Analysis of clinical characteristics and risk factors of cervical intraepithelial neoplasia complicated with vaginal intraepithelial neoplasia

doi:10.21203/rs.3.rs-3444264/v1

Objective: This study aims to explore the clinical characteristics and risk factors of cervical intraepithelial neoplasia (CIN) complicated with vaginal intraepithelial neoplasia (VaIN)

Methods: A total of 229 patients diagnosed with CIN complicated with VAIN, who received treatment at the Gynecology Department of the Third Xiangya Hospital of Central South University between January 2013 and January 2023, were included in this study. Additionally, 458 CIN patients treated during the same period were randomly selected as a control group. The clinical characteristics of both groups were compared and analyzed to identify risk factors for CIN complicated with VAIN.

Results: The mean age of patients in the CIN complicated with VAIN group was 49.84±14.03 years, with 55.5% being postmenopausal. In the CIN group, the mean age was 45.79±11.41 years, with 31.4% being postmenopausal. The difference between the two groups was statistically significant (p < 0.05). Among the CIN complicated with VAIN group, 97.4% of patients had HPV infection, with 46.7% being infected with multiple HPVs and 20.1% being infected with three or more HPVs. The infection rate was significantly higher than that in the CIN group (91.0%, 29.5%, 10.5%). There was a significant positive correlation between the grade of CIN and VAIN (Kappa=0.274, p < 0.05), indicating that as the grade of cervical lesions increased, so did the degree of vaginal lesions. Multifactor analysis revealed that menopause (OR=2.522), multiple HPV infections (OR=1.491), and three or more categories of HPV infections (OR=1.529) were independent risk factors for CIN complicated with VAIN (p < 0.05).

Conclusion: In order to provide early intervention and avoid missed diagnosis and treatment, it is important to pay attention to vaginal examination during colposcopy for postmenopausal and CIN patients with multiple HPV infections. This will help in timely detection of vaginal lesions.

Vaginal intraepithelial neoplasia

Cervical intraepithelial neoplasia

Human papillomavirus

Risk factors

it is important to pay attention to vaginal examination during colposcopy for postmenopausal and CIN patients with multiple HPV infections. This will help in timely detection of vaginal lesions.

Cervical intraepithelial neoplasia (CIN) is closely associated with the development of cervical cancer. High-grade squamous intraepithelial neoplasia (HSIL) is a precancerous lesion that can progress to cervical cancer. Thanks to advancements in cervical cancer screening, timely detection and treatment of patients with cervical precancerous lesions have resulted in favorable prognoses. Vaginal intraepithelial neoplasia (VAIN) is a rare vaginal lesion caused by human papillomavirus (HPV) infection^[1]. Its incidence is lower than CIN and accounts for only 0.4% of vaginal lesions in the lower reproductive tract^[2]. Although the incidence rate is low, VAIN2/3, as a precancerous lesion of vaginal cancer, carries the risk of progression. VAIN often coexists with CIN, but due to its low occurrence, colposcopy doctors tend to focus more on cervical lesions and may overlook the observation of the vagina, particularly the fornix. This can lead to missed or delayed diagnosis and treatment in some patients^[3].

1.1 Study Population: A total of 4,766 patients with cervical lesions treated at Xiangya Third Hospital of Central South University from January 2013 to January 2023 were included in this study. Among them, 229 cases had cervical intraepithelial neoplasia (CIN) complicated with vaginal intraepithelial neoplasia (VAIN). A control group of 458 CIN patients was randomly selected in a 1:2 ratio. The inclusion criteria for this study were patients who underwent cervical or vaginal biopsy under vaginal colposcopy examination at our hospital and were pathologically diagnosed with CIN or VAIN. The exclusion criteria included patients who had previous uterine surgery, gynecological malignancies, previous vaginal surgery, previous laser therapy, or those who refused cervical or vaginal biopsy.

1.2 Study Methods

1.2.1 Data Collection Clinical data of the enrolled patients were collected, including age, clinical manifestations, menopausal status, time since menopause, obstetric history, thinprep cytologic test (TCT) results, HPV types (single type infection, multiple infections, triple or more infections), CIN and VAIN grades.

1.2.2 TCT Cervical canal and cervical vaginal squamous cells were collected, and after sample preparation, the slides were stained using the Papanicolaou staining method and diagnosed by pathologists. The TCT results were interpreted according to the 2001 TBS (the Bethesda System) reporting system, including: normal (NILM), atypical squamous cells of undetermined significance (ASCUS), atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H), low-grade squamous intraepithelial lesion (LSIL), and high-grade squamous intraepithelial lesion (HSIL).

