| Premature rupture of membranes (preterm premature rupture of membrance, PPROM) is one of the key factors leading to preterm delivery, accounting for 25% of preterm birth. Moreover, approximately 30-40% of preterm birth is related with PPROM. Of which before 36 weeks of gestation PPROM will appear the more serious complications of pregnancy, the incidence increasing trend in recent years. Preterm premature rupture of membranes accounted for the reasons as 1/3 of obstetric complications, early prediction of major clinical significance of early prevention, to some extent reduce the preterm birth rate, and improve maternal and newborn survival.For a long time, premature rupture of membranes is a potenially crisis before onset of premature labor, which is often combinated with it. Furthermore, premature labor become inevitable in preterm premature rupture of membranes, all both of each other. Therefore, it is particularly important to analysis of risk factors on the PPROM and its early diagnosis, as well as the early preventional treatment, which plays a significantly role in clinical The previous studies indicate that matrix metalloproteinases (MMPs) belong to endogenous enzyme which could degrade all protein components in extracellular matrix and the corresponding components of extracellular matrix. It also can control content and fetal membrane extracellular matrix structure, causing weakening of the structure of fetal membranes, leading to premature rupture of membranes. With further study, the etiology of premature rupture of membranes has step in the genomic era. As polymorphisms of MMPs gene are associated with PPROM between the mother and the fetus, pregnant women who carry susceptibility genes also increased the risk of occurrence of PPROM. However, susceptibility genes is characterized by genetic mutations itself does not directly lead to diseases, but a potential risk of the disease increased. That is to say diseases are occurred when the external harmful factors are involved in. The reason why different susceptibility, resistance and other biological characteristics of diseases between different groups and individuals are based on the variation of human genome DNA sequence, and single nucleotide polymorphism (SNP) is the most common one. Recently, the studies show that fetal MMP-1, MMP-9 and MMP-8 polymorphisms among MMP family in certain gene promoter region are related with PPROM.However, at present the research on SNP screening in MMPs which is related to mechanisms of PPROM and maternal gene polymorphism analysis are poorly known. Furthermore, it is limited to a particular gene and the study always aim at the Americas and African-American race. While asian ethnic origin (China) research in this area has not yet reported.Objectivethis study is in based on preterm premature rupture of membranes, from the molecular and protein level to verify correlation between of MMP expression on fetal membranes and PPROM, then further studies at the genetic level, to verify correlation between of MMPs gene polymorphism and PPROM and to analysis the revelance of them by combinning with maternal factors.at last,we analysis the relationship of the affect of MMPs gene polymorphism and maternal factors and it's common affect to the susceptibility of PPROM.my objective is provide scientific basis for early prevention and early prediction of PPROM.It will be profit for improving maternal and child survival rate and improving quality of life.It is a major clinical significance work.Methods一,Analysis of risk factors related to premature rupture of membranesSelect the cases of premature rupture of membranes group of pregnant women, premature rupture of membranes in pregnant women, pregnant women in spontaneous preterm labor and normal term natural delivery pregnant women as a case-control group in seven Guangdong hospitals delivery from May 2009 to January 2010.二,Fetal membranes MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) expression Differences1, collection of peripheral blood of objects to extract DNA, application of quantitative PCR reaction (SYBR Green method Real-time PCR) method to analysis MMPs mRNA content.2, collection of fetal membranes of objects to extract RNA, application of Western-blot analysis protein expression of MMPs on fetal membranes.三,study the relevance of premature rupture of membranes and matrix metalloproteinase gene polymorphisms and environmental factorsApplication of matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) detection of MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) polymorphic locus and then analysis the genotype distribution of case-control group by Hardy Weinberg, calculate the genotype, allele frequency distribution and then analysis the relevance of PPROM.in that and the combination of environmental factors.å››,Statistical analysisBy SPSS16.0 for statistical analysis. Statistical description of measurement data used x±s, Statistical description of count data used frequency and percentage. The differences between the two groups of measurement data used to compare the group t test, if the missing variance with Mann-Whitney U test; multiple groups were compared with analysis of variance compared between groups using LSD-t test, if uneven variance using the Kruskal-Wallis H test was used to compare two groups, Mann-Whitney U test, multiple testing correction using Bonferroni correction. Count data was used to compare differences between groups tested, compared with the separate tests, univariate and multivariate Logistic regression analysis. P<0.05 considered statistically significant difference. Generous with Chen Da Fang,Chen Chang Zhong secondary procedures with the SAS macro genetic Hard-weinberg equilibrium test, and calculate genotype and allele frequency distribution. Use of test used to compare with the separate tests, univariate and multivariate Logistic