Focused ultrasound: a novel therapy for improving vaginal microecology in patients with high-risk HPV infection

Abstract Objectives To investigate changes in vaginal microecology in women with high-risk human papillomavirus (HR-HPV) infection after focused ultrasound (FU) treatment. Materials and methods We collected vaginal secretions at the time of admission and 3 months after FU treatment from 169 women who received FU treatment for cervical HR-HPV infection between July 2020 and September 2022. Among them, there were 101 patients with cute vaginitis, we also collected their vaginal secretions after one week of drug treatment. These samples were evaluated for vaginal microecology and HPV-DNA examination. Results Of the 169 patients, 101 (59.7%) suffered from acute vaginitis at the time of admission. After one week of targeted antibiotics drug treatment, there were no pathogens or pus cells on the field of microscopic vision, but there was no significant difference(p > 0.05) in the diversity and density of vaginal flora, the proportion and function of Lactobacillus (H2O2 negative rate) between one week after treatment and at the time of admission. At the time of admission of the 169 patients, the normal flora rate was 40.3%, which increased to 93.5% three months after FU treatment. The differences in vaginal secretion parameters at the time of admission and 3 months after FU treatment were as follows: H2O2 negative rate (37.3% vs. 3.6%), leukocyte esterase positive rate (54.4% vs. 5.9%), sialidase positive rate (38.5% vs. 4.1%), bacterial vaginitis positive rate (55% vs. 4.7%), fungal vaginitis positive rate (44.4% vs. 5.9%), and trichomonal vaginitis positive rate (7.1% vs. 0). The difference was statistically significant (p < 0.01). The pH value and Nagent score at the time of admission were significantly higher than those three months after FU. Three months after FU, the positive rate of HPV was 5.8% in the group of patients with normal vaginal microecology at the time of admission and post-FU; it was 6.7% in the group of patients with abnormal vaginal microecology at the time of admission and normal vaginal microecology post-FU; and it was 100% in the group patients with abnormal vaginal microecology at the time of admission and post-FU. A significant difference was observed among the three groups (p < 0.01). Conclusion FU is an effective treatment for patients with cervical HR-HPV infection. FU does not interfere with the vaginal microecology of HR-HPV positive patients with normal vaginal microecology.FU followed by antibiotic drug therapy for pathogens is beneficial to restore the function of Lactobacillus vaginalis in HR-HPV positive patients with acute vaginitis,so as to improve the vaginal microecology of HR-HPV positive patients with abnormal vaginal microecology.


Introduction
The female vaginal microecosystem comprises microecological flora, local immunity, and special anatomical structure, which interact with the host and environment in a coordinated and balanced way.Imbalance of the vaginal microecosystem may increase the risk of vaginitis and cervical cancer caused by High-risk human papillomavirus (HR-HPV) infection [1], which is the 'chief culprit' of cervical cancer.Colonization and genetic recombination of HR-HPV-infected cervical epithelial cells leads to the development of cervical intraepithelial lesions, with only 80þ% of women can rely on their own immunity to eliminate the infection [2][3].Therefore, the colonization conditions of HR-HPV have become a research hotspot.
Recent studies have revealed that HR-HPV infection can worsen the disorder of vaginal microecology and reduce the local cellular immune function of the cervix, ultimately accelerating the disease progression [4][5][6].Persistent high-risk HPV infection is a significant risk factor for cervical carcinogenesis.Therefore, early detection of cervical precancerous lesions and HPV infection, followed by early intervention and routine patient follow-up, have played a positive role in the prevention of cervical cancer [7].
While current clinical guidelines recommend follow-up for patients with HPV infection without intraepithelial lesions, there is no recommendation for the treatment of persistent HPV infection [8][9][10].Focused ultrasound (FU) is a noninvasive treatment that has been used to treat high-risk cervical lesions associated with human papillomavirus infection [11].Fu et al. found that FU therapy could regulate cell proliferation and increase apoptosis by reducing the expression of p16 and Ki-67 and increasing the expression of Fas in treated cervical tissues, thus preventing them from progressing into high-grade lesions [12].However, significant differences in the prognosis of different patients were observed under the same ultrasound treatment condition.Identifying the cause of these differences can improve the effectiveness of FU in treating patients with HR-HPV infection.Vaginal microecology may be one of the important factors affecting the prognosis of HR-HPV-infected patients treated with FU.
This study aims to investigate whether FU treatment can improve the vaginal microecological status of patients with HR-HPV infection, find a way to block persistent HR-HPV infection, explore a new treatment model for HR-HPV infection, and prevent its pathological changes.

