The Immunomodulation Role of Vaginal

Microenvironment On Human Papillomavirus

Infection

Lingyan Sun1, Li Li1, Wenxin Xu1, Cen Ma1

1 Department of Obstetrics and Gynecology Laboratory, e First Aliated Hospital of Soochow University, Suzhou, Jiangsu 215000,

China.

GMJ.2023;12:e2991

www.gmj.ir

 Correspondence to:

Cen Ma, Department of Obstetrics and Gynecology

Laboratory, The First Aliated Hospital of Soochow

University, No. 188, Shizi Street, Gusu District, Suzhou,

Jiangsu 215000, China.

Telephone Number: +86 512 6522 3637

Email Address: macen_edu@outlook.com

Received 2023-04-03

Revised 2023-05-14

Accepted 2023-05-18

Abstract

Background: Evidence suggests the role of the vaginal microbiome and microenvironment

in the immunity state. The human papillomavirus (HPV) infection is widely dependent

on the healthy vaginal microenvironment. Hence, this study aimed to investigate the

role of the vaginal microenvironment in the rate of high-risk HPV (hr-HPV) infection.

Materials and Methods: This cross-sectional study was performed on 512 women with hr-HPV

positive (n=212) or negative (n=300) infection. The vaginal samples of women were examined

regarding yeas and Gardnerella vaginalis infection. Also, Lactobacillus acidophilus, pH, and

enzyme activity (such as catalase, proline aminopeptidase, and leucocyte esterase) were compared

between the two groups. Also, the histopathological study was performed on the vaginal samples.

Results: The higher rate of yeast and G. vaginalis infections as well as decreased L. acidophilus,

were signicantly observed in women with hr-HPV positive infection (P<0.001). Also,

histopathological ndings indicated that cervical intraepithelial neoplasia grade I-III and cervical

cancer lesions were markedly higher in hr-HPV positive group compared with control women.

Conclusion: The hr-HPV infection was markedly correlated to vaginal microenviron-

ments, and it could a risk factor for the elevation of the rate of high-grade cervical lesions.

[GMJ.2023;12:e2991] DOI:10.31661/gmj.v12i0.2991

Keywords: Human Papillomavirus; Vaginal Microenvironment; Lactobacillus acidophilus;

Cervical Cancer; Cervical Intraepithelial Neoplasia

Introduction

In the vagina of healthy women, coexist of

multiple microorganisms provides the natu-

ral micro-ecological environment [1]. Vaginal

dysbiosis is a common condition that disrupts

the immune balance, leading to a breakdown

in the protective layer of cells and enhanced

sexually transmitted infections [2]. Indeed,

vaginal dysbiosis is an imbalance in the types

and amounts of bacteria in the vagina, which

can lead to various diseases [2]. In a normal

microenvironment vagina, Lactobacillus aci-

dophilus is the main microorganism that bal-

ances the vaginal environment [3]. When the

vagina is aected by exo- and/or endogenous

factors, the imbalance of vaginal microecolo-

gy occurs [3]. The common vaginal infectious

GMJ

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Sun L, et al. Role of Vaginal Microenvironment On HPV Infection

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diseases in women include bacterial vaginosis

(BV), vulvovaginal candidiasis, Trichomonas

vaginitis, and aerobic vaginitis (AV) [3, 4].

The human papillomavirus (HPV) is a small

spherical DNA virus with no envelope, which

can lead to abnormal proliferation of squa-

mous epithelium of cervical mucosa and then

develop into cervical lesions and even cer-

vical cancer (CC) [4]. The HPV infection of

most susceptible people is transient and can

be eliminated by itself, but when the vaginal

microecology is impaired, the high-risk HPV

(hr-HPV) persistent infection may occur [5].

HPV infection can cause local immune chang-

es in the cervix and vaginal mucosa, change

the composition of vaginal ora, and lead to

oxidative stress reaction, which impairs the

antioxidant enzyme system [6-8]. Chao et al.

showed that some vaginitis diseases play an

important role in the gradual transformation

of normal cervical tissues into cancer [9].

Hence, in this study, we aimed to investigate

the role of the microenvironment of the va-

gina on hr-HPV infection and its association

with cervical lesions.

