Comparison of Topical 5-Aminolevulinic Acid Photodynamic Therapy With the Loop Electrosurgical Excision Procedure for Patients With Cervical High-Grade Squamous Intraepithelial Neoplasia: A Prospective and Propensity-Matched Analysis.

Yao Yang: writing – original draft, formal analysis, data curation, visualization. Bingying Sun: data curation, resources. Dan Cao: methodology. Ying Xu: resources, investigation. Yi Chen: writing – review and editing, validation, resources, investigation. Dan Wu: writing – review and editing, supervision, project administration, funding acquisition, conceptualization.

This prospective study enrolled 144 patients with histologically confirmed CIN 2 who underwent PDT or LEEP between March 2023 and March 2024 at the International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University. The inclusion criteria were: (1) pathologically confirmed CIN 2 and (2) high‐risk HPV positivity. The exclusion criteria included: (1) menopause; (2) a history of CIN 2 or higher, LEEP, vulvar or vaginal cancer; (3) other antiviral therapy during treatment and follow‐up; (4) pregnancy or breastfeeding; (5) immunosuppression or presence of other immune system disorders; (6) Type 3 transformation zone or lesions within intracervical canal; and (7) hypersensitivity to 5‐aminolevulinic acid (Figure  1 ). Flow chart. All patients provided written informed consent before treatment. Patients were randomly assigned to either the PDT group or the LEEP group using a computer‐generated randomization sequence. This study was approved by the Ethics Committee of the International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University (GKLW‐A‐2023‐015‐01), and registered with the Chinese Clinical Trial Registry (ChiCTR2300074684). In accordance with the journal's guidelines, we will provide our data for independent analysis by a selected team by the Editorial Team for additional data analysis or for the reproducibility of this study in other centers, if requested. Based on previous studies, the lesion clearance rate of the LEEP for CIN 2 ranged from 79.3% to 98.2% [ 19 , 20 ]. Using an estimated effective rate of 90.0% and a dropout rate of 10%, we calculated a required sample size of n  = 114 ( n 1  =  n 2  = 57) patients. A non‐inferior experimental sample size was calculated in the PASS software 15 (NCSS, LLC) with the statistical hypothesis: H0: p 1– p 2 ≤ Δ versus H1: p 1– p 2 > Δ. Outcomes included lesion clearance, regression, persistence, and progression, as well as HPV clearance, persistence, and reinfection at the 6‐ and 12‐month follow‐up assessments. Lesion clearance : No histologically diagnosed intraepithelial lesions or normal colposcopy results during follow‐up colposcopy. Lesion regression : Histological diagnosis of CIN 1 during follow‐up colposcopy. Lesion persistence : Histological diagnosis of CIN 2 during follow‐up colposcopy. Lesion progression : Histological diagnosis of CIN 3 or invasive carcinoma by colposcopy during follow‐up. HPV clearance : Negative for the original type of HPV during follow‐up. HPV reinfection : Testing positive for HPV again after prior clearance. The 6‐month follow‐up data were used as the primary outcome endpoints, and the 12‐month follow‐up data were used as secondary outcome endpoints. All patients returned 1 week after treatment to assess posttreatment complications. First follow‐up : HPV testing, TCT, and colposcopy were performed 6 months after the final treatment in the PDT group and 6 months after surgery in the LEEP. Endocervical curettage was conducted in both groups at each follow‐up. Second follow‐up : The same tests were repeated in each group at the 12‐month follow‐up. Cervical biopsies were performed when colposcopy results were abnormal at the 6‐ and 12‐month follow‐ups, with the histological diagnosis confirmed by two senior pathologists. PDT : Application and Fixation‐A 20% 5‐aminolevulinic acid thermosensitive