Journal of Infection and Public Health 13 (2020) 1961–1966

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Journal of Infection and Public Health

journal homepage: http://www.elsevier.com/locate/jiph

Original Article

The Correlation between the Determination of Vaginal

Micro-Ecological Composition and the Outcome of HPV Infection by
High-Throughput Metagene Sequencing Information Technology on
the Illumina Platform
Weiye Cheng a,1 , Fei Xu b,1 , Leilei Gao a , Jinwei Liu a,∗
a
Department of Gynecology, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medicine College, Hangzhou, 310014, Zhejiang Province,
China
b
Department of Gynecology, The Affiliated Hospital of Hangzhou Normal University, Hangzhou, 310000, Zhejiang Province, China

a r t i c l e i n f o a b s t r a c t

Article history: Objective: To study the correlation between vaginal micro-ecological composition and the outcome of
Received 13 February 2020 human papillomavirus (HPV) infection by High-Throughput Metagene Sequencing Information Technol-
Received in revised form 21 May 2020 ogy on the Illumina Platform, and to improve the efficiency of clinical infection detection. Methods: With
Accepted 28 May 2020
the aid of Illumina high-throughput sequencing platform and sequence research method, the composi-
tion and diversity of vaginal microorganisms in high-risk HPV (HR-HPV) infected women and healthy
Keywords:
women were analyzed. The differences in vaginal flora of HR-HPV infected women and healthy women
HPV infection
were compared to explore the correlation between HR-HPV infection and vaginal flora. Results: The
vagina microecology
vaginal flora
structure of vaginal flora in healthy women was relatively single, with Lactobacillus as the dominant
High-Throughput Metagene Sequencing genus, accounting for more than 80%. The structure of vaginal flora in women infected with HR-HPV was
Information Technology significantly different from that in non-infected women. The former had a significantly increased species
gene sequencing diversity, which was mainly manifested by a decrease in Lactobacillus and an increase in Gardnerella
vaginalis. Mycoplasma and Ureaplasma urealyticum might play a synergistic role in the initial stage of
cervical lesions caused by HR-HPV infection. Conclusion: The prevention and treatment of mycoplasma
and Ureaplasma urealyticum should be valued clinically to prevent the occurrence of HR-HPV infection
and cervical lesions.
© 2020 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for
Health Sciences. This is an open access article under the CC BY-NC-ND license (http://creativecommons.
org/licenses/by-nc-nd/4.0/).

Introduction
Human papillomavirus (HPV) is one of the identified risk factors
for cervical diseases. Various research has confirmed that persis-
tent infection of high-risk HPV (HR-HPV) is the necessary condition
for the occurrence of cervical intraepithelial neoplasia and cervical
cancer [1]. HPV has multiple subtypes, of which more than 100
subtypes have been discovered and more than 40 subtypes are
related to cervical lesions. Researches have detected multiple HPV
infections in 304 women by flow-through hybridization and found
that the top five HPV subtypes are HPV-16 (19.2%), HPV-52 (13.7%),
HPV-58 (12.3%), HPV-18 (8.2%), and HPV-53 (6.8%). All these sub-
∗ Corresponding author.
E-mail address: liujinweihznu@yeah.net (J. Liu).
1
These authors contributed equally to this work.
types belong to the high-risk subtypes [2]. Other research has tested
586 samples by the rapid hybridization of nucleic acid molecules.
The results have indicated that the high-risk subtypes account for
86.7% of total cases, and the low-risk subtypes account for 9.09%.
The most common HPV infection subtypes are 52, 53, 58, and 16
[3]. After infection, HPV exists in host cells under both integrated
and non-integrated states. Most healthy women can automatically
remove the HPV when they are infected for the first time. How-
ever, if the in vivo mechanism is impeded, or multiple consecutive
HPV infections occur cervical diseases, even cervical cancer, will
occur [4]. Also, scholars have explored that the positive rates of HR-
HPV infection in patients with low-grade squamous intraepithelial
lesions, high-grade squamous intraepithelial lesions, and cervical
cancer reached 34.7 %%, 85.4%, and 100%, respectively [5,6].
HR-HPV infection can cause vaginal micro-ecological distur-
bances and changes in flora structure, such as a decrease in the
abundance of Lactobacillus vaginalis, an increase in the abundance