Materials and Methods

1. Participants and Study Design

This cross-sectional study was performed

on 512 women who visited at the outpatient

department of Obstetrics and Gynecology of

the aliated hospital of Soochow Universi-

ty, Suzhou, China, during 2019-2022. All the

women were evaluated regarding HPV status

and divided into hr-HPV-positive group and

controls (hr-HPV-negative). HPV infection

diagnosis was performed based on standard

methods. Briey, the exfoliated cells were

centrifuged at room temperature at 12000 R /

minute for 1 minute. HPV-DNA was extract-

ed with DNA Extraction Kit (Wuhan Boruida

Biotechnology, China). HPV genotypes were

detected by polymerase chain reaction based

on hybridization microarray. This genotyping

test can qualitatively detect 18 high-risk types

(such as HPV16, 18, 31, 33, 35, 39, 45, 51, 52,

53, 56, 58, and 59) and three low-risk types

(such as HPV6, 11, and 81). In this study,

HPV-DNA positivity was dened as the di-

agnostic standard of HPV infection positivity,

and HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53,

56, 58, and 59 were considered as 13 high-risk

types.

2. Sample Size Calculation

Regarding Lv et al. [10], vaginal infections

were observed in 0.36 % of controls, and with

α=0.05 and power of 0.8, the sample size was

calculated as 187 for each group.

3. Inclusion and Exclusion Criteria

All the women aged over 18 years were in-

cluded in this study. Also, women with preg-

nancy, history of genitalia dysplasia and/or

malignancy, human immunodeciency virus

(HIV) infection, immunosuppressive diseas-

es, history of chemotherapy and/or radiother-

apy, multi-partner, and history of previous

HPV infection were excluded from the study.

4. Data Collection

4.1. Vaginal Sampling

To assess the vaginal microenvironment, se-

cretions on the lateral wall of the vagina were

collected with three sterile cotton swabs by

trained midwives based on a similar protocol

[11].

4.2. Assessment AV

One swab was used by the chemical reaction

method for AV. The AV was considered pos-

itive in the case of Donders diagnostic scale

score ≥3 points.

4.3. Determine pH of Vaginal Secretion

The pH value of vaginal secretions of the two

groups was examined using a joint inspection

analyzer (Anhui Anchang Biotechnology,

China) with the vaginitis ve-link Kit (Guang-

zhou Ruihui Biotechnology, China), and the

results showed that blue means pH≥4.8, and

yellow and green means pH<4.8.

4.4. Normal Flora

One swab was placed into a test tube contain-

ing a small amount of 0.9% sodium chloride

for routine microscopic examination, includ-

ing leukocytes, epithelial cells, T. vagina-

lis, Candida, L. acidophilus, miscellaneous

bacteria, and clue cells. L. acidophilus was

considered absent if its counts were ≤30/ in

each high power eld (HPF), and counts of

Role of Vaginal Microenvironment On HPV Infection Sun L, et al.

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>30/HPF were considered normal. Observa-

tion of candida spores and hyphae under the

microscope was considered positive; micro-

scopic evaluation also detected T. vaginalis. 

4.5. Measurement Proline Aminopeptidase,

Catalase, and Leucocyte Esterase Activity

To evaluation of the proline aminopeptidase

activity, a microplate was used, and GBC fast

garnet salt (Sigma, Germany) was added after

4 hours of incubation at 35°C. After an addi-

tional 5 minutes of incubation with the dye, a

pink-red color indicated a positive test, while

a yellowish-amber color indicated a negative

test [12].

Based on the manufacturer’s instructions,

Chemstrip Leucocyte esterase was used (Bo-

eringer Mannheim Diagnostics, China). A

dipstick test with a trace color was considered

positive [13]. Also, catalase enzyme activity

was evaluated based on changes in measured

absorbance at 240 nm of wavelength.

4.6. Identication of BV

Certain criteria, such as the presence of vag-

inal discharge, pH >4.5, amine production,

and a decrease in Lactobacillus morphotypes,

were considered to diagnose BV, along with

specic bacterial morphotypes.

Also, in the case of Nugent ≥7 points, the

sample was considered positive for BV. Al-

though recovering Gardnerella vaginalis by

culture was not a specic indicator of BV, it

was considered as additional test [14].

4.7. Histopathological Study

Cervical tissues were collected by colposco-

py biopsy or cervical circumcision for his-

topathological examination. Two expert pa-

thologists who were blinded to the results of

high-risk HPV-DNA testing read the lms and

nally reported the pathological sections ac-

cording to the Bethesda System classication

recommended by the International Cancer In-

stitute in 2001 [4].

Pathological examination results were divided

into normal or inammatory, cervical intraep-

ithelial neoplasia (CIN) I-III, and CC. Normal

or inamed cervix was dened as chronic

cervicitis (NC), CIN grade I was dened as

a low-grade squamous intraepithelial lesion

(LSIL), and CIN grade II and III were dened

as high-grade SIL (HSIL).

5. Ethical Issues

This study was approved by the First Aliat-

ed Hospital of Soochow University (approval

code: 201906001). Also, the written informed

constant was obtained from all the partici-

pants.

