| ObjectiveCharacterizing clearly the evolution of the microbiota in the birth canal during the period from the late pregnancy to the rupture of the fetal membrane at the first stage of the labor,and optimizing the preparation parameters of the birth-canal microecology transporter(Birth-canal Microecology Transporter,BMT),to improve the transfer technology of the birth-canal microecology and provide the experimental data support for the application of the new birth-canal microecology transporter in the following research.Materials and MethodsThis research was approved both by the Life Science Ethics Review Committee of Zhengzhou University and the Medical Ethics Review Committee of the Fifth Affiliated Hospital of Zhengzhou University,and all subjects selected had signed informed consents before the starting of the study.From March to October in 2018,48 pregnant women satisfying the inclusion criteria from the Department of Obstetrics in the Fifth Affiliated Hospital of Zhengzhou University were selected.According to the clinical contrast principle,samples of microflora from the birth canal were collected at the admitted hospitalization,and the clinical trials of the“birth-canal microecology transporter” made of the new material in six different conditions were completed in the cesarean group,meanwhile,the samples collected again during the time within three minutes after the fetal membrane rupture at the first stage of labor in the natural delivery group.Each sample was taken three times in parallel and cryopreserved at-80 °C refrigerator.The genomic DNA of all the samples were extracted by the method of CTAB(Cetyltrimethylammonium Ammonium Bromide),and the V3 + V4 variable regions of 16 S rRNA gene were amplified using the PCR,the products of which were quantified and purified,then the Illumina Hiseq2500 platform was used for sequencing.Finally,the composition and the diversity of the microbiome were analyzed by the software,such as Mothur,Lefse,Metastat,as well as the statistical methods Welch’s t-test and ANOSIM nonparametric test.Results:1.All of the 96 samples collected from 48 pregnant women included in this study,were qualified by DNA extraction,sequenced and data processed,none of which was left off.The number of all the samples’ OTUs(Operational Taxonomic Units)was270 to 589 by microbial annotated and classified.2.It was showed that the sequencing depth of the samples was sufficient to cover all the taxa from the dilution curve of the diversity.According to the OTU analysis,the microbes of all the samples,in the level of order,family and genus,were dominated by the Lactic acid bacteria community.3.The composition of the bacteria communities was quite similar between the groups before and after the rupture of the fetal membrane,and the microbial abundance of the two samples were no statistical difference in genus and species level(P>0.05,Welch’s t-test).The same as above,the microbial composition of the group M45-70 was similar with the control group in the level of order,family and genus.Moreover,it showed that there was no significant difference in the abundance of the benefit and the neutral microflora at the genus level between the M45-70 group and the control group(P>0.05,Welch’s t-test),but it was significant lower on the abundance of the harmful bacteria in the group M45-70 than the control group at the genus level(P<0.05,Welch’s t-test).4.The alpha diversity indexes of the groups above were numerically closer,moreover,there was no statistical difference between them(P>0.05,Welch’s t-test).Similarly,the alpha diversity indexes of the M45-70 group were all closer to the NC group.Furthermore,there was no difference between the two groups after the statistical analysis(P>0.05,Welch’s t-test).5.According to the beta diversity analysis,the Unifrac distance among the samples of the groups reflected on the heat map was quite near,and the median lines of the two groups were closer to each other between the groups before and after the rupture of the fetal membrane,similar with the same result between the M45-70 group and the control group in the optimal tests.Moreover,there was no significant difference between the groups above(P>0.05,Welch’s t-test).Conclusion:1.The applicable conditions for the technique of the "birth-canal microecology transfer" are not limited to whether or not the fetal membrane is ruptured.2.It is completely consistent with the experimental significance to choose the subjects who have no rupture of the fetal membrane to complete the optimizing trail.3.The birth-canal microecology transporter prepared by the new silk film can transfer the original microbes in the birth canal.Moreover,the treatment factors,such as the 0.45% hypotonic saline solution and the 70%soaking level,are the optimal parameters on the transfer.4.The new “birth-canal microecology transporter” can be used effectively and safely to transfer the microecology of the birth canal,so as to restore the intergenerational symbiotic microbiome for newborns delivered by cesarean section. |
PDF Full Text Request