Title: Clinical Characteristics, HPV Involvement, and Demographic Risk Factors in Women
with Cervical Intraepithelial Neoplasia Complicated by Vaginal Intraepithelial Neoplasia
Version: 0 Date: 28 Jan 2024
Reviewer:  Sveinung Wergeland Sørbye
Reviewer's report:
Xu et al. have conducted a study to explore the clinical characteristics and risk factors associated
with cervical intraepithelial neoplasia (CIN) when complicated with vaginal intraepithelial
neoplasia (VAIN). The research involved a comparative analysis of clinical data from 212 CIN
patients, including 50 cases also presenting with VAIN. They found a notable age difference
between patients with combined CIN and VAIN (median age of 57) and those with CIN
only (median age of 41), with age proving to be an independent risk factor for CIN with
VAIN. Furthermore, a higher prevalence of human papillomavirus (HPV) infection, particularly
multiple HPV infections, was observed in the combined CIN and VAIN group. Educational
levels also differed significantly, with more patients below the undergraduate level in the
combined group. The study highlights the importance of vigilant vaginal examinations during
colposcopies in older patients, especially those with multiple HPV infections and cytological
abnormalities, to ensure early detection and treatment of vaginal lesions. This approach is critical
for avoiding missed diagnoses and improving patient outcomes.
The claims are properly placed in the context of the previous literature. The experimental data
support the claims. The manuscript is written clearly enough that most of it is understandable to
non-specialists. The authors have provided adequate proof for their claims, without overselling
them. The authors have treated the previous literature fairly. The paper offers enough details of
methodology so that the experiments could be reproduced.
Comments
1. To ensure accurate and efficient peer reviews and simplify the post-review editing procedures,
it is recommended to use a line numbering system for all submitted manuscripts.
2. As a non-native English speaker, I have suggested some reformulations to improve the
language in the manuscript. However, I recommend considering a review by a native English
speaker to further enhance sentence construction, readability, coherence, and accuracy, ensuring
a polished final presentation.
3.  In the discussion, the authors mention the existence of over 130 HPV types. However, it
is important to note that current research has identified more than 200 distinct HPV types.
This discrepancy should be addressed to ensure the accuracy and relevance of the information
presented in the manuscript.
https://www.cdc.gov/vaccines/pubs/pinkbook/hpv.html
https://www.cancer.gov/about-cancer/causes-prevention/risk/infectious-agents/hpv-and-cancer
https://www.hpvcenter.se/human_reference_clones/
Burd EM. Human papillomavirus and cervical cancer. Clin Microbiol Rev. 2003 Jan;16(1):1-17.
doi: 10.1128/CMR.16.1.1-17.2003. PMID: 12525422; PMCID: PMC145302.
Minor revisions
Title,  "Clinical Characteristics, HPV Involvement, and Demographic Risk Factors in Women
with Cervical Intraepithelial Neoplasia Complicated by Vaginal Intraepithelial Neoplasia"
Abstract, "Abstract
Purpose:
This study aimed to explore the clinical characteristics and risk factors associated with cervical
intraepithelial neoplasia (CIN) when coexisting with vaginal intraepithelial neoplasia (VAIN).
Methods:
We analyzed the clinical data of 212 patients diagnosed with CIN, including 50 patients
with concurrent VAIN. The groups were compared to identify distinct clinical features and
independent risk factors for the co-occurrence of CIN and VAIN, using logistic regression
analysis.
Results:
Patients with both CIN and VAIN had a median age of 57, significantly older than the 41-year
median age of patients with CIN only (P<0.05). A higher prevalence of HPV infection (98.0%)
was observed in the CIN and VAIN group, with a notable rate of multiple HPV infections
(67.3%) compared to the CIN-only group (P<0.05). Educational levels were significantly
lower in the combined CIN and VAIN group (P<0.05). HPV16, 33, and 52 were identified
as significant types for single and multiple infections. Multivariate analysis confirmed age as
an independent risk factor for CIN with VAIN (P<0.05). VAIN3 patients were more likely to
exhibit HSIL and ASC-H, whereas VAIN1 cases tended to correspond with ASCUS and LSIL
diagnoses.
