Important Inflammatory Mediator Gene Polymorphisms Associated With Susceptibility To Spontaneous Preterm Birth

Background:Preterm birth(PTB), defined as birth before 37 complete weeks of gestation, is one of major causes of prenatal morbidity and mortality. PTB is significantly associated with neonatal long-term complications, such as cerebral palsy, bronchopulmonary dysplasia, and developmental delay, retinopathy of prematurity, hearing and vision problems. PTB can be divided into two main groups: spontaneous preterm birth(SPTB) and therapeutic preterm birth(TPTB). SPTB accounts for approximately 70% of all preterm births and preterm premature rupture of membranes(PPROM) accounts for 50% of all SPTBs. Epidemiological studies suggest that SPTB clusters in families. Moreover, there are significant racial/ethnic disparities and recurrences in the incidence of SPTB. Genetic association studies have identified several genetic polymorphisms related to infection, inflammation, and the innate immune system, which are risk factors for SPTB. Genetic factors are known to contribute to SPTB. The etiology and pathogeny of SPTB remain unclear. However, evidence illustrates that intra-uterine infection and inflammation play a role in the pathophysiology of SPTB and PPROM. SPTB and PPROM are closely related with the level of inflammatory mediators which are involved in the regulation of infection and inflammation, including inflammatory cytokines, chemokines and the matrix metalloproteinases. Given that single nucleotide polymorphisms(SNPs) in inflammatory mediators gene expression contribute to the risk of SPTB and PPROM, these genes are plausible candidates. However, the present association studies about polymorphisms and susceptibility to SPTB are few. Moreover, some shortages, including small sample size, inadequate adjustment for confounding factors and the inconsistent results, had been presented in these studies. Additionally, there are considerable racial disparities in genetic polymorphisms. The results of genetic association for other racial populations may not be inapplicable to Chinese population. So, well-designed case-control studies are needed to comprehensively research the correlation between the polymorphism of inflammatory mediator and susceptibility to SPTB in Chinese population. Identification of susceptibility genes and SNP contributing to SPTB would assist in predicting individuals and populations at risk of SPTB and help to clarify the pathogenesis of this disorder.Objective: To investigate the association between the genetic polymorphisms of MCP-1, RANTES, TNF-α, IL-15, IL-1β, MMP-8 and susceptibility to SPTB. It is expected that these results can be applied as new biomarkers to identify high-risk subgroups of preterm neonates who might benefit from personalized prevention and treatment in the future.Methods: The strategy was the genetic association study based on the candidate gene. This case-control study enrolled 753 SPTB singleton neonates and 681 term neonates. We stratified the preterm neonates according to PROM status. Based on gestational age, SPTB were divided into three sub-categories: extremely preterm(≤28 weeks), very preterm(28 to 32 completed weeks of gestation) and moderate preterm(33 to 36 completed weeks of gestation). extremely preterm and very preterm neonates could be combined to severe preterm neonates(≤32 weeks). All polymorphisms were genotyped using PCR-based restriction fragment length polymorphism analysis, polymerase chain reaction(PCR)-sequencing and Sequenom Mass ARRAY?SNP. The single allele and genotype frequencies in each of the two groups were analyzed using Chi-square(χ2) tests. The associations between each polymorphism and the risk of SPTB were estimated using logistic regression analysis.The odds ratios(ORs) and 95% confidence intervals(CIs) were used to measure the statistical strength of the correlation. We adjusted two confounding factors,including maternal age and neonatal sex in five genetic models for analysis of susceptibility to SPTB. A correlation was considered to be significant as P<0.05. The serum concentrations of RANTES were measured by RANTES ELISA kit and all measurements were performed in duplicates.Results:1. There was no significant association between the risk of SPTB and the MCP-1G-2518 A