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\begin{document}

\title{HPV prevalence and Detection Sensitivity in Vaginal Intraepithelial
Neoplasia: a hospital-based study}



\author[1]{Yusha Chen}%
\author[1]{Qiaoyun Chen}%
\author[1]{Huifeng Xue}%
\author[1]{Jinwen Zheng}%
\author[1]{Jiancui Chen}%
\author[1]{Xiangqin Zheng}%
\affil[1]{Fujian Provincial Maternity and Children's Hospital}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Vaginal intraepithelial neoplasia (VaIN) always diagnosed through
coloscopy accidentally when the cervical cancer screening was abnormal.
A precise estimate of the detection rate of cervical cancer screening
for VaIN is limited. This study to investigated the characteristics and
screening history of VaIN, and compared the sensitivity of cytology and
human papillomavirus tests on the cervix against vaginal and cervical
high-grade squamous intraepithelial lesion (HSIL) or cancer. A total of
1200 patients with definitive histopathologic diagnoses of VaIN were
included in this study. Among them, 22.5\% were diagnosed with VaIN2+,
and 50.4\% were concomitant with cervical lesions. The median age of
VaIN2+ patients was 41.5 years old , while VaIN1 reported a median age
of 53 years old , p<0.001. This study reported that VaIN was
significantly and positively correlated with cervical lesions (r=0.387).
The high-risk human papillomavirus (hr-HPV) detection rate was 88.5\%
(883/998) in VaIN and 95.4\% in VaIN2+. HPV 16 was the most prevalent
HPV type in VaIN2+, which accounted for 54.5\%, followed by HPV58
(17.0\%), HPV52 (14.8\%), HPV51 (11.4\%), and HPV18 (10.2\%). The
sensitivity of hr-HPV and cytology tests on the cervix for detecting
VaIN2+ was 95.0\% and 84.9\%, respectively. Both tests were not
significantly different from detecting CIN2+. When the cervical cancer
screening is abnormal and referring to colposcopy, acetic acid and
Lugol's iodine need to cover the whole vaginal mucosa as well as the
fornix, attention need to be paid for the abnormal images of vagina in
order to find VaIN.%
\end{abstract}%



\sloppy


\textbf{1. Introduction}

Vaginal intraepithelial neoplasia (VaIN) is an uncommon gynecologic
disease that accounts for only 1\% of all lower genital tract
intraepithelial neoplasia.\textsuperscript{1}Human papillomavirus (HPV)
infection is implicated as a causative agent of VaIN.\textsuperscript{2}
As with cervical lesions, persistent high-risk HPV (hr-HPV) infection
can induce the progression of VaIN to vaginal cancer. Other risk factors
include first sexual intercourse at age younger than 17, having more
than 5 sexual partners, immunosuppression, smoking, pelvic radiation
therapy, exposure to diethylstilbestrol in utero, and history of
cervical precancer or cancer.\textsuperscript{3,4}

Epidemiologic information about VaIN is limited, with only one
population-based study in the United States in 1977 estimating that VaIN
may occur in 0.2- 0.3 per 100,000 women.\textsuperscript{5} VaIN is
often discovered during colposcopy when cervical cancer screening is
abnormal. A missed diagnosis of VaIN may occur if colposcope
practitioners solely concentrate on examining the cervix and neglect to
observe the vagina for potential vaginal lesions. There are currently no
screening programs for VaIN except for women who have had a hysterectomy
for cervical precancerous lesions or cervical cancer. Thus, the true
incidence rates of VaIN may be higher than reported. According to the
2014 WHO classification of tumors of the female reproductive organs
(4\textsuperscript{th} Edition)\textsuperscript{6},VaIN was classified
into the low-grade squamous intraepithelial lesion (LSIL), which refers
to VaIN1, and high-grade squamous intraepithelial lesion (HSIL), which
includes VaIN2 and VaIN3. Compared with cervical intraepithelial
neoplasia (CIN), the possibility of VaIN progression to invasive vaginal
cancer is much lower, and most VaIN will regress. However, the risk of
progression to invasion vaginal cancer remains, especially for
HSIL.\textsuperscript{7-9} Hence, it is crucial to discover vaginal HSIL
and treat it promptly prevent vaginal cancer.

Currently, there is no FDA-approved hr-HPV test available for use in
vaginal cancer screening. In general population screening, excluding
women post-hysterectomy with vaginal stump, both cytological tests and
HPV test sample above the surface of cervix. Assessing the sensitivity
of cervical cancer screening against VaIN can be helpful in developing
an effective screening protocol and managing screen-detected
abnormalities. Additionally, in the post-HPV-vaccine era, knowing the
prevalence and distribution of HPV genotypes can aid in selecting the
most suitable vaccine among the available options to protect against
VaIN and vaginal cancer. Due to its rarity, the estimate of accuracy of
cervical cancer screening to detect VaIN2 is limited by the availability
of few studies.

The present study aims to analyze the clinical data of 1200 VaIN
patients at the largest cervical disease center in Fujian province,
China. The characteristics and screening history of VaIN were revealed,
the result may help better understand and detect VaIN.

