Cohort Study And Mechanism Exploration Of Focused Ultrasound In The Treatment Of High-risk Human Papillomavirus Infection Related Cervical Low-grade Lesions

Worldwide,cervical cancer is the fourth most common female malignant tumor,and in low-and middle-income countries,cervical cancer ranks second among female malignant tumors.Persistent high-risk human papillomavirus（HR-HPV）infection is closely related to the occurrence and development of cervical precancerous lesions and cervical cancer.Cervical low-grade squamous intraepithelial lesion（LSIL）is the histological manifestation of HPV infection.At present,ablation and excision are the most commonly used modalities for the treatment of cervical intraepithelial neoplasia,but both have their own advantages and disadvantages.In the past ten years,Focused Ultrasound（FU）has also been used in the treatment of cervical lesions in China,and has attracted attention due to its unique treatment mode,good curative effect and fewer side effects.However,the efficacy of FU in the treatment of HR-HPV infection with cervical low-grade lesions needs to be comprehensively and systematically evaluated.At the same time,the mechanism of FU in the treatment of cervical lesions is rarely reported.For the above reasons,we carried out this research.Objectives1.A retrospective cohort study was conducted to compare the efficacy of FU and interferon drugs in the treatment of HR-HPV infection related low-grade cervical lesions.2.A prospective cohort study was conducted to compare HR-HPV clearance rate and LSIL lesion regression rate between FU and observation groups,and to analyze the related factors affecting HR-HPV clearance.3.To explore the mechanism of FU from the changes of cervical local immune status,the changes of vaginal microbial communities and the relationship between the two factors.Materials and methods1.According to the inclusion and exclusion criteria,the clinical data of patients were extracted from the electronic medical record system of gynecological outpatient department of the Affiliated Hospital of North Sichuan Medical College and selected into the study cohort.Kaplan-Meier curve and Cox regression model were used to compare the difference of HR-HPV clearance rate between the two groups,and the relationship between HR-HPV clearance and age,HR-HPV infection type and treatment methods were also analyzed.2.From October 2020 to November 2021,the patients were selected in the gynecology clinic of the Affiliated Hospital of North Sichuan Medical College according to the sample size calculation results and the inclusion and exclusion criteria,and then enrolled in the prospective cohort.The follow-up time was 6 months.Two independent sample t-test,nonparametric test and chi-square test were used to compare the differences in baseline data,HR-HPV clearance rate and LSIL lesion regression rate between the two groups.Univariate and multivariate logistic regression were used to analyze the correlation between clinical characteristics,HR-HPV infection types and intervention methods and HR-HPV clearance.3.（1）The patients in the FU group were selected according to the inclusion and exclusion criteria.The vaginal lavage fluid was collected before and 3 months after treatment.The concentrations of seven cytokines in the vaginal lavage fluid were detected by flow cytometry,including IL-2,IL-4,IL-5,IL-6,IL-10,tumor necrosis factor（TNF）and gamma interferon（IFN-γ）.（2）The patients in the FU group were selected according to the inclusion and exclusion criteria.The vaginal microbial samples were collected before and 3 months after treatment,and microbial 16S ribosomal-RNA gene sequencing and bioinformatic analyses were performed.Results1.A total of 592 patients were included in the retrospective cohort study,of which 300（50.7%）received FU and 292（49.3%）received interferon drugs.The median time of HR-HPV clearance was 6.0 months（95%CI,5.24–6.76）in FU group and 26.0 months（95%CI,22.32–29.68）in medication group.There was a significant difference between the two groups（P=0.000）.The cumulative clearance rate of HR-HPV was 47.3%in FU group and 5.8%in medication group at 6 months,with a significant difference between the two groups（P=0.000）.The cumulative clearance rate of HR-HPV was 72.3%in FU group and 31.5%in medication group at 24 months,with a significant difference between the two groups（P=0.000）.Multivariate Cox regression analysis showed that the clearance rate of HR-HPV treated with FU was 4.93times higher than that treated with interferon drugs（95%CI,3.84-6.32,P=0.000）,and the