6. Statistical Analysis

Data were presented as number and percent

and/or mean and standard deviations. The

IBM SPSS Statistics for Windows, version

21 (IBM Corp., Armonk, NY, USA) using the

chi-square test was applied for data analysis.

Also, logistic regression analysis was used for

multivariate analysis to determine the Odds

ratio (OR). A P-value<0.05 was considered as

statistically signicant.

Results

Regarding HPV testing, 212 women had

positive results, and 300 were considered

HPV-negative. The median age of participants

in hr-HPV-positive and -negative groups were

38 years (ranged 20-65) and 37 years (ranged

22-65).

There were no signicant dierences among

studied women in terms of age (P=0.056).

Regarding Table-1, there was no signicant

dierence between the two groups in terms of

pH (OR= 0.84, P=0.67).

However, the presence of proline aminopepti-

dase (OR=4.83), catalase (OR=2.41), and leu-

cocyte esterase (OR=2.531) were signicant-

ly associated with HPV positivity (P˂0.001,

Table-1). As shown in Table-2, yeast, G. vag-

inalis, and L. acidophilus were signicantly

more observed among women in hr-HPV pos-

itive group (P<0.001).

Indeed, HPV positivity reduced L. acidophilus

in 70.28% of women compared with 52.33%

in controls (OR: 1.71, P=0.017, Table-2).

Multivariate logistic regression analysis

showed that yeast infection, G. vaginalis in-

fection, catalase positivity, proline aminopep-

tidase activity, leucocyte esterase positivity,

and decreased L. acidophilus were indepen-

dent risk factors for hr-HPV-positive infection

(P<0.05).

4GMJ.2023;12:e2991

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Sun L, et al. Role of Vaginal Microenvironment On HPV Infection

Table1. The Baseline Characteristics of Studied Women

Parameters

hr-HPV OR (95%

CI) P-value**

Positive

(n=212)

Negative

(n=300)

pH, n(%)

≥ 4.6 14 (6.6) 17 (5.6) 0.84 (0.39 to

1.8) 0.673

<4.6 198 (93.4) 283 (94.4)

P-value* 0.774

Proline aminopeptidase, n(%)

Absent 175 (82.5) 283 (94.4) 4.83 (2.17 to

11.02) ˂0.001

Present 37 (17.5) 17 (5.6)

P-value* 0.01

Leucocyte esterase, n(%)

Absent 52 (24.5) 93 (31) 2.531 (1.28

to 3.65) <0.001

Present 160 (75.5) 207 (69)

P-value* 0.323

Catalase, n(%)

Absent 21 (28.3) 154 (51.4) 2.41 (1.26 to

4.62) 0.008

Present 152 (71.7) 146 (48.6)

P-value* <0.001

* Between two groups based on the Chi-square test

**Logistic regression

HPV: Human papillomavirus; CI: Condence interval

Table 2. Vaginal Microbial Features of Women

Parameters

hr-HPV OR (95%

CI) P-value**

Positive

(n=212)

Negative

(n=300)

L. acidophilus, n(%)

Normal 63 (29.7) 143 (47.6) 1.71 (1.15 to

2.53) 0.017

Decreased 149 (70.3) 157 (52.3)

P-value* 0.009

Yeasts, n(%)

Absent 100 (47.1) 227 (75.6) 2.7 (1.04 to

7.03) 0.041

Present 112 (52.9) 73 (24.4)

P-value* <0.001

G. vaginalis, n(%)

Absent 112 (52.9) 237 (79) 3.35 (1.44 to

7.8) 0.005

Present 100 (47.1) 63 (21)

P-value* <0.001

* Between two groups based on Chi-square

**Logistic regression

HPV: Human papillomavirus; OR: Odds ratio; CI: Condence interval

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Role of Vaginal Microenvironment On HPV Infection Sun L, et al.

Histopathological Study

The histopathological ndings revealed that

352 (68.7%) of all the individuals had NC

results; however, 11 (2.1%) women have

malignant lesions (Table-3). The LSIL,

HSIL, and CC were signicantly observed

in the women of the hr-HPV positive group

(P<0.001, Table-3). Indeed, HPV infection

leads to malignant changes in the vagina and

cervix of women.

Discussion

The results of the current study showed

that HPV infection was correlated with the

presence of proline aminopeptidase, catalase,

and leucocyte esterase. Also, high-grade

lesions were signicantly observed among

women with hr-HPV infection.

Our ndings in line with previous studies

indicated that L. acidophilus was more

frequent in women with HPV-negative

infection [15-17]. In contrast, yeast and

G. vaginalis were signicantly present in

women with HPV-positive infection. Santella

et al. [18] demonstrated that the microbiota

composition in patients with HPV infection

diered signicantly from control women.

