In 2015, the International Society for the Study of Vulvovaginal Disease proposed a revised classification of vulvar intraepithelial neoplasia (VIN) terminology. This classification included subtypes such as low-grade squamous intraepithelial lesion (LSIL) and high-grade squamous intraepithelial lesion (HSIL), alongside the VIN differentiated type (1). LSIL and HSIL correspond to the former VIN1 and VIN2/3 nomenclature, respectively. VIN2/3 is more prevalent in younger women (2) and is considered a premalignant condition. This condition is related to invasive vulvar squamous cell carcinomas (VSCC), which account for over 80% of vulvar malignancies.

The management of VIN remains challenging owing to the lack of a clear consensus regarding the best treatment modality. VIN therapy must be individualized; therefore, comparing therapies to determine the optimal treatment is often difficult. Patients with VIN1 are recommended to undergo observation without treatment owing to the high rate of spontaneous regression. For patients with visible VIN1 lesions or those whose VIN1 lesions do not improve during observation, drugs, physical therapy, and surgical procedures can be considered. Current treatment options for VIN2/3 include local surgical excision (consisting of the removal of all visible lesions using a scalpel and electrosurgery), chemotherapy (including cidofovir, photodynamic therapy, and imiquimod), photodynamic therapy, laser ablation, and vaccination (3). Local surgical excision, often in the form of vulvectomy, can be disfiguring, emotionally distressing, and cause sexual problems in many women. Additionally, the incidence of VIN among younger women has been increasing, prompting the consideration of conservative therapy as a viable option. Alternative conservative topical chemotherapy, utilizing immune-modulating agents, and antiviral therapy have varying disadvantages. These include high rates of ulceration and unclear success response rates, ranging 26–70% (4–6). Photodynamic therapy has demonstrated numerous limitations, including a high rate of treatment failure, immunosuppressive effects, and consequent increases in direct and non-direct costs (7). Laser surgery, which uses a high-energy light beam, has been proposed as a surgical intervention for various cases of VIN. This approach has yielded mixed success, with reports indicating generally favorable tolerance, satisfactory healing, and minimal sexual dysfunction (8, 9).

However, the risk of residual disease or VIN recurrence exists in different treatment methods owing to unclear identification of the lesion’s macroscopic characteristics. Notably, different areas of the vulva exhibit variations in skin structure. Few studies have investigated the depth of epithelial and involved appendages and yielded consistent results. These studies suggested that depths of 1.0 mm and 2.0–2.5 mm in non-hairy and hairy sites, respectively, were appropriate for successful treatment (10, 11). However, these studies included only a small number of patients. Thus, a study with a large sample size focusing on VIN lesions with a more precise thickness is required to help guide clinical treatment. In this study, we aimed to describe the depth of involved and noninvolved vulvar epithelium and appendages in women with VIN and recommend the optimal depth for epithelial ablation during laser surgery.

A total of 1,474 women who underwent vulvar biopsy or local surgical vulvectomy between January 1, 2019 and December 30, 2021 were diagnosed with VIN ( Table 1 ), with an average of 42.72 ± 14.31 years. Of them, 1,139 (80%) and 335 (20%) were diagnosed with VIN1 and VIN2/3, respectively. In addition, women in the VIN2/3 group were significantly older than those in the VIN1 group (P<0.01). We also found a significant difference in HPV infection rate (P<0.01) but not in cytology (P = 0.499) between the VIN1 and VIN2/3 groups. For the VIN lesions, 90% (1,325 of 1,474) were unifocal, and 10% (149) were multifocal. In our study, VIN was commonly found on the posterior fourchette (76.85%), labia majora (11.61%), and labia minora (9.92%). We recorded 1,374 of 1,678 (77.83%) VIN lesions in non-hairy areas and 372 (22.17%) in hairy areas. Cervical squamous intraepithelial neoplasia (CIN) and/or vaginal squamous intraepithelial neoplasia (VaIN) were observed in 38.85% (481 of 1,238) of VIN cases without a history of CIN/VaIN/any other disease. We noted a significantly higher positive rate for concurrent CIN and VaIN in the VIN2/3 group (56.14%, 160 of 285) compared with that in the VIN1 group (33.68%, 321 of 953) (P=0.000).

We randomly selected 285 VIN1 patients who were diagnosed during the same period as VIN-2/3 patients, all of whom had no history of CIN/VaIN. The clinical characteristics of the women with VIN1 and VIN2/3 are shown in Supplementary 1 . In the VIN1 group, six cases had two lesion sites, specifically the posterior fourchette and labia majora. In the VIN2/3 group, 20 cases had 2 lesion sites, and 3 cases had 3 lesion sites, specifically the posterior fourchette, labia majora, and perianal areas. In the VIN1 group, CIN/VaIN 1 and CIN/VaIN 2/3 were detected in 35 (12.28%) and 8 (2.81%) cases, respectively. Two patients (0.70%) also had squamous cell carcinoma of the cervix (SCC). In the VIN2/3 group, CIN/VaIN 1, CIN/VaIN 2/3, SCC, and vaginal squamous carcinoma (VaSCC) were detected in 90 (31.6%), 59 (20.7%), 8 (2.8%), and 3 (1.1%) patients, respectively.