Conclusion:
The co-occurrence of CIN and VAIN is significantly influenced by patient age and educational
level. The findings advocate for more diligent vaginal examination during colposcopy in older
patients, particularly those with multiple HPV infections and cytological abnormalities, to
enhance the early detection of vaginal lesions and prevent missed diagnoses and treatments.
Additionally, the high prevalence of HPV infection, especially with certain types, underscores
the importance of HPV monitoring in this patient population."
Introduction, "Vaginal intraepithelial neoplasia (VAIN) represents a precancerous condition
of the vagina, characterized by epithelial dysplasia and carcinoma in situ without invasion
beyond the basal membrane. This condition, largely attributed to human papillomavirus
(HPV) infection, is classified into low-grade and high-grade intraepithelial lesions based
on the differentiation potential of squamous cells and associated clinical risks. Low-grade
lesions, featuring mature and differentiable squamous cells, pose a lesser risk of recurrence or
progression to invasive cancer. In contrast, high-grade lesions are marked by the proliferation
of immature squamous epithelial cells post-HPV infection, which, if untreated, are more likely
to recur or develop into invasive cancer.
VAIN predominantly affects women aged 35 to 55. Although it is a relatively rare condition
with an incidence of only 0.2 to 0.3 per 100,000, it is important to note that the number of
VAIN cases represents approximately 0.6% to 1% of the total number of cervical intraepithelial
neoplasia (CIN) cases. This comparison highlights the lesser prevalence of VAIN compared
to CIN, yet underscores its significance as part of the spectrum of female lower reproductive
tract intraepithelial neoplasias. The low incidence of VAIN could be attributed to its often
asymptomatic nature and reliance on singular diagnostic methods like the ThinPrep cytologic
test (TCT) or colposcopic biopsies.
Recent studies, however, indicate a rising incidence of VAIN, particularly among patients with
cervical lesions. This trend may be linked to high-risk HPV infections or persistent infections,
alterations in the vaginal microenvironment, and the severity of concurrent cervical lesions.
Moreover, the treatment approach for cervical lesions appears to influence VAIN incidence,
with a noted increase in VAIN cases in the vaginal stump post-hysterectomy.
Despite these insights, VAIN often goes undetected due to its lack of specific symptoms, the
oversight of the vaginal site in colposcopic diagnoses, limited disease awareness, and small-
scale studies. This situation underscores the need for heightened vigilance and comprehensive
examination strategies to improve VAIN detection and management."
Materials and methods, "Study Population:
This study involved 212 patients diagnosed with cervical intraepithelial neoplasia (CIN) from
May 2019 to May 2023. Among these, 50 patients were diagnosed with CIN complicated by
vaginal intraepithelial neoplasia (VAIN), while the remaining 162 had CIN only.
Inclusion Criteria:
Patients were included if they underwent a cervical or vaginal wall biopsy via colposcopy at our
hospital, with subsequent histopathological confirmation of CIN or VAIN.
Exclusion Criteria:
Patients were excluded if they had a current or past history of any malignant tumor, uterine
prolapse, endometrial atypical hyperplasia, or other significant gynecological diseases.
Data Collection:
Clinical data were collected for all enrolled patients. This included age, clinical symptoms,
menopausal status, smoking history, marital history, results from the ThinPrep cytologic test
(TCT), and the grading of CIN and VAIN."
Materials and methods, TCT, "The ThinPrep Cytology Test (TCT)
Procedure:
For the TCT, exfoliated cells were collected from the cervical canal and cervicovaginal area.
Following sample preparation, Pap staining was applied, and a pathologist performed the
diagnostic evaluation.