Study design
This prospective study was conducted at the Liuzhou Maternal and Child HealthCare Hospital in Guangxi Province, China, between July 2020 and September 2022.The study was approved by the Ethics Committee of Liuzhou Maternal and Child HealthCare Hospital(Reference:KS-KY-2021-007), and all participants were provided with information about the study's objectives, methods, and significance.Written consent was obtained from those who agreed to participate in the study.The study was conducted in accordance with the ethical principles outlined in the Helsinki Declaration and was registered with the clinical trials registry in China under registration number ChiCTR2200058129.

Patients
At the beginning of the study, according to the inclusion criteria, a total of 198 patients with HR-HPV infection who were treated in Liuzhou Maternal and Child Health Hospital from July 2020 to September 2022 were included.In the course of the trial, a total of 169 patients were actually included in accordance with the inclusion criteria and treatment follow-up.
The inclusion criteria were as follows: (1) women aged 18-45 years with a history of sexual activity, (2) at least 6 months after the first discovery of HR-HPV infection, reexamination found that the same type of HR-HPV DNA was still positive, and (3) women with low-grade cervical intraepithelial neoplasia ( LSIL).
The exclusion criteria were: (1) patients with atypical squamous cells or suspected high-grade squamous intraepithelial lesions (ASC-H), high-grade squamous intraepithelial lesions (HSIL), atypical glandular cells (AGC), or squamous cell carcinoma (SCC); (2) patients with a history of cervical physiotherapy, (3) patients with autoimmune or immunodeficiency diseases, (4) patients with severe heart, liver, kidney, or blood coagulation dysfunction, (5) patients who could not complete follow-up, (6) pregnant and lactating women, (7) patients who had received vaginal drug release treatment within 1 month, and(8)patients with acute vaginal inflammation who were treated with targeted antibiotics for one week still had at least one of the following indicators of inflammation: ‹ Any kind of pathogenic microorganism, such as fungi, bacteria, trichomonas, etc., was positive, › Nagent score >3, fi White blood cell count >10 per high magnification visual field, fl Leukocyte esterase positive.
After obtaining patients' consent and signature on the operation consent form, they were treated with FU.Vaginal secretions were collected at the time of admission, after one week of drug treatment and 3 months after FU treatment for detection and comparison (Figure 1).

Specimen collection
The patients were instructed to abstain from sexual intercourse, vaginal injection, and flushing for at least two weeks before sampling.The patients were positioned in a bladder lithotomy position, followed by regular cleaning and disinfection.During the first visit, after one week of drug treatment and 3 months after FU treatment, the patient's vagina and cervical orifice were fully exposed through the vaginal speculum.A specific HR-HPV sampling brush was rotated 3 times in the cervical orifice, the secretion was scraped, then immersed in the preservation solution and stored at À80 C for HR-HPV detection [13].Vaginal secretions were scraped with a cotton swab and properly stored in a sterile test tube for vaginal microecological detection [14].

Microscopic examination of brine wet film
(1) Slice preparation: The collected vaginal secretion sample was applied directly and evenly onto a slide that was dripping with normal saline.
(2) Microscopic examination: the presence or absence of trichomonas and Candida hyphae were observed under low magnification, while the number of leukocytes, epithelial cells, bacilli and cocci was observed under high magnification to identify the presence of Candida bacteria and spores.

Comet-800 vaginal microecology detection and analysis system
Samples of vaginal secretions were collected for vaginal microecological detection.Vaginal microecology detection using Comet-800 vaginal microecology detection kit(Shandong ShiDaSi Biological Industry Co., Ltd.China).The detection was divided into two parts: (1) Dry chemical method to detect catalase, leukocyte lipase, sialidase and pH value; (2) Gram staining.Vaginal secretion specimens were dried and fixed and their microflora was analyzed under an oil microscope after Gram staining.The analysis included the proportion of Lactobacillus, flora density, flora diversity and Nagent score to allow the evaluation of the microecological status of the specimen.

