Correlation Between Vaginal Flora Disturbance And Cervical Cancer Recurrence

Aims:This study intends to use 16 s rDNA high-throughput sequencing technology to compare and analyze the differences in vaginal dysbacteriosis diversity between patients with primary cervical cancer and recurrent cervical cancer,and combined with clinical indicators to analyze the relationship between vaginal dysbacteriosis and cervical cancer recurrence,The relationship between vaginal dysbacteriosis and recurrent cervical cancer is supposed to provide clinical treatment measures guidance to delay the recurrence of cervical cancer and prolong the survival time of recurrent cervical cancer patients.Methods:1.In this study,I selected patients with 35 cases of primary cervical cancer(named Group PC)and recurrent cervical cancer(named Group RC)who were treated in the Second Affiliated Hospital of Nanchang University from February 2022 to February 2023.Among them,The patients were required to have pathological evidence but had not received treatment.All the subjects included in the study signed informed consent.2.Vaginal secretions were collected at the time of visit of all patients.,2 cases of PC group and 3 cases of RC group were excluded due to lack of clinical data,2cases of PC group and 4 cases of RC group were excluded due to failure of vaginal flora DNA extraction.Finally,31 patients in the PC group and 18 patients in the RC group were sent to Shanghai Paisonol Company for 16 s rDNA high-throughput sequencing.This technology used Illumina platform to sequence the V4(a)region of microbial 16 s rDNA and then,vaginal microflora is classified according to the classification method of "kingdom,phylum,class,order,family,genus and species" and analyze species diversity of vaginal microflora,vaginal microflora that are beneficial to human health are defined as "probiotics".such as Lactbacilus 、bifidobacterium and Streptococcus,vaginal microflora that cause harm to human health are called "pathogenic bacteria",such as Gardnerella,Prevobacteria,Bacteroides,Firmicutes etc.3.Clinical data such as number of abortions,number of births,age,height,weight,Body Mass Index,HPV,Lymphocyte ratio,Neutrophil ratio,hemoglobin,SCCA,Clinical FIGO stage,pathological grade,pathological type,lymph node metastasis and tumor size were collected at the first visit of PC patients and RC patients.Result:1.Clinical data of the subjects were analyzed: There are no significant differences in the clinical data of age,weight,height,BMI,gestational time,HPV infection,tumor size and pathological type between the two groups(P > 0.05),while There were statistically significant differences in lymph node metastasis(P< 0.05),pathological grade and FIGO stage(P<0.01).The results showed that lymph node metastasis,pathological grade and FIGO stage were correlated with cervical cancer recurrence.2.Alpha and Beta diversity analysis of vaginal microflora bewteen PC group and RC group :Diversity analysis includes Alpha diversity and Beta diversity.Alpha diversity makes a statistical analysis of Chao1 index,Shannon index and Simpson index.The results showed that compared with the PC group,Chao1 index(136.25 vs 84.21,P<0.01),Shannon index(3.18 vs 2.41,P<0.05)and Simpson index(0.78 vs 0.63,P<0.05)in RC group increased significantly.There was no significant difference in Beta diversity,indicating that the Alpha diversity of vaginal flora in RC group was higher than that in PC group.VENN diagram shows 1104 specific outs in RC group and 921 in PC group,indicating significant differences in the composition of vaginal flora between the two groups.3.Comparison of differences in vaginal microbial bewteen PC group and RC group at phylum level:The top five dominant bacteria phyla in PC group and RC group were pathogenic bacteria,which were Bacteroidetes,Fusobacterium,Firmicutes,Proteobacteria and actinomyces,respectively.The top five dominant bacteria phyla in PC group accounted for 96.64% of the total sequencing volume,and the top five dominant bacteria phyla in RC group accounted for 97.14% of the total sequencing volume.Compared with PC group,the relative abundance of Bacteroides(73.06% vs 59.54%)and Proteobacteria(6.24% vs 4.16%)in RC group was increased.The relative abundance of Fusobacteria(11.03% vs 22.49%),Firmicutes(6.18% vs7.76%)and actinobacteria(0.63% vs 2.69%)decreased,but the differences were not statistically significant.4.Comparison of differences in vaginal microbial bewteen PC group and RC group at genus level:The top five dominant bacteria genera in PC group and RC group were pathogenic bacteria,which were Prevoella,porphyromonas,cilium,Bacteroides and pseudomonas,respectively.The top five dominant bacteria genera in PC group accounted for 77.09% of the total sequencing volume,and the top five dominant bacteria genera in RC group accounted for 74.47% of the total sequencing volume.As for pathogenic bacteria,compared with PC group,patients in RC group were Prevoella(35.49% vs 31.27%),Bacteroides(14.03% vs 2.90%,P<0.05),Pseudomonas(4.80% vs 1.67%)increased relative abundance,but decreased relative abundance of porphyromonas(14.9% vs 18.9%)and cilium(5.25% vs 22.35%,P<0.05).The difference between Bacteroides and cilium was statistically significant.For probiotics,the relative abundance of Lactobacillus(1.12% vs 3.72%)was lower in RC group than in PC group.5.Correlation analysis between vaginal microflora and clinical test indexes:Correlation studies showed that the relative abundance of Prevotella was positively correlated with SCCA(r= 0.2952,p<0.05),while Prevotella was positively correlated with the proportion of neutrophils(r= 0.1288,p>0.05)and lymphocyte(r=-0.0377,p>0.05)the correlation was not statistically significant.The relative abundance of Lactobacillus is positively correlated with lymphocyte ratio(r= 0.3334,p<0.05),while the Lactobacillus with SCCA(r=-0.1283,p>0.05)and neutrophil ratio(r=-0.1499,p>0.05)The correlation was not statistically significant.6.Diversity analysis of vaginal microflora in two groups of patients: The comparative analysis of Chao1 index,Shannon index and Simpson index of patient with HPV infection(positive vs negative),clinical FIGO stage(stage I-II vs III-IV),lymph node metastasis(no vs yes),pathological grade(low-medium vs highly differentiated),pathological type(squamous cell carcinoma vs adenocarcinoma),and tumor size(< 4cm vs ≥4cm)showed that the average value of HPV infection(positive vs negative)in the PC group was 90.62,40.92,P<0.05,respectively.The mean values of lymph node metastasis(no vs)in RC group were 0.73,0.85,P<0.05,and the mean values of tumor size(< 4cm vs ≥4cm)in RC group were 105.36,184.78,P<0.05.The differences were statistically significant.These results indicated that the patients with HPV infection in the primary cervical cancer group had more vaginal flora disorders than those without HPV infection.In the recurrent cervical cancer group,the patients with lymph node metastasis,tumor larger than 4cm,compared with the patients without lymph node metastasis,tumor < 4cm vaginal flora disorder.Conclusion:(1)Compared with patients with primary cervical cancer,patients with recurrent cervical cancer had a disorder of vaginal microflora,which was manifested as increased Alpha diversity,and the relative abundance of Lactobacillus was decreased and the relative abundance of pathogenic bacteria was increased at the genus level.(2)Correlation analysis revealed that the disturbance of vaginal flora was correlated with the recurrence of cervical cancer,and The mechanism may be that the disturbance of vaginal flora reduces the abundance of probiotics and increases the number of pathogenic bacteria,resulting in the decrease of the local resistance of the body to the vagina,and eventually leads to the recurrence of cervical cancer.However,the specific mechanism still needs to be further explored.