2. Materials and Methods

\textbf{2.1 Study Population}

This study included patients with a definitive histopathologic diagnosis
of VaIN who were referred for colposcopy from February 2013 to November
2022 at the Cervical Disease Diagnosis and Treatment Health Center at
Fujian Maternity and Child Health Hospital dueto abnormal cytology
results and/or a positive hr-HPV test, and underwent biopsy from
suspicious regions. The pathological diagnosis of tissue and cytology
specimens were independently performed by two senior pathologists. The
interval between cytological tests/ HPV test and colposcopy were all
less than 3 months. Patients with a history of VaIN or cervical cancer
metastasis were excluded from the study. Clinical information, including
age, gravidity, parity, HPV genotypes, cytology results, cervical lesion
history, hysterectomy history cervical pathology and vaginal pathology,
was obtained from the medical records of the department.

This study was conducted in compliance with the Declaration of Helsinki
(as revised in 2013) and was endorsed by the Ethics Committee at Fujian
Maternity and Child Health Hospital, Affiliated Hospital of Fujian
Medical University (2023KY003). An exemption of informed consent was
obtained considering the retrospective nature of the study.

\textbf{2.2 Specimen Collection}

For patients with a cervix, a special TCT brush and/or HPV special brush
was used to scrape the cervical exfoliated cells at the junction of the
squamous column epithelium and the cervical canal. For patients who had
undergone total hysterectomy, brush-cell samples were from vaginal
stump. The cell samples were stored in the corresponding cell preserve
solution for cytology and HPV testing.

\textbf{2.3 Cytology Screening Testing}

Liquid-based method was used as cytology screening test. The results
were reported based on the 2001 Bethesda
system.\textsuperscript{10}Atypical squamous cells of unknown
significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL),
high-grade squamous intraepithelial lesion (HSIL), atypical squamous
cells, cannot rule out HSIL (ASC-H), atypical glandular cells (AGC),
endocervical adenocarcinoma in situ (AIS), squamous cell carcinoma (SCC)
and adenocarcinoma were defined as abnormal cytology results.

\textbf{2.4 HPV Testing}

Three types of HPV testing were performed: Hybrid capture-2 testing
(Qiagen, Hilden, Germany) which detecting HPV DNA of 13 high-risk
oncogenic types (i.e.,HPV-16, -18, -31, -33, -35, -39, -45, -51, -52,
-56, -58, -59, and -68), PCR-RDB HPV genotyping (Yaneng Biotech) which
detecting 18 types of hr-HPV (i.e.,HPV-16, 18, 31, 33, 35, 39, 45, 51,
52, 53, 56, 58, 59, 66, 68, 73, 82, and 83) and five types of low-risk
HPV (lr- HPV) (6, 11, 42, 43, and 81), and Aptima (Gen-Probe Inc., San
Diego, CA)~ which detecting HPV E6/E7 mRNA from 14 high-risk oncogenic
types (i.e., HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58,
-59, -66, and -68).

\textbf{2.5 Statistical Analysis}

Statistical analyses were performed using SPSS 23.0 (IBM Corp).
Two-tailed p-values less than 0.05 were considered statistically
significant. Categorical variables were presented as frequencies
(percentages) and analyzed using chi-squared tests. Numerical variables
were tested for normality using the Shapiro--Wilk normality test. Date
with normal distribution were expressed as mean \selectlanguage{ngerman}± standard deviation and
analyzed with t-test. Non-normal data distribution was expressed as
median (interquartile range) and analyzed with the Mann-Whitney test.
The correlation of VaIN and CIN was evaluated using Kendall's
correlation test.

\textbf{3. Results}

\textbf{3.1 Characteristics of study objects}

A total of 1200 patients who met the inclusion and exclusion criteria
and were included in the study. The characteristics of patients are
presented in Table 1. The overall median age was 44.17 years old from a
range between 17 and 90 years old. Of the total, 930 (77.5\%) patients
had VaIN 1, 194 (16.2\%) patients had VaIN 2, 67 (5.5\%) patients had
VaIN3 and 9 (0.8\%) patients had vaginal cancer.