Evaluation criteria
(1) Vaginal microecological balance was defined as follows: the proportion of Lactobacillus !70%, while other miscellaneous bacteria <30%; pH value 3.8-4.5;Nagent score 0-3; leukocyte count 0-10/high magnification field and no pus cells or other special pathogens; leukocyte lipase and sialidase negative; H 2 O 2 positive (Concentration !2 lmol/L).The proportion of Lactobacillus, the concentration of catalase and pH value represent the functional level of Lactobacillus. (

Anti-inflammatory therapy
After collecting the first vaginal discharge, patients with acute vaginitis received anti-inflammatory treatment.According to the treatment standards for vaginitis [15], bacterial vaginitis was treated with one-week metronidazole, fungal vaginitis was treated with one-week clotrimazole suppository plus fluconazole, and trichomonal vaginitis was treated with one-week oral ornidazole plus metronidazole gel.The results of vaginal microecology were reexamined using the above methods, and FU treatment was performed after confirming the absence of any inflammatory reaction.

Focused ultrasound therapy
In this study, a FU system (Ultrasound Therapeutic Device model-CZF300, manufacturer: Chongqing Haifu Medical Technology Co.Ltd., China) was used.The therapeutic energy was produced by a transducer with the focal length of 4 mm and size of 10 mm in diameter, operating at a frequency of 10.0 MHz.
All patients underwent FU treatment 3-7 days after menstruation without anesthesia.First, a sterile speculum was placed in the vagina to provide a clear view of the cervix, and cotton balls were used to remove cervical mucus and discharge.The vaginal wall and cervical surface were then sterilized with iodophor.The hand-held ultrasonic transducer was placed in contact with the cervix using a coupling medium, and the spiral scanning track was used for treatment at a power of 3-5 watts of power and a scanning speed of 5 $ 10mm/s.The treatment typically lasted for 5-10 min until the lesion and the squamocolumnar junction shrunk with moderate inward depression of the external cervical os [16].Participants were asked to stop all sexual intercourse, vaginal irrigation and vaginal drug delivery within the next 3 months after FU treatment.

Statistical analyses
The statistical analyses were performed using SPSS version 21.0 (IBM Corp., Armonk, NY, USA).Normal distributions were described as mean ± standard deviation.The independent sample and paired t-test were used for analysis.McNemar's paired Chi-square test and direct Chi-square test were used to calculate categorical variable changes.The rank sum test of rank data was used to analyze changes in the vaginal diversity and density calculation.The paired t-test was used to analyze changes in pH value and Nagent point.A p-value <0.05 was considered statistically significant.

Correlation between social demography and vaginal microecology
Among  1).

Comparison of the function of lactic acid bacteria and the density and diversity of vaginal flora at the time of admission and one week after drug treatment
Among the total 169 patients with HR-HPV infection, 101 patients (59.7%) suffered from acute vaginitis.There were no pathogens or pus cells on the field of microscopic vision after one week of targeted antibiotic treatment, but there was no significant difference in the diversity and density of vaginal flora and the related indexes representing the function of Lactobacillus, such as the proportion of Lactobacillus, pH value and H 2 O 2 negative rate between at the time of admission and one week after drug treatment, as shown in Tables 2 and 3.

Comparison of vaginal microecological balance at the time of admission and 3 months after FU treatment
Among the total 169 patients with HR-HPV infection, 101 patients (59.7%) had vaginal microecological disorders at the time of admission and received antibiotics for specific pathogens before FU treatment.However, three months after FU was combined with drug therapy, vaginal microecological disorders were detected only in 11 patients, accounting for 6.5%.In patients without vaginal microecological disorders at the time of admission, no one developed any disorder within 3 months after FU treatment.The proportion of vaginal microecological disorders 3 months after FU treatment was lower than that at the time of admission, and the difference was statistically significant (v 2 ¼ 88.0111, p < 0.01), as shown in Tables 4  and 5.

Comparison of abnormal vaginal secretions at the time of admission and 3 months after FU
Three months after FU, the proportion of H 2 O 2 negative, leukocyte esterase positive, sialic anhydrase positive, bacterial vaginitis, fungal vaginitis, trichomonal vaginitis were lower than that at the time of admission, and the difference was statistically significant (p<0.01)(Table 6).

Comparison of abnormal vaginal microenvironment at the time of admission and 3 months after FU
As shown in Table 7, the levels of pH value and Nagent score of vaginal secretion in patients at 3 months after FU were significantly lower than those observed at the time of admission (p < 0.01).

