| Research Background:Intrauterine adhesions(IUA)refers to the repair disorder of endometrial basal layer caused by mechanical injury and/or infection,resulting in partial or total adhesion occlusion of the uterine cavity,seriously affecting the physical and mental health of women of childbearing age.Existing studies have pointed out that there is a significant vaginal microecological imbalance in patients with IUA.Female reproductive tract is a complex microecological system.The changes in host and environmental factors can lead to reproductive tract microecological imbalance,leading to a variety of inflammatory reactions and causing the occurrence of reproductive tract diseases.More and more scholars have affirmed the importance of endometrial inflammation in the occurrence and development of IUA.The uterine cavity is not aseptic,but there are few reports about the role of microbiota in the uterine cavity in the occurrence and development of IUA.Research Objective:In this study,Illumina sequencing method was used to analyze the endometrial microbiota of patients among the three IUA subgroups and the control group,understanding the diversity and composition of endometrial microbiota and exploring the composition and possible role of endometrial microbiota in the pathogenesis of IUA.Methods:1.Research subjects:①There were 20 patients with low-grade(IUA-L),12 patients with middle-grade(IUA-M),and 14 patients with high-grade(IUA-H)according to the American Fertility Society(AFS)scoring system who were diagnosed for the first time by hysteroscopic surgery because of infertility,secondary amenorrhea or less menstruation in the department of gynecology of Zhujiang Hospital of Southern Medical University from March 2016 to March 2019.②Control group(C):twenty-one women of childbearing age who underwent hysteroscopic surgery at the same time were enrolled and the endometrial histopathology showed that there were no endometrial lesions during the operation.2.Specimen collection and experimental methods:The patient was examined by hysteroscopy after general anesthesia.During the operation,the sheath of the hysteroscope avoided contact with the vaginal wall.The electric cutting ring gently scraped a little endometrium without electricity,taking 1 or 2 samples.The endometrial tissue was placed in a sterile and enzyme-free frozen tube,and the cryopreservation tube was quickly stored it in a liquid nitrogen tank or in a refrigerator at-80℃.It was used for bacterial gene extraction,16SrRNAV4 amplification,Illumina highthroughput sequencing and biological information analysis to compare the composition and diversity of endometrial microbiota among the four groups.3.Statistical processing:Statistical analyses were performed with Statistical Product and Service Solutions(SPSS)(version 20.0).Kolmogorov-Smirnov was used to test the normality.If the distribution is normal,one-way ANOVA was used for comparison between multiple groups,and the two groups are compared in pairs.If the variance is homogeneous,the LSD method is used.If the variance is not uniform,the Dunnett’s T3 method is used.And if the distribution is non-normal,the comparison between multiple groups should be based on multiple independent sample nonparametric tests,the Kruskal-Wallis test was used.And two independent sample nonparametric tests(Mann-Whitney U)were used for pairwise comparison.Statistical significance was set at P<0.05.Results:The sequencing results of endometrial tissue microorganism group showed that:1.A total of 10,089,655 sequences were obtained in this study,with an average of 150,591.9±86,852.5 per sample.2.Compared with the control group,the Alpha diversity of endometrial microbiota decreased in the three subgroups of IUA,respectively(P=0.012;P=0.811;P=0.002).There was no significant difference in the richness of endometrial microbiota in each group.With the deepening of uterine adhesion,the uniformity of endometrial microbiota distribution in IUA patients were increased,but the overall uniformity was lower than that in the control group.3.The endometrial microbiota in IUA group was significantly different from that in control group(P<0.05).4.At the phylum level,the endometrial microbiota of patients in the three subgroups of IUA and the control group were mainly composed of Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria,of which Proteobacteria accounted for more than 64.48%.At the genus level,the proportions of Klebsiella,Shewanella,and Lactobacillus were higher in Group IUA than those in Group C,respectively(20.67%&8.77%,P=0.006;13.37%&4.53%,P=0.175;12.74%&6.95%,P=0.882).The proportion of Acinetobacter was statistically lower in Group IUA than that in Group C(P=0.005).Conclusion:1.Microbiomes present in all intrauterine samples.2.Compared with the control group,the distribution of endometrial microbiota in IUA group was less uniform,and the similarity of bacterial community structure of the bacterial flora was lower.3.With different degree of uterine adhesion,the community structure of endometrial microbiota was also different,and it changed the composition proportion.But intrauterine adhesions did not change the main phylum and genus species of endometrial microbiota. |
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