover, they reported that NRG-4 concentration was positively correlated with BMI, and triglyceride and low-density lipoprotein cholesterol. Jiang et al. (22) suggested that low NRG-4 levels were associated with increased subclinical atherosclerosis and increased carotid intima thickness in obese patients. They also showed that among the obese patients, those with high NRG-4 levels had lower BMI and systolic blood pressure levels than those with low NRG-4 levels. In the study conducted by Dai at al. (16) decreased NRG-4 levels were found in NAFLD and yet they did not find any relationship between NRG-4 and BMI. Similarly, Sato and Minatsuki (17) showed that NRG-4 is a predictor of the severity of CAD but they did not find any correlation between NRG-4 and BMI, cholesterol levels or high sensitive C-reactive protein. Similar to these studies, we did not detect any correlation between the demographic, clinical and laboratory parameters and NRG-4 levels and the severity of PE was also not related with NRG-4 levels. Therefore, it may be assumed that NRG-4 levels are independent of general measures of obesity or that BMI does not accurately reflect the presence and activity of brown adipose tissue, the main source of NRG-4. It is also possible that NRG-4 levels may be increased in PE pregnancies earlier than the third trimester, when samples were taken in our study.

It should be noted that brown adipose tissue plays a role in energy metabolism by regulating the production of ATP and thermogenesis (10,11). Similarly, Wang et al. (10) demonstrated that NRG-4 stimulates liver lipogenesis by activating Erb B3/B4 receptors. This evidence suggests that the endocrine role of NRG-4 in metabolic diseases, such as type 2 DM, gestational DM, NAFLD and obesity may be more closely associated with vascular and inflammatory pathways. All of these factors, particularly the low levels of NRG-4 associated with an atherogenic process, which has similarity with the etiopathogenesis of PE and, conversely, elevated levels in gestational diabetes mellitus, which also has some similarities to PE, may be the reason why we could not detect any significant variation in NRG-4 levels among the study and control groups. We therefore suggest that NRG-4 is an unsuitable biomarker for third trimester PE.

Study limitations

However, there were limitations of this study that should be noted. One problem lies with sampling time and the lack of data for NRG-4 levels prior to the onset of clinical PE. Samples in our study were taken only in the third trimester and longitudinal sampling throughout preganacy could have provided more enlightening results. These data collected in a longitudinal fashion may provide a greater understanding of the significance of NRG-4 during the development of PE. A further limitation was the relatively small sizes of the PE sub-groups. Future studies should recruit sufficient patients with PE to sub-divide them on the basis of both obesity and severity of disease. These data may add more detailed and reliable information about the pathogenesis of PE and the role of NRG-4, if any, in this.


In conclusion, despite the limitations, to our knowledge, this is the first study to have investigated maternal levels of NRG-4 in PE. Though we could not find any difference in NRG-4 levels in PE pregnancies compared to healthy pregnancies, future investigations of the role of NRG-4 in PE should address the physiological changes of pregnancy, metabolic pathways known to be affected by NRG-4 and the different stages in the development of PE.

Ethics Committee Approval: The study design was approved by the institutional research ethics committee (approval number: 28/2019).

Informed Consent: Written informed consent was obtained from all participants.

Peer-review: Externally peer-reviewed.

Author Contributions: Surgical and Medical Practices: K.Y., D.F.Ö.; Concept: K.Y., F.H.Ö.; Design: K.Y.; Data Collection or Processing: F.D.Y.Y., B.Y.; Analysis or Interpretation: T.Ç.; Literature Search: B.Y.; Writing: K.Y.

Conflict of Interest: No conflict of interest is declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.


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