【作者】 房学强；

【导师】 李会庆；

【作者基本信息】 山东大学 ， 流行病与卫生统计学， 2008， 博士

【摘要】 研究背景胎膜早破(premature rupture of fetal membranes,PROM)是指在临产前胎膜破裂,国内发生率占分娩总数的2.7%～17%,国外为5%～15%,而且近年来其发病率呈逐年升高趋势。胎膜早破一方面容易导致孕产妇宫内感染、产褥感染、难产,增加羊水栓塞的机率,另一方面诱发宫内窘迫、脐带脱垂、胎婴儿感染和早产等。研究报道,约30%～40%的胎膜早破可并发早产,临床上有近1/3的早产被证实由此引起,从而增加了围生期母婴各种并发疾病的发病率和死亡率。生殖道感染、吸烟、微量元素与维生素缺乏等是胎膜早破的危险因素,其中生殖道病原微生物上行性感染是引起胎膜早破的主要原因。常见的病原体有β-溶血性链球菌、淋球菌、沙眼衣原体及某些厌氧菌等。病原体经宫颈口感染胎膜,也可经血行播散至子宫、胎盘,发生羊膜炎、绒毛膜羊膜炎。生殖道感染中亟待解决的问题是细菌性阴道病(bacterial vaginosis,BV)菌群分类与胎膜早破的关系。随着经济的发展和城市化进程的加快,人们的生活水平有了提高,但同时也带来了微环境的污染。研究发现室内装修与自然流产和胎儿畸形等有关联,但与胎膜早破是否有关尚未见报道。近年来研究表明,基因多态性与其表达的蛋白酶活性存在关联,同一基因的某些亚型导致了其表达的酶活性增强或降低,对外界进入体内物质的代谢能力发生改变,由此增加疾病的易感性或者减弱其抵抗力。流行病学研究发现,胎膜早破有家族聚集性,在不同种族人群中的发病率不同:非洲籍美国妇女比白人妇女发生胎膜早破的危险性大两倍,这些差异说明遗传因素在胎膜早破的发生中也起重要作用。近年来关于基因多态性与早产、低体重儿关系的报道较多,而与胎膜早破关系的研究很少,据我们所知仅有研究报道了MMP-2基因和TNF基因与胎膜早破的关联,目前胎膜早破易感基因的探讨是一个国内外研究的热点。BV是阴道内正常菌群失调或菌群漂移,高浓度的需氧菌或厌氧菌群代替了乳酸杆菌而成为优势菌所致,发病率为10%～41%。BV是妊娠妇女最主要的并发疾病,可引起胎儿低体重、早产、新生儿死亡及妇女继发不孕等并发症,也是胎膜早破的重要危险因素。目前临床常用的BV实验室检查并非根据病原菌检测作出诊断,且有30%以上的患者治疗后复发,主要原因是BV复杂菌群中很多难于培养,菌群分类难于检测,阻碍了对BV病原体的研究进展。随着细菌分子生物学技术的发展,在微生物分子生态学研究领域应用的16S rRNA PCR变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术为探讨BV菌群分类提供了新的契机。研究目的采用传统流行病学和分子流行病学方法,探讨与胎膜早破发生有关联的危险因素。通过病例对照研究、分子流行病学和基因.环境交互作用分析,从易感基因和环境因素两方面分析胎膜早破的危险因素,并建立不依赖培养16S rRNAPCR-DGGE检测BV菌群分类方法,为胎膜早破防治提供科学依据。研究方法1.胎膜早破危险因素研究采用前瞻性队列观察方法,从妊娠开始建立观察队列,跟踪随访至分娩。对2006年4月至2007年11月在济南市妇幼保健院孕期保健并分娩的妇女,进行问卷调查和随访检查记录,收集孕妇及胎儿的监测资料。调查内容包括孕妇的一般特征,妇科和孕期检查记录,分娩情况,生殖道感染情况,孕妇、丈夫烟酒和接触毒物情况(妊娠期住在新装修房间的时间)及流产史等。描述监测孕产妇的流行病学特征。以发生胎膜早破者作为指示病例组,以未发生并发症者(包括胎膜早破)作为指示对照组,应用非条件Logistic回归分析胎膜早破发生的危险因素。2.易感基因与胎膜早破关联研究采集研究对象的静脉血提取DNA,采用PCR-RFLP法检测维生素D受体(Vitamin D receptor,VDR)和白细胞介素1β(interleukin-1β,IL-1β)基因多态性;采用焦磷酸测序法检测亚甲基四氢叶酸还原酶(Methylenetetrahydrofolatereductase,MTHFR)和基质金属蛋白酶-9(matrix metalloproteinase,MMPs)的基因多态性。以基因野生型纯合子为比较的基线(OR=1.0),采用Logistic回归分析突变基因与胎膜早破的关联强度(OR)。将基因与其他危险因素(生殖道感染、BV、被动吸烟、室内装修)相结合,分析环境-基因间的交互作用。3.细菌性阴道病菌群检测方法的研究采集阴道分泌物提取微生物的DNA;使用针对细菌保守区设计的通用引物(正向引物带“GC”夹),对提取的DNA进行PCR扩增,DGGE电泳对细菌进行分类,再次PCR扩增的产物纯化测序。测序结果使用Vector NTI9.0等软件进行对接,同时与Genbank、欧洲核糖体数据库和自建的16S rRNA数据库进行比对,按照97.5%同源性标准确定微生物种类。为进一步验证结果的准确性,建立针对常见菌株的单一PCR和同时检测多个已知菌株的多重PCR技术。4.统计学处理以FOXPRO 8.0建立数据库,使用SPSS 15.0软件进行数据分析,计数资料采用相对数指标,显著性检验用χ~2检验。采用非条件Logistic回归分析危险因素,观察指标为比值比(OR)及其95%可信区间(95%CI)。研究结果第一部分胎膜早破危险因素研究1.共调查了2900例孕产妇,其中445例发生胎膜早破,发生率为15.34%。2.以455例发生胎膜早破的孕产妇作为病例组,以2135例未发生胎膜早破及其他并发症者作为对照组,应用非条件Logistic回归分析胎膜早破的危险因素,将户籍作为混杂因素调整后关联有显著性的因素是:怀孕初期RTI,OR为2.1(95%CI:1.04-4.40);怀孕后期RTI,OR为2.1(95%CI:1.61-2.86);室内装修时间2个月、3个月和3个月以上,OR分别为2.6(95%CI:1.38-4.71)、2.4(95%CI:1.15-4.88)和2.3(95%CI:1.72-3.17);流产史(流产2次),OR为1.7(95%CI:1.18-2.32);丈夫吸烟,OR为1.6(95%CI:1.31-2.01)。第二部分基因多态性与胎膜早破关联研究1.将MMP-9的C/C基因型作为比较的基线(OR=1.0),(T/C+T/T)基因型的OR为1.0(95%CI:0.66-1.54)。与室内是否装修结合分析,(T/C+T/T)基因型合并室内装修比C/C基因型合并室内未装修发生胎膜早破的危险性增加2.5倍,OR为3.5(95%CI:1.26-9.47)。2.将MTHFR的C/C基因型作为比较的基线(OR=1.0),(T/C+T/T)基因型的OR为1.6(95%CI:0.93-2.82)。