1.2.3 HPV Typing Detection was performed using the GenPlex microfluidic automatic nucleic acid detection technology. The sample extraction was conducted using the HPV test kit (biochip method), and the nucleic acid chip detection instrument was utilized to detect 24 types of HPV. These include 18 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, and 83) and 6 low-risk types (HPV6, 11, 42, 43, 44, and 81).

1.2.4Histopathological Examination Two attending physicians conducted colposcopy using an aEDNA C6 stereomicroscope (EDAN Instruments, Inc., Wuhan, China). Both doctors had at least 3 years of colposcopy experience and conducted at least 400 examinations annually. This study followed a standard colposcopy protocol, which included acetic acid testing and Lugol's iodine testing^[4]. The classification and preliminary diagnosis of colposcopy abnormalities were described according to the 2003 International Federation for Cervical Pathology and Colposcopy (IFCPC) terminology ^[5]. The cervical and vaginal biopsies of the most suspicious lesion identified during colposcopy were analyzed by two pathologists (4 cervical slides and 1-2 vaginal slides), and the pathological diagnosis was based on the 2020 World Health Organization (WHO) classification of female genital tract tumors ^[6]. The pathological diagnosis was graded based on the worst pathological result, with CIN divided into two groups: LSIL (CIN1) and HSIL (CIN2-3). Similarly, VAIN was histologically divided into two groups: LSIL/VAIN1 and HSIL/VAIN2~3.

1.3 Statistical Analysis Statistical analysis was performed using SPSS 25.0 software. Quantitative data that followed a normal distribution were expressed as means ± standard deviations and tested using a t-test. Quantitative data that did not follow a normal distribution were expressed as medians (P) and compared using the Mann-Whitney U test. Count data were expressed as numbers and percentages, and intergroup comparisons were conducted using the chi-square test. Concordance was tested using the Kappa test. A P-value <0.05 was considered statistically significant.

2.1 Basic information of patients

In the CIN group, the average age was 45.79 ± 11.41 years old. In the CIN complicated with VAIN group, the average age was 49.84 ± 14.03 years old, which was significantly higher than that of the CIN group (P < 0.05) (Table 1).

2.2 Analysis of clinical characteristics of the two groups

Postmenopausal patients accounted for 55.5% (127/229) in the CIN complicated with VAIN group, while only 31.4% (144/458) in the CIN group, showing a significant difference between the two groups (P < 0.05).The number of pregnancies was 3 (2, 4) times in the CIN complicated with VAIN group and 3 (2, 5) times in the CIN group, with no significant difference between the two groups. Similarly, the number of births and abortions showed no significant difference between the two groups.In the CIN complicated with VAIN group, 31.9% (73/229) of patients had symptoms of contact bleeding or abnormal vaginal bleeding/discharge, while others were discovered during clinical examination or colposcopy. In the CIN group, 27.3% (125/458) of patients had corresponding symptoms.Among the patients in the CIN complicated with VAIN group, 97.4% (223/229) had HPV infection, with a multiple HPV infection rate of 46.7% (107/229). Additionally, 20.1% (46/229) of patients were infected with three or more types of HPV. In the CIN group, 91.0% (417/458) of patients had HPV infection, with a multiple HPV infection rate of 29.5% (135/458). Furthermore, 10.5% (48/458) of patients were infected with three or more types of HPV. The incidence of HPV infection and multiple HPV infections was higher in patients with CIN complicated with VAIN compared to patients with only CIN, with significant differences (P < 0.05).In the analysis of HPV genotypes in two groups of patients, it was found that both groups had the highest prevalence of HPV16 infection. The infection rate was 55.9% (128/229) in the group with CIN complicated with VAIN and 50.0% (229/458) in the CIN group. The HPV16 infection rate was significantly higher in the CIN complicated with VAIN group compared to the CIN group (P < 0.05) (Table 1).

Logistic regression multivariate analysis revealed that menopause, multiple HPV infections, and three or more categories of HPV infections were identified as risk factors for concurrent VAIN in CIN (OR = 1.529, P0.05)(Table 2).

Among patients with multiple HPV infections, 56.9% (62/109) in the concurrent VAIN group were infected with HPV16 type in addition to other types, while it was 59.9% (91/152) in the CIN group, with no significant difference between the two groups (p > 0.05) (Table 3).

2.3 TCT results analysis

TCT results analysis for the two groups of patients showed that 76.9% (176/229) of patients in the concurrent VAIN group had abnormal cytology results, while it was 73.1% (335/458) in the CIN group, with no significant difference between the two groups (p > 0.05) (Table 4).