gel (118 mg/vial; Fudan Zhangjiang Biopharmaceutical Co. Ltd. Shanghai, China) was prepared by dissolving each vial in 0.5 mL of thermosensitive hydrogel at room temperature. Thin cotton strips soaked in the 5‐aminolevulinic acid gel were inserted 1.5–2 cm into the cervical canal, leaving a tail at the external os for easy removal. Cotton pads, customized to the cervical size and lesion condition, were soaked in the 5‐aminolevulinic acid gel and applied to the cervical surface for complete coverage. A gauze‐filled condom was inserted into the vaginal cavity to secure the medicated cotton pads, with the open end left outside the vaginal introitus for easy removal before illumination. Sealing and Illumination—Vulva was sealed with cling film, gauze, or medical dressings to prevent medication loss. Illumination was performed using an LED illuminator (LED‐IB, Wuhan Yage Photo‐Electronic Co. Ltd.) for the cervical surface and a 2‐cm cylindrical diffuser fiber connected to a laser instrument (Ld600‐c, Wuhan Yage Photo‐Electronic Co. Ltd.) for the cervical canal, emitting red light at 635 nm. The cervical surface and canal were irradiated for 30 min at 100 J/cm² 3–4 h [ 21 ] after application. Six treatments were administered, each spaced 7–14 days apart. LEEP : The length of the resected cervix is determined by the type of transformation zone: 7–10 mm for the TZ1 and 10–15 mm for the TZ2 [ 9 ], and all excised tissues were sent to the pathology department for histopathological diagnosis. Sexual intercourse was prohibited for 3 months after the PDT and LEEP. HPV‐DNA detection : HPV testing was performed using a Roche Cobas 4800 system (Roche Diagnostics, Shanghai, China) which was categorized into HPV 16/18 or 12 other high‐risk HPV infections (including types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) based on the results [ 3 , 22 ]. TCT results : Cervical cytology was classified using the 2001 Bethesda System criteria: (1) negative for intraepithelial lesion or malignancy (NILM); (2) atypical squamous cells of undetermined significance (ASCUS); (3) atypical squamous cells, cannot exclude high‐grade squamous intraepithelial lesion (ASC‐H); (4) low‐grade squamous intraepithelial lesion (LSIL); (5) high‐grade squamous intraepithelial lesion (HSIL); and (6) squamous cell carcinoma (SCC); glandular epithelial abnormalities were not addressed here. Pathology : The pathological diagnoses of all patients were confirmed by two senior pathologists, and the pathological findings were categorized as inflammation, LSIL (CIN 1), HSIL (CIN 2, CIN 3), and SCC. Baseline data, including age, gravidity, parity, HPV subtypes, TCT results, colposcopy results, pathological findings (with or without vaginal intraepithelial neoplasia (VaIN), and glandular involvement). Follow‐up data, including repeated HPV tests, TCT results, re‐colposcopy results, and pathological findings were collected for all patients at each follow‐up. Logistic regression was conducted to screen for possible factors influencing the choice of treatment modality. Patients in the PDT and LEEP groups were matched 1:1 using a propensity score, estimated based on the baseline data above, with the nearest neighbor matching method. Logistic regression and propensity score analyses were conducted using Stata MP 15. Other statistical analyses were performed with IBM SPSS Statistics 27 (IBM Corp., Armonk, N.Y., USA). Normally distributed measures are presented as mean ± standard deviation (SD), with t ‐tests applied for group comparisons. Non‐normally distributed measures are expressed as the median (P50) with interquartile ranges (P25–P75), and comparisons between groups were conducted using the non‐parametric rank‐sum test. Statistics results were expressed as N (%), with group comparisons evaluated using the chi‐square test. Factors influencing treatment outcomes were analyzed using logistic regression. p  < 0.05 was deemed significant.