与丈夫是否吸烟结合分析,(T/C+T/T)基因型合并丈夫吸烟比C/C基因型合并丈夫不吸烟发生胎膜早破的危险性增加2.2倍,OR为3.2(95%CI:1.57-6.61);与室内是否装修结合分析,(T/C+T/T)基因型合并室内装修比C/C基因型合并室内未装修发生胎膜早破的危险性增加3.6倍,OR为4.6(95%CI:2.00-10.54)。3.将VDR的T/T基因型作为比较的基线(OR=1.0),(T/t+t/t)基因型的OR为1.4(95%CI:0.71-2.66)。与室内是否装修结合分析,(T/t+t/t)基因型合并室内装修比T/T基因型合并室内未装修发生胎膜早破的危险性增加4.1倍,OR为5.1(95%CI:1.02-25.86)。4.将IL-1β的C/C基因型作为比较的基线(OR=1.0),T/C基因型的OR为1.7(95%CI:0.81-3.51)。与丈夫是否吸烟结合分析,T/C基因型合并丈夫吸烟比C/C基因型合并丈夫不吸烟发生胎膜早破的危险性增加10.7倍,OR为11.7(95%CI:1.42-96.08);与BV检测结果结合分析,T/C基因型合并BV阳性比C/C基因型合并BV阴性发生胎膜早破的危险性增加13.8倍,OR为14.8(95%CI:1.61-136.53)。第三部分细菌性阴道病菌群检测方法的研究对已知病原菌建立了多重PCR检测方法,可以同时检测阴道加德纳菌、阴道奇异菌与动弯杆菌的合并感染,三种菌扩增出的片段大小分别为570bp、420bp和1164bp。对BV菌群建立了不依赖培养的广谱16S rRNA PCR-DGGE分类方法。使用16S通用引物扩增阴道分泌物DNA得到的片段大小约200bp;进行DGGE电泳后,分离条带,再次扩增,然后测序,与GenBank序列比对分析,确定菌株种类。本文列出了7份标本的检测结果,标本出现3～7条带;使用Vector NTI9.0和Identifire软件对测序后的序列进行比对,正常乳酸菌群有惰性乳酸杆菌和卷曲乳酸杆菌,致病微生物有阴道奇异菌和解脲支原体。同时建立了可进行BV菌群种类比对的16S rRNA数据库。结论1.室内装修、生殖道感染、流产史和被动吸烟是胎膜早破的危险因素。2.单因素分析发现MTHFR C677T、MMP-9 C-1562T、VDR TaqI和IL-1β+3593四个基因位点多态性与胎膜早破无显著性关联。3.基因-环境交互作用分析发现:MMP-9、MTHFR和VDR基因与室内装修有协同作用;MTHFR和IL-1β基因与被动吸烟有协同作用;IL-1β基因与BV感染之间存在协同作用,增加了胎膜早破发生的危险性。4.建立了阴道加德纳菌、阴道奇异菌和动弯杆菌的多重PCR方法;建立了不依赖细菌培养的16S rRNA-PCR-DGGE对BV菌群检测的新方法,可以对BV菌群进行分类和动态变化监测。多重PCR方法也可以对PCR-DGGE检测结果进行验证。意义与创新1.本研究运用传统流行病学研究方法,国内首次发现妊娠期居住环境中有害物质(苯和甲醛)增加发生胎膜早破的危险性。2.本研究发现MTHFR C677T、MMP-9 C-1562T、VDR TaqI和IL-1β+3593四个基因位点多态性与胎膜早破无显著性关联;但基因-环境交互作用分析发现与室内装修有协同作用的基因位点有MTHFR C677T、VDR TaqI、MMP-9C-1562T;与被动吸烟有协同作用的有MTHFR C677T、IL-1β+3593;与BV感染有协同作用的基因位点是IL-1β+3593,环境因素增加了这些基因型携带者胎膜早破发生的危险性。3.建立了16S rRNA PCR-DGGE检测BV菌群分类方法,并建立了16S rRNA数据库,为测序结果的比对分析提供了基础。该方法可用于BV的快速诊断和菌群分类及动态漂移的监测,为BV有效防治措施的制定提供依据。不足与建议不足:1.易感基因检测样本量尚不足,未分析基因-基因间交互作用。2.使用16S rRNA-DGGE方法按照测序比对结果确定BV菌群种类,除此之外,如果比对结果符合率在97.5%-99%之间,并且一条带对应几种菌株都符合时,需要从中选择已知细菌株进行培养或生化实验鉴定,由于时间关系,此部分内容未进行。建议:1.采用基因芯片等高通量技术,对影响胎膜早破的候选基因进行大范围的筛选。2.采用16S rRNA PCR-DGGE技术对BV患者进行菌群分类,并进行验证实验与临床动态观察。更多还原

【Abstract】 Premature rupture of fetal membranes(PROM)occurs before delivery.Its domestic incidence is 2.7%-17%and abroad 5%-15%,and the incidence is increasing in recent years.PROM can increase the probability of intrauterine infection,puerperal infection,difficult labour and amnionic fluid embolism,and can induce fetal distress, prolapse of the cord,fetus and infant infection and premature birth.It has been reported that about 30%to 40%of PROM is accompanied by premature birth and about one third of premature birth result from PROM.As a result,the incidence and mortality of various concurrent diseases increase at the perinatal stage.It has been demonstrated that genital tract infection,smoking,microelement and vitamin deficiency are risk factors for PROM.The ascending infection of pathogenic microorganism is the main reason for PROM.The frequent pathogens are beta streptococcus,neisseria gonorrhoeae,chlamydia trachomatis and some anaerobes. The pathogens can infect fetal membranes through cervix,and can also infect the womb and placenta through blood and cause amnionitis and chorioamnionitis.It