Introduction

Relaxin-2 is an important gestational hormone in shaping the endometrium in early pregnancy and its secretion peaks during the first trimester [1]. The relaxin family of peptides are structurally found to be similar to insulin and insulin-like growth factors with A-chains and B-chains making up the bioactive molecule [2, 3] and a C-peptide connecting the pre-molecule [4].

A study investigating relaxin levels in women with type 2 diabetes found a correlation between relaxin and insulin sensitivity. Contrarily, a negative correlation between relaxin and beta cell function has been suggested [5]. Another study reported lower levels of relaxin-2 but not of relaxin-3 in patients with recent onset type 2 diabetes. This data combined would suggest that relaxin-2 could be involved in the pathophysiology of type 2 diabetes and beneficial in increasing insulin sensitivity in patients with insulin resistance [6].

Pregnancy is a normal state of insulin resistance which ensures continuous glucose transport to the fetus. Women are however not normally hyperglycemic during pregnancy. If hyperglycemic, the women are diagnosed with gestational diabetes mellitus [7].

There are currently no reports on levels of relaxin-2 in gestational diabetes mellitus. The aim of this study was to investigate plasma levels of relaxin-2 in patients with gestational diabetes mellitus, in comparison to pregnant controls without diabetes.

Materials and methods

Women included in this study were diagnosed with gestational diabetes mellitus (GDM) at Skåne University Hospital in Lund as part of a general screening program offered to all pregnant women in the region. The diagnostic criterion was a capillary plasma glucose value ≥ 10 mmol/L (≥ 180 mg/dL) after a 2-h 75  g oral glucose tolerance test (OGTT) following overnight fasting [8]. When a family history of diabetes was present or the woman’s body mass index (BMI) was above 30  kg/m2, the OGTT was offered at the 12th week of gestation, otherwise the OGTT was offered at the 28th week of gestation. Pregnant women without diabetes were included as controls and recruited from nearby maternity care centers (Lindängen Malmö, Staffanstorp and Dalby). Venous blood samples were drawn into ethylenediaminetetraacetic acid-plasma tubes and arrived at the laboratory the next day. Plasma was separated from blood cells by centrifugation at 2000 x g and was stored at −70 °C until use. Since relaxin levels peak during the first trimester we included only women with GDM (n = 137) diagnosed already at 12 weeks of gestation and controls (n = 114) recruited at the same gestational age.

Plasma samples were analyzed for human relaxin-2 and adiponectin with DuoSet enzyme linked immunosorbent assay (ELISA) (R&D Systems, Minnesota, MN, USA) according to the manufacturer’s instructions. Plasma samples were diluted 1:5 for relaxin-2 and 1:2000 for adiponectin and run in duplicates. The intra-coefficient of variation for relaxin-2 and adiponectin was 3.2 and 2.2%, respectively. The relaxin-2 antibody was reported not to cross-react with recombinant human insulin, pro-insulin or C-peptide.

C-peptide levels were measured in duplicates with ELISA (Mercodia, Uppsala, Sweden) according to manufacturer’s instructions. The intra-coefficient of variation ranged between 2.9–4.8%. The detection limit of the assay was reported to be 25 pmol/L.

Data following a normal distribution were reported as mean  ±  standard deviation (SD). Non-parametric data were ranked and reported as median followed by the inter-quartile range. Student’s t-test or the Mann–Whitney U-test was used to compare differences between groups. Spearman’s rank correlation coefficient was used to test for associations. Logistic regression was used to analyze the effect of several independent variables on GDM. A p-value < 0.05 was considered statistically significant and MedCalc Statistical Software version 16.4.3 (MedCalc Software bvba, Ostend, Belgium) was used to perform all the statistical analyses.

Results

Clinical and laboratory data are presented in Table 1.

