Introduction

Leptin, an adipokine is produced and secreted by white adipose tissue as well as by other tissues, including, skeletal muscle, bone marrow, placenta, ovaries, mammary epithelium, stomach, liver and lymphoid tissues [1, 2]. It is well established that leptin modulates whole-body energy homeostasis [3, 4] but overwhelming evidence shows the existence of relationship between leptin and reproductive functions like oocyte maturation, regulation of embryo development as well as implantation and placentation [5, 6]. Leptin deficiency/ resistance has been found to be associated with various diseases including type 2 diabetes mellitus and pregnancy induced diabetes mellitus. It is estimated that in India at any given point of time, over 4 million women are affected with diabetes during pregnancy, commonly known as pregnancy induced diabetes or gestational diabetes mellitus [7]. The prevalence of GDM varies from 4 to 18% in Indian population due to difference in age and/or socioeconomic status of the women or incomplete implementation of screening programs [8]. Timely detection and treatment of gestational diabetes may prevent various complications like spontaneous abortion, still birth, congenital anomalies, macrosomia, etc. [9]. The pathogenesis of GDM is still controversial but the basic theory of GDM involve insulin insensitivity and raised level of anti-insulin hormones like human placental lactogen, prolactin, glucocorticoid, and progesterone which are secreted by placenta during pregnancy [10].

Maternal leptin level increases and remain elevated during the pregnancy but highest concentration is achieved in the second trimester between 24 and 28 weeks of pregnancy [11]. Recently, leptin emerged as a first trimester biomarker to predict diabetes during pregnancy [12]. Evidence from previous studies suggest that expression of leptin and its receptor increases in placental tissue of women with GDM [13] along with the increase in soluble form of leptin receptor (LEPRe). Despite high level of serum leptin in GDM, leptin resistance may occur due to the formation of leptin and LEPRe complex which decreases the availability of free leptin thereby preventing the binding of leptin to the bound form of leptin receptor [14]. The exact mechanism by which leptin regulate glucose homeostasis is still under experiment. However, few studies proposed that in leptin sensitive individuals, leptin secreted from adipose tissue inhibit the secretion of insulin from the pancreatic β- cells while individuals who are leptin resistant, secretion of insulin increases due to diminished leptin signalling resulting in hyperinsulinemia. In pregnancy, both insulin sensitivity and responsivity decreases but in most of the pregnant women it get adjusted with the increase in insulin secretion. While in some pregnant women secretion of insulin does not increase or insulin is not able to act on target tissues, leading to insulin resistance and hyperglycaemia [15, 16].

It is well known that pregnancy is a state of low-grade inflammation which gets exaggerated in GDM. The production of proinflammatory cytokines including interleukin-6 (IL-6) is known to be elevated in GDM women as compared to healthy pregnant women [17, 18]. IL-6 is also secreted by the placenta during pregnancy, the concentration of which may further increases in response to hyperleptinemia in GDM [19]. Role of IL-6 in the pathophysiology of glucose intolerance has been studied in type 2 diabetes mellitus and recommended that cytokine may serve as a potential biomarker for screening glucose intolerance in early stage [20]. Gestational diabetes which is similar to type 2 diabetes may be characterized by elevated level of IL-6 leading to insulin resistance. In addition, leptin increases the production of inflammatory cytokines like IL-6 by activating monocytes via jak2/stat3 and p-38 MAPK/ERK1/2 signalling pathways [21]. Gestational diabetes is a state of leptin resistance characterized by a vicious cycle of hyperinsulinemia and hyperleptinemia aggravating an inflammatory situation. Despite the availability of sufficient literature, the exact molecular mechanism which causes insulin resistance in GDM remains unclear. The aim of our study was to estimate the level of serum leptin, insulin and IL-6 among GDM, non-GDM and healthy non- pregnant women and to find out the correlation among study groups.

Materials and methods

Study participants and ethical approval

This cross-sectional study was conducted from April 2017 to March 2019 in the Department of Biochemistry and Department of Obstetrics and Gynaecology, King George’s Medical University, Lucknow, Uttar Pradesh India. Ethical clearance for this study was obtained from Institutional Ethical Committee (Ref code:83rd ECM IIA/P5 dated 04/02/2017). Informed and written consent was obtained from all the enrolled study subjects. The flowchart of enrollment of pregnant women and their sample collection is shown in Fig. 1. In this study DIPSI criteria was adopted for screening GDM [22].