Classification:
The results were categorized according to the 2001 Bethesda classification of cervical cytology
(TBS) reporting system, also known as the Bethesda 3-tier system. The categories included:
1. Negative for intraepithelial lesion or malignancy (NILM)
2. Atypical squamous cell of undetermined significance (ASCUS)
3. Atypical squamous cells-cannot exclude HSIL (ASC-H)
4. Low-grade squamous intraepithelial lesion (LSIL)
5. High-grade squamous intraepithelial lesion (HSIL)"
Materials and methods, HPV detection, "HPV Detection
Methodology:
HPV detection was performed using microfluidic automatic nucleic acid detection technology.
Specimen extraction was facilitated by an HPV detection kit, and a nucleic acid chip detector
was employed for identifying HPV types.
HPV Types Detected:
The detection covered a range of HPV types, categorized into high-risk and low-risk groups:
High-risk types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, and 83.
Low-risk types: 6, 11, 42, 43, 44, and 81."
Materials and methods, Histopathological examination,  "Histopathological Examination
Procedure:
The diagnosis of cervical and vaginal lesions was established based on the histopathological
findings from cervical and vaginal wall biopsies obtained during colposcopy.
Classification of CIN (Cervical Intraepithelial Neoplasia):
According to the 2014 WHO classification of female genital organ tumors, CIN is categorized
into:
LSIL: Includes CIN1 and immunochemically P16-negative CIN2.
HSIL: Comprises CIN3 and P16-positive CIN2.
Classification of VAIN (Vaginal Intraepithelial Neoplasia):
Histologically, VAIN is divided into:
LSIL (VAIN1)
HSIL (VAIN2-3)"
Materials and methods, Statistical analysis, "Statistical Analysis
Software Used:
The data were analyzed using SPSS version 19.0 statistical software.
Analysis Methods:
Quantitative data that conformed to a normal distribution were expressed as mean ± standard
deviation (x±s) and analyzed using the t-test.
For comparisons among groups, the chi-square test (#2 test) was utilized.
Significance Threshold:
A p-value of less than 0.05 (P<0.05) was considered to indicate statistical significance."
Results, General information, "Results
General Information:
Table 1 presents a comparison of the general information and clinical data between the CIN
group and the CIN combined with VAIN group. This comparison includes variables such as
age, smoking status, alcohol consumption, HPV status, age at first sexual intercourse, number
of sexual partners, number of pregnancies, and educational level.
Significant Findings:
Notably, significant differences were observed between the two groups in terms of age, HPV
status, and educational level (P<0.05)."
Material and methods, "HPV Types:
HPV16 infection was the most prevalent in both groups. In the CIN combined with VAIN group,
the most common infections, in order of frequency, were HPV16 (22 cases), followed by HPV18,
HPV52, HPV33, and HPV56. In the CIN group, HPV16 led with 71 cases, followed by HPV18,
HPV56, HPV33, and HPV52. Notably, the rates of HPV16, HPV33, and HPV52 infection were
significantly lower in the CIN group compared to the CIN with VAIN group (P < 0.05, as detailed
in Table 2).
TCT Results:
Among patients with VAIN3, all TCT results were abnormal. Specifically, 81.8% of these
patients exhibited either HSIL or ASC-H, a proportion that was significantly different from
patients with VAIN1. Further details and comprehensive data can be found in Table 3."
Discussion, first paragraph, "With the widespread implementation of cervical cancer screening
programs, a significant number of precancerous cervical lesions are being detected and
effectively treated, leading to favorable prognoses. VAIN, characterized histologically by
dysplasia of the vaginal epithelium without stromal invasion, is one such lesion that can be
reliably diagnosed. Although VAIN and CIN share similar risk factors, the incidence of CIN
is notably higher than that of VAIN. However, in recent years, advancements in cytology and
HPV detection, combined with increased vigilance by colposcopists towards vaginal lesions,
have led to a rise in the detection rates of VAIN."