{[}insert Table 1{]}

\textbf{3.2 Clinical characteristics comparison between VaIN1 and
VaIN2+}

The median age of VaIN2+ patients was 53 years (range: 42-59.25
years)which was older than that of VaIN1 patients with a median age of
41.5 years (range: 32-52 years) (p\selectlanguage{english}<0.001). Moreover, VaIN2+ patients
were more likely to have concomitant cervical lesions (67.7\% vs.
46.2\%, p\selectlanguage{english}<0.001), higher gravidity (p=0.021) and parity (p\selectlanguage{english}<0.001)
compared to VaIN1 patients. There were also more post-menopausal women
in VaIN2+ group the VaIN1 group (52.3\% vs. 24.8\%, p\selectlanguage{english}<0.001). The
hr-HPV positive rate among VaIN2+ patients was higher than VaIN1
patients (95.4\% vs.86.8\%, p\selectlanguage{english}<0.001), with HPV16/18 detected more
frequently in VaIN2+ patients(62.2\%) than in VaIN1 patients (27.9\%)
(p\selectlanguage{english}<0.001).There was no statistically significant difference in single
and multiple hr-HPV infections between VaIN1 and VaIN2+ patients. VaIN1
group was more likely to have a history of low-grade cervical
intraepithelial neoplasia than VaIN2 group (6.9\% vs. 3.0\%, p\selectlanguage{english}<0.001),
while VaIN2 group was more likely to have a history of high-grade
cervical intraepithelial neoplasia than VaIN1 group (29.3\% vs. 14.4\%,
p\selectlanguage{english}<0.001). Furthermore, ASCUS and LSIL were more frequantly detected in
VaIN1 patients, while
ASC-H\textbackslash{}HSIL\textbackslash{}SCC\textbackslash{}AGC were
more likely to be detected in VaIN2 patients, the difference is
statistically significant (p\selectlanguage{english}<0.001) (Table 2). Additionally, the
analysis showed a weak correlation between vaginal and cervical lesions
(Kendall's Tau correlation=0.387, p\selectlanguage{english}<0.001).

{[}insert Table 2{]}

\textbf{3.3 Distribution of hr-HPV among different grades of VaIN}

A total of 832 cases underwent HPV genotyping, with the most common HPV
genotypes detected in VaIN being
HPV16\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV52\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV58\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV51\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV56\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV18,accounting for 26.9\%, 21.0\%,
17.7\%, 12.7\%, 11.7\%, 10.6\%, 10.1\%, respectively(Figure 1A).The
detail numbers was displayed in Supplementary Table S1. In VaIN 1, the
most frequently detected HPV types were
HPV52\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV16\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV58\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV53\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV56\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV51\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV59\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV18, accounting for
22.7\%, 19.5\%, 17.8\%, 13.6\%, 12.0\%, 11.7\%, 10.2\%, 10.1\%,
respectively (Figure 1B).While in VaIN2+, the most common HPV types were
HPV16\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV58\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV52\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV51\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV18\begin{CJK}{UTF8}{gbsn}、\end{CJK}\selectlanguage{english}HPV53, accounting for 54.5\%, 17\%,
14.8\%, 11.4\%, 10.2\%, respectively(Figure 1C).

{[}insert Figure 1{]}

\textbf{3.4 Comparison of the sensitivity of cervical cancer screening
against VaIN and CIN}

The sensitivities of cytology, hr-HPV and cotesting is displayed in
Table 3. Among patients without total hysterectomy, cytology had a
sensitivity of 84.9\% for VaIN2+, which was slightly lower than the
sensitivity of 87.4\% for CIN2+; however, this difference was not
statistically significant (P=0.495). The sensitivities of hr-HPV testing
for detecting VaIN2+ and CIN2+ were 95.0\% and 96.7\%, respectively
(P=0.480). The cotesting sensitivities for detecting VaIN2+, CIN2+, and
VaIN2+ after hysterectomy were all 100\%. The sensitivity of hr-HPV
testing for detecting VaIN2+ was significantly higher than that of
cytology (95.4\% vs. 88.1\%, p=0.007). However, there was no statistical
difference between the two tests for detecting VaIN2+ after hysterectomy
(97.1\% vs. 96.4\%, p=0.829).

{[}insert Table 3{]}

\textbf{4.Discussion}

In the present study, the HR-HPV detection rate was 88.5\% (883/998) in
all cases of VaIN. Specifically, the detection rate was 95.4\% in cases
of VaIN2+.The most common hr-HPV genotypes found in VaIN were HPV16,
HPV52, HPV58, HPV53. The median age of patients with VaIN2+ was 11.5
years older than VaIN1. The proportion of postmenopausal women in the
population with VaIN2+ disease was higher than that in the population
with VaIN1 disease. Patients with VaIN2+ tended to have more pregnancies
and deliveries than those with VaIN1. Additionally, they were more
likely to have a history of CIN. VaIN grade was significantly positive
correlated with cervical lesions, but correlation was weak (r=0.387).
HPV 16 was present in 54.5\% cases of VaIN2+ making it the most
prevalent HPV type in VaIN2+, followed by HPV58 (17.0\%), HPV52
(14.8\%), HPV51 (11.4\%), and HPV18 (10.2\%).. The sensitivity of hr-HPV
testing for VaIN2+ and CIN2+ were significantly higher than that of
cytology testing. However, for VaIN of vaginal stumps, they had no
difference. Furthermore, the sensitivity of cervical cytology and hr-HPV
testing for detecting VaIN2+ was not significantly different from their
sensitivity for detecting CIN2+.