Comparison of HPV positive rate in different vaginal microecological groups at the time of admission and 3 months after FU
Three months after FU treatment, the positive rate of HPV was 5.8% in patients with both normal vaginal microecology at the time of admission and 3 months after FU, 6.7% in patients with abnormal vaginal microecology at the time of admission and returned to normal 3 months after FU, but 100% in patients with both abnormal vaginal microecology at the time of admission and 3 months after FU.The difference among the three groups was statistically significant (v 2 ¼ 38.41, p < 0.01) as shown in Table 8, Figure 2.

Discussion
In the present study, The vaginal bleeding of all patients after FU treatment ended within two weeks.During the follow-up period, no adverse reactions such as postoperative wound infection, ulcer, cervical adhesion, and unbearable pain were found, indicating that FU treatment is safe.We found that most of the patients with HR-HPV infection became negative 3 months after FU treatment, and the negative conversion rate was 87.57% (148/169).Sixty-eight patients with normal vaginal microecology before FU treatment all remained normal after FU treatment, which proved that FU did not interfere with the vaginal microecology of HR-HPV-positive patients with normal vaginal microecology.In the ninety patients with abnormal vaginal microecology, after treatment with antibiotics for pathogens, the inflammatory indexes turned negative, but the diversity and density of vaginal flora, the proportion and function of Lactobacillus were not significantly different from those before treatment.The results showed that the use of antibiotics alone had no significant effect on the morphological characteristics and function of Lactobacillus.The indexes of vaginal microecological balance with these patients were improved 3 months  after FU treatment, including the positive rate of bacterial vaginitis, trichomonal vaginitis and fungal vaginitis, as well as Nagent score and indicators reflecting the function of Lactobacillus, such as the proportion of Lactobacillus, pH value and H 2 O 2 concentration.Our results indicated that FU followed by antibiotic drug therapy for pathogens is beneficial to restore the function of Lactobacillus vaginalis in HR-HPV positive patients with acute vaginitis,so as to improve the vaginal microecology of HR-HPV positive patients with abnormal vaginal microecology.
Our results also showed that both older age and younger age at first delivery are risk factors for abnormal vaginal microecology.In addition, living in rural areas, low education level, low-income level, smoking, no fixed occupation, low age of first sexual life, multiple sexual partners are also the risk factors for no improvement in vaginal microecology after FU.Health education programs may benefit those with lower socioeconomic status to improve the treatment of HPV infection.
The female vagina flora is relatively simple, in which Lactobacillus is the dominant bacteria to maintain the normal microecology of the vagina, which helps to inhibit the invasion of pathogenic microorganisms and the occurrence of reproductive tract tumors, and can activate the local cervical immune system to eliminate pathogenic microorganisms in time [4].Several studies compared the vaginal microecological status of HR-HPV positive and negative patients and concluded that the vaginal microecological imbalance was closely related to persistent HR-HPV infection [17][18][19].These studies also indicated that early correction of microecological imbalance might shorten the course of HPV infection and accelerate its regression, which is consistent with the results of this study.
FU has been used to treat HR-HPV infection and lowgrade cervical intraepithelial neoplasia, and its safety and efficacy have been confirmed [20][21][22].A study has confirmed that the necrotic tissue produced by FU can induce the body to produce corresponding immune cells and stimulate the immune response, a process called the 'shedding effect' [23].
The principle of FU in the treatment of cervical lesions is to use a specific ultrasonic transducer to safely locate the ultrasound in a specific target area after passing through the epidermal tissue, and convert the sound energy into heat energy, so that the local temperature of the target area will instantly rise to more than 60 C, thus killing the HPV host cells.At the same time, its thermal effect can also improve local tissue microcirculation, promote tissue reconstruction, eliminate the HPV virus without changing the normal morphology of the cervix, and treat cervical squamous intraepithelial neoplasia [20][21][22].Zeng and his colleagues compared the immune factors in cervical tissue before and after FU treatment.The results showed that the expression of ERAP1 increased after FU treatment.The expression of Immunoglobulin A (IgA) and IL-10 was significantly downregulated (p < 0.05) [22].It revealed that FU could improve the local immune milieu of the cervix to some extent.
HPV infection has been the focus in the field of cervical cancer prevention.FU treatment can be used to treat patients with HR-HPV infection to improve the vaginal microecological status, and shorten the time of HPV-negative conversion [17][18][19].Our study found that FU treatment can improve the vaginal microecological status of patients with HR-HPV infection, which provides an opportunity to reduce the further development of HPV infection.The advantages of this study include standardized sampling (prohibition of sexual intercourse, vaginal administration and irrigation at least two weeks before sampling) [13][14], combined with the changes of the vaginal microbiota, the relationship between vaginal microbiota and negative conversion of HPV was expounded.
Recently, a study attempted to use ultrasound super-resolution microangiography (US-SR) to reconstruct local vascular imaging in vivo using microbubble contrast agent micron localization [24].We hypothesized that it would be interesting to use US-UR to evaluate cervical microvascular changes in patients with HPV infection and to determine whether cervical microvascular intervention can reduce HPV infection and put HPV into a clinical 'latent' state in future studies.Our study suggested that the vaginal microecology of HR-HPV patients with abnormal vaginal microenvironment will be significantly improved after FU treatment, which might be linked to the revascularization of the cervix, changes in blood circulation after FU treatment, stimulation of immune cell proliferation by necrotic infected cell fragments, activation of T lymphocyte immune regulation, and overall improvement in the local microenvironment [20][21][22].A future study will evaluate the local cervical microvascular remodeling after FU treatment, understand the microvascular factors related to these vaginal microecological changes, examine the possible changes of blood flow, and develop new intervention measures to reduce the clinical related HPV persistence (that is, long-term detectable HPV infection).
Although statistically significant, the limitations of this study included a small sample size in a single center and the requirement to treat vaginal inflammation before FU treatment, which limited some of our inferences.
In conclusion, after FU treatment, no significant change was observed in the vaginal microecology of HR-HPV-positive patients with normal vaginal microecology, which proved that FU did not interfere with the vaginal microecological level of HR-HPV-positive patients with normal vaginal microecology.However, FU followed by antibiotic drug therapy for pathogens is beneficial to restore the function of Lactobacillus vaginalis in HR-HPV-positive patients with acute vaginitis,so as to improve the vaginal microecology of HR-HPV positive patients with abnormal vaginal microecology, suggesting a potential approach to prevent the further development of HPV infection.