Table 1 Clinical and laboratory data of women included in the study. Spearman’s rank correlation coefficient was applied to test for associations
Full size table

Plasma levels of relaxin-2 were significantly higher in women with GDM at 12 weeks of gestation (0.83 [0.40–1.53] ng/mL) compared to controls (0.47 [0.3–1.0] ng/mL; p  = 0.001). Also the C-peptide levels were higher in women with GDM (1.12 [0.85–2.17] nmol/L) compared to controls (0.60 [0.40–0.83] nmol/L; p   < 0.001). Relaxin-2 was positively correlated to C-peptide (r s = 0.198; p  = 0.002).

Plasma levels of adiponectin were significantly lower in women with GDM at 12 weeks of gestation (2.8 [0.9–4.4] µg/mL) compared to controls (5.2 [3.5–6.8] µg/mL; p  <  0.001). Adiponectin was negatively correlated with C-peptide (r s = −0.365; p  <  0.001) but not with relaxin-2.

BMI was higher in women with GDM (29.0 [23.4–32.4] kg/m2) compared to controls (24.7 [22.1–27.6] kg/m2; p  <  0.001). There was no significant correlation between BMI and relaxin-2, but a positive correlation with C-peptide (r s = 0.319; p  <  0.001) and a negative correlation with adiponectin (r s = −0.216; p  <  0.001).

Women with GDM were older (32.1  ±  5.7 years) compared to controls (30.4  ±  5.1 years; p = 0.019). Maternal age was correlated with BMI (r s = 0.180; p = 0.002) and C-peptide levels (r s = 0.128; p = 0.024) but not relaxin-2 or adiponectin.

In a logistic regression model with GDM as the dependent variable and relaxin-2, adiponectin, BMI, C-peptide, and maternal age as independent variables, relaxin-2 and maternal age lost their statistical significance while it was retained by the other variables.

Discussion

In this study, we report higher levels of relaxin-2 in the first trimester of pregnancy complicated by GDM than in pregnant women without diabetes. Relaxin-2 levels were also correlated with plasma C-peptide levels but not with adiponectin, BMI or age.

A strength in this study is that both women with GDM and controls were analyzed in the first trimester to avoid the normal pregnancy variation of relaxin-2. Also, all samples were received and stored within 1 day.

Limitations of the study include the lack of insulin resistance measurements. There was also an age difference between the GDM and control group, age did however not correlate to neither relaxin-2 nor adiponectin.

Relaxin-2 did not retain statistical significance as an independent variable explaining presence of GDM in the multivariate analysis. The role of relaxin-2 as an independent predictor for GDM must still be considered to be uncertain, and addressed in future studies.

Women diagnosed with GDM already at 12 weeks of gestation will have an inherent increased risk of developing type 2 diabetes in the future [9]. Thus, it would seem reasonable to expect findings regarding relaxin-2 levels similar to those previously reported in type 2 diabetes [5, 6]. In contrast, we found higher plasma levels of relaxin-2 in women with GDM in our study. Pregnancy itself, since relaxin-2 is an important reproductive hormone, might explain the difference in results. Currently, there is a lack of knowledge regarding plasma relaxin-2 levels in pregnant women with type 2 diabetes. However, a few reports of pregnant patients with type 1 diabetes [10, 11] showed markedly elevated levels of relaxin. The authors suggest a possible connection to the diabetic pregnancy but call for further studies.

Elevated levels of relaxin-2 in the third trimester of 26 women with GDM has been reported, though not reaching statistical significance [12]. The study suggested that increased levels of relaxin-2 might be to compensate for the increased insulin resistance.

Plasma levels of adiponectin were significantly lower in women with GDM in this study, which was expected and well in agreement with other reports [13–15]. To our knowledge, there are no reports on the relationship between relaxin-2 and adiponectin, neither in pregnancy nor in diabetes.

In conclusion, this is the first study to report significantly higher levels of relaxin-2 in patients with GDM. Relaxin-2 may be a possible confounder to the known parameters influencing more severe insulin resistance in these pregnant women, as reflected by higher C-peptide levels and a higher BMI, as well as lower adiponectin values. We suggest a possible role for increased plasma levels of relaxin-2 in compensating for increased insulin resistance in GDM.