Efficacy and pregnancy outcomes of focused ultrasound for cervical high-grade squamous intraepithelial lesions

Abstract Objective To investigate the efficacy and adverse effects of focused ultrasound (FU) in the treatment of high-grade squamous intraepithelial lesions (HSIL) and follow up on pregnancy outcomes in patients. Methods This retrospective study recruited 57 patients aged 20–40 years with cervical HSIL combined with HR-HPV infection who received FU treatment between September 2019 and April 2022. Clinical data of the patients were obtained from hospital records. HSIL cure rate and cumulative HR-HPV clearance rate were assessed after treatment. Patients were followed up on fertility and pregnancy outcomes after treatment by telephone interviews until April 1, 2023. Results During a 6-month follow-up, the HSIL cure rate was 73.7%, and a statistical difference between CIN2 and CIN3 (75.6% vs. 66.7%, p = 0.713) was not present. HSIL -recurrence was not observed during the follow-up period, and the median follow-up duration was 12 months. The cumulative HR-HPV clearance rates at the 6- and 12-month follow-ups were 56.1% and 75.4%, respectively. The median clearance time of HR-HPV was 6 (95% confidence interval, 5.46–6.54) months. The clearance rate was higher in HPV16/18 than in non-HPV16/18 (86.7% vs. 62.9%, p = 0.038). After treatment, the successful pregnancy rate in patients with fertility intentions and spontaneous abortion rate were 73.9% and 5.9%, respectively. Preterm birth, preterm premature rupture of membranes, or low-birth-weight infants were not observed. Conclusion FU treatment can regress HSIL and accelerate HR-HPV clearance in young women of childbearing age with cervical HSIL associated with HR-HPV infection, and has no significant adverse effects on pregnancy outcomes.


Introduction
Cervical cancer is the fourth most common cancer in women worldwide.Approximately 99% of cervical cancers are associated with high-risk human papillomavirus (HR-HPV) infection [1,2].According to statistics from the International Agency for Research on Cancer (IARC), approximately 340,000 women worldwide died of cervical cancer in 2020 [3].Standardized cervical cancer screening, accurate diagnosis, and subsequent treatment have reduced the morbidity and mortality associated with cervical cancer in countries with high human development index [4].
According to the 2019 American Society for Colposcopy and Cervical Pathology (ASCCP) Risk-Based Management Consensus Guidelines, cervical high-grade squamous intraepithelial lesions (HSIL) are precancerous lesions, including cervical intraepithelial neoplasia 2 (CIN2) with diffuse P16-positive immunohistochemistry and CIN3 [5].The treatment methods include cervical conization and ablation.Traditional ablation treatments, including cryotherapy and thermal coagulation, have strict indications and contraindications.Although cryotherapy is the most commonly employed ablation therapy, its implementation is often limited owing to difficulties in the procurement and transportation of refrigerant gas, especially in low and middle-income countries [6].Moreover, studies have shown a high rate of CIN2 þ recurrence after cryotherapy [7].Conization of cervical lesions, including the loop electrosurgical excision procedure (LEEP) and cold knife conization (CKC), reduces the risk of progression to invasive cancer but may increase the risk of adverse pregnancy outcomes such as second-trimester miscarriage, preterm labor, preterm premature rupture of membranes, low birth weight infants, and other related obstetrical complications [8,9].Although the exact mechanism is unclear, studies have revealed that this risk increases with cone length [10].
Therefore, it is imperative to develop an effective and minimally invasive treatment for young women who intend to conceive.Focused ultrasound (FU) is a noninvasive physiotherapy technique that has rapidly developed in recent years and has been used to treat cervical lesions associated with highrisk human papillomavirus infection [11].A focused transducer allows the deposition of penetrating ultrasound energy in the deep target tissue under the skin, which is an inside-out treatment that can lead to immune stimulation and improvement of vaginal microecology [12][13][14].Numerous studies have revealed a high success rate of FU treatment for LSIL and HR-HPV infections [15][16][17], but only a few have highlighted its efficacy, fertility, and pregnancy outcomes for HSIL.
Herein, we conducted a retrospective study to evaluate the efficacy and safety of FU in the treatment of HSIL and to follow up on treated patients to determine fertility and pregnancy outcomes.