After applying the inclusion and exclusion criteria and conducting propensity score matching, 120 patients were included. The mean age of the patients in the PDT and LEEP groups did not significantly differ (29.78 ± 1.27 years (range: 20–46 years) and 31.38 ± 1.16 years (range: 25–45 years); t  = –1.865, p  = 0.065). No statistically significant differences were found between the two groups in HPV infection subtypes, TCT results, glandular involvement, or the presence of VaIN ( p  > 0.05) (Table  1 ). Baseline characteristics of patients in the two groups (matched cases). PDT n  = 60 ( n , %) LEEP n  = 60 ( n , %) Note: p value: comparison between groups. Other type: including types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. In the 6‐month follow‐up, there were no significant differences between the groups in the lesion regression rate ( X 2  = 0.069, p  = 0.793), lesion clearance rate ( X 2  = 0.154, p  = 0.695), clearance rate of HPV 16/18 ( X 2  = 0.042, p  = 0.838), or the clearance rate of the other 12 high‐risk HPV infections ( X 2  = 2.672, p  = 0.102). (Table  2 ). Follow‐up in the two groups. 86.7% (52/60) 85.0% (51/60) 91.7% (55/60) 93.3% (56/60) 66.7% (40/60) 70.0% (42/60) 86.7% (52/60) 90.0% (54/60) 13.3% (8/60) 13.3% (8/60) 8.3% (5/60) 6.7% (4/60) 0.0% (0/60) 0.0% (0/60) 0.0% (0/60) 0.0% (0/60) 55.0% (33/60) 65.0% (39/60) 76.7% (46/60) 78.3% (47/60) 68.0% (17/25) 65.2% (15/23) 80.0% (20/25) 73.9% (17/23) 45.7% (16/35) 64.9% (24/37) 74.3% (26/35) 81.1% (30/37) 9.1% (3/33) 12.8% (5/39) In the 12‐month follow‐up, there were no significant differences between the groups in the lesion regression rate ( X 2  = 0.120, p  = 0.729), lesion clearance rate ( X 2  = 0.323, p  = 0.570), clearance rate of HPV 16/18 ( X 2  = 0.251, p  = 0.616), or the 12 other HPV infections ( X 2  = 0.481, p  = 0.488). In addition, three patients (9.1%) in the PDT group and five patients (12.8%) in the LEEP group experienced HPV reinfection (Table  2 ). The results indicated that there was no lesion progression or recurrence in either the PDT or LEEP groups during the 12‐month follow‐up. Five patients in the PDT group and four patients in the LEEP group exhibited persistent CIN 2 lesions in the 12‐month follow‐up. We observed that six out of the nine patients with persistent lesions exhibited an TCT result of ≥ LSIL. Additionally, we present the images of one successful case before and 6 months after PDT (Figure  2 ). Colposcopy and pathology view before and after treatment with PDT. (A, B) Colposcopic image of the cervix before PDT, with an acetowhite epithelium after the acetic acid test and non‐stained lesions by Lugol's solution (×08). (C) Histological image of the cervix before treatment: local low‐grade squamous intraepithelial lesion (Grade 1), high‐grade squamous intraepithelial lesion (Grade 2) at the 12 o'clock position, and chronic mucositis on endocervical curettage. Hematoxylin and eosin stain (×20). (D, E) Colposcopy image of the cervix after 9–12 months, with no acetowhite epithelium after the acetic acid test, and stained epithelium at the 12 o'clock position using Lugol's solution (×08). (F) Histological image of the cervix after treatment: chronic mucositis at 12 o'clock position. Hematoxylin and eosin stain (×20). Regarding the 1‐week posttreatment complications, the adverse effects of PDT were minimal, and the principal manifestation was increased vaginal discharge, mild distension, and pain in the lower abdomen. No severe adverse effects were observed. In contrast, the incidence of postoperative vaginal bleeding was higher in the LEEP group. Increased vaginal discharge occurred in four patients in the PDT group and five patients in the LEEP group. There were no cases of vaginal bleeding exceeding the amount of menstruation in the PDT group while three cases were observed in the LEEP group. Lower abdominal distension was observed in three cases in the PDT group and five cases in the LEEP group. No discharge odor was observed in the PDT group, whereas one case of odor was observed in the LEEP group. Factors that may have influenced lesion regression in the PDT group at 6 months of follow‐up included age, gravidity, parity, TCT results, HPV infection subtype, the presence of VaIN, and glandular involvement. Logistic regression analysis revealed that cytological findings indicating a TCT result of ≥ LSIL were the independent risk factors for lesion regression ( p  = 0.032, 95% confidence interval (CI): –0.024 to 0.847) (Table  3 ). The regression analysis of influencing factors for lesion regression in the PDT group. We analyzed the correlation between positive resection margins following LEEP and the rate of HPV clearance at 12‐month follow‐up. Positive resection margins were statistically significant ( p  < 0.001), indicating that they influenced the HPV clearance rate in patients of the LEEP group at 12‐month follow‐up. More specifically, the HPV clearance rate for patients with positive margins was 0.48 times that of patients with negative margins (95% CI: 0.009–0.245).