is urgent to be resolved to make it clear of the relationship between BV(bacterial vaginosis)flora and PROM.With the developing of economy and speeding up of urbanization process,the living levels increase,but also it brings pollution at the same time.It has been found that room decoration is related to spontaneous abortion and fetal anomaly but not to PROM.Recently,it has been reported that the gene polymorphisms are associated with the activity of protease.Some subtypes of one gene lead to increase or decrease of the enzymatic activity,and change of the metabolic ability of alien substance.As a result, the susceptibility to diseases is increased or the power of resistance is weakened. Epidemiology studies find that PROM has familial aggregation and the incidences are different among different ethnic populations.The risk is twice in African American women as in white women.The difference indicates the effect of genetic factor in PROM.The reports about relations between gene polymorphisms and preterm delivery and low birth weight are more while between gene polymorphisms and PROM are less.To our knowledge there are only reports of MMP-2 gene and TNF gene associated with PROM,so it is a hot issue to approach the predisposing genes involved in PROM.BV is dysbacteria or drift of bacteria in the vagina where high concentration of aerobia and anaerobian take place of lactobacillus and become the dominant bacteria. Its incidence among women is 10%to 41%.Chiefly,BV is the main concurrent disease among pregnant women which can cause severe complications such as low birth weight of foetus,premature birth,neonatal death and secondary inferitility of the women and is also the most important risk factor for PROM.At present,the laboratory diagnosis of BV is not based on determination of pathogenic bacteria.It recurs in more than 30%of the patients after treatment because some of the BV complicated bacterias are hard to culture and some are difficult to detect,which hamper the development of the BV flora study.With the development of molecular biology techniques,16S rRNA PCR-DGGE(Denaturing gradient gel electrophoresis)makes it possible to approach the classification of BV flora.ObjectivesTo investigate the risk factors associated with PROM by a combined method of traditional epidemiology and molecular epidemiology.To analyze the risk factors for PROM from both predisposing genes and environmental factors by means of case-control study,molecular epidemiology and gene-environment interaction analysis.To establish methods for detection and classification of BV flora. Methods1.Study on risk factors for PROMA prospective cohort observation method was taken;the follow-up investigation period was from pregnancy to childbirth.Data was obtained by questionnaire and follow-up tests records of the pregnant women who took gynecologic examination and gave birth to a child at Jinan Hospital of