2.4 Analysis of CIN and VAIN levels

Analysis of CIN and VAIN grades in the concurrent VAIN group revealed a positive correlation between VAIN grade and CIN grade when using the traditional classification of grades 1–3 (Kappa = 0.274, P < 0.05). As the cervical lesion level increased, the vaginal lesion level also increased. However, there was no correlation between CIN and VAIN grades when using the LSIL/HSIL classification method (Table 5.1, 5.2).

2.5 VAIN lesion site analysis

In the concurrent VAIN group, 216 cases had lesions located in the upper half of the vagina, among which 89 cases were located in the fornix, 2 cases were located in the lower half of the vagina, 2 cases had lesions spread throughout the entire vagina, and 9 cases had unknown lesion locations.

With the promotion of cervical cancer screening, there has been an increase in the detection and treatment of cervical precancerous lesions. The incidence of CIN (cervical intraepithelial neoplasia) is significantly higher than that of VAIN (vaginal intraepithelial neoplasia) ^[7]. In recent years, advancements in cytology, HPV testing, and colposcopy have led to an upward trend in the detection rate of VAIN ^[8]. However, diagnosing VAIN can be challenging due to difficulties in visual examination caused by the upper vagina or vaginal folds. Additionally, VAIN often lacks specific symptoms, leading to missed diagnoses. In this study, only 31.9% of CIN patients with VAIN exhibited related symptoms such as intrauterine bleeding, abnormal vaginal bleeding, and external genital/vaginal itching.

A study by Lamos et al ^[9] found that VAIN primarily occurs in the upper one-third of the vagina, particularly in the fornix. However, our research results differ slightly from theirs. We observed that in the CIN complicated with VAIN group, vaginal lesions were mainly located in the upper vaginal segment (94.3%), with only 38.9% in the fornix. The lesions were primarily found in the upper vaginal segment outside the fornix. Our study also revealed a correlation between the severity of cervical lesions and vaginal lesions. As the grading of CIN increases, the grading of VAIN also increases (Kappa = 0.225, P < 0.05), which aligns with existing literature ^[10]. Therefore, when examining cervical lesions under colposcopy, especially in cases of high-grade cervical lesions, special attention should be given to the upper vaginal segment and vaginal dome. If necessary, multiple vaginal biopsies should be performed to rule out vaginal lesions.

According to literature reports, age has been identified as an independent risk factor for combined CIN and VAIN ^[11]. A study conducted by Zhang et al^[12] further suggests that menopausal status is also an independent risk factor for combined VAIN and CIN, with VAIN levels increasing as age advances. However, the research conducted by Yu et al ^[13] did not conclude that the risk of high-grade vaginal lesions increases with age. Instead, their study found that menopausal status is a risk factor for CIN complicated with VAIN (OR = 2.522), and it is not associated with patient age or menopausal time. Postmenopausal women have a higher risk of developing VAIN compared to women of childbearing age. This can be attributed to the decrease in estrogen levels, thinning of the vaginal epithelium, and changes in the microbiota, which make them more vulnerable to HPV infection. Furthermore, their ability to clear the virus is weakened, leading to persistent infection and the development of vaginal lesions.

The occurrence of VAIN is closely related to HPV infection. The literature reports a range of HPV infection rates in VAIN, varying from 85–98%. Among the different types of HPV infections in VAIN, HPV16 is the most common ^[14–15]. However, there are variations in the results, which could be attributed to differences in sample size or testing methods among different ethnic groups. Currently, the exact mechanism by how HPV leads to VAIN is not fully understood. Zhang et al. ^[16] reported that various types of HPV virus infections can cause VAIN, and multiple HPV infections are considered risk factors for VAIN. Previous studies have shown that the types of HPV vary at different levels of VAIN, with a significant increase in HPV16 infection rate as the disease level increases ^[17]. Another study suggests that HPV-31 positivity and persistent HPV infection are associated with the occurrence of VAIN ^[18]. This study also found that multiple HPV infections, especially three or more, are risk factors for CIN complicated with VAIN. Among the patients with CIN complicated with VAIN, 46.7% had multiple HPV infections, 20.1% had three or more HPV infections. In comparison, among the patients with CIN alone, 29.5% had multiple HPV infections, and 10.5% had three or more types of HPV infections. The rate of multiple HPV infection was significantly higher in patients with CIN combined with VAIN, with a statistically significant difference (P < 0.05). However, contrary to other research findings, this study did not find that CIN patients infected with HPV16 (whether single infection or multiple infections) are more likely to develop VAIN.