In previous studies, the HPV detection rate in patients with VaIN2/3
ranged from 50\% to 100\%, and from 25\% to 89\% in vaginal
cancer.\textsuperscript{11-13} The different results may be attributed
to variations in study sample sizes, HPV detection methods, and sample
site selections (cervix or vaginal wall). HPV 16 was the dominated HPV
type in VaIN especially in VaIN2+ in all former
studies.\textsuperscript{13-17} In a global multicenter
study\textsuperscript{17} that performed HPV DNA detection and typing in
597 vaginal precancerous and cancerous lesions, HPV 16 accounted for
59\% of VaIN2+, which is very close to the result of this study.. Aside
from HPV 16, the following HPV genotypes were not consistent in
different studies, which may due to the regional differences in HPV type
prevalence. HPV18, 33, 45, 31 were the following common HPV types in
VaIN2+ in Europe and North America\textsuperscript{13}, while HPV 58,
52, 39, 33 were the following common in Taiwan,
China\textsuperscript{14}. The result of this study show the HPV
prevalent in VaIN2+ is similar to HPV prevalent in CIN2+ in our previous
study\textsuperscript{18}, but HPV 58 exceeded HPV 52 to become the
second most common HPV type in VaIN 2+ in present study, however, the
difference was not great. HPV vaccination provides an excellent
opportunity to prevent VaIN2+. Denmark, a country with high HPV
vaccination coverage, found that the incidence rate of high-grade VaIN
decreased by nearly 16\% per year among women younger than 30 years old
after the introduction of HPV vaccine.\textsuperscript{19} As China is a
country with poor HPV vaccine coverage\textsuperscript{20}, the results
of HPV prevalent presents near baseline distribution and inspire the use
of HPV vaccines that cover HPV 16, 58, 52, 51, and 18 to provide
excellent protection against VaIN2+.

Research has shown that a history of high-grade cervical lesions and
cervical cancer increases the risk of developing noncervical HPV-related
anogenital premalignancies and carcinomas for at least 20
years\textsuperscript{21, 22}, even after total
hysterectomy\textsuperscript{23-25}. Consistent with these findings, the
present study revealed that patients with VaIN2+ were more likely to
have a history of high-grade cervical lesions or cervical cancer than
those with VaIN1. Moreover, postmenopausal women, those with gravidity
and parity more than three times, and those with hr-HPV infection are
all at increased risk of developing VaIN2+. These results underscore the
importance of screening high-risk populations for VaIN2+. Currently,
both cytology and HPV testing are performed on the cervix except for
vaginal stumps. It is important to evaluate the sensitivity of cervical
cancer screening for detecting VaIN2+ and how it compares to CIN2+.
Previous studies have shown that the sensitivity of cytology for
detecting CIN2+ ranged from 47\% to 72.9\%, while the sensitivity of HPV
testing ranged from 89.9\% to 95\%.\textsuperscript{26, 27} Meanwhile,
the sensitivity of cytology for detecting VaIN2+ ranged from 61.5\% to
83.8\%, the sensitivity of HPV testing ranged from 82.5\% to 97.3\%, and
cotesting had a sensitivity ranging from 95.2\% to
100\%.\textsuperscript{28, 29} Although these findings suggest that
cervical cancer screening has a higher sensitivity for detecting VaIN2+
compared to CIN2+, direct comparison between studies is not possible due
to differences in HPV testing methods and cytologic quality, as well as
the object of study. This study has a unique advantage in that it
directly compares the sensitivity of cervical cancer screening (cytology
or HPV test) for VaIN2+ and CIN2+ within the same study. The results
indicate that the sensitivity of cervical cytology and hr-HPV testing
are equivalent for detecting both VaIN2+ and CIN2+, with cotesting
having the highest detection rate. However, for patients who have
undergone hysterectomy, the sensitivity of cytology for VaIN2+ was
comparable to that of HPV testing, but the small sample size may have
biased the results. Vaginal cytology testing has a reported sensitivity
of 83\% for predicting vaginal HSIL\textsuperscript{30}, while vaginal
hr-HPV testing has a sensitivity of 100\%\textsuperscript{31}, but a
direct comparison between these tests is not appropriate. Thus, future
well-designed controlled studies are necessary to further investigate
these findings.

Most of the vaginal lesions occur in the upper one-third of the vagina.
A colposcopy is a necessary procedure for diagnosing VaIN. Acetic acid
should be applied to the entire vaginal mucosa, including the fornix,
for 1-2 minutes, followed by the application of Lugol's iodine to
confirm the presence or absence of abnormal epithelium throughout the
vaginal mucosa and fornix. In this study, 32.3\% of VaIN2+ cases were
not accompanied by CIN. Therefore, when cervical cancer screening is
abnormal and there are no abnormal images on the cervix, attention
should be paid to the vaginal wall. According to the 2019 American
Society of Colposcopy and Cervical Pathology (ASCCP)
guidelines\textsuperscript{32}, for non-pregnant patients [?]25 years old
with HSIL cytology, expedited treatment (treatment without colposcopic
biopsy) for cervix is preferred. However, this type of management of
abnormal cervical cancer screening may lead to a missed diagnosis of
VaIN. Therefore, when choosing expedited treatment, colposcopic
examination should also be performed not only on the cervix but also on
the vaginal wall, and it is important to take a biopsy where a lesion is
identified on the vagina.

