| Aims:By using the popular high-throughput sequencing technique,this study analyzed the correlation of vaginal flora diversity among normal healthy women,patients with benign ovarian tumors and ovarian cancer,and analyzed the relationship between vaginal flora and the occurrence and development of benign ovarian tumors and ovarian cancer by combining the clinical biochemical indicators among them,so as to provide new thinking for revealing the mechanism of ovarian cancer occurrence and development.Method:1.vaginal secretions of study subjects were collected and included in the study:Under the condition of informed approval and signature consent of the patients,the vaginal secretions of normal healthy women,benign ovarian tumors and ovarian cancer patients in the Second Affiliated Hospital of Nanchang University from November 2019 to November 2021 were collected and designated as healthy control group(Group C),ovarian benign tumor group(Group BP),and ovarian cancer group(Group OC).The collected vaginal secretions were stored at minus 80 C for subsequent DNA extraction and sequencing.2.collect clinical biochemical indicators of three groups of subjects:The Hemoglobin concentration(hemoglobin),Neutrophil ratio,Lymphocyte ratio,carbohydrate antigen-125(CA-125)and carbohydrate antigen-199(CA-199)in the serum of three groups of subjects were extracted from the electronic medical record system of our hospital.3.DNA sequencing of vaginal flora by high-throughput sequencing technique:A total of 96 vaginal secretion samples were included in this study,which were divided into three groups,namely,group C with 32 cases,group BP with 32 cases and group OC with 32 cases.By matching the collected vaginal secretions with their clinical and biochemical indicators,it was found that the clinical and biochemical indicators of some subjects in the healthy control group,benign ovarian tumor group and ovarian cancer group were not completely included in the electronic medical record system of the Second Affiliated Hospital of Nanchang University,so 5 subjects in group C,1 in BP group and 8 in OC group were removed.In the subsequent DNA extraction,6 subjects in group C and 9 in OC group were removed because of the failure of DNA extraction.The final number of included studies is C group: 21 cases,BP group: 31 cases,OC group: 15 cases,a total of 67 cases.In the end,the DNA of vaginal flora from 67 subjects was sequenced by high-throughput sequencing method on Illumina platform.In this paper,the bacteria which are beneficial to human body and harmful to human body are defined as "probiotics" and "pathogenic bacteria" in the specific genera of bacteria from high-throughput sequencing.According to the sequencing results,the diversity of vaginal flora was analyzed.4.statistical methods:Adopting R software system was used to statistically analyze the clinical biochemical indexes of patients.Graphpad prism 8.0 software was used to provide data analysis on the diversity of vaginal flora,and the correlation between them was studied.Result:(1)Comparison of three groups of clinical biochemical indicators:(1)As far as hemoglobin concentration is concerned,the average values of the three groups are C group: 126.7 g/L;Group BP: 120.3g/l;OC: 114.8 g/L,and the hemoglobin concentration among the three groups showed a downward trend,among which the P value of C group and BP group was < 0.01;The P values of group C and OC were < 0.05,both of which were statistically significant,but the P values of BP and OC were > 0.05,with no statistical significance.(2)For lymphocyte ratio,the average values of the three groups were C group:40.6%;Group BP: 28.9%;OC group: 19.3%.It can be seen that the lymphocyte ratio also showed a downward trend among the three groups,among which the P value of C group and BP group was < 0.0001;P < 0.0001 in group C and group OC;P value of BP group and OC group was less than 0.001,and the difference was statistically significant.(3)In terms of neutrophil ratio,the average values of the three groups were C group: 55.8%;Group BP: 63.6%;OC group: 71.9%.It can be seen that the neutrophil ratio among the three groups showed an upward trend,among which the P value of C group and BP group was < 0.01;P < 0.0001 in group C and group OC;P value of BP group and OC group was less than 0.01,and the difference was statistically significant.(4)For carbohydrate antigen-199(CA-199),the average values of the three groups were C group: 16.98 u/ml;Group BP: 40.45 u/ml;Group OC: 68.91 u/ml,among which the p value of group c and BP group was < 0.01,and the difference was statistically significant.However,the P values of group C and group OC were >0.05;P > 0.05 between BP group and OC group,and there was no significant difference between them.