In this study, at the 6‐ and 12‐month follow‐ups, no significant differences were observed in the rate of lesion regression between the PDT and LEEP groups, nor the HPV clearance rate. Neither group exhibited any lesions that progressed to Grade 3 or invasive cervical carcinoma during follow‐up. Furthermore, the TCT results were found to influence lesion regression in the PDT group. Natural regression of CIN occurs in three directions: regression, persistence, and progression. High‐grade squamous intraepithelial lesions are associated with a significant risk of progression to cervical cancer and, in most patients, should be treated rather than merely monitored. For CIN 3, the expected natural regression rate ranges from 32% to 47%, while 12% to 40% of cases may progress to invasive carcinoma if left untreated. For CIN 2, approximately 40%–58% will regress without treatment, whereas 22% will progress to Grade 3, and 5% will advance to invasive carcinoma [ 23 , 24 ]. A recent Danish study found that 33% of CIN 2 cases progressed to CIN 3 or higher within 24 months [ 24 ], which is higher than the previously reported rate of 22%. Therefore, the risk of CIN 2 progressing to CIN 3 or even invasive cervical cancer may be underestimated. The LEEP compromises the integrity of the uterine cervix, resulting in a lack of mechanical support for the cervix, reduced elongation of the repaired tissues, and alterations in the vaginal immune microenvironment. These changes have been found to significantly increase the rate of mid‐trimester miscarriage in postoperative pregnancies, as well as elevated risks of preterm labor, low birth quality, premature rupture of membranes, and perinatal morbidity and mortality [ 5 ]. A positive correlation has been observed between the depth of conization and the incidence and severity of preterm labor [ 25 ]. In the United Kingdom, 2.5% of the total preterm births annually (840 preterm births, including 196 at a gestational age of less than 32 weeks) were attributed to prior treatment for squamous intraepithelial lesions or CIN [ 26 ]. One study reported a 6% increase in the risk of preterm birth for each additional millimeter of cone length exceeding 12 mm [ 27 ]. Cervical regrowth length 6 months after cervical conization has been negatively correlated with cone excision depth [ 28 ]. Additionally, endometriosis in the cervix or cervical canal may develop after loop electrosurgical excision, which can be a source of atypical glandular epithelium in the cervix during postoperative follow‐up, leading to unnecessary misclassification [ 29 ]. Consequently, exploring noninvasive treatment options for CIN 2 in women of childbearing age to reduce cervical damage has become a popular research topic worldwide [ 8 ]. Relevant studies have reported that the maximum necrosis depth of oral tumors induced by PDT was 1.3 mm, while the average thickness of normal cervical squamous epithelium was 212.8 μm, 245.3 μm for Grade 1 CIN, 191.4 μm for Grade 2, and 218.5 μm for Grade 3, suggesting that the effective depth of PDT treatment is theoretically applicable to all cervical squamous intraepithelial lesions [ 30 , 31 ]. Additionally, the efficacy and safety of PDT for CIN and cervical HPV infection were evaluated in a 2018 systematic review and meta‐analysis of randomized clinical trials, further confirming the effectiveness of PDT [ 32 ]. Our results demonstrated that the lesion regression rates in the LEEP group were 85.0% (51/60) and 93.3% (56/60) at 6 and 12 months of follow‐up, respectively, which aligns with previous studies [ 19 , 20 ]. In terms of lesion regression, PDT achieved an efficacy comparable to the LEEP. Moreover, no lesion progression was observed in the PDT group during follow‐up, indicating that PDT could effectively treat CIN 2. Additionally, no significant difference in HPV infection clearance was noted between the two groups, corroborating a previous study [ 20 ]. In the PDT group, apart from increased vaginal discharge and mild lower abdominal distension, no complications of vaginal bleeding greater than the menstrual flow were observed, as in the LEEP group. PDT is a painless, minimally invasive, or noninvasive treatment for CIN 2 [ 12 ]. Additionally, PDT may be an effective supplementary treatment for patients with positive resection margins after cervical conization. Compared with regular follow‐up, Zhang et al. found that PDT can reduce residual and recurrence rates [ 33 ]. CIN is associated with a high recurrence rate. Previous studies reported that among 1000 patients with Grade 2 or higher, 81 experienced recurrence within 5 years after the LEEP, resulting in a 5‐year recurrence rate of 8.1% [ 34 ], This highlights the necessity for regular monitoring and long‐time observation after surgery. Conversely, Wang