Table 2 , Figures 1 , 2 show the epithelial thickness of VINs in different sites. We examined 291 and 309 sections of tissue from 285 patients with VIN1 and 285 patients with VIN 2/3, respectively. Of the 600 tissue sections, VIN was detected on the posterior fourchette (45.33%), labia majora (18.83%), and labia minora (14.83%). The maximum depths of epithelial lesions were 1.6 mm and 2.75 mm in the VIN1 and VIN 2/3 groups, respectively. In the VIN2/3 group, significant differences in the thickness of involved and noninvolved epithelia were detected across all vulvar sites (P<0.05). Moreover, in the VIN1 group, the thickness of involved epithelia was greater than that of the noninvolved epithelia, except in the clitoris, urethral opening, and navicular fossa. The thickness of involved epithelium were 0.69 ± 0.44 mm and 0.49 ± 0.23 mm in the VIN2/3 and VIN1 groups, respectively (P=0.000). However, the depth of noninvolved epithelia was projected to be consistent across all VIN grades. We found that 32.81% (187 of 570) of VINs were involved in hairy areas. The rates of involvement in hairy areas were 28.07% (80 of 285) and 37.54% (107 of 285) in the VIN1 and VIN2/3 groups, respectively. The most common lesion site in non-hairy areas was the posterior fourchette in both groups. Conversely, the labia majora was the most common lesion site in hairy areas. We noted significant differences in the epithelial thickness between VINs in non-hairy and hairy areas (0.52 ± 0.30 mm vs. 0.78 ± 0.45 mm, P<0.001).

Table 3 shows the depth of involved epithelial and skin appendages in VIN and noninvolved tissue. Compared with nondysplastic samples, we found no significant difference in the depth of stratum corneum between the VIN groups. The thickness of involved skin appendages in VIN ranged 0.91–5.44 mm (mean depth, 1.98 ± 0.64 mm), whereas that of noninvolved skin appendages ranged 0.26–4.38 mm (mean depth, 1.66 ± 0.85 mm). VIN appeared to affect hair follicles in only one patient, with the depth reaching 5.44 mm. The thickness of epithelium of the involved skin appendages in VIN was consistently greater than that of the involved epithelium at the same section (1.98 ± 0.61 mm vs. 1.01 ± 0.52 mm, P<0.001). Hair follicles represented the most commonly involved appendage, followed by sebaceous glands. The involvement of sweat glands was not detected in any VINs ( Figure 3 ).

As shown in Supplementary 2 , the thickness of involved epithelia in all VIN grades was consistently greater than that of the noninvolved epithelia across all age groups. We observed a significant decrease of thickness with age in both noninvolved and involved epithelia in all VIN grades (P<0.001 for all comparisons). In comparisons between the VIN2/3 and VIN1 groups, the differences in thickness of the involved epithelia were statistically significant across the different age groups, including pre- and postmenopausal women (P<0.001 for all comparisons). Comparisons of the thickness of noninvolved epithelia showed no significant differences between the VIN2/3 and VIN1 groups in subgroup-level (age, premenopausal and postmenopausal group; P>0.05 for all comparisons).

To compare the vulvar epithelium thickness before and after FFPE treatment, we analyzed 21 pairs of frozen and corresponding FFPE-treated sections. The epithelial thickness was 0.32 ± 0.18 mm and 0.31 ± 0.11 mm for the frozen and FFPE sections, respectively, indicating no significant difference in size changes due to tissue fixation (P=0.56).

This study aimed to investigate the depth of involved and noninvolved vulvar epithelium and appendages in women with VIN to provide evidence for laser surgery. Our study showed that the value of cytology results was limited in identifying the severity of VIN lesions as no differences was observed between cytology results of the VIN1 and VIN2/3 groups. However, when the cytology results are positive for HSIL, care should be taken to avoid missing serious lesions during colposcopy. In our study, most VINs were associated with HPV infection, consistent with other studies that reported HPV positivity rates >80% (12–15), confirming the cause-and-effect relation.