regression analysis group genotype and allele distribution between tests. P<0.05 considered statistically significant difference.Result一,Analysis of risk factors related to premature rupture of membranes1,study population age, pre-pregnancy body mass index and weight gain during pregnancy situation.The age of premature rupture of membranes group is larger than premature rupture of membranes group, there was significant difference between the two (P= 0.003); weight in duration of pregnancy of premature rupture of membranes is less than the normal full-term group, there was statistically significant differences between them (P<0.001).2,Analysis of clinical data in the general population:Premature rupture of membranes group compared with the premature rupture of membranes, chorioamnionitis, it's distribution of the two groups, there were statistically significant differences (P=0.030); premature rupture of membranes group and the normal full-term group comparison, there were statistically significant differences in two group of medical examination of duration of pregnancy, past history of preterm or premature rupture of membranes, history of vaginal bleeding during pregnancy chorioamnionitis, smoking or passive smoking, twin pregnancy, in vitro fertilization and embryo transplantation in treatment. (P<0.05).3,analysis the risk factors related to premature rupture of membranes by Logistic regression analysis of single factor:age is a risk factor for remature rupture of membranes group and premature rupture of membranes group, premature rupture of membranes group compared with term normal production group, the protective factors are:weight gain during pregnancy, medical examination of duration of pregnancy, the number of abortions less than two times;the related risk factors are:previous history of preterm or premature rupture of membranes, history of vaginal bleeding during pregnancy,chorioamnionitis, smoking or passive smoking.二,MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) expression difference in fetal membranes.Premature rupture of membranes group compared with the premature rupture of membranes group, MMP2, MMP7 expression decreased, MMP3, MMP8 expression increased, and there were statistically significant differences between them (P <0.001), MMP1, MMP9 expression was not significantly significant difference; with spontaneous preterm birth group, MMP2, MMP7 expression increased, MMP8 expression decreased, and there were statistically significant differences between them (P<0.001), MMP1, MMP3, MMP9 expression was not significant statistical difference; and premature premature rupture of membranes group compared with the normal full-term, MMP1, MMP2, MMP3, MMP7,MMP9 expression increased,and there were statistically significant differences between them (P<0.001), MMP8 there was no significant statistical difference. Western-blot to further verify the differences in protein expression:MMP-1 in the group of premature rupture of membranes, spontaneous preterm birth group, premature rupture of membranes group was higher than the normal full-term group; MMP-2 in premature premature rupture of membranes group, premature rupture of membranes group was higher than spontaneous preterm group and the normal full-term group, premature rupture of membranes group was higher than premature rupture of membranes group; MMP-3 in premature rupture of membranes group, spontaneous preterm premature rupture of membranes group was higher than the normal full-term group and the group; MMP-7 expression in premature rupture of membranes group than premature rupture of membranes group, spontaneous preterm labor group and the normal full-term group; MMP-8 expression in the spontaneous preterm group than premature rupture of membranes group, premature rupture of membranes group and the normal full-term group; MMP-9 in the normal full-term group was less than spontaneous preterm birth group, premature rupture of membranes group and premature rupture of membranes group.三,relevance of premature rupture of membranes and matrix metalloproteinase gene polymorphisms associated with maternal factors.1, use Hardy Weinberg equilibrium to test the genotype of case-control group:This study used statistical software SAS9.1 to to carry out Hardy Weinberg equilibrium test for normal full-term group. in addition to MMP-2 (rs243866) MMP-8 (rs11225395), the genotypes all passed the balancing test in the remaining MMPs-SNP locus, it shows the collected samples in this study is the basic representation of certain groups, it suitable to become the candidate susceptibility genes.2, genotype frequency of MMPs gene SNPs, allele frequency comparison in spontaneous preterm group,premature rupture of membranes group, premature rupture of membranes group and full-term normal control group.(1) genotype frequency comparison MMPs in preterm premature rupture of membranes group, spontaneous preterm group, premature rupture of membranes group and normal control group.Premature rupture of membranes group compared with the premature rupture of membranes group, there were statistically significant differences in that genotype frequencies of MMP1 (rs1799750), MMP3 (rs617819, rs35068180); compared with spontaneous preterm birth group, there were statistically significant differences in that genotype frequencies of MMP2 (rs243865, rs243866, rs2285053, rs17859821), MMP3 (rs35068180); compared with the normal full-term, there were statistically significant differences in that genotype frequencies of MMP1 (rs1799750), MMP2 (rs2285053, rs17859821), MMP3 (rs617819, rs35068180), MMP8 (rs11225395).(2) allele frequency comparisons MMPs in preterm premature rupture of membranes group, spontaneous preterm group, premature rupture of membranes group and normal control group.Premature rupture of membranes group compared with the premature rupture of membranes group, there were statistically significant differences in that allele frequency of MMP3 (rs617819, rs35068180); compared with spontaneous preterm group, there were statistically significant differences in that allele frequency of MMP2 (rs243865, rs243866), MMP3 (rs35068180); compared with the normal full-term group, there were statistically significant differences in that allele frequency of MMP2 (rs2285052, rs2285053), MMP3 (rs35068180).3,relevance of risk factors of premature rupture of membranes by Logistic regression analysis:â… , single-factor analysis(1) Analysis the relevance of MMPs frequency distribution of the genotypes associated with premature rupture of membranesMMP1 (rs 1799750) G. DEL genotype frequency in premature rupture of membranes group is higher than that is in the normal full-term group, the difference was statistically significant, carry G. DEL genotype will increase risk of premature rupture of membranes. MMP2 (rs2285052) CA genotype frequencies lower than the normal full-term group, the difference was statistically significant, carrying CA genotype does not increase the risk of premature rupture of membranes. MMP2 (rs2285053) CT genotype frequency was higher than the normal full-term group, the difference was statistically significant, carry CT genotype will increases the risk of premature rupture of membranes.(2) Analysis the relevance of MMPs frequencies distribution of the allele associated with premature rupture of membranesPremature rupture of membranes group compared with normal full-term group, carrying MMP2 (rs243865) T allele does not increase the risk of premature rupture of membranes, it is the protective factors for premature rupture of membranes.Premature rupture of membranes group compared with normal full-term group, compared with MMP2 (rs243866) G allele carried an increased risk of preterm premature rupture of membranes (OR=2.007,95% CI:1.035-3.891, P= 0.039). preterm premature rupture of membranes group compared with normal full-term group, MMP2 (rs2285052) A allele is the protective factor for preterm premature rupture of membranes, does not increase the risk of premature rupture of membranes. preterm premature rupture of membranes group compared with normal full-term group, it carrying with MMP2 (rs2285053) T allele frequency was higher than the normal full-term group, the difference was statistically significant, increased risk of preterm premature rupture of membranes.â…¡, multi-factor analysis(1) MMPs genotype combination of maternal factorsanalysised the factor of MMPs genotype combination of maternal factors in preterm premature rupture of membranes group and normal full-term group by used multi-factor multi-factor comparison Logistic regression analysis, such as showed that risk factors for preterm premature rupture of membranes were:a history of vaginal bleeding during pregnancy, there chorioamnionitis, history of smoking or passive smoking, carrying MMP1 (rs1799750) GG genotype, carrying MMP2 (rs2285053) CC genotype; while carrying MMP2 (rs2285052) CC genotype, medical examination of duration of pregnancy are likely the protective factor for preterm premature rupture of membranes.(2) MMPs allele combination of maternal factorsanalysised the factor of MMPs allele combination of maternal factors in preterm premature rupture of membranes group and normal full-term group by used multi-factor multi-factor comparison Logistic regression analysis, such as showed that risk factors for preterm premature rupture of membranes were:previous history of premature delivery or premature rupture of membranes, the history of vaginal bleeding during pregnancy, chorioamnionitis, history of smoking or passive smoking,, carrying MMP8 (rs11225395) A allele; carrying MMP2 (rs243866) A allele, carrying MMP2 (rs2285053) C allele, carrying MMP3 (rs617819) C allele, medical examination of duration of pregnancy may be a protective factors for preterm premature rupture of membranes. Conclusion1,the protective factors related to preterm premature rupture of membranes are: weight gain during pregnancy, medical examination of duration of pregnancy, abortion times≤1, the risk factors related to preterm premature rupture of membranes are past history of preterm or premature rupture of membranes, vaginal bleeding during pregnancy history of chorioamnionitis, smoking or passive smoking,.2,preterm premature rupture of membranes group compared with normal full-term group, the expression of MMP1, MMP2, MMP3, MMP7,MMP9 in preterm premature rupture of membranes group is increased, there was no significant statistical difference in the expression of MMP8 in that.3, there is relevance in preterm premature rupture of membranes with MMP1 (rs1799750) G. DEL genotype, MMP2 (rs2285053) CT genotype, MMP2 (rs2285052) CA genotype. there is relevance in preterm premature rupture of membranes with MMP2 (rs243865) T allele, MMP2 (rs2285052) A allele, carrying MMP2 (rs243866) G allele, MMP2 (rs2285053) T allele.4, MMP1 (rs1799750) GG genotype, MMP2 (rs2285053) CC genotype were affect-ed maternal factors,such as:history of vaginal bleeding during pregnancy, history of chorioamnionitis, history of smoking or passive smoking.they were risk factors and protective factors of premature rupture of membranes is include:carrying MMP2 (rs2285052) CC genotype, medical examination of duration of pregnancy;5, MMP8 (rs11225395) A allele were affect-ed maternal factors,such as:past history of preterm labor or premature rupture of membranes, history of vaginal bleeding during pregnancy, chorioamnionitis, history of smoking or passive smoking.they were risk factors for preterm premature rupture of membranes; and protective factors of premature rupture of membranes is include:carrying MMP2 (rs243866) A allele, MMP2 (rs2285053) C allele, MMP3 (rs617819) C allele, medical examination of duration of pregnancy;... |
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