The limitations of this study are as follows: firstly, this is a single
hospital-based retrospective analysis, and selection bias may exist.
Secondly, three types of HPV testing were used in this study, which may
have resulted in heterogeneity of the results. Thirdly, since all the
patients included in this study were diagnosed because of abnormal
cervical cancer screening, VaIN without abnormal TCT or positive hr-HPV
results were not taken into account, so the sensitivity of cervical
cancer screening may be over-estimated.

\textbf{Conclusion}

Vaccines that cover HPV 16, 58, 52, 51, and 18 are likely to provide
excellent protection against VaIN2+. The rate of multiple HPV infection
in VaIN1 is not statistically different from that in VaIN2+. Cervical
cancer screening has similar sensitivity for VaIN2+ as for CIN2+, with
hr-HPV testing showing higher sensitivity than cytology, and cotesting
showing the highest detection rate. However, in populations after
hysterectomy, cytology and hr-HPV testing have similar sensitivity for
detecting VaIN2+. During colposcopy, acetic acid and Lugol's iodine
should be applied to cover the entire vaginal mucosa and fornix, and
attention should be paid to abnormal images in the vagina.

\textbf{Acknowledgments}

The authors thank Liyu Dai for supporting in the clinical management,
Home for Researchers editorial team (www. home-for-researchers.com) for
language editing service and Dafeng Huang for encouragement.

\textbf{Author Contributions}

Yusha Chen and Qiaoyun Chen were involved in the design of the study.
Huifeng Xue and Jinwen Zheng carried out data collection and analysis.
Yusha Chen assisted in drafting the manuscript. Xiangqin Zheng and
Jiancui Chen revised the manuscript. All the authors reviewed and
approved the final manuscript.

\textbf{Reference}

1. Sillman FH, Sedlis A, Boyce JG. A review of lower genital
intraepithelial neoplasia and the use of topical
5-fluorouracil.\emph{Obstetrical \& gynecological survey} . Apr
1985;40(4):190-220. doi:10.1097/00006254-198504000-00002

2. Sugase M, Matsukura T. Distinct manifestations of human
papillomaviruses in the vagina. \emph{International journal of cancer} .
Jul 29 1997;72(3):412-5.
doi:10.1002/(sici)1097-0215(19970729)72:3\textless{}412::aid-ijc7\textgreater{}3.0.co;2-s

3. Daling JR, Madeleine MM, Schwartz SM, et al. A population-based study
of squamous cell vaginal cancer: HPV and cofactors. \emph{Gynecologic
oncology} . Feb 2002;84(2):263-70. doi:10.1006/gyno.2001.6502

4. Strander B, Andersson-Ellstr\selectlanguage{ngerman}öm A, Milsom I, Sparén P. Long term risk
of invasive cancer after treatment for cervical intraepithelial
neoplasia grade 3: population based cohort study. \emph{BMJ (Clinical
research ed)} . Nov 24 2007;335(7629):1077.
doi:10.1136/bmj.39363.471806.BE

5. Henson D, Tarone R. An epidemiologic study of cancer of the cervix,
vagina, and vulva based on the Third National Cancer Survey in the
United States. \emph{American journal of obstetrics and gynecology} .
Nov 1 1977;129(5):525-32.

6. Kurman RJ, Carcangiu ML, Herrington CS, et al. WHO classification of
tumours of female reproductive organs. 4th eds. IARC Press; 2014:

8\selectlanguage{english}-253.

7. Ao M, Zheng D, Wang J, Gu X, Xi M. Risk factors analysis of
persistence, progression and recurrence in vaginal intraepithelial
neoplasia. \emph{Gynecologic oncology} . Sep 2021;162(3):584-589.
doi:10.1016/j.ygyno.2021.06.027

8. Gunderson CC, Nugent EK, Elfrink SH, Gold MA, Moore KN. A
contemporary analysis of epidemiology and management of vaginal
intraepithelial neoplasia. \emph{American journal of obstetrics and
gynecology} . May 2013;208(5):410.e1-6. doi:10.1016/j.ajog.2013.01.047

9. Aho M, Vesterinen E, Meyer B, Purola E, Paavonen J. Natural history
of vaginal intraepithelial neoplasia. \emph{Cancer} . Jul 1
1991;68(1):195-7.
doi:10.1002/1097-0142(19910701)68:1\textless{}195::aid-cncr2820680135\textgreater{}3.0.co;2-l

10. Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System:
terminology for reporting results of cervical cytology. \emph{Jama} .
Apr 24 2002;287(16):2114-9. doi:10.1001/jama.287.16.2114

11. Srodon M, Stoler MH, Baber GB, Kurman RJ. The distribution of low
and high-risk HPV types in vulvar and vaginal intraepithelial neoplasia
(VIN and VaIN). \emph{The American journal of surgical pathology} . Dec
2006;30(12):1513-8. doi:10.1097/01.pas.0000213291.96401.48