(5)The analysis of carbohydrate antigen-125(CA-125)showed that the average values of three groups were C group: 14.20 u/ml;Group BP: 36.89 u/ml;Group OC:412.55 u/m,with p value of group c and BP < 0.001;P < 0.0001 in group C and group OC;P value of BP group and OC group was < 0.0001,and the difference was statistically significant.There are significant differences in neutrophil ratio,lymphocyte ratio and carbohydrate antigen-125 in clinical biochemical indexes of the research subjects,which may be caused by the state of the disease itself.(2)Comparison of the diversity of vaginal flora among three groups:high-throughput sequencing is used to analyze the diversity of vaginal flora,and Chao 1 index,Shannon index and Simpson index are all research indicators for studying the Alpha diversity of vaginal flora.Inter-group difference analysis and principal component analysis(PCA)were used to investigate the Bate diversity of vaginal flora.(1)As far as Chao 1 index is concerned,the average values of three groups are C group: 107.53;Group BP: 125.45;OC group: 162.89,the Chao 1 index showed an upward trend,and the difference among the three groups was statistically significant(P =0.017);(2)For Shannon index,the average values of three groups are C group: 0.93;BP group: 1.76;OC: 2.04,Shannon index also showed an upward trend,and the P value of the three groups was 0.044,the difference was statistically significant;(3)For Simpson index,the average values of three groups are C group: 0.25;BP:0.40;OC: 0.48,Simpson index also showed an upward trend,but there was no significant difference among the three groups(P =0.054).The above Alpha diversity data showed that the diversity of vaginal flora in OC group was significantly higher than those in BP group and C group,and the diversity of vaginal flora in BP group was significantly higher than that in C group.(4)Bate diversity analysis method based on inter-group difference analysis and principal coordinate analysis(PCA)was adopted,and the results showed that the vaginal flora among the three groups had significant differences.(3)Analysis of genus level of difference of vaginal flora among three groups:The difference in the structure of the vaginal flora of the three groups was significant as a whole,and the strains were Lactobacillus sp.at the genus level.The vaginal flora of the three groups had significant advantages,but the abundance of the rest of the strains was lower in the three groups and they were all different.In this study,we compared the abundance of "probiotics"(Lactobacillus)that all had significant advantages over that of some "pathogenic bacteria"(Megasphaera,Sneathia and Prevotella,and Streptococcus).The relative abundance of "probiotics" in the three groups was: group C: 84.5%;BP group: 52.2%;OC group: 40.9%,the trend is downward,the group C and BP group P value < 0.001,the difference was statistically significant;P<0.0001 for group C and OC,and the difference was statistically significant;P values of BP group and OC group were < 0.0001,and the difference was also statistically significant.For partial "pathogen" analysis,there was no significant trend in the relative abundance of Megasphaera,Sneathia,Prevotella,and Streptococcus,and their differences were not statistically significant.(4)Correlation analysis between the difference of vaginal flora diversity and clinical biochemical indexes:(1)Compared with carbohydrate antigen-125,the relative abundance of Lactobacillus was-0.203 in R and 0.097 in P;The R value was-0.271 and the P value was 0.025 for the relative abundance and neutrophil ratio of Lactobacillus,showing a negative correlation.(2)Compared with the relative abundance of streptococcus and carbohydrate antigen-125,the R value and P value were 0.051 and 0.684 respectively,and compared with the relative abundance of streptococcus and the ratio of neutrophils,the R value and P value were 0.093 and 0.457 respectively,showing a positive correlation.Conclusion:(1)In the comparison of the vaginal flora diversity among the normal healthy women,the ovarian benign tumor and the ovarian cancer patients,it is found that there are significant differences in the vaginal flora diversity of the three groups,and the vaginal flora diversity of the ovarian cancer patients is the highest,the ovarian benign tumor patients are the second,and the normal healthy women are the lowest.(2)The decrease of the relative abundance of "probiotics"(Lactobacillus)in the vaginal flora has a positive correlation with benign and malignant ovarian tumors;(3)The inflammatory reaction after the vaginal flora disorder can promote the development of benign and malignant ovarian tumors,but the specific mechanism remains to be confirmed by further research. |
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