polymorphism whether in the overall sample, preterm neonates with PROM and without PROM. However, the-2518 AA genotype was found to be significantly associated with an increased susceptibility to extremely(OR 2.74; 95%CI, 1.10-6.72; P = 0.014)and very SPTB(OR 2.07; 95%CI, 1.27-3.36; P = 0.005) compared with-2518 G positive genotypes(GG + GA genotypes). In addition, as extremely preterm and very preterm neonates were combined, the-2518 AA genotype was still significantly associated with an increased susceptibility to SPTB(OR 2.23; 95%CI, 1.40-3.54; P<0.001).2. Compared with the T-positive genotypes(TT + TC genotypes), the RANTESIn1.1 T/CCC genotype was statiscally associated with increased SPTB susceptibility(OR 1.44; 95%CI, 1.01-2.05; P = 0.043). It was also noted that the In1.1CC genotype was marginally associated with increased SPTB susceptibility in preterm neonates without PROM(OR 1.48; 95%CI, 1.00-2.19; P = 0.05).There was no association between the RANTESIn1.1T/C polymorphism and susceptibility to extreme or moderate SPTB. However, the In1.1CC genotype was significantly associated with an increased susceptibility to very SPTB(OR 1.88; 95%CI, 1.05-3.38; P = 0.044). Moreover, RANTES concentration was lower in the preterm neonates than in the controls(P = 0.035) and that the In1.1CC genotype was associated with decreased serum RANTES level in preterm neonates(P<0.001).3. It was found that the distribution of the TNF-α G-308 A allele and genotype frequencies in the SPTB patients and the controls were not significantly different in the overall sample. There was no significant association between the risk of SPTB and TNF-αG-308 A the polymorphism in the overall sample(OR 0.85; 95%CI, 0.61-1.19; P = 0.35), preterm neonates with PROM(OR 0.77; 95%CI, 0.45-1.31; P = 0.32) and without PROM(OR 0.88; 95%CI, 0.62-1.27; P =0.51), extremely preterm neonates(OR 0.39; 95%CI, 0.12-1.28; P = 0.078), very preterm neonates(OR 0.68; 95%CI, 0.38-1.19; P = 0.16) and moderate preterm neonates(OR 1.02; 95%CI, 0.71-1.46; P = 0.92).4. There was no significant association between the genetic polymorphisms of IL-15rs10833 and susceptibility to SPTB in the overall sample, extremely preterm neonates, very preterm neonates and moderate preterm neonates. However, in preterm neonates with and without PROM, a significant association was observed between SPTB susceptibility and the A-positive genetypes(GA+AA genetypes)(OR 0.46; 95%CI, 0.23-0.92; P = 0.017; OR 1.47; 95%CI, 1.04-2.09; P = 0.031).5. Compared with the CC genotypes, the IL-1β +3953T-positive genotype(CT+TT genotype) was significantly associated with a increased susceptibility to SPTB(OR 1.63; 95%CI, 1.02-2.59; P = 0.036) and moderate SPTB(OR 1.79; 95%CI, 1.09-2.94; P = 0.022).The +3953T-positive genotype was also significantly associated with increased SPTB susceptibility in preterm neonates with PROM(OR 2.36; 95%CI, 1.32-4.23; P = 0.005).6. Compared with the CC genotypes, MMP-8-799T-positive genotypes(CT+TT genotypes) were significantly associated with a decreased susceptibility to SPTB(OR 0.79; 95%CI, 0.63-0.9; P = 0.027) and moderate SPTB(OR 0.78; 95%CI, 0.61-0.99; P = 0.037). The-799T-positive genotypes(CT+TT genotype) were also significantly associated with decreased SPTB susceptibility in preterm neonates with PROM(OR 0.67; 95%CI, 0.49-0.92; P = 0.014). Comparing with the CC + CT genotypes,-799 TT genotype was marginally associated with a decreased SPTB susceptibility in preterm neonates without PROM(OR 0.71; 95%CI, 0.50-1.00; P = 0.049).Conclusions:1. Important single nucleotide polymorphisms(SNPs), including MCP-1 G-2518 A, RANTESIn1.1T/C, IL-15rs10833, IL-1β C+3953T and MMP-8C-799 T, contribute to the SPTB susceptibility.2. RANTES concentration was lower in the preterm neonates than in the controls(P = 0.035). The RANTESIn1.1CC genotype was associated with the decreased serum RANTES levels in preterm neonates. It was suggested that there was a significant association between the In1.1T/C genotype and the expression of RNATES in the sera from preterm neonates.3. Among Chinese population, IL-1β C+3953T polymorphism was associated with significantly susceptibility to SPTB. Identifying the SNP would advance the development of solutions to preventing SPTB and would help to clarify the causes and mechanisms of this disease.