Associations Of Interleukin-1 Gene Polymorphisms And Their Protein Production With Endometriosis Risk

BACKGROUNDEndometriosis is a common benign disease with increasing morbidit. More and more investigation indicated that immune, heredity, environmental factor and hormonal factor contribute to the development and progression of endometriosis, however, its pathogenesis is still unknown. Recently, researches pay more attention to the immunological-inflammatory balances in endometriosis patients. Increasing evidence showed that cytokines and growth factors play important roles in this disease.Interleukin-1 (IL—1) family is consisted of IL-1α, IL-1β, IL-1 receptor antagonist (IL-1ra), IL-1 receptors and so on. IL—1β is an important pre-inflammation cytokine. Many studies have shown that the concentrations of IL-1β were significantly higher in peritoneal fluid, ectopic and eutopic endometrial cells from patients with endometriosis as comparison with controls. It indicated that IL-1β may break the equilibrium of microenvironment in endometriosis patients through its immune-inflammatory effects. Besides, IL-1β may also augment theproduction of other cytokines and trigger the growth, adhesion, invasiveness and angiogenesis of endometrial fragments in peritoneal fliud. It strongly suggested that IL-lp plays an important role on the development and progression of endometriosis. IL-lra, by binding to the IL-1 receptor types I without exerting an effector function, acts as an endogenous competitive antagonist for IL-1 p. However, research showed that eutopic endometrium of patients with endometriosis expressed less IL-lra, which was completely absent in ectopic endometrium. The imbalance of IL-1 P and its natural receptor antagonist provide a clue that IL-lra may be the protective cytokine of patients from endometriosis. And there is little report about the levels of IL-lra in the serum and peritoneal fluid of patients with endometriosis up to now. Thus, the first part of our research is to investigate the role of IL-lra on endometriosis by detecting the levels of IL-lra in the serum and peritoneal fluid of patients with and without endometriosis.In 1997, Dr.Kennedy indicated that endometriosis, like diabetes and asthma, may be a complex genetic disease (with multiple gene loci interact with each other) interacting with environmental factors. Due to the important roles of cytokines play on endometriosis, more studies began to investigate the association of their polymorphisms and the susceptibility of endometriosis. It is found that the polymorphisms of many cytokines, such as IL-2 receptor, IL-4, IL-10, VEGF, TNF-a, IFN and TGF-P, were associated with susceptibility of endometriosis. Moreover, studies showed that the concentrations of these cytokines were significantly different in peritoneal fluid, ectopic and eutopic endometrial cells from women with endometriosis as comparison with healthy controls. In fact, the synthesize of cytokines is precisely regulated in vivo, including pre-transcription, gene transcription, translation and the modification after translation. Any change in this chain may influence their expression. As the vital part of the process of proteinexpression, any changed in genes sequences will remodel protein synthesize accordingly.The genes for IL-1 have several polymorphisms. Single nucleotide polymorphisms (SNP) such as -511 of the promoter and +3953 of exon 5 regions of the IL-ip gene (C-*T), as well as the variable number of tandem repeat in intron 2 of IL-lra gene, have been described. More excitingly, those polymorphisms may influence IL-ip or/and IL-lra protein production. Judging from above studies, we supposed that these polymorphisms might contribute to the development of endometriosis through changing genetic sequence, influencing transcription and translation and resulting in the abnormality of protein expression or function.In the present study, we detected the concentrations of interleukin-1 receptor antagonist (IL-lra) in peritoneal fluid and serum in patients with endometriosis to explore the relationship between IL-lra and endometriosis. Furthermore, the polymorphisms of IL-ip-511 promoter, exon 5 and IL-lra were detected by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) in the patients with and without endometriosis, in order to investigate whether the three IL-1 gene polymorphisms may be one of the risk factors for the susceptibility of endometriosis in Chinese Han population.MATERIALS AND METHODS1. Clinical specimen and ONA isolationGenomic DNA was extracted from blood lymphocytes of 138 Chinese women of Han nationaltity, undergoing laproscopy and laprorectomy for endometriosis in Women's hospital, School of Medicine, Zhejiang University. The control group consisted of 100 women without pelvic endometriosis and adenomyosis evidenced by laproscopy or laprorectomy (tubal anastomosis n=55, benign ovianian cysts n=29,infertility n=7, IUD malposition n=6, uterus septus n=3). 2. Determination the serum and peritoneal fluid IL-lra levels by ELISAThe IL-lra levels of serum and peritoneal fluid were determined in 74 EMs patients and 44 controls by ELISA.3. GenotypingPCR (Polymerase chain reactions) was performed to detect polymorphisms of IL-lra;PCR-RFLP was used to determine the genotypes in IL-ip-511 and +3953 sites.4. Statistical analysisKruskal-Wallis H, chi-square, Mann-Whitney U, Wilcoxon and Spearman rank correlation analysis were used for statistical analysis, two-tailed test with p<0.05 were considered significant. All data were managed with SPSS 11.5 for windows.RESULTS1. IL-lra level in peritoneal fluid of patients with endometriosis was significantly lower than that of controls (P <0.05). According to staging criteria of r-AFS, the levels of IL-lra in peritoneal fluid of patients with III-FV stage endometriosis was statistically lower than that of controls (P<0.05). But there was no significant difference between EQ-IV stage and I - II stage endometriosis or I -II stage endometriosis and controls (P>0.O5). Whereas, the level of IL-lra in serum showed no significant difference between the two groups (P > 0.05).2. The concentration of IL-lra in peritoneal fluid and serum was not correlated with the phase of the menstrual cycle in the women with endometriosis (P > 0.05). And the level of serum IL-lra in secretory phase was much higher thanthat of proliferative phase in controls (P <0.05).3. There were no significant differences in the allele and genotype frequencies of IL-1P-51K +3953 sites in endometriosis patients compared with controls (PX).O5).4. There were significant differences in the allele and genotype frequencies of IL-lra in endometriosis patients compared with controls (PO.05). Moreover, in comparison with the reference genotype, the wild Al/Al homozygote and Al allele, the odds ratio for A1/A2 and A2 allele was 3.48 (95%CI: 1.13—10.69) and 3.66 (95%CI: 1.23—10.94), respectively.5. No significant differences are found in the serum and peritoneal fliud IL-lra levels between Al/Al homozygote and A1/A2 heterozygote (PX).O1). However, there is significant difference in the level of peritoneal fliud IL-lra with Al/Al homozygote between patients with and without endometriosis (PO.01).CONCLUSIONS1. The levels of IL-lra in peritoneal fluid in the patients with endometriosis were significantly lower compared with controls. Moreover, the concentrations of peritoneal fluid IL-lra were decreased with the severity of endometriosis. It indicates that IL-lra may play an important role on the development of endometriosis and show negative correlative with severity of endometriosis.2. There was no significant difference of serum IL-lra levels between the patients with and without endometriosis.3. The IL-lra levels of serum and peritoneal fluid both in endometriosis patients were not correlated with the menstruation cycle.4. The polymorphisms of IL-lf5-51K +3953 sites may be not the risk for the susceptibility to endometriosis of Chinese women.5. Polymorphisms of IL-lra may be associated with the susceptibility to endometriosis of Chinese women. Moreover, the A2 allele and A1/A2 genotype of IL-lra gene may enhance the risk for the Chinese women to suffer endometriosis.