TCT has been widely used for screening cervical lesions and the correlation between cytological screening abnormalities and CIN has been extensively studied. However, there is limited research on cytology in VAIN screening. Ao et al. ^[17] found that 68.9% of patients with multiple lesions in the lower reproductive tract had abnormal cytological results. ASC-H and HSIL were more common in VAIN3, while ASCUS and LSIL were more common in VAIN1 and VAIN2. Liquid-based cytology plays a crucial role in screening for VAIN. In this study, 76.9% of CIN patients with VAIN had abnormal cytological results, slightly higher than the 73.1% in the CIN group, but the difference was not statistically significant. Therefore, cytological abnormalities can only indicate the presence of lower reproductive tract lesions in patients, but cannot indicate the presence of multiple lesions in the lower reproductive tract.

VAIN complicated with CIN often exhibits a low spontaneous regression rate ^[19], making it prone to vaginal lesion progression. Therefore, it is important to thoroughly evaluate whether patients with CIN, especially those who are postmenopausal and have multiple HPV infections, have vaginal lesions under colposcopy, with special attention to the vaginal dome and upper segment. If necessary, biopsy and pathological examination of the vaginal wall should be performed to promptly detect vaginal lesions and provide early intervention, thus avoiding missed diagnosis and treatment. This study is a retrospective study and did not include factors such as HPV viral load and HPV infection time, which are important limitations. Future studies should aim to expand the sample size and conduct more in-depth research.

Author contributions statement: H.S.L. assembled the data and drafted the manuscript. J.F.J conceived the idea for the study, carried out the analyses and reviewed and revised the manuscript.

Conflict of interest statement:None declared. Completed disclosure of interest forms are available to view online as supporting information.

Financial Support: None

Data availability statement:The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICAL APPROVAL

This study was approved by The Third Xiangya Hospital of Central South University Review Board: 2023-R23038 on June 16, 2023.

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Table 1.Clinical characteristics of the two groups

Clinical characteristics	CIN complicated with VAIN group	CIN group	T/Z/Chi-Square value	p
Age（y）	49.84±14.03	45.79±11.40	4.054	＜0.001
G	3（2,4）	3（2,5）	2.279	0.023
P	2（1,2）	2（1,2）	0.911	0.362
A	1（0,2）	1（0,3）	3.920	＜0.001
menopausal Yes No	127 102	144 314	36.868	＜0.001
HPV infection Yes No	223 6	417 41	9.604	0.002
Multiple HPV infections Yes No Uninfected	107 116 6	135 282 41	24.982	＜0.001
three or more categories of HPV infections Yes No Uninfected	46 177 6	48 369 41	19.451	＜0.001
HPV16 infection Yes No No HPV infection	128 95 6	229 188 41	9.975	0.007
symptom Vaginal bleeding Intrasexual bleeding Vaginal discharge Vulva/vaginal itching Asymptomatic	24 33 10 6 156	38 60 15 12 333	1.951	0.745

Table 2. Logistic regression multivariate analysis

	B	S.E,	Wals	Sig.	Exp (B)	95%CI
menopausal	.925	.170	29.606	.000	2.522	1.807	3.519
Multiple HPV infections	.399	.195	4.204	.040	1.491	1.018	2.184
three or more categories of HPV infections	.425	.169	6.341	.012	1.529	1.099	2.129

Table 3. postmenopausal time and multiple HPV infection types

CIN complicated with VAIN group

CIN group

T/Chi-Square value

p

postmenopausal time（year）

9.84±6.51

8.50±7.05

1.615

0.107

Multiple HPV infections

Contains HPV16 infection

Yes

No

62

47

91

61

0.234

0.629

Table 4. TCT results analysis

TCT

CIN complicated with VAIN group

CIN group

T/Chi-Square value

p

NILM

Abnormal

27

176

80

335

4.029

0.133

Unknown

26

43

Table 5.1 Analysis of CIN and VAIN levels

CIN	VAIN				Kappa	p
	VAIN1	VAIN2	VAIN3
Cin1	20	28		10	0.274	＜0.001
Cin2	25	27		6
Cin3	28	22		63

Table 5.2 Analysis of CIN and VAIN levels

CIN	VAIN		Kappa	p
	LSIL	HSIL
LSIL	20	38	0.026	0.690
HSIL	53	118

Analysis of clinical characteristics and risk factors of cervical intraepithelial neoplasia complicated with vaginal intraepithelial neoplasia

Status:

Version 1

Abstract

Take-home message

Introduction

Materials and Methods

1.2 Study Methods

Results

2.1 Basic information of patients

2.2 Analysis of clinical characteristics of the two groups

2.3 TCT results analysis

2.4 Analysis of CIN and VAIN levels

2.5 VAIN lesion site analysis

Discussion

Declarations

References

Tables

Status:

Version 1