https://doi.org/10.1016/j.jiph.2020.05.024
1876-0341/© 2020 The Author(s). Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1962 W. Cheng et al. / Journal of Infection and Public Health 13 (2020) 1961–1966
of other pathogenic bacteria. In turn, an increase in the abun-
dance of other bacterial species will promote the expression and
development of HR-HPV [7]. Since the vagina is a relatively closed
environment, the structure of the flora is complex, and most of
the flora is anaerobic bacteria. Traditional methods of pure cul-
ture, isolation, and identification have limitations. Moreover, the
traditional methods mainly observe the morphological and phys-
iological characteristics of cultured bacteria through microscopy.
With the development of technology and the improvement of
requirements, it is necessary to identify almost all flora in the
vagina to determine the abundance and structure of bacteria for
the diagnosis and treatment of diseases. Therefore, it is neces-
sary to apply the most advanced technology to replace traditional
methods [8]. The Barcoded Paired-End Illumina Sequencing (BIPES)
method is a novel generation sequencing method based on the Illu-
mina Solexa high-throughput sequencing platform combined with
16SrRNA tag sequence analysis of microbial communities. It has
many advantages, such as simple operation, low cost, and excellent
reproducibility. Therefore, BIPES can quickly and systematically
obtain systematic, structured, and comprehensive flora informa-
tion [9,10].
Although there have been discussions on vaginal microecology,
studies combining high throughput metagene sequencing informa-
tion technology with vaginal microecology are rare. Based on the
Illumina high-throughput sequencing platform and BIPES sequence
research methods, this study analyzed the composition and diver-
sity of vaginal microorganisms in HR-HPV-infected women and
healthy women. Then, it compared the differences in vaginal flora
between HR-HPV-infected women and healthy women to explore
the correlation between HR-HPV infection and vaginal flora. By ana-
lyzing the vaginal flora of women with different levels of cervical
lesions, the distribution characteristics and community structure
of vaginal microbes at different stages of cervical lesions were
understood. Also, the dynamic changes in various microbial popu-
lations during the occurrence and development of cervical lesions
were tracked, thereby exploring the correlation between the occur-
rence and development of cervical lesions and dynamic changes
in vaginal flora. This study provided a theoretical basis for further
research on the etiology of cervical lesions, the development of
vaginal micro-ecological therapies, and the development of pro-
biotic preparations.
Method
Cervical cancer is one of the most serious diseases that threaten
the health of women all around the world. Persistent HR-HPV
infection has been identified as a risk factor for cervical pre-
cancerous lesions and cervical cancer. After continuous infection
with HR-HPV, the vaginal flora structure of healthy women may
become imbalanced, causing the disturbance of the vaginal micro-
ecological environment. If the viruses are not removed by the body
automatically, the infection may progress to cervical precancer-
ous lesions or cervical cancer after several years. Therefore, the
study of HR-HPV infection and vaginal micro-ecology has become
increasingly significant. Fig. 1 showed human cervical cells that
were infected with HPV.
Research objects and grouping
A total of 131 women who were examined or diagnosed in
our hospital from September 2016 to June 2019 were selected.
They were divided into two groups according to the results of
HR-HPV screening (the hC2 method): (1) the HR-HPV negative
group (HPV- group/group A), including a total of 33 healthy women
with negative HR-HPV test results; (2) the HR-HPV-positive group
