**4. Discussion**

Our findings confirmed that smoking, parity, previous hysterectomy for CIN2+ and abnormal cytology should be considered as potential risk factors for VAIN, and a significant association is maintained by histologic grade. In addition, abnormal colposcopic findings, including grade G2, papillary and vascular patterns, are predictive of the development of high-grade VAIN, even at multivariate analysis.

According to our results, current or former smoking was significantly associated with the risk of VAIN, as already well-known in previous literature [23–25]. Sherman et al. also showed that smoking is significantly associated with the occurrence of high-grade VAIN in women infected by HR-HPV [6], as a possible consequence of a biological interaction between smoke and the viral protein of HR-HPV genotypes. Due to the large number of missing data regarding HR-HPV status, it was not possible to investigate the same correlation in our study population.

In our analysis, parity was related with a significantly increased risk of developing VAIN and, in particular, high-grade VAIN, as opposed to previous findings [26]. However, it was not possible to exclude a selection bias due to the significant distribution of missing data for parity by VAIN grade at diagnosis.

It is well-established that women with a previous history of CIN or cervical cancer, who underwent hysterectomy, remain at a higher life-time risk of VAIN and should be carefully screened for HPV-related vaginal and vulvar disease throughout their lives [26,27]. Our study confirmed that prior hysterectomy for CIN2+ should be considered as a risk factor for high-grade VAIN. Indeed, VAIN after hysterectomy usually arises near the vaginal cuff [7], since HPV infection is often multifocal and may affect other sites of the female lower genital tract. Moreover, the grade of VAIN may be affected by the severity of previous cervical disease [26] and women with a history of CIN2+ should be extensively counselled regarding the future risk of VAIN before hysterectomy. Previous hysterectomy for HPV-related cervical lesions has also been recognized as a risk factor for progression to vaginal cancer [28].

Unlike other authors [22], we did not find any correlation between age at diagnosis and the histological grade of VAIN. However, Zhou et al. also reported a poor rank correlation [22], whereas Boonlikit et al. did not show any significant distribution of patients' age among different VAIN grade groups [17]. The mean age of our patients was 52.4 ± 12.8 years. Therefore, we did not investigate whether the post-menopausal status correlated with an increased risk of VAIN because of a thinner vaginal epithelium that results in more susceptibility to changes in the vaginal microbiome and HPV infections [26].

Even immunosuppression was not associated with the development of VAIN in our cohort, as opposed to previous studies [29], probably due to the small proportion of immunosuppressed patients (11.5%).

Most diagnoses of VAIN were preceded by an abnormal pap smear result, thus supporting the assumption that cytology, in combination with a HR-HPV test, is an effective tool for early diagnosis of VAIN, even after hysterectomy, since its sensitivity is not inferior to that for CIN2+ detection [5]. We did not investigate whether cytology positivity was higher in patients with a previous hysterectomy, as recently shown by Zhang et al. in a large retrospective series of VAIN. However, the combined use of cytology and HPV testing could curb this issue, since no statistically significant difference in co-testing positivity was identified in women with or without a history of previous hysterectomy [30]. HR-HPV status was known only in 160 out of 255 enrolled patients. Most of the missing data were found in women with a first diagnosis of VAIN in the early 2000s, when HPV testing was neither applied for primary screening nor routinely performed as a triage test after abnormal cytology. In our cohort with an available Cobas result, 83.8% of cases were affected by HR-HPV infection, with or without HPV 16 and 18, as reported by previous literature [26]. Most of the women affected by VAIN3 were positive for HR-HPV with 16 and/or 18 (53.5%). As already explained, this association was only borderline significant, due to the large number of missing Cobas results, but is in agreement with previous data [31]. HPV 16, 52, 56 and 58 have been identified as the most prevalent genotypes in high-grade VAIN [30], while many LR and HR genotypes have been linked to the development of low-grade VAIN. HPV type distribution is even more heterogeneous in case of coexisting cervical lesions, although a recent study by Zhang et al. showed that different HPV genotypes are independent causative agents of coexisting CIN and VAIN [32]. Furthermore, specific HPV genotypes, particularly HPV 16, have been related to a greater risk of VAIN persistence, progression and recurrence [28]. Therefore, HPV genotyping could be a useful tool for risk-stratification of patients affected by VAIN.

