A Study On Risk Factors Associated With PROM And Methods For Detection Of BV Flora

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.