Wei et al. [19] found that there were microbial

perturbations in the early phase of hr-HPV

infection, with a decrease in L. acidophilus

and an increase in bacteria related to BV, such

as G. vaginalis.

Their ndings are consistent with our study.

Also, Wei et al. [19] suggested that the

predominance of some BV-associated bacteria

during hr-HPV infection may increase the risk

for cervical neoplasia.

In our study, the detection rates of yeasts and

G. vaginalis in the hr-HPV-positive group

were also higher than those in the HPV-

negative group.

In line with our study, Dareng et al. [20]

evaluated the association between vaginal

microbiota and persistent hr-HPV infection in

vaginal microbiota of Nigerian women, which

showed a high diversity vaginal microbial

community with a paucity of Lactobacillus

species was associated with persistent hr-HPV

infection.

The normal vaginal microbiota usually

maintains the acidic environment in the

vagina by producing lactic acid and maintains

the self-purication eect of the vagina

[21,22]. In the normal vaginal environment,

the quantity and function of L. acidophilus

play an important role in maintaining the

vaginal microenvironment [22]. When the

number of L. acidophilus decreases, the

normal acidic environment of the vagina

is impaired, and the antimicrobial eect is

reduced. Pathogenic bacteria could invade the

vagina and proliferate, resulting in infection

of the reproductive system and changes in the

microenvironment of bacteria in the vagina

[23].

Table 3. Histopathological Findings of Studied Women

Parameters

hr-HPV OR (95%

CI) P-value* P-value**

Positive

(n=212)

Negative

(n=300)

NC 80 (37.74) 272 (90.6) 0.35 (0.23

to 0.53) 0.002 <0.001

LSIL 32 (15.09) 12 (4) 0.23 (0.11

to 0.46) 0.008 <0.001

HSIL 90 (42.45) 15 (5) 0.07 (0.04

to 0.12) <0.001 <0.001

CC 10 (4.72) 1 (0.4) 0.06 (0.009

to 0.53) 0.024 0.01

* Between two groups based on Chi-square test

**Logistic regression

HPV: Human papillomavirus; NC: Non-cancer, LSIL: Low-grade squamous intraepithelial lesion; HSIL:

High-grade squamous intraepithelial lesion; CC: Cervical cancer; OR: Odds ratio; CI: Condence interval

Indeed, reproductive system infection could

lead to abnormal levels of sialidase and

other enzymes and increase the risk of HPV

infection. For example, G. vaginalis infection

may disrupt the protective mucosal barrier,

cause micro damage and/or change of epithelial

cells, and increase HPV susceptibility [23]. In

our study, G. vaginalis infection was more

observed among women with HPV-positive

infection. Hence, it seems that one of the

possible roles of the microenvironment of the

vagina in the protection of HPV infection is

related to the increase of G. vaginalis.

Fungal infection can cause inammatory

reactions, increase tissue permeability,

produce invasive enzymes, destroy epithelial

cells, and increase HPV susceptibility [5].

Hydrogen peroxide has antibacterial activity,

while catalase inhibits hydrogen peroxide;

consequently, the susceptibility to HPV

infection dramatically increased. Our results

showed that the catalase activity in the HPV-

positive group was markedly higher than in

the control group.

The LSIL, especially in young women, is

usually due to transient HPV infection, while

persistent HPV infection is associated with

HSIL and CC [24, 25]. Our study indicated

that the rate of HPV infection in patients with

high-grade cervical lesions (LSIL, HSIL, CC

groups) was higher than that in the NC group.

Chan et al. [26] showed that the rate of HPV-

positive infection in patients with CIN grade

I was signicantly lower than that in patients

with CIN II- III and invasive CC. In the early

stage of HPV infection, the virus is generally

in an active replication period, but the immune

system can recognize it. However, when

the vaginal environment is abnormal, and

the vaginal mucosa and cervical epithelium

are damaged, it may enhance the persistent

infection of HPV, reduce the clearance rate of

HPV, and ultimately increase the risk of high-

grade CIN [27].

Limitations

In this study, we only examined some

important microbial parameters, while other

microbial species as well as a larger sample

size, could provide better evidence regarding

the exact role of the vaginal microenvironment

in the increase of HPV infection.

Conclusion

The hr-HPV infection is closely related to the

vaginal microenvironment, and G. vaginalis

and yeast infections play an important role

in increasing the rate of HPV infection. Also,

HPV infection could lead to high-grade

cervical lesions and elevate the rate of CC.

Conict of Interest

The authors of the study declare there were no

conicts of interest.

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