Regarding the diagnostic accuracy of colposcopy in relation to the histological grade of VAIN, our study confirmed that colposcopic grade G2 and vascularity were significantly associated with VAIN3, including VAIN3 with stromal microinvasion. These associations have been widely demonstrated by other authors [17,19,20], that already observed specific abnormal colposcopic findings, such as grade 2 and vascular punctuation, more commonly in women diagnosed with VAIN3 rather than with VAIN2 or VAIN1. Interestingly, our study included a larger proportion of women diagnosed with VAIN3, when compared to previous studies, and also considered microinvasive VAIN3 as a separate category [17,19]. Moreover, in our cohort we found colposcopic grade 2 in 46.6% of women, that is a prevalence roughly double that previously reported by Sopracordevole et al. (22.7%) [19].

The correlation between vascularity and high-grade VAIN has been already explained by Boonlikit et al., as a consequence of the lack of vascular structure in very mature squamous vaginal epithelium. Thus, vascular patterns appear later, as distinct from the cervical dysplastic process, in which vascular punctuation appears early due to the immature squamous metaplasia of the transformation zone [17].

Conversely, our results showed a significant association between papillary lesions and VAIN3, and in particular, microinvasive VAIN3. This is totally different from the evidence of other authors who detected micropapillary patterns more frequently in women affected by low-grade VAIN [19,20,22]. The exact meaning of this colposcopic feature is still unclear and lacking. According to our experience, if the papillary pattern is caused by a persistent HPV infection, as already suggested [19,20,22], it should be considered as the expression of dysplastic progression towards high-grade VAIN. Another possible explanation of this relevant difference could derive from the absence of this specific feature in the 2011 IFCPC colposcopic terminology [21]. In fact, a recent study found poor concordance between the diagnosis based on the 2011 IFCPC colposcopic classification and vaginal histology for the high-grade VAIN category (only 35.71%), with a substantial false negative rate (42.86%), thus suggesting that the IFCPC nomenclature should be improved and better standardized for vaginal lesions [22].

Notably, we did not find any significant difference among VAIN grade groups regarding lesions number, as against that sustained by Zhou et al. [20]. Even vaginal localization was not significantly associated with the histological grade of VAIN. Nevertheless, the prevalence of VAIN in the vaginal vault (38.7%) and in the upper third of vaginal walls (45.1%) was much higher than in the lower two thirds (16.2%), in agreement with previously reported frequencies [3,22,33,34].

To the best of our knowledge, this is the first study to investigate and portray colposcopic characteristics of not only low- and high-grade VAIN, but also VAIN3 with stromal microinvasion, that should always be correctly identified before choosing a therapeutic approach.

The main strengths of the study are related to the higher proportion of high-grade VAIN in our population and the data homogeneity, because all colposcopies were performed at a referral oncologic center, only by trained colposcopists, with particular expertise in the diagnosis and treatment of vaginal lesions. On the contrary, limits of the study include selection bias related to the single center retrospective design of the study and the amount of missing data in old medical reports.

A better defined and standardized application of the 2011 ICFPC colposcopic terminology for vaginal lesions could be useful for correct diagnosis and management of VAIN. Indeed, identifying risk factors and colposcopic patterns predictive for high-grade VAIN would help the colposcopist to sample the area most likely to contain VAIN3 or stromal invasion, especially in large and multifocal lesions, which could simultaneously hold different grades of VAIN.

Appropriate diagnosis of VAIN3, with or without stromal microinvasion, is mandatory to choose the optimal management, which still remains challenging and controversial for high-grade VAIN. Several therapeutic regimens, including conservative surveillance, ablative procedures and surgical excisions, have been proposed over time, due to a high recurrence rate of VAIN2-3 despite the type of treatment [4,35–38]. Hence, proper and accurate diagnosis could allow for more personalized risk-based management, based on risk factors, colposcopic patterns and the histologic grade of VAIN.