Matemal and Child Health from Aprial 2006 to November 2007.The data of the mother and fetal was collected.The investigation contents included the characteristics of the subjects,the records of gynecologic examination and tests during pregnancy,status of childbirth,genital tract infection,smoking and poison contact of women and their husbands(the time living in newly decorated room),and abortion history.The epidemiologic features of women were described.Those who encountered PROM were regarded as patients group and those who didn’t control group.The risk factors for PROM were analyzed by Logistic regression.2.Study on associations between predisposing genes and PROMVenous blood samples were taken and DNA was extracted.Gene polymorphisms were analyzed for VDR(Vitamin D receptor)and IL-1β(interleukin-1β)by PCR-RFLP,and for MTHFR(Methylenetetrahydrofolate reductase)and MMP-9 (matrix metalloproteinase)by pyrosequencing.The homozygote of wild type was treated as reference baseline(OR=1.0)and Logistic regression model was performed to analyze the effect of mutant gene on PROM.The gene-environment interaction was analyzed by combination gene with other risk factors,such as gential tract infection, BV,passive smoking,room decoration.3.Establishment of methods for detection and classification of BV floraMicroorganism DNA was extracted by vaginal swab and amplified by PCR using universal primers(with a "GC" clamp on the forward primer)aimed at the conservative regions of bacterium.The product was separated by DGGE.After that, PCR re-amplification of the bands was conducted and the product was sequenced. Sequencing results was connected by Vector NTI9.0 and compared with Genbank,the ribosome database of Europe and self-established 16RNA database.The species of microorganism were identified by 97.5%homology standards.To confirm the result, we also established single and multiple PCR detecting known bacterias.4.Statistical analysisThe database was constructed by FOXPRO8.0 and SPSS15.0 was used to calculate the statistical index.The numeration data were described as relative ratios and chi-square test was used to evaluate the significance.The risk factors were analyzed by non-conditional Logistic regression model.The observation indexes were odds ratios(ORs)and 95%confidence intervals(95%CI).Results1.Risk factors for PROM1.1.A total of 2900 pregnant women were investigated and 445 PROM developed with the incidence rate of 15.34%.1.2 445 PROM were regarded as patient group and 2135 pregnant women without any complication as control group.To analyze risk factors associated with PROM,non-conditional logistic regression model was used and censue register was adjusted as confounding factor.The factors with significant association with PROM included:RTI in early pregnancy with OR of 2.1(95%CI:1.04-4.40),RTI in late pregnancy with OR of 2.1(95%CI:1.61-2.86),room decoration 2 months,3 months and