| Objectives:High-throughput sequencing technology and fluorescence quantitative PCR were used to study the bacterial abundance and pregnancy outcome of placenta and fetal membranes of pregnant women with premature rupture of membranesMethod:Pregnant women with regular prenatal examination and no complications were selected as the research objects in the first Affiliated Hospital of Kunming Medical University.There were 5 pregnant women with premature PROM,25 pregnant women in the TPROM group,and 30 pregnant women in the normal control group.All placental specimens were collected within one hour after cesarean section The v3-V4 regions of the bacterial 16sRNA gene were amplified and analyzed by high-throughput sequencing.With 97%sequence consistency,the obtained OTUs cluster analysis and annotation were performed to compare the differences in the distribution of bacterial communities in the fetal membrane and placenta among the three groups,and then QPCR verification was conducted based on the sequencing results and relevant literature.Results:1.High-throughput sequencing results①The placenta of pregnant women is mainly Firmicutes,Bacteroidetes,Spirochaetes,Proteobacteria and Actinobacteria.②Comparison between the PROM group and the normal group showed that Shannon index(3.33±0.05 vs.3.38±0.10)and Simpson index(0.11±0.008 vs.0.12±0.001)had no statistical difference(p>0.05),while Chaol index(407.98±6.10 vs.450±11.30)and ACE index(404.46±6.22 vs.449.89±11.75)had statistical difference(p<0.05).PPROM group and TPROM group showed no significant differences in Shannon index(3.38±0.066 vs.3.29±0.07),Simpson index(0.10±0.007 vs.0.11±0.014),Chaol index(413.21±10.31 vs.403.61±7.30),and ACE index(410.01±10.46 vs.399.83±7.45)(p>0.05).③Difference in phylum level flora abundance:There was no significant difference in placental flora abundance between PPROM group and TPROM group.Placental bacteria in the PROM group and the normal group showed significant differences in Firmicutes,Bacteroidetes,Spirochaetes,Actinobacteria,Cyanobacteria,Spirochaetes(p<0.05),while Proteobacteria(p>0.05).④Differences in the abundance of endemic bacteria:there were no significant differences in Lactobacillus between the three groups(p>0.05).The cultured bacterium,Clostridium,Streptococcus,actinporobacillus,Catenibacterium,terrisporspiraceae,Lachnospiraceae are significantly different from the normal group of placentin bacteria(p<0.05).2.QPCR verification results①The abundance of lactic acid bacteria in the PPROM group was 1/2 that of the control group,and the TPROM group was also less than the control group.The ratio of clostridium in PPROM group and TPROM group was 1.7 and 1.5 times respectively.The abundance of Streptococcus in PPROM group and TPROM group was 2.2 times of the control group.②Lactobacilli in the PPROM group and TPROM group had little difference compared with the control group.The ratio of clostridium in PPROM group and TPROM group was 1.5 and 1.1 times respectively.The abundance of Streptococcus in PPROM group and TPROM group was 1.7 times and 2.3 times of the control group.③Lactobacilli in PPROM group and TPROM group had little difference compared with the control group.The proportion of clostridium in PPROM group was 1.5 times that in control group,and 1.1 times that in TPROM group.The abundance of Streptococcus in PPROM and TPROM were 1.7 times and 2.3 times respectively.Conclusions:1.Placentas in the normal group,the preterm fetal membrane rupture group and the full-term fetal membrane rupture group were not sterile,and the distribution of placental flora was diverse2.The diversity of placental flora was consistent between the premature rupture of membranes group and the normal group,but the abundance of placental flora was significantly different.3.In the premature RUPTURE of membranes group,the contents of Lactobacillus were negatively correlated with c-reactive protein levels,while the contents of Streptococcus and Clostridium were positively correlated with C-reactive protein levels. |