Study population
This study was approved by the Research Ethics Committee of the Affiliated Hospital of North Sichuan Medical College (protocol number:2020ER124-1).Ninety patients with cervical biopsy-confirmed HSIL and HR-HPV infection were treated by FU at the Gynecology Clinic of the Affiliated Hospital of North Sichuan Medical College between September 2019 and April 2022.All patients had signed the informed consent of FU therapy before treatment.
The inclusion criteria were as follows:(1) women aged 20-40 years old with HPV-DNA test-confirmed HR-HPV infection and cervical histopathology-confirmed HSIL; (2) satisfactory colposcopy with a fully visible transformation zone, upper margin of the lesion and pathologically negative endocervical curettage (ECC); and (3) no antiviral treatment within 1 month before treatment and no cervical physical or surgical treatment within 1 year prior to treatment.(4) patients with complete follow-up data The exclusion criteria were as follows:(1) pregnant and lactating women; (2) patients with recurrent HSIL/CIN2þ; and (3) patients with severe hepatic, renal, cardiac, hematologic, or autoimmune disease.Baseline data included age, body mass index (BMI), HR-HPV type, histopathology, number of sexual partners, age at first sexual intercourse, and maternity history.

Focused ultrasound treatment
The patients were treated using a Model-CZF300 FUS therapeutic device (Haifu Technology Co., Ltd., Chongqing, China) by a trained professional gynecologist.Three to seven days after menstruation, the patients were placed in the lithotomy position.After disinfecting the vulva and vagina, the cervix was fully exposed by the vaginal speculum.The treatment area was reconfirmed with a 5% acetic acid and 1% iodine solution, and the scope of treatment exceeded the lesion area by 3-5 mm.A special coupling agent was applied to the surface.The FU treatment probe covered the cervical transformation zone and cervical lesions.A continuous scan was performed from the inside to the outside at a speed of 5-10 mm/s, centered on the external os of the cervix.The power of ultrasonic treatment was 3.5-4.5Wand the working frequency and pulse frequency were 10 MHz and 1000 Hz, respectively.Treatment was stopped according to the cervical surface characteristics as follows: the treatment area shrank and the external cervical os was slightly depressed inward and appeared slightly white.The mean treatment time for the patients was approximately 200-300 s.

Follow-up
Patients were advised not to have sexual intercourse for 2 months after treatment and to use contraception for 3 months.Vaginal bleeding, wound infection, and wound healing (cervical adhesions and cervical scarring) were observed for 3 months after treatment.Colposcopy and multi-point cervical biopsies were performed at 3-6 months after the treatment.HR-HPV detection was performed every 6 months, and colposcopy and multi-point cervical biopsies were repeated if necessary.The participants were followed up via telephone interviews for fertility and pregnancy outcomes after treatment.The last date of the telephone interview was 1 April 2023.

Colposcopy and cervical biopsy and cervical cytology testing
ThinPrep2000 (Holocyte, USA) was used to process the cervical cells, and the results were reported by pathologists according to the 2014 Bethesda system [18].Colposcopies were performed by gynecologists at colposcopy clinics.Targeted biopsies were performed using colposcopy and ECC, if necessary.The pathological diagnoses of all patients in this study were provided by experienced pathologists at our hospital.

Definitions
The primary outcomes were HSIL cure rate and HR-HPV clearance rate.A multi-point cervical biopsy under colposcopy was performed at 3-6 months after the treatment.Outcomes were classified into the following three states [19]: disease persistence or progression (HSIL or invasive cancer), disease improvement (histological LSIL), and disease cure (no cervical intraepithelial lesions on histopathological examinations).
Disease recurrence [16] was defined as the histological reappearance of HSIL among patients who were cured at 3-6 months of follow-up.HR-HPV clearance was considered negative for all HR-HPV subtypes.HR-HPV infections were divided into two types [20]: HPV16/18 and non-HPV16/18 infections.The secondary outcomes were safety and adverse effects of FU treatment, as well as pregnancy outcomes after treatment, including intraoperative pain score, postoperative bleeding, postoperative infection, and cervical healing (cervical adhesion and cervical scar).The mean pain score was measured using a visual Analogue Scale [21].Vaginal bleeding after treatment was divided into three grades as follows [17]: very minor bleeding (spotting bleeding), minor bleeding (<1/2 menstrual bleeding), and major bleeding (! menstrual bleeding).Postoperative local cervical infection was defined as a foul-smell, purulent secretion, fever, and/or abnormal white blood cells [16].

Statistical analysis
The data were analyzed using SPSS26.0software.Continuous variables with a normal distribution were described as mean ( v) ± standard deviation (S), while those with non-normal distribution were described by median (range).Categorical variables were expressed as frequencies (percentages) and analyzed using Pearson's chi-square test or Fisher's exact test.The clearance time of HR-HPV was evaluated using the Kaplan-Meier model and nonparametric rank-sum (log-rank) test.All tests were bilateral (p < 0.05).

Results
In total, 90 patients were recruited, of whom 63 met the inclusion criteria and 6 were lost.Therefore, 57 patients were successfully followed-up, with a dropout rate of 9.5%.Twenty women became pregnant after treatment, and three women with unplanned pregnancies underwent artificial abortions.Seventeen women were included in the pregnancy outcome observation (Figure 1).