(HPV + group), including a total of 98 patients with positive HR-
HPV test results. The HR-HPV positive group was divided into three
sub-groups further:(1) the low-grade group (group B), including 26
cases who had positive HPV test results and/or low-grade cervi-
cal intraepithelial neoplasia confirmed by cervical biopsy; (2) the
high-grade group (group C), including 40 cases with high-grade
cervical intraepithelial neoplasia, which was confirmed by cervi-
cal biopsy; (3) the cervical cancer group (group D), including 32
cases with cervical cancer, which was confirmed by cervical biopsy.
Inclusion criteria: women aging 21-65 years old; women with a
history of sexual life for 3 years and above; women who were non-
menstrual, non-pregnant, and non-puerperal. Exclusion criteria:
women aging >65 years old; women with no history of sexual life;
women with immunodeficiency diseases; women with systemic
diseases, such as diabetes mellitus, and hormone therapy diseases,
such as acute and chronic nephritis. Meanwhile, the following con-
ditions must be met: (1) no vaginal flushing within 48 hours; (2) no
sexual encounter and vaginal drug administration within 3 days;
(3) no systematic use of antibiotics or antifungals within 1 month.
Sample collection
Before the sample collection, the included objects were
informed of the experimental plans and the research significance.
The informed consent forms of included objects were obtained.
Then, the samples were collected in the gynecological examination
room. The objects were told to urinate before sample collection.
Then, the objects laid their legs apart in a lithotomy position on
a gynecological examination bed to fully expose the genitals and
perineum. A sterile speculum was used to open the vagina. Two
sterile vaginal cotton swabs were used to collect the secretions
at the vaginal fornix or the middle side of the vagina. The swabs
were rotated for 10-15 seconds. After they fully absorbed the secre-
tions, the swabs were carefully put into separate dry and sterile
test tubes. The samples must be avoided from contacting with the
vaginal opening and the vulva to prevent sample contamination.
The cotton swabs were put into dry and sterile test tubes for the
extraction of total bacterial DNA. The vaginal swab genomic DNA
kits (Beijing Bioteke, China) were used to extract the total bac-
terial genomic DNA from secretions on vaginal swabs. The main
reagents included binding solution, buffer solution, rinse solution,
protease, and elution buffer. The total bacterial DNA was extracted
from vaginal secretions according to the specific instructions of the
kit.
Amplification of 16SrRNA-V4 region gene fragments
The 16SrDNA is the gene encoding 16SrRNA, which is the most
common in bacterial taxonomy research. In the evolutionary pro-
cess of bacteria, the evolution of rRNA is relatively conservative.
Therefore, rRNA is considered to be the most ideal ruler to explain
the history of life evolution. Especially, 16SrRNA is the most con-
servative, which is often used to identify and classify bacteria. It
has a moderate length of 1.5 Kb and is divided into the variable
region and the conserved region. The conserved region can be used
to amplify 16SrDNA, and the variable region can be used to identify
the flora type [11].
The extracted DNA was used as a template, the gene frag-
ment of its 16SrRNA-V4 region was amplified, and the primer
sequences used were 5’-GAGTGCCAGCMGCCGCGGTAA-3’ and 3’-
GGACTACHVGGGTWTCTAAT-5’. The PCR amplification conditions
were: denaturation at 94 ◦ C for 2 minutes, denaturation at 94 ◦ C for
30 seconds, annealing at 54 ◦ C for 30 seconds, extension at 72 ◦ C for
45 seconds, extension at 72 ◦ C for 5 minutes after 30 consecutive
cycles, and stored at 4 ◦ C.
 W. Cheng et al. / Journal of Infection and Public Health 13 (2020) 1961–1966 1963