12. De Vuyst H, Clifford GM, Nascimento MC, Madeleine MM, Franceschi S.
Prevalence and type distribution of human papillomavirus in carcinoma
and intraepithelial neoplasia of the vulva, vagina and anus: a
meta-analysis. \emph{International journal of cancer} . Apr 1
2009;124(7):1626-36. doi:10.1002/ijc.24116

13. Smith JS, Backes DM, Hoots BE, Kurman RJ, Pimenta JM. Human
papillomavirus type-distribution in vulvar and vaginal cancers and their
associated precursors. \emph{Obstetrics and gynecology} . Apr
2009;113(4):917-924. doi:10.1097/AOG.0b013e31819bd6e0

14. Chao A, Chen TC, Hsueh C, et al. Human papillomavirus in vaginal
intraepithelial neoplasia. \emph{International journal of cancer} . Aug
1 2012;131(3):E259-68. doi:10.1002/ijc.27354

15. Bertoli HK, Rasmussen CL, Sand FL, et al. Human papillomavirus and
p16 in squamous cell carcinoma and intraepithelial neoplasia of the
vagina. \emph{International journal of cancer} . Jul 1
2019;145(1):78-86. doi:10.1002/ijc.32078

16. Serrano B, de Sanjose S, Tous S, et al. Human papillomavirus
genotype attribution for HPVs 6, 11, 16, 18, 31, 33, 45, 52 and 58 in
female anogenital lesions. \emph{European journal of cancer (Oxford,
England : 1990)} . Sep 2015;51(13):1732-41.
doi:10.1016/j.ejca.2015.06.001

17. Alemany L, Saunier M, Tinoco L, et al. Large contribution of human
papillomavirus in vaginal neoplastic lesions: a worldwide study in 597
samples. \emph{Eur J Cancer} . Nov 2014;50(16):2846-54.
doi:10.1016/j.ejca.2014.07.018

18. Chen Y, Li S, Zheng J, Xue H, Chen J, Zheng X. Prevalence of
multiple human papillomavirus infections and association with cervical
lesions among outpatients in Fujian, China: A cross-sectional
study.\emph{Journal of medical virology} . Dec 2022;94(12):6028-6036.
doi:10.1002/jmv.28062

19. Bertoli HK, Baandrup L, Aalborg GL, Kjaer AK, Thomsen LT, Kjaer SK.
Time trends in the incidence and survival of vaginal squamous cell
carcinoma and high-grade vaginal intraepithelial neoplasia in Denmark -
A nationwide population-based study. \emph{Gynecologic oncology} . Sep
2020;158(3):734-739. doi:10.1016/j.ygyno.2020.05.683

20. Deng C, Chen X, Liu Y. Human papillomavirus vaccination: coverage
rate, knowledge, acceptance, and associated factors in college students
in mainland China. \emph{Human vaccines \& immunotherapeutics} . Mar 4
2021;17(3):828-835. doi:10.1080/21645515.2020.1797368

21. Ebisch RMF, Rutten DWE, IntHout J, et al. Long-Lasting Increased
Risk of Human Papillomavirus-Related Carcinomas and Premalignancies
After Cervical Intraepithelial Neoplasia Grade 3: A Population-Based
Cohort Study. \emph{Journal of clinical oncology : official journal of
the American Society of Clinical Oncology} . Aug 1
2017;35(22):2542-2550. doi:10.1200/jco.2016.71.4543

22. Sand FL, Munk C, Jensen SM, Svahn MF, Frederiksen K, Kjaer SK.
Long-Term Risk for Noncervical Anogenital Cancer in Women with
Previously Diagnosed High-Grade Cervical Intraepithelial Neoplasia: A
Danish Nationwide Cohort Study. \emph{Cancer epidemiology, biomarkers \&
prevention : a publication of the American Association for Cancer
Research, cosponsored by the American Society of Preventive Oncology} .
Jul 2016;25(7):1090-7. doi:10.1158/1055-9965.epi-15-1291

23. Ciavattini A, Di Giuseppe J, Marconi C, et al. Hysterectomy for
cervical intraepithelial neoplasia: A retrospective observational
multi-institutional study. \emph{International journal of gynaecology
and obstetrics: the official organ of the International Federation of
Gynaecology and Obstetrics} . Dec 2022;159(3):679-688.
doi:10.1002/ijgo.14233

24. Schockaert S, Poppe W, Arbyn M, Verguts T, Verguts J. Incidence of
vaginal intraepithelial neoplasia after hysterectomy for cervical
intraepithelial neoplasia: a retrospective study. \emph{American journal
of obstetrics and gynecology} . Aug 2008;199(2):113.e1-5.
doi:10.1016/j.ajog.2008.02.026

25. Alfonzo E, Holmberg E, Sparen P, Milsom I, Strander B. Risk of
vaginal cancer among hysterectomised women with cervical intraepithelial
neoplasia: a population-based national cohort study. \emph{BJOG : an
international journal of obstetrics and gynaecology} . Mar
2020;127(4):448-454. doi:10.1111/1471-0528.16028