Fig. 1
figure 1

Flowchart representing the selection of GDM, non-GDM and healthy non-pregnant women

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Study procedure

Blood sample was collected under aseptic precaution for estimation of all the biochemical parameters. 2-hour plasma glucose was estimated by glucose oxidase-hydrogen peroxide method on fully autoanalyzer (ELITech Selectra). Serum was separated for the analysis of insulin, IL-6 and leptin by centrifuge at 1,000–2,000 x g for 10 min and stored at -80◦C until batch analysis. All the samples were thawed at room temperature before analysis. Serum leptin and insulin were measured by commercially available ELISA kit according to guidelines of manufacturer (DRG International, Inc. U.S.A) [23, 24] and IL-6 was measured by DIACLONE ELISA kit manufactured in France [25].

Statistical analysis

All statistical analysis was performed by using MS-excel and Graph pad prism 5 (version 5.01). Continuous variables were expressed as mean ± standard deviation. For comparison of mean between three groups, one-way ANOVA analysis with post-hoc by Tukey’s adjustment for group differences was used. Unpaired student t-test was used for the comparison of mean between two groups. To find out the correlation, Pearson correlation was applied. p-value < 0.05 was considered significant.

Results

The baseline characteristics of GDM, non-GDM healthy pregnant women and healthy non-pregnant women

The mean age of women was similar in all the three groups. Diastolic and systolic pressure (mmHg) was significantly higher among GDM women and healthy non-pregnant women as compared to non-GDM pregnant women (p < 0.0001). Pulse rate (/min) was significantly higher (p < 0.04) among non-GDM pregnant women as compared to non-pregnant women. Body mass index (BMI) was higher among GDM women as compared to non-pregnant women which was statistically significantly (p < 0.002). In addition, haemoglobin levels were significantly lower among GDM and non-GDM pregnant women as compared to healthy non-pregnant women (p < 0.0001). Family history of diabetes mellitus was 13.6%, 12% and 9.5% in GDM, non-GDM pregnant women and healthy non-pregnant women respectively. 7.4% GDM women and 6% of non-GDM women had family history of hypertension while it was 14.2% in non-pregnant women (Table 1).

Table 1 Baseline and clinical characteristics of GDM, Non-GDM pregnant women and healthy non-pregnant women
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Mean serum leptin, insulin and IL-6 level in GDM, non-GDM pregnant women and healthy non-pregnant women

Mean level of serum leptin, insulin and IL-6 were higher among GDM as compared to non-GDM pregnant and non-pregnant women (Fig. 2a and b) however, statistically significant difference was observed only in IL-6 levels.

Fig. 2
figure 2

a Mean serum leptin and insulin level in GDM, non-GDM pregnant and healthy non-pregnant women b Mean serum IL-6 level in GDM, non-GDM pregnant and healthy non-pregnant women

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Comparison of serum leptin, insulin and IL-6 level among GDM, non-GDM pregnant and healthy non-pregnant women

Mean serum leptin level was higher among GDM women as compared to non-GDM pregnant women and healthy non-pregnant women though the difference not statistically significant as shown in Fig. 3a. Serum insulin level was almost similar among GDM, non-GDM pregnant and healthy non-pregnant women as shown in Fig. 3b. However, serum IL-6 level was significantly higher in GDM women as compared to non-GDM and healthy non-pregnant women (p < 0.0001) (Fig. 3c). Post hoc analysis was done to confirm the finding of IL-6 in the study groups as shown in supplementary Table 1.

Fig. 3
figure 3

(a) Serum leptin (b) insulin and (c) IL-6 levels among GDM, non-GDM pregnant women and healthy non-pregnant women

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Trimester-wise comparison of mean serum leptin, insulin and IL-6 level of GDM and non-GDM women

GDM and non-GDM pregnant women were further classified into 3 trimesters (1st trimester: ≤12weeks; 2nd trimester: 13–26 weeks; 3rd trimester: ≥27 weeks) depending upon weeks of pregnancy. Mean level of serum leptin, insulin and IL-6 are shown in Fig. 4a and b.