Baseline characteristics
Baseline demographics and medical histories are presented in Table 1.The mean age of the patients was 28.2 ± 3.8 years.

Treatment outcomes
As presented in Table 2, most patients reported mild to moderate pain that was tolerable.The mean pain score was 4 (3-6).Three patients had secondary major bleeding 2 weeks after treatment that lasted for 2-3 days, and improved after vaginal gauze packing, while no patient had a local cervical infection requiring antibiotics or hospitalization [16].Three months after treatment, 54 patients healed completely, three had mild cervical hyperplasia, and none showed cervical adhesion or scar formation.
All patients underwent a multi-point cervical biopsy 3-6 months after the treatment (Figure 2).As presented in Table 3, 42 patients were cured (cure rate, 73.7%), and 13 showed improvement.Statistical significant difference was not observed in the cure rate between CIN2 and CIN3 (75.6% vs. 66.7%, p > 0.05).Treatment was ineffective in two patients, both of whom showed persistent HSIL; the disease persistence rate was 3.5%.According to the patient's condition and wishes, one patient received FU retreatment and the other patient received LEEP.Both patients were deemed a CIN1 after 6 months of treatment.All patients were followed up for over 6 months; the median follow-up time was 12 (interquartile range 9.5-14.5)months.Recurrences were not observed.

Fertility and pregnancy outcomes
As presented in Table 5, 23 women intended to conceive; 17 of these 23 women succeeded, resulting in a pregnancy rate of 73.9%.One patient experienced spontaneous abortion in the first trimester, which led to a miscarriage rate of 5.9% (1/17).Among the women who did not intend to conceive, three conceived and had an induced abortion in the first trimester.The median time between treatment and pregnancy was 16.5  months, as calculated from the last menstrual period.

Obstetric outcomes
Among all pregnant women, one had premature rupture of membranes (PROM), and one had gestational diabetes.Among the eight women who delivered, two delivered   vaginally and six underwent the cesarean section.Among all women undergoing cesarean sections, two women had a history of cesarean section), two women were suspected to have macrosomia in the third trimester, and two women were because of social factors.There were no complications such as preterm delivery or preterm premature rupture of membranes (PPROM).All the fetuses were delivered smoothly.

Neonatal outcomes
Eight newborns were born, all of which were full-term single-live births, and there were no premature babies or low-birth-weight infants.The mean (±SD) gestational age of newborns was 39.37 ± 1.03 (median ¼ 39.57) weeks.The mean (±SD) birth weight was 3253.1 ± 470.4 g, and the mean Apgar score at birth was 9.5 ± 0.7.