VINs tend to be multifocal and multicentric, with approximately 18–56% VIN patients simultaneously having cervix, vaginal, and anal lesions (2, 16–19). In our study, 31.58% of VIN2/3 cases were concurrent with LSIL, 20.27% with HSIL, 2.81% with SCC, and 1.05% with VaSCC; VIN2/3 cases were more likely to be accompanied by cervical and vaginal lesions. Our data supported the concept that HPV-related disease can manifest as multicentric lesions in the lower female genital tract rather than being confined to one particular organ. Thus, detecting VIN on clinical examination should prompt a thorough examination from the cervix to the perianal area (20). Meanwhile, owing to the lack of effective screening methods for VIN2/3, a comprehensive and careful examination of the vulvar and perianal areas remains the main method for the early detection of VIN2/3.

The thickness of involved epithelium was ≤1 mm in 87.67% (526 of 600) of VIN cases. However, 61 (of 301, 20.37%) VIN2/3 and 13 (of 291, 4.47%) VIN1 cases showed an epithelial thickness >1 mm. Further, the number of VIN2/3 lesion sites with depths >1 mm in hairy sites (including the labia majora and perianal areas) was higher (at 49 of 97 cases). Meanwhile, 53.85% (7 of 13) VIN1 cases occurred in hairy sites (including the labia majora and perianal areas). Therefore, a treatment depth of 1 mm may be sufficient for most VIN cases, although patients with lesions in hairy areas should be monitored. According to our results, more than half of the patients with lesions in hairy sites had lesion depths of 1–3 mm.

In our results, lesions in hairy sites were thicker than those in non-hairy areas. Histologically, non-hairy sites include the clitoris, labia minora, and posterior fourchette, which are characterized by the absence of hair follicles and sweat glands. Hair-bearing skin of the inter-labial grooves, labia majora, lateral perineal, and perianal areas consist of skin appendages, including hair follicles, sebaceous glands, and apocrine and exocrine sweat glands. The upper parts of hair root sheath and lining of the sebaceous gland duct are susceptible to the extension of epithelial lesions owing to their contiguity with the surface epithelium and the presence of similar cell types. Exocrine and apocrine gland ducts are lined by their own independent epithelium, which are not contiguous with the surface epithelium. Involvement of hair root sheaths to depths of 0.8–2.5 mm has been documented (21). Involvement of the sebaceous duct occurs less often, secondary to that of the sheath.

Baggish and Dorsey suggested a uniform depth of 3 mm for laser vaporization for all areas of the vulva (22). Buckley et al. conducted a study involving 28 patients with involved skin appendages and reported that CO2 laser eradication of the skin to a depth of 5 mm can eliminate all atypical epithelium in skin appendages. However, it is unclear whether tissues within the 5–10 mm depth should also be destroyed to ensure that no appendages remain, considering that the appendages may penetrate deeper than 5 mm (21). Based on our results, the depth of lesions extending into the appendages was much deeper than that of the involved epithelium in the same section. We found that superficial lesions in non-hairy areas were vaporized by the laser to a ≤1 mm depth; however, lesions extending onto hairy areas were vaporized to 3 mm to destroy involved skin appendages. Our results were similar to previous findings of a study involving only 29 patients, of which only 5 cases had involved skin appendages (23). However, in our study, we detected only one case with involved appendages with a lesion depth >5 mm. Therefore, we were unable to provide evidence supporting laser surgery with a >5 mm depth for VIN patients.

VIN treatment aims to completely destroy the lesion, improve symptoms, exclude invasion, preserve normal vulvar anatomy and function, and avoid recurrence (3). VIN has a recurrence rate of 20–36.7% despite treatment (24, 25), with 2–15% of cases progressing to vulvar cancer (26, 27). The risk factors for recurrence and progression of VIN remain poorly understood. To eliminate VIN and avoid recurrence, health-care professionals must understand the structure of the skin and recognize the involvement of appendages.

In summary, the epithelium of VIN2/3 lesions was thicker than that of VIN1 lesions, especially in hairy areas. The depth of involvement of appendages was greater than the thickness of epithelial lesion in the same section. The lesion depth in hairy areas was 1–3 mm, with or without appendage involvement. This was deeper than the lesion depth in non-hairy areas, which was approximately 1 mm. Hence, the removal of involved epithelium and appendages would be advisable for laser surgery.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

The studies involving humans were approved by Fudan University Obstetrics and Gynecology Hospital Ethical committee approval. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

JX: Writing – original draft, Writing – review & editing, Formal Analysis, Investigation, Methodology. ZC: Methodology, Writing – original draft. YX: Formal Analysis, Writing – original draft. LS: Conceptualization, Formal Analysis, Methodology, Writing – original draft, Writing – review & editing, Investigation. CW: Conceptualization, Formal Analysis, Writing – original draft, Writing – review & editing. QC: Conceptualization, Formal Analysis, Methodology, Writing – original draft, Writing – review & editing, Data curation, Investigation.