Fig. 4
figure 4

a Serum leptin and insulin level in 1st, 2nd and 3rd trimester in GDM and non-GDM pregnant women *1st trimester serum leptin GDM versus non-GDM (p < 0.027) **3rd trimester serum leptin GDM versus non-GDM (p < 0.002) b Mean serum IL-6 level in 1st, 2nd and 3rd trimester between GDM and non-GDM pregnant women

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Comparison of serum leptin, insulin and IL-6 level in 1st, 2nd and 3rd trimester between GDM and non-GDM women

Serum leptin

In 1st trimester serum leptin levels were lower in GDM women as compared to non-GDM women which was statistically significant (p < 0.027) as shown in Fig. 5a. While in 2nd and 3rd trimester serum leptin levels were higher among GDM women as compared to non-GDM pregnant women however, statistically significant difference was found only in 3rd trimester (p < 0.002) as shown in Fig. 5b.

Fig. 5
figure 5

Serum leptin level in 1st trimester (a) and 3rd trimester (b) of GDM and non-GDM pregnant women

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Serum insulin

Serum insulin levels were almost similar in all the three trimester when compared between GDM and non-GDM women (p > 0.374).

Serum IL-6

Serum IL-6 levels were significantly higher in 1st trimester of GDM women (p < 0.017) as compared to non-GDM pregnant women (Fig. 6).

Fig. 6
figure 6

Serum IL-6 level in 1st trimester of GDM and non-GDM pregnant women

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Correlation between serum leptin, insulin and IL-6 in the study groups (GDM, non-GDM pregnant women and healthy non-pregnant women)

No significant correlation was found between serum leptin, insulin and IL-6 in GDM women. Negative correlation was observed between serum insulin and IL-6 in 2nd trimester of non-GDM women (p < 0.033, r= -0.312) (Fig. 7a). While in healthy non-pregnant women positive association was observed between serum insulin and IL-6 (p value 0.016, r = 0.339) (Fig. 7b). Leptin was not found to be associated with insulin and IL-6 in non-GDM as well as healthy non-pregnant women.

Fig. 7
figure 7

a Correlation of IL-6 with insulin in 2nd trimester of non-GDM pregnant women

b Correlation of IL-6 with insulin in non-pregnant women

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Discussion

In the present study, serum leptin levels were higher in GDM women compared to non-GDM and healthy non-pregnant women although the difference was not statistically significant. Furthermore, leptin levels were higher in 2nd and 3rd trimesters as compared to the 1st trimester in GDM women, but the difference was not statistically significant. However, in non-GDM women, serum leptin levels were almost similar in all three trimesters. Regarding leptin levels between GDM and non-GDM women, statistically significant differences were observed in the 1st and 3rd trimesters. In the 1st trimester, serum leptin levels were lower while in the 3rd trimester, the levels were significantly higher in GDM women. In our study, 27 GDM and 23 non-GDM pregnant women constituted the 1st trimester and significantly higher leptin levels were observed in the non-GDM group. This difference can be explained by the fact that three non-GDM women had serum leptin levels of 37.88, 34.28, and 31.28 ng/ml in the 1st trimester, which possibly affected the mean leptin levels. In the literature, the highest maternal leptin concentration has been reported around 24–28 weeks of gestation and in our study, pregnant women with ≥ 27 weeks of gestation comprised the 3rd trimester which might have resulted in significantly higher serum leptin levels. Serum insulin levels were higher in GDM women compared to non-GDM and healthy pregnant women but the difference was not statistically significant. Furthermore, in all three trimesters serum insulin levels were similar in both GDM and non-GDM women. We found significantly higher levels of serum IL-6 in GDM women. Post hoc analysis showed that IL-6 levels were significantly higher in GDM women than in non-GDM women as well as healthy non-pregnant women. Although IL-6 levels were higher in all three trimesters of GDM women, significantly higher levels were observed only in the 1st trimester of GDM women compared to non-GDM women.

Furthermore, IL-6 showed a negative correlation with insulin during the 2nd trimester of non-GDM women while a positive correlation was observed between IL-6 and insulin in healthy non-pregnant women. No correlation was observed between serum IL-6 and leptin levels in all three study groups.