et al. found that among 62 patients with CIN 2 followed up for 12 months after PDT, only one patient had pathologically confirmed recurrence, yielding a 1‐year recurrence rate of 1.6% [ 35 ]. In our study, no recurrence was observed in either group, which is lower than previously reported rates. We attribute this discrepancy to the relatively small sample size and short duration of follow‐up. Our statistical results indicate that cytological findings (TCT ≥ LSIL) were independent risk factors for lesion regression in patients with CIN 2. Specifically, a higher TCT grade correlates with a poorer response to PDT and regression was associated with low‐grade or lower cytology. Similarly, Chen et al. identified continuous cervical cytology results of ≥ ASCUS and persistent prototype HPV positivity as factors influencing the clearance of cervical lesions through PDT [ 36 ]. These results suggest that PDT may not be appropriate for patients with CIN 2 and higher‐grade cytology, providing valuable insights for predicting the efficacy of PDT in the future. Our study distinguishes itself from previous research by prospectively analyzing factors affecting the efficacy of PDT and discussing the correlation between positive resection margins after the LEEP and the rate of HPV clearance. However, this study has limitations. First, the prospective analysis was conducted over a relatively short follow‐up period. Therefore, randomized controlled trials, multicenter studies, and longer follow‐up durations are necessary to explore the factors influencing the therapeutic effects of PDT and to provide a more theoretical basis for the clinical treatment of patients with CIN 2. This study demonstrated that PDT could effectively treat patients with CIN 2 and may be more beneficial for women of reproductive age to prevent the risk of obstetric complications. Additionally, cytological findings may serve as a potential indicator for determining the suitability of PDT for treating CIN 2.

PDT is an effective and safe treatment for CIN 2, preserving the anatomical structure of the cervix and thereby safeguarding the fertility of young women. Consequently, it can be a preferred choice for women of childbearing age. However, for patients with specific cytological findings before treatment, an individualized treatment plan should be formulated based on each patient's unique condition.

Cervical intraepithelial neoplasia (CIN), a group of cervical lesions closely associated with invasive carcinoma of the uterine cervix, is classified into low‐ or high‐grade squamous intraepithelial lesions (LSIL or HSIL) [ 1 ]. HSIL includes CIN Grades 2 and 3 (CIN 2 and CIN 3). Persistent high‐risk human papillomavirus (HPV) infection is the key factor in the development of cervical precancerous lesions and cervical cancer, with HPV 16 and 18 causing 50%–60% of high‐grade cervical lesions [ 2 ]. The primary treatment for HSIL is cervical resection, also known as cervical conization [ 3 ]. Cervical conization includes cold knife conization and the most widely used method, the loop electrosurgical excision procedure (LEEP) [ 4 ]. However, evidence published over the past 15 years also indicates that these treatments are associated with increased reproductive morbidity, such as preterm labor and midterm miscarriage [ 5 , 6 ], after treatment [ 7 , 8 ]. Therefore, for young women of childbearing potential, CIN 2 with satisfactory colposcopy and negative endocervical curettage for cervical canal lesions can be treated with physical therapies, including cryotherapy, laser therapy, and electrocoagulation [ 9 ]. Photodynamic therapy (PDT) is a modern, noninvasive drug‐device combination treatment for various non‐neoplastic diseases and cancers, including CIN [ 10 ]. PDT works through interactions between photosensitizers, appropriate light‐activated wavelengths, and oxygen [ 11 ]. These reactions generate reactive oxygen species (ROS), which either interact with exogenously administered photosensitizers or are produced endogenously, leading to cell death [ 12 , 13 ] through necrosis or apoptosis. Second‐generation photosensitizers, such as 5‐aminolevulinic acid, a protoporphyrin IX precursor, are widely used in PDT [ 14 ]. Due to its lesion‐specific properties, the potential of topical 5‐aminolevulinic acid PDT for treating lesions of the female lower genital tract has been explored [ 14 , 15 , 16 , 17 ]. Wu et al. used PDT in 31 patients with Grade 2 lesions with a 6‐month efficacy rate of 77.78% [ 18 ]. This prospective study aimed to compare the rates of lesion regression and HPV clearance between PDT and LEEP in patients with CIN 2 combined with high‐risk HPV infection and to analyze the relevant factors affecting the effectiveness of PDT.

The authors declare no conflicts of interest.