more than 3 months with OR of 2.6(95%CI:1.38-4.71),2.4(95%CI:1.15-4.88) and 2.3(95%CI:1.72-3.17)respectively,history of abortion(twice)with OR of 1.7 (95%CI:1.18-2.32)and husband smoking with OR of 1.6(95%CI:1.31-2.01).2.Study on associations between predisposing genes and PROM2.1.When MMP-9 C/C genotype was used as the baseline(OR=1.0),the OR of MMP-9(T/C+T/T)genotype associated with PROM was 1.0(95%CI:0.66-1.54).To analyze the combined effect of MMP-9 gene and room decoration,MMP-9 C/C+non-decoration was used as baseline(OR=1.0)and MMP-9 (T/C+T/T)+decoration was significantly associated with PROM(OR 3.5, 95%CI:1.26-9.47). 2.2.When MTHFR C/C genotype was used as the baseline(OR=1.0),the OR of MTHFR(T/C+T/T)genotype associated with PROM was 1.6(95%CI:0.93-2.82).To analyze the combined effect of MTHFR gene and passive smoking,MTHFR C/C+non-smoking was used as baseline(OR=1.0)and MTHFR(T/C+T/T)+smoking was significantly associated with PROM(OR 3.2,95%CI:1.57-6.61).To analyze the combined effect of MTHFR gene and room decoration,MTHFR C/C+non-decoration was used as baseline(OR 1.0)and MTHFR(T/C+T/T)+decoration was significantly associated with PROM(OR 4.6,95%CI:2.00-10.54).2.3.When VDR T/T genotype was used as the baseline(OR=1.0),the OR of VDR(T/t+t/t)genotype associated with PROM was 1.4(95%CI:0.71-2.66).To analyze the combined effect of VDR gene and room decoration,VDR T/T+non-decoration was used as baseline(OR=1.0)and VDR(T/t+t/t)+decoration was significantly associated with PROM(OR5.2,95%CI:1.02-25.86).2.4.When IL-1βC/C genotype was used as the baseline(OR=1.0),the OR of IL-1β(T/C+T/T)genotype associated with PROM was 1.7(95%CI:0.81-3.51).To analyze the combined effect of IL-1βgene and passive smoking,IL-1βC/C+non-smoking was used as baseline(OR=1.0)and IL-1β(T/C+T/T)+smoking was significantly associated with PROM(OR=11.7,95%CI:1.42-96.08).To analyze the combined effect of IL-1βgene and BV infection,IL-1βC/C+non-BV was used as baseline(OR=1.0)and IL-1β(T/C+T/T)+BV was significantly associated with PROM(OR=14.8,95%CI:1.61-136.53).3.Establishment of methods for detection and classification of BV floraA multiple PCR method was established for known bacteria detection.It could simultaneously detect of Gardnerella vaginalis,Atopobium vaginalis and Mobiluncus which produce 570bp,420bp and 1164bp fragment respectively.A culture-independent broad range 16S rRNA method was established for classification of BV flora.Using universal primers to amplify bacterial 16S rRNA gene by PCR,we obtained a 200bp DNA fragment.The product was separated by DGGE,cut the bands,reamplified and sequenced.The result was compared to the sequences of GenBank database and ascertain.the type of the bacterium.This paper showed the electrophoresis result of 7 specimens,with 3 to 7 bands for each sample. Vector NTI9.0 and Identifire were used to compare the sequences with 16S rRNA database.Four species were verified as Lactobacillus iners and Lactobacillus crispatus that belong to nomal vaginal microflora,Atopobium vaginalis and M.urealyticum that belong to pathogenic bacteria.We constructed a 16S rRNA database which can be used for BV flora determination and classification.Conclusions1.In this study,room decoration,infections of reproductive tract,abortion and passive smoking were found to be risk factors for PROM.2.After univariate analysis,no significant associations were observed between polymorphisms of four gene loci of MTHFR C677T,MMP-9 C-1562T,VDR TaqI and IL-1βC+3593T and PROM.3.The environment-gene interaction analysis showed synergia effect on the development of PROM in following combinations:MMP-9 gene,MTHFR gene and VDR gene combined with room decoration;MTHFR gene and IL-1βwith passive smoking;IL-1βgene with BV infection.4.Methods of multiplex PCR towards G.vaginalis,A.vaginae and Mobiluncus and culture-independent broad range 16S rRNA-PCR-DGGE were established which can be used for detection and classification of BV flora and its dynamic changes. MPCR method could be used to confirm the results of 16S rRNA PCR-DGGE.Significance and creativity1.We are the first who found that poisonous gas(benzene and formaldehyde compounds)that pregnant women contact in the living environment during their gestational period increased the risk of PROM.2.No significant association was found between loci of MTHFR C677T,MMP-9 C-1562T,VDR TaqI,IL-1β+3593 and PROM.After gene-environment interaction analysis,synergitic effect were found to increase the risk of PROM by following combinations:MTHFR C677T,VDR TaqI,MMP-9 C-1562T with room decoration; MTHFR C677T,IL-1β+3593 with passive smoking;IL-1β+3593 with BV infections. The environmental factors increased the risk of PROM for these genetype carriers..3.We established a method of 16S rRNA-PCR-DGGE to determine and classify BV flora and constructed a 16S rRNA database which could be used for sequence matching.This method can be used for rapid diagnosis of BV,classification and dynamic drift observation of BV flora,the results of which provide robust evidence for BV management.Shortcomings and suggestionsShortcomings:1.The sample size used for predisposing genes study was still insufficient and the interaction between genes was not analyzed.2.This study ascertained the type of BV bacteria according to the sequence matching result.If the coincidence rate of the matching result ranges from 97.5%to 99.0%,methods of culture or biochemistry identification should be used to confirm the result,especially when several type of bacteria consistent with one band.For reason of time,this part did not carry out.Suggestions:1.To screen the candidate gene in a larger range using the high-throughput technology of genechip.2.To analyze the BV microbial flora in a large clinical sample and observe their dynamic changes use the method of 16S rRNA PCR-DGGE.更多还原