Fig. 1. HPV infection process of human cervical cells (Fig. 1A to 1D) indicated that human cervical cells changed from normal to HPV-infected and canceration.

Illumina sequencing

With DL2000 as the marker, the yield and characteristics of the

obtained PCR products were detected by 0.8% agar gel electrophore-
sis. The gel after electrophoresis was detected with a UV gel system
and photographed. The final PCR product volume required for
each sample was calculated by the relevant analysis software. The
PCR product of each sample was added to the same clean Eppen-
dorf (EP) tubes according to the calculated volume. Finally, the
mixed sample was frozen and sent to Shenzhen BGI for double-
end 100 bp sequencing. The sequencing platform was Illumina
HiSeq2000. During the amplification process of PCR, due to the dif-
ferent amplification efficiency between samples, the concentration
of PCR products varied, which would directly lead to differences
in sequencing depth during the analysis of later sequencing data.
Therefore, to avoid the existence of such errors, the gel analysis soft-
ware BandScan and Invitrogen fluorometer were used for detection Fig. 2. Comparison of PD index of vaginal flora between the HPV + and HPV- groups.

and analysis. First, the BandScan was used to perform quantification

of each sample, balance the differences in sequencing depth that
might be caused by the above reasons, and gradually monitor the
quality of the PCR amplification products. Then, the Invitrogen flu-
orescence quantifier was used to perform absolute quantification
of the PCR amplification products to reduce possible errors further.
Finally, according to the values calculated after relative quantifi-
cation and absolute quantification, each PCR product was mixed
uniformly at the same final concentration.

Statistical methods

The data were analyzed by using SPSS 22.0 statistics software.

The measurement data were expressed as mean ± standard devia-
tion. The comparison between multiple groups was performed by
using the One-way ANOVA. The comparison of rates was performed
by using the Chi-square test. P < 0.05 was considered statistically Fig. 3. Comparison of the Shannon index of vaginal flora between the HPV + and
significant. All data collected would be summarized and tabulated HPV- groups.
by using Microsoft Excel software.

The 16SrRNA-V4 region sequences of vaginal microorganisms

Results
Analysis results of vaginal flora of HPV + and HPV- groups
Alpha diversity describes the number and distribution of sam-
ples within a micro-ecosystem, which is usually analyzed by using
the PD whole tree (PD) index and Shannon index. After the normal-
ity test, the data of the PD index and Shannon index did not conform
to the normal distribution. Therefore, the two independent samples
non-parametric test (Mann-Whitney Test) was used for statistical
analysis. As shown in Figs. 2 and 3, both the PD index and the Shan-
non index of the HPV + group increased, and the difference in the
Shannon index was statistically significant (P = 0.000), indicating
that the species diversity of the vaginal flora in the HPV + group
was significantly different from that of the HPV- group.
in all collected samples were sequenced by Illumina, and a total
of 22 bacteria were detected. For the kinds of bacteria, the major-
ity of vaginal flora in the HPV- group was the Phylum Firmicutes,
accounting for 90.69%, and the remaining 21 kinds of bacteria
accounted for less than 10%. In addition, bacteria whose relative
abundance was then 1% included Actinobacteria, Bacteroides, Pro-
teobacteria, and Fusobacteria, accounting for 3.95%, 1.33%, 1.98%,
and 1.27%, respectively. In the HPV + groups, the relative abun-
dance was significantly different. The most significant change
was the reduction of the Phylum Firmicutes, which was reduced
to 55.16%. The relative abundance of Actinobacteria, Bacteroides,
Proteobacteria, Phylum Tenericutes, and Fusobacteria increased,
accounting for 24.03%, 8.94%, 6.31%, 3.38%, and 1.92% of total vagi-
nal flora, respectively. The bacteria of each group were shown in
Fig. 4.
1964 W. Cheng et al. / Journal of Infection and Public Health 13 (2020) 1961–1966
Fig. 4. Schematic diagram of vaginal bacteria in both groups.
Fig. 5. Schematic diagram of bacterial genera in both groups.
The genera of the vaginal flora in both groups were ana-
lyzed. In the HPV- group, Lactobacillus was the dominant genus,
accounting for 81.54%. Other genera were also included, such
as Gardnerella vaginalis, streptococcus, and Prevotella Shan and
Collins. However, their contents were less. In the HPV + group, the
content of Lactobacillus decreased significantly, accounting for only
40.48%. Meanwhile, the relative abundance of Gardnerella vaginalis
increased significantