Gestational diabetes mellitus (GDM) is a metabolic disorder that arises during pregnancy and is characterized by glucose intolerance with onset or first recognition during pregnancy [1]. It poses significant risks to both maternal and fetal health, including an increased risk of preeclampsia, macrosomia and future development of type 2 diabetes mellitus (T2DM) [2]. The hormonal changes that occur during pregnancy, including placental hormone secretion, contribute to insulin resistance, which is a key feature in the pathophysiology of GDM [3]. Calcitonin gene-related peptide (CGRP) is a neuropeptide widely distributed in the central and peripheral nervous systems and is known to exert multiple biological functions, including vasodilation, inflammation modulation and metabolic regulation [4]. Several studies have demonstrated that CGRP can impair insulin-mediated glucose metabolism in the liver and skeletal muscles [5,6]. Additionally, elevated CGRP levels have been implicated in obesity and insulin resistance, suggesting a potential link to the development of GDM [7,8]. Adiponectin, an adipose-derived hormone, plays a central role in enhancing insulin sensitivity and exhibits anti-inflammatory properties. Low circulating levels of adiponectin have been associated with increased insulin resistance and the pathogenesis of GDM [9].

Several   studies  have   reported   significantly   decreased   adiponectin     levels    in     women    with     GDM compared   to healthy pregnant controls [10,11]. Another crucial marker of glucose control is glycated hemoglobin (HbA1c), which reflects average blood glucose levels over the previous two to three months. While it is commonly used in the diagnosis and monitoring of diabetes, its role in screening for GDM remains under debate [12,13]. Nonetheless, recent studies have suggested that HbA1c may be useful for predicting adverse pregnancy outcomes and postpartum diabetes in women with GDM [14–16]. Given the interplay between neuropeptides, adipokines and glycemic markers, evaluating serum levels of CGRP, adiponectin and HbA1c in pregnant women may enhance our understanding of the pathophysiology of GDM and identify potential biomarkers for its diagnosis and management.

The case-control study was carried out in Tikrit city (Salah Al-Din General Hospital) from 15th of January to the end of December 2022. The number of pregnant women understudy were 100 women in the 3rd trimester of pregnancy whose ages were between 20-45 years old who attended to Salah Al-Din General Hospital /Obstetric part. The study also included 50 healthy non-diabetic pregnant women in 3rd trimester of pregnancy ages were between 20-45 years as control group.

Inclusion criteria

• Pregnant women (3rd trimester)

• Age 20-45 year

Exclusion criteria

• Patients who take treatment affecting blood glucose like corticosteroid drugs

• Type 1 and type 2 diabetic patients

The gestational age of each pregnant woman was determined based on the first day of the last menstruation and confirmed with prior medical records and the results of routineultrasonographic measurements. Height and weight measurements were conducted for each pregnant woman included in the study. Maternal BMI was calculated using the standard equation (weight [kg]/height2 [m2]) at the time of sample collection. Medical information, such as maternal age, gravida, parity, number of abortions, reproductive and medical history was obtained from patient files.Five ml of blood was collected by vein puncture using five ml syringe from each patient in fasting state. Blood samples were placed into two tubes, one of them containing anticoagulant: Ethylenediaminetetraacetic acid (EDTA) for hemoglobin and HbA1c assessment (i-chroma, Korea). The second part of the sample was 3 ml which placed in plane tubes, left for 30 minutes at 37 °C for clotting and centrifuged at 3000 rpm for 15 minutes, sera from were then aspirated and transferred into Eppendorf tubes for late biochemical for assessment of blood sugar and calcitonin gene-related peptide (CGRP) and adiponectin by Enzyme linked immunosorbent assay (ELISA).

 Statistical Analysis

Computerized statistically analysis was performed using Minitab version 17 statistic program. Comparison was carried out using t-Test probability. The P value> 0.05 was considered statistically significant and for result which its P value was less than 0.01 was considered highly significant, while for those which its P value was greater than 0.05 considered non-significant statistically.

In Table 1, There was not a significant difference between the studied cases and the control group in terms of patient age, gestational age at the time of sampling or parity (P>0.05).

The study showed the mean of CGRP was elevated significantly in GDM group (8.04 pg/ml) compared with women without GDM control group (2.91 pg/ml) (P:0.001), (Table 2).

The study showed the mean of adiponectin was reduced significantly in GDM group (77.89) compared with women without GDM control group (99.72 pg/ml) (P:0.001), (Table 3).

Table 4 shows the fasting blood sugar and glycosylated Hb in GDM group. It was found that HbA1c was elevated significantly in GDM women (5.91%) and the lowest mean was non-GDM women (5.21%) and fasting blood sugar was also elevated significantly in GDM women (135.3 mg/dl) and the lowest mean was non-GDM women (91.35 mg/dl).

The study showed positive correlation of CGRP serum level with each of FBS, HbA1c and Gestational age and negative correlation with serum Adiponectin, (Table 5).

Table 1: Clinical characteristics of studied women

Table 2: Levels of CGRP in the studied groups

Figure 1: Levels of CGRP in the studied groups

Table 3: Levels of Adiponectin in the studied groups

Table 4: The blood sugar and HbA1c level between studied groups

Table 5: Correlation between CGRP and different parameters among GDM women

* r value ≥ 0.2: positive correlation

** r value ≥ -0.2: negative correlation

The synthesis and release of CGRP through extensive neural networks of motor and sensory nerves indicate that CGRP has multiple metabolic functions [4]. For instance, it acts as an anti-insulin agent, disrupting insulin's action on hepatic glycogen metabolism and inhibiting insulin-mediated glycogen synthesis in skeletal muscle both in vivo and in vitro [5]. Additionally, CGRP is associated with the development of obesity-induced insulin resistance and studies have shown that mice lacking CGRP were protected against diet-induced obesity [6,7]. These findings suggest that CGRP may influence both obesity and insulin resistance, supporting the results of the present study, which found significantly increased serum CGRP levels in pregnant women with GDM compared to controls. In our study, CGRP levels were significantly elevated in the GDM group (mean: 8.04 pg/ml). Koşar et al. [8] similarly found higher serum CGRP levels in GDM patients than in healthy pregnant women, aligning with our findings. Their results suggested CGRP could serve as a predictive factor for insulin treatment in GDM. Additionally, Aveseh et al. [9] proposed that CGRP might contribute to the pathogenesis of T2DM by facilitating adipose tissue lipolysis during exercise. However, Zelissen et al. [8] reported that CGRP levels were independent of BMI in GDM women, highlighting possible discrepancies due to varying study designs or sample sizes. The small number of participants in the current study might also limit its generalizability. Thus, further studies across different BMI strata are warranted to clarify the role of CGRP as a predictor for GDM development or insulin requirement.

Correlation analysis in the present study revealed that CGRP levels were negatively associated with adiponectin concentrations. Adiponectin, a polypeptide hormone secreted by adipose tissue, is thought to play a vital role in the pathogenesis of GDM. It is influenced by endocrine changes during pregnancy, particularly those related to placental and ovarian steroid hormones [10]. Pala et al. [11] and Altinova et al. [12] both reported significantly lower adiponectin levels in GDM patients, findings echoed in our study. Cseh et al. [13] also emphasized the contribution of low adiponectin levels to insulin resistance in GDM, further substantiating our results. Moreover, we found that adiponectin negatively correlated with BMI. This observation is consistent with earlier studies [14–16] that reported an inverse relationship between adiponectin and BMI. For instance, Pala et al. [17] and Cseh et al. [13] demonstrated that maternal adiponectin concentrations negatively correlated with pregnancy BMI in GDM cases. For over three decades, researchers have investigated the clinical utility of HbA1c during pregnancy as a screening tool for GDM [18,19]. Claesson et al. [20] found that HbA1c levels in late pregnancy could predict postpartum diabetes in women with GDM. Sengupta et al. [21] also observed elevated HbA1c levels in the third trimester among women with poorly controlled diabetes. However, Nielsen et al. [22] reported that HbA1c levels are generally lower in late pregnancy, possibly due to physiological hemodilution, whereas Maresh et al. [23] found elevated levels in both the second and third trimesters. Sengupta et al. [24] further demonstrated that third-trimester HbA1c levels could predict glycemic control in diabetic pregnancies. Additionally, longitudinal studies have shown that HbA1c levels decline slightly in the second trimester and rise again in the third trimester [25,26], likely due to increasing insulin resistance and erythrocyte turnover during pregnancy [27,28]. Some studies have also explored the use of HbA1c in diagnosing diabetes and prediabetes in women with a history of GDM [29–31] and very few have investigated its value in predicting postpartum diabetes [32,33]. Together, the findings of this study support previous evidence that CGRP and adiponectin levels, along with HbA1c, may serve as useful biomarkers for the identification and management of GDM. Nonetheless, more comprehensive, multicenter prospective studies are necessary to validate their clinical applicability.

Serum CGRP levels were increased significantly in pregnant women with GDM compared with women without GDM control group and the outcomes of this investigation suggest that high CGRPlevels in pregnant women may be a pivotal role in the natural history of GDM.

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8. Pala, H.G. et al. "Adiponectin levels in gestational diabetes mellitus and in pregnant women without glucose intolerance." Advances in Clinical and Experimental Medicine, vol. 24, no. 1, 2015, pp. 85–92.

9. Altinova, A.E. et al. "Circulating concentrations of adiponectin and tumor necrosis factor-α in gestational diabetes mellitus." Gynecological Endocrinology, vol. 23, no. 3, 2007, pp. 161–165.

10. Cseh, K. et al. "Plasma adiponectin and pregnancy-induced insulin resistance." Diabetes Care, vol. 27, no. 1, 2004, pp. 274–275.