Our findings are consistent with those of Mokhatri et al. [26] who also observed no significant differences in the levels of serum leptin between GDM and non-GDM women. In their case-control study, serum leptin levels were higher in the non-GDM group. Similarly, Saucedo et al. [27] found no significant difference in the plasma leptin concentration between GDM and non-GDM women. Simmons et al. [28] observed no difference in the maternal leptin levels among Polynesians, South Asians, and GDM women, although mean leptin levels were higher among GDM women. Goel et al. [29] reported higher serum leptin levels in GDM women during 28–32 weeks of pregnancy, which is consistent with our observation in the 3rd trimester GDM women. Similarly, Gao et al. [10] found significantly higher levels of serum leptin during 14–20 weeks and 24–32 weeks of pregnancy among GDM women. Kautzky-Willer et al. [30] also observed significantly higher leptin levels at 28 weeks of gestation among GDM women compared to non-GDM as well as patients with type 1 diabetes mellitus. In contrast to our findings, Saini et al. [23] observed significantly higher serum leptin levels in 30 GDM women compared to 60 non-GDM women. Noureldeen et al. [31] reported lower serum leptin levels in the 3rd trimester among GDM women compared to non-GDM women, which is in contrast with our observations. Consistent with our study, Shalayel et al. [32] found no significant difference in serum insulin levels during 3rd trimester among GDM, non-GDM, and IGT (Impaired Glucose Tolerance) women. Contrary to our results, Fakhrul-Alam et al. [33] in a recent study measured serum insulin levels during 24–40 weeks of gestation and found higher insulin levels in GDM women compared to non-GDM. Wang et al. [34] measured serum insulin levels in 55 GDM and 87 non-GDM women during late pregnancy and found significantly higher levels in GDM women. Similarly, Sarma et al. [35] and Wang et al. [36] found significantly higher serum insulin levels during 24–28 weeks of gestation and Kautzky-Willer et al. [30] found significantly higher insulin levels at 28 weeks of gestation among GDM women. Yang Y et al. [37] observed significantly higher IL-6 levels during the 1st trimester of GDM women compared to non-GDM women, which is similar to our observation. Similarly, Hassiakos et al. [38] found significantly raised IL-6 levels in GDM women during 11–14 weeks of pregnancy. Sarma et al. [35] reported significantly higher IL-6 levels in 52 East Indian women with GDM at 24–28 weeks of pregnancy compared to 48 non-GDM women. Contrary to our findings, Abell et al. [39], and Lain et al. [40] found no difference in IL-6 levels between GDM and non-GDM women during 12–15 weeks and 9.3 weeks of gestation, respectively. Similarly, Braga et al. [41] observed no significant difference at 24–28 weeks of gestation. Simjak et al. [42] included 12 GDM, 12 non-GDM, and 10 healthy non-pregnant women, and reported lower IL-6 levels in non-pregnant women and higher levels in GDM women during 28–32 weeks and 36–38 weeks of gestation, though the difference was not statistically significant.

Similar to our findings, Sarma et al. [35] found no correlation between IL-6 and insulin levels in GDM women. In contrast to our findings, Kautzky-Willer et al. [30] found a positive correlation between fasting leptin and fasting insulin levels in GDM women.

This study was conducted on the North Indian population and compared the levels of leptin, insulin, and IL-6 trimester-wise in GDM and non-GDM women. Several studies on GDM women measured the levels of leptin, insulin, and IL-6 independently, although very few focused on their relationship. Our study tried to explore whether any association exists between inflammation, hyperleptinemia, and hyperinsulinemia in gestational diabetes. There are a few limitations of our study. First, the population screened for gestational diabetes was restricted to women from the city and adjoining areas only. Hence, the outcomes of this study cannot be generalized. Also, women in all three trimesters of GDM and the non-GDM groups were different. To study the changes in adipokines and inflammatory markers, longitudinal studies are required so that the same group of women enrolled in the 1st trimester is followed up during 2nd and 3rd trimesters as well as after the delivery.

Conclusion

Serum leptin and IL-6 levels are altered during pregnancy, a condition that further worsen in patients with gestational diabetes mellitus. The absence of any significant correlation between leptin and IL-6 levels signifies that leptin is not associated with inflammation during pregnancy. Altogether, our study suggests that IL-6 may be used as a biomarker, especially during the first trimester, for screening gestational diabetes mellitus. Further study with a larger sample size may pave the way for the discovery of an early diagnostic marker for gestational diabetes mellitus.