26. Liang LA, Einzmann T, Franzen A, et al. Cervical Cancer Screening:
Comparison of Conventional Pap Smear Test, Liquid-Based Cytology, and
Human Papillomavirus Testing as Stand-alone or Cotesting
Strategies.\emph{Cancer epidemiology, biomarkers \& prevention : a
publication of the American Association for Cancer Research, cosponsored
by the American Society of Preventive Oncology} . Mar
2021;30(3):474-484. doi:10.1158/1055-9965.Epi-20-1003

27. Koliopoulos G, Nyaga VN, Santesso N, et al. Cytology versus HPV
testing for cervical cancer screening in the general
population.\emph{The Cochrane database of systematic reviews} . Aug 10
2017;8(8):Cd008587. doi:10.1002/14651858.CD008587.pub2

28. Ao M, Zheng D, Wang J, Gu X, Xi M. A retrospective study of cytology
and HPV genotypes results of 3229 vaginal intraepithelial neoplasia
patients. \emph{Journal of medical virology} . Aug 30
2021;doi:10.1002/jmv.27311

29. Zhang L, Wang Q, Zhang H, Xie Y, Sui L, Cong Q. Screening History in
Vaginal Precancer and Cancer: A Retrospective Study of 2131 Cases in
China. \emph{Cancer management and research} . 2021;13:8855-8863.
doi:10.2147/cmar.S335701

30. Frega A, French D, Piazze J, Cerekja A, Vetrano G, Moscarini M.
Prediction of persistent vaginal intraepithelial neoplasia in previously
hysterectomized women by high-risk HPV DNA detection. \emph{Cancer
letters} . May 8 2007;249(2):235-41. doi:10.1016/j.canlet.2006.09.003

31. Khan MJ, Massad LS, Kinney W, et al. A common clinical dilemma:
Management of abnormal vaginal cytology and human papillomavirus test
results. \emph{Gynecologic oncology} . May 2016;141(2):364-370.
doi:10.1016/j.ygyno.2015.11.023

32. Perkins RB, Guido RS, Castle PE, et al. 2019 ASCCP Risk-Based
Management Consensus Guidelines for Abnormal Cervical Cancer Screening
Tests and Cancer Precursors. \emph{Journal of lower genital tract
disease} . Apr 2020;24(2):102-131. doi:10.1097/lgt.0000000000000525

Figure 1 The prevalence of hr-HPV genotype in total VaIN(A) , VaIN1(B)
and VaIN2+(C). hr-HPV, high-risk human papillomavirus

Table 1 Clinical characteristics of patients with vaginal
intraepithelial neoplasia\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
Characteristics & Total Number (\%)\tabularnewline
\textbf{Age, median (range)} & 44.17(17 to 90)\tabularnewline
\textbf{Menstrual status} &\tabularnewline
premenopausal & 708(59.0\%)\tabularnewline
menopausal & 305(25.4\%)\tabularnewline
hysterectomy(subtotal/total) & 187(15.6\%)\tabularnewline
\textbf{History of cervical lesions} &\tabularnewline
hysterectomy for benign disease & 56(4.7\%)\tabularnewline
hysterectomy for CIN/SCC & 111(9.3\%)\tabularnewline
hysterectomy cancers other than cervical cancer &
13(1.0\%\%)\tabularnewline
LEEP for HSIL & 100(8.3\%)\tabularnewline
LSIL & 72(6\%)\tabularnewline
hysterectomy for unknown reasons & 8(0.7\%)\tabularnewline
none & 840(70.0\%)\tabularnewline
\textbf{Cytology results} &\tabularnewline
NILM & 162(13.5\%)\tabularnewline
ASCUS & 330(27.5\%)\tabularnewline
LSIL & 492(41.0\%)\tabularnewline
HSIL & 110(9.2\%)\tabularnewline
ASC-H/ AGC/AIS & 51(4.2\%)\tabularnewline
SCC/ACC & 3 (0.3\%)\tabularnewline
not performed & 52(4.3\%)\tabularnewline
\textbf{HR-HPV resutls} &\tabularnewline
positive & 883(73.6\%)\tabularnewline
negative & 115(9.6\%)\tabularnewline
Not tested & 202(16.8\%)\tabularnewline
\textbf{HR-HPV infection pattern} &\tabularnewline
single type & 489 (40.8\%)\tabularnewline
multiple types & 340 (28.3\%)\tabularnewline
untyped & 371(30.9\%)\tabularnewline
\textbf{Concomitant CIN} &\tabularnewline
no & 504(49.6\%)\tabularnewline
CIN1 & 340(33.5\%)\tabularnewline
CIN2 & 101(9.9\%)\tabularnewline
CIN3 & 47(4.6\%)\tabularnewline
Cervical cancer & 24(2.4\%)\tabularnewline
\textbf{VaIN} &\tabularnewline
VaIN1 & 930(77.5\%)\tabularnewline
VaIN2 & 194 (16.2\%)\tabularnewline
VaIN3 & 67 (5.5\%)\tabularnewline
vaginal cancer & 9 (0.8\%)\tabularnewline
\bottomrule
\end{longtable}