Disclosure statement
No potential conflict of interest was reported by the author(s).

)
Vaginal microecological imbalance was judged as follows: the presence of clue cells or leukocytes with positive lipase and sialidase and Nagent score !7 indicates bacterial vaginitis; the presence or detection of candida mycelium, bacteria, or spores indicates fungal vaginitis; the presence or detection of trichomonas vaginalis indicates trichomonal vaginitis; the identification of dominant bacteria other than Lactobacillus is suggestive of flora imbalance.The absence of dominant bacteria, flora density 1 'þ', and diversity 1 'þ' indicate flora inhibition, while Lactobacillus as the dominant bacteria, flora density is 4 'þ', and diversity is 4 'þ' indicate excessive flora proliferation [14].

Figure 2 .
Figure 2. Images of patients with acute vaginitis when at the time of admission、one week after drug therapy and 3 months after FU treatment.

Table 2 .
Comparison of the function of H 2 O 2 negative rate and the density and diversity of vaginal flora at the time of admission and one week after drug treatment [n (%)].
N: number of total subjects; n: number of subjects in the given category.a By McNemar Chi-square test; b By McNemar paired Chi-square test.

Table 3 .
Comparison of PH value and the proportion of Lactobacillus between at the time of admission and one week after drug treatment [ x± s].
N: number of total subjects; a By Paired t-test.

Table 4 .
Comparison of normal rate of vaginal flora at the time of admission and 3 months after FU[n (%)].

Table 5 .
Comparison of vaginal flora density and diversity at the time of admission and 3 months after FU [n (%)].

Table 6 .
Comparison of abnormal vaginal secretions at the time of admission and 3 months after FU[n (%)].
N: number of total subjects; n: number of subjects in the given category.a By McNemar paired Chi-square test.

Table 7 .
Comparison of abnormal vaginal microenvironment at the time of admission and 3 months after FU[ x± s].
Abbreviations:N: number of total subjects; a By Paired t-test.

Table 8 .
Comparison of HPV positive rate in different vaginal microecological groups at the time of admission and 3 months after FU[n(%)].
a By direct Chi-square test.

Table 9 .
Socio-demographic analysis of people with different vaginal microecological conditions.By independent-sample t-test; b by direct Chi-square test.