clearance rate of single HR-HPV infection was 1.53 times higher than that of multiple HR-HPV infection（95%CI,1.16-2.02,P=0.002）.In the FU group,no significant difference was observed in HR-HPV clearance rate between HR-HPV persistent and non-persistent infections(X²⁼0.751,P=0.386).2.A total of 185 patients were enrolled in the prospective cohort study,including 95 in the FU group and 90 in the observation group.At the 6-month follow-up,9 people in the FU group and 12 in the observation group were lost,with a total loss rate of 11.4%.Except for contraceptive methods and pathological results,there was no significant difference between the FU group and the observation group in all clinical baseline data（P>0.05）.The pathological results showed that the lesions in the FU group were wider than those in the observation group（P<0.05）.At the 6-month follow-up,the HR-HPV clearance rate in the FU group was 75.6%and 25.6%in the observation group,with a significant difference between the two groups（P=0.000）.The regression rate of LSIL was 89.5%in the FU group and 56.4%in the observation group,with a significant difference between the two groups（P=0.000）.Multivariate logistic regression analysis found that only the intervention methods and HPV infection types had a significant impact on the clearance of HR-HPV.The HR-HPV clearance rate in the FU group was9.03 times higher than that in the observation group（95%CI,3.75-21.73,P=0.000）,and the clearance rate of single HR-HPV infection was 5.28 times higher than that of multiple HR-HPV infection（95%CI,1.83-15.23,P=0.002）.86.3%（82/95）of the patients treated with FU for 3-5 minutes,the mean intraoperative blood loss was 1.8±0.6（1-3）m L,and the mean intraoperative pain score was 2.6±1.0（1-6）,97.9%（93/95）of the patients had no other discomfort during the operation.The quality of life score in the FU group was improved at 6-month follow-up compared with the baseline,with a significant difference（P=0.000）,but there was no significant difference in the observation group before and after（P=0.083）.3.（1）The comparison of Th1/Th2 cytokines in the vaginal lavage fluid before and 3 months after FU treatment showed that the concentrations of Th2 cytokines IL-6 and IL-5 decreased,and the difference was statistically significant（P<0.05）.The concentration of IL-4 in HR-HPV-cleared patients was lower than that in non-cleared patients,and the difference was statistically significant（P<0.05）.（2）The bioinformatics analysis results of16S ribosomal-RNA gene sequencing showed that there was no significant difference in alpha diversity index before and 3 months after FU treatment（P>0.05）,but the rarefaction curve showed that the diversity of vaginal microbiome before treatment was higher than that after FU treatment.LEf Se analysis showed that Bifidobacterium contributed the most to the difference between the two groups at the genus level,and the abundance after FU treatment was significantly higher than that before treatment（P=0.000）.The results of the partial Mantel test showed that there was a significant correlation between cytokines and microbial communities（P=0.002）.Linear regression analysis showed that Th2 cytokine IL-10 was significantly positively correlated with Sobs index and Shannon index（P<0.05）.Conclusions1.The HR-HPV clearance rate of FU therapy is significantly higher than that of interferon drug therapy,and the median time of HR-HPV clearance is significantly shorter than that of interferon drug therapy.At the same time,FU therapy also has a good effect on persistent HR-HPV infection.Interferon drugs are not recommended as a treatment method for HR-HPV infection-related cervical lesions.2.For patients with HR-HPV infection combined with LSIL,FU can eliminate HR-HPV infection and make the lesions regress in a short time,with few adverse effects and good tolerance,which provides a better choice for patients who urgently require treatment.3.（1）FU can inhibit the production of Th2 cytokines IL-6 and IL-5,shift and stimulate cervical local immunity,and contribute to the clearance of HR-HPV infection and the regression of cervical lesions.（2）The lower diversity of vaginal microbiome is related to the higher clearance rate of HR-HPV infection.FU may contribute to the low diversity of vaginal microbiome.The increase of Bifidobacterium abundance in vaginal microbiome is related to the clearance of HR-HPV infection.FU may contribute to the increase of Bifidobacterium abundance.The increase in the diversity of the vaginal microbiome is related to the cervical immunosuppressive microenvironment.