Discussion
In this study, we evaluated the efficacy and safety of FU therapy for HSIL and determined pregnancy outcomes.Histopathological outcome after FU treatment is an important strategy for measuring complete regression of the lesion and has rarely been described.We observed that the histopathological cure rate of HSIL was 73.7% after FU therapy.A previous study from Kenya [7] reported a cure rate of 77.2% for CIN2þ at 6 months after cryotherapy; however, the residual CIN2þ rate was 22.8%.A thermal ablation study conducted in Cameroon [22] revealed a cervical cure rate of 70.2% for HSIL/CIN2þ, which was consistent with the value observed in our study.Generally, the cure rate of HSIL treated with FU was equivalent to that observed with other ablation treatments.We also observed that the cumulative HR-HPV clearance rates were 56.1% and 75.4% at 6 and 12 months after FU treatment, respectively.A previous study showed that the clearance rates of HR-HPV in patients with histological LSIL were 5.8% and 16.1% at 6 and 12 months after interferon treatment, respectively [14].In contrast, our study revealed that FU significantly accelerated HR-HPV clearance.Qin et al. [20] reported a clearance rate of 54.6% for HSIL with FU treatment at a 6-month follow-up, which is consistent with that observed in our study.Another study [20] observed an HR-HPV clearance rate of approximately 79.2% at 12 months for HSIL after LEEP, whereas a meta-analysis [23] revealed that the median HR-HPV clearance rate at 12 months after LEEP treatment was 79% (range 62.2-88.2).In addition, most patients (94.7%) had very minor or minor bleeding after FU treatment, and only three (5.3%)patients required vaginal packing for secondary bleeding.A previously published study [24] on cervical intraepithelial lesions (CIN) by conization revealed that 37 of 85 patients (45.1%) had major bleeding (! menstrual bleeding) after conization, and 14 (18.2%)experienced heavy bleeding than expected.Another meta-analysis [25] reported that the incidence of cervical stenosis was 4% (10/240) and 8.3% (18/215) and that of hemorrhage after treatment was 6.5% and 7.4% after LEEP and CKC, respectively.In our study, most patients (94.7%) showed good cervical healing, and the incidence of local infection, cervical scarring, or adhesions was not present.Generally, these results show that the efficacy of FU was similar to that of the LEEP, but the incidence of adverse complications after FU treatment was lower than that observed with cervical conization.This result may be attributed to the mechanism of action of FU therapy, which has a minor impact on cervical anatomy and function.
Physical or ablation therapy causes little damage to the cervix because it does not remove the diseased tissue.However, recurrence and residual lesions are the greatest challenges encountered after treatment.A meta-analysis [26] involving indirect comparisons showed that the HSIL persistence/recurrence rates were 4.7% after LEEP and 1.4% after CKC at 12 months; however, the incidence of complications was higher after CKC.Chen et al. [27] also found that the lesion persistence rate within 12 months after LEEP was 7.6% (27/355), which was closely related to positive margins.The positive-margin rate was 40.3% (145/355).In our study, we did not observe HSIL recurrence in any patients during the follow-up period.However, the treatment was ineffective in two patients, and the persistence rate of HSIL was 3.5%.A recent study [16] that treated CINs with FU, including HSIL and LSIL, reported recurrence in one patient in the LSIL group, but recurrence was not observed in the HSIL group at 12 months after treatment.Therefore, we speculated that the persistence/recurrence rate after FU therapy was not higher than that observed after LEEP.However, this observation may be attributed to the small sample size and short followup time; thus, further studies with larger sample sizes and longer follow-up times are required.
In an Italian study [28], persistent HPV16 infection was associated with a higher recurrence rate of CIN2þ than other HR-HPV subtypes (odds ratio ¼ 11.33 [95% CI, 1.25-102.93;p ¼ 0.0126).In the present study, the clearance rate of HPV16/18 was higher than that of the other HR-HPV types after FU therapy (86.7% vs. 62.9%, p ¼ 0.038), and the clearance of HPV16/18 was faster (v 2 ¼ 4.817, p ¼ 0.028).These findings are supported by previous studies [20,29].Therefore, we speculate that the low HSIL recurrence rate may be related to the faster clearance of HPV16/18, which can be attributed to the therapeutic mechanism of action of FU and the activation of locally relevant immunity.Some studies [30][31][32] have shown that persistent HR-HPV infections are associated with immunosuppression.Focused ultrasound therapy can regulate immune factors secreted by immune cells and enhance local immune functions [15,33,34], thereby effectively contributing to HR-HPV clearance and cervical intraepithelial lesion regression.
Conization may shorten cervical length and volume, and the excision of additional cervical tissue is associated with worse pregnancy outcomes, according to studies [9,35,36].This risk may be related to scar formation, glandular destruction, and an altered immune environment [37] after resection.However, pregnancy following FU treatment for HSIL has rarely been reported.In this study, we followed up on fertility and pregnancy-related outcomes and found a pregnancy rate of 73.9% among women who intended to conceive after FU treatment.The median time from treatment to pregnancy was 16.5  months.Among the women who delivered, preterm birth, PPROM, and low-birthweight infants were not observed.This observation indicates that FU therapy does not have a significant effect on cervical function or pregnancy outcomes.Further studies with longer follow-up periods and larger numbers of pregnancies are warranted to confirm this finding.
This study has a few limitations: (i) it was a retrospective study and lacked a control group, which may have led to biased results.(ii) the small sample size of the study population did not allow for an in-depth assessment of the factors that influence efficacy and pregnancy outcomes.We are currently conducting a multicenter, prospective, randomized clinical trial to assess patient prognosis and the impact of this treatment on fertility.
In conclusion, FU can effectively regress HSIL and accelerate the clearance of HR-HPV infection with few adverse effects.Our preliminary results showed no significant adverse effects of FU on pregnancy outcomes after HSIL treatment.However, the indications for FU treatment should be strictly controlled and patients should be closely followed up after treatment.

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

Figure 2 .
Figure 2. Colposcopy images of CIN2 before FU treatment (A/B/C) and at 6 months after FU treatment (D/E/F).(A) the cervix was exposed and the squamocolumnar junction was fully visible.(B) With a 5% acetic acid test, thin acetowhite soon appeared in the transformation area, and the edge thickened, especially in the direction of 12 o'clock, as indicated by the arrow.(C) With a 1% iodine test, the transformation area and local edges were unstained, especially at the point indicated by the arrow.(D) The cervix was completely covered with mature squamous epithelium.(E) With a 5% acetic acid test, an obvious abnormality was not observed.(F) With a 1% iodine test, the entire cervical surface was stained.

Table 2 .
Adverse effects of FU treatment.

Table 1 .
Baseline demographics and edical history.

Table 4 .
HR-HPV clearance rate after FU treatment.By Log-rank (Mantel-Cox) test. b

Table 5 .
Fertility and pregnancy outcomes after FU.