Discussion, second paragraph, "VAIN and CIN represent precancerous lesions of the vaginal
and cervical epithelia, respectively. Given that these tissues are homologous, some studies
suggest that VAIN may be an extension of CIN. Research on patients with CIN and cervical
cancer indicates a significant overlap, with up to 14.2% (99 out of 699 patients) presenting with
concurrent VAIN. Furthermore, data reveal that 72.7% of these patients have VAIN, and those
with CIN or cervical cancer are 82 times more likely to develop VAIN compared to those without
these conditions.
The severity of VAIN is closely related to the level of CIN or cervical cancer. Generally, a
higher grade of CIN correlates with a higher incidence and severity of VAIN. Interestingly, some
studies have found no significant difference in the age or timing of VAIN development post-
surgery in patients with high-grade CIN or cervical cancer, suggesting that the occurrence of
VAIN post-surgery is not age-dependent. Moreover, there appears to be a correlation between
the grades of VAIN and CIN. Specifically, when the incidence of CIN I and CIN III is higher
than that of CIN II, a similar pattern is observed in VAIN, with the number of VAIN I and VAIN
III cases surpassing those of VAIN II. This suggests a parallelism in the distribution of lesion
severity between the cervical and vaginal epithelia."
Discussion, third paragraph, "Over 200 HPV types have been identified, with more than 40
known to infect the cervix. HPV types vary in terms of the tissue they infect and their associated
risk. For skin infections, HPV types can be categorized into low-risk (e.g., 1, 2, 3, 4, 7, 10, 12,
15) and high-risk types (e.g., 5, 8, 14, 17, 20, 36, 38). Similarly, for mucosal infections, low-
risk types include 6, 11, 32, 34, 40, 42, 43, 44, 53, 54, and high-risk types include 16, 18, 30,
31, 33, 35, 39.
Low-grade VAIN is commonly associated with both high-risk HPV subtypes (16, 18, 31, 33, 35,
45, 51, 52, 56) and low-risk types (6, 11, 42, 43, 44). In cases of high-risk HPV infection, there
is often co-infection with subtypes 16 and 18, accounting for more than 50% of these cases.
Furthermore, some studies indicate that vaginal lesions post-hysterectomy for high-grade CIN
are predominantly high-grade or invasive, with HPV binding sites in these vaginal or vulvar
tissues being consistent with those found in previous cervical lesions."
Discussion, last part,  "Implications and Future Directions:
Our study's findings emphasize the significant association between the co-occurrence of CIN
and VAIN with patient age and educational level. This underscores the critical need for thorough
vaginal examinations during colposcopy, especially in older patients or those with multiple HPV
infections and cytological abnormalities, to facilitate early detection and treatment of vaginal
lesions.
The current trends in bioinformatics and meta-analysis offer promising tools for future research
in CIN and VAIN. There is a notable gap in the application of these advanced analytical
methods in understanding the genetic and molecular aspects of these conditions. Future research
should focus on exploring gene expression levels, transcriptomics, proteomics, and genetic
polymorphisms associated with vaginal intraepithelial neoplasia. Such investigations could
provide valuable insights for developing more precise diagnostic and therapeutic strategies."
You should add a conclusion, "Conclusion:
This study contributes to the understanding of the complex relationship between CIN and VAIN,
particularly highlighting the role of HPV infection and its impact on these conditions. Our
analysis reveals:
1. A significant correlation between the incidence of VAIN and the presence and severity of CIN.
2. The pivotal role of HPV, especially high-risk types, in the development of both CIN and
VAIN.
3. The influence of age and educational level on the occurrence of VAIN in patients with CIN.
These findings advocate for enhanced vigilance in the detection of VAIN among CIN patients,
emphasizing the importance of patient demographics and HPV status in clinical evaluations.
The potential for future research, particularly utilizing bioinformatics and meta-analysis, is
immense and could substantially contribute to the fields of gynecological cancer prevention and
treatment."