| BACKGROUNDPolycystic ovary syndrome (PCOS) is an endocrine disorder of unknown etiology, often accompanined with genital function disturbance and abnormal glucose metabolism. It is characterized by hyperandrogenism, chronic anovulation and polycystic ovaries. In addition, women with PCOS frequently exhibit insulin resistance (IR), obesity, the inverted ratio of luteinizing hormone (LH) to follicle stimulating hormone (FSH).Calcitonin gene-related peptide (CGRP) is a37-amino acid regulatory neuropeptide that is encoded by an alternative processing of calcitonin gene and mainly synthesized in dorsal root ganglia. CGRP mediates its effects through receptor composed of a G protein-coupled receptor called calcitonin receptor-like receptor and receptor activity-modifying protein1. It has been well documented that CGRP exerts complex beneficial cardiovascular effects including potent vasorelaxation and protective effects on cardiomyocytes and endothelial cells, and contributes to pain transmission and inflammation responsible for the mechanisms of migraine and skin pain. CGRP has also been linked to metabolic disorder including IR and obesity. The role of CGRP in the female reproductive system has not been well understood. It has been shown that CGRP inhibits myometrial contractility and regulates uterine blood flow. However, the regulating effect of CGRP on ovary function remains unclear. It has been reported that CGRP immunoreactive fibers enter ovary via plexus nerves. Interestingly, it has also been demonstrated that by immunolabelling more CGRP-positive nerve fibers exist in dihydroepiandrosterone-induced polycystic rat ovaries than in controls. These findings suggested that CGRP may play a role in the genesis and development of PCOS.The mainly infertile reason of patients with PCOS is oocyte immaturation. The maturation course is regulated by multiple factors and cyclic adenosine monophosphate (cAMP) is one of the key inhibitory factors that affect oocyte maturation. cAMP is mainly generated by oocyte or transported through gap junction from neighboring cells. Oocyte maturation is arrested by either promoting cAMP synthesis or by inhibiting cAMP decomposition. CGRP activates adenylate cyclase resulting in elevated intracellular cAMP content. These findings suggest that CGRP may participate in the regulation of oocyte maturation.The aim of the present study was therefore to evaluate the plasma level of CGRP in patients with PCOS and its relationship to IR, body mass index (BMI), LH/FSH ratio, plasma testosterone (T) level. We further observed the effects of CGRP on mouse oocyte maturation in vitro. The intracellular cAMP content and concentrations of T and E2in the cultured medium were determined.METHODSTwo hundred and fifteen women patients diagnosed with PCOS admitted between August,2009and August2011in CITIC-Xiangya were included in this study. One hundred and three age-matched healthy childbearing age women with husband of azoospermatism or severe oligoasthenospermia were admitted as controls. Control subjects were enrolled according to the criteria as follows:(1)22-38years old;(2) normal menstrual cycle between28±3days;(3) infertile because of male factors. PCOS subjects were enrolled according to the following criteria:(1)22-38years old;(2) irregular menstrual cycle,(3) diagnosed by Rotterdam criterion. Blood samples were obtained in the early morning in days2-5of menstrual cycle in two groups. The plasma concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), testosterone (T) and CGRP were measured. In addtion, the PCOS subjects underwent glucose tolerance test and insulin releasing test for measuring plasma glucose and insulin levels and calculating homeostasis model assessment-insulin resistance (HOMA-IR), area under the curve of insulin (AUC-insulin), area under the curve of glucose (AUC-glucose). The relationship between plasma level of CGRP with hormonal and metabolic parameters was analysed. Ovaries were collected from6week-old Kunming female mice. By immunolabelling, expression and location of CGRP in ovaries were observed. Cumulus oocyte complexes (COCs) were collected and cultured, and then treated with CGRP at the concentrations of0,10-10,10-9,10-8M for24h to observe the percentage of germinal vesicle breakdown (GVBD) and first polar body (PBI). Luteinizated human granule cells were collected and cultured, and then treated with CGRP (0,10-10,10-9,10-8M) for24h. The intracellular cAMP content and concentrations of T and estradiol (E2) in the cultured medium were determined. The mRNA expression of CGRP receptor was determined by RT-PCR analysis.RESULTS(1) There was no significant difference between these two groups with respect to age, body height or the plasma levels of E2. The control subjects had a higher FSH levels (6.98±0.16vs6.51±0.10mlU/ml, P <0.01), but lower body weight (50.7±0.5vs56.3±0.6kg), body mass index (BMI,20.46±0.21vs22.73±0.22kg/m2, P<0.01), plasma LH level (4.57±0.20vs8.48±0.37mIU/ml), LH/FSH ratio (0.66±0.03vs1.29±0.05) and plasma T levels (0.34±0.16vs0.43±0.01ng/ml,) compared with PCOS patients (P<0.01).(2) The plasma levels of CGRP in PCOS patients were significantly higher than that of control subjects (6.14±0.29ng/L vs4.48±0.25, P<0.05).(3) There was a significantly positive correlation between the plasma CGRP concentration with HOMA-IR, AUC-insulin and AUC-glucose in PCOS subjects. There was also a significantly positive correlation between the plasma CGRP concentration with LH/FSH ratio and the plasma T concentration in control and PCOS groups separately, or in two groups together. In contrast, there was no correlation between the plasma CGRP concentration and BMI.(4) The stratified analysis about the ratio of LH/FSH and BMI was performed. The plasma CGRP concentration was markedly increased with the elevated ratio of LH/FSH in PCOS subjects. The plasma CGRP concentration was higher in PCOS subjects with the ratio of LH/FSH>2than that of PCOS subjects with the ratio<2. As to BMI, there was no difference in the plasma concentration of CGRP between BMI>24and BMI<24PCOS subjects.(5) CGRP was expressed in mouse ovaries, especially in the oocyte and ovarian stroma. The percentages of GVBD and PBI in mouse oocytes were significantly inhibited by CGRP in concentration-dependent manner. Human granule cells expressed CGRP receptor mRNA. The intracellular cAMP content and concentrations of T and E2in the cultured medium were significantly increased by CGRP in concentration-dependent manner.CONCLUSIONTo the best of our knowledge, this is the first study to investigate plasma CGRP level in relationship to hormonal and metabolic parameters in women with PCOS. The main findings of the present study are as follows:(a) the plasma CGRP level was increased in the PCOS subjects and it was positively correlated with HOMA-IR, AUC-insulin, AUC-glucose, the ratio of LH/FSH and T levels;(b) human granule cells expressed CGRP receptor and exogenous CGRP inhibited oocyte maturation which was related to an increase in intracellular of cAMP content and in release of T and E2in granule cells. CGRP may be used as a novel biomarker or a prognostic factor for PCOS and as a potential therapeutic target in the clinical setting. These findings suggest that CGRP may participate in the pathophysiological process of PCOS. CGRP may be used as a biomarker or a prognostic factor for PCOS and as a potential therapeutic target in the clinical setting. |
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