Abbreviations: CIN, cervical intraepithelial neoplasia; SCC, cervical
squamous cell carcinoma; LEEP, loop electrosurgical excision procedure;
LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade
squamous intraepithelial lesion; NILM, negative for intraepithelial
lesion or malignancy (NILM); ASC-US, atypical squamous cells of unknown
significance; ASC-H, atypical squamous cells, cannot rule out HSIL; AGC,
atypical glandular cells; AIS, endocervical adenocarcinoma in situ; SCC,
squamous cell carcinoma; hr-HPV, high-risk HPV; VaIN, vaginal
intraepithelial neoplasia

Table 2 Comparison of clinical characteristics between VaIN1 and VaIN2
patients.\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& VaIN1 & VaIN2+ & Z/x & P\begin{CJK}{UTF8}{gbsn}值\end{CJK}\selectlanguage{english}\tabularnewline
\textbf{Age} & 41.5(32,52) & 53(42,59.25) & -9.806 &
<0.001\tabularnewline
\textbf{Concomitant CIN} & & & &\tabularnewline
no & 440(53.8\%) & 64(32.3\%) & 29.386 & <0.001\tabularnewline
yes & 378(46.2\%) & 134(67.7\%) & &\tabularnewline
\textbf{Cervical lesions histogy} & & & &\tabularnewline
none & 729(78.7\%)\textsuperscript{a} &
180(67.7\%)\textsuperscript{a} & 34.654 & <0.001\tabularnewline
LSIL & 64(6.9\%)\textsuperscript{a} & 8(3.0\%)\textsuperscript{b} &
&\tabularnewline
HSIL & 133(14.4\%)\textsuperscript{a} &
78(29.3\%)\textsuperscript{b} & &\tabularnewline
\textbf{Menstrual status} & & & &\tabularnewline
premenopausal & 614(75.2\%) & 94(47.7\%) & 57.151 &
<0.001\tabularnewline
menopausal & 202(24.8\%) & 103(52.3\%) & &\tabularnewline
\textbf{Cytology results} & & & &\tabularnewline
NILM & 132(14.7\%)\textsuperscript{a} &
30(11.9\%)\textsuperscript{a} & 191.936 & <0.001\tabularnewline
ASCUS/LSIL & 703(78.5\%)\textsuperscript{a} &
119(47.0\%)\textsuperscript{b} & &\tabularnewline
ASC-H/AGC/HSIL/SCC & 60(6.7\%)\textsuperscript{a} &
104(41.1\%)\textsuperscript{b} & &\tabularnewline
\textbf{HR-HPV results} & & & &\tabularnewline
negative & 106(13.2\%) & 9(4.6\%) & 11.343 & 0.001\tabularnewline
positive & 697(86.8\%) & 186(95.4\%) & &\tabularnewline
\textbf{HR-HPV infection pattern} & & & &\tabularnewline
single & 381(58.2\%) & 108(62.1\%) & 0.865 & 0.352\tabularnewline
multiple & 274(41.8\%) & 66(37.9\%) & &\tabularnewline
\textbf{Gravidity} & & & &\tabularnewline
<3 & 407(52.7\%) & 86(43.4\%) & 5.358 & 0.021\tabularnewline
[?]3 & 366(47.3\%) & 112(56.6\%) & &\tabularnewline
\textbf{Parity} & & & &\tabularnewline
<3 & 674(87.2\%) & 140(70.4\%) & 32.973 & <0.001\tabularnewline
[?]3 & 99(12.8\%) & 59(29.6\%) & &\tabularnewline
\textbf{HPV genotype} & & & &\tabularnewline
HPV 16/18 & 183(27.9\%) & 108(61.4\%) & 68.342 & <0.001\tabularnewline
non-type 16/18 hr-HPV & 473(72.1\%) & 68(38.6\%) & &\tabularnewline
\bottomrule
\end{longtable}

Note: \textsuperscript{a,b} different alphabet means statistically
significant (p<0.05)

Table 3 Comparison of Sensitivity for Cervical Cancer Screening against
VaIN2+ and CIN2+\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
VaIN2+ (without total hysterectomy)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
CIN2+\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
P-value (row)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
VaIN2+ (after total hysterectomy)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Cytology\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
84.9\%(157/285)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
87.4\%(139/159)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.495\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
97.1\%(66/68)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Hr-HPV\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
95.0\%(133/140)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
96.7\%(119/123)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
0.480\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
96.4\%(53/55)\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
P-value (column)\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
0.004*\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
0.005*\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.19\columnwidth}\raggedright\strut
0.829\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
Cotesting\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
100\%(134/134)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
100\%(91/91)\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.17\columnwidth}\raggedright\strut
100\%(50/50)\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

Note: * denotes statistical significance (p \textless{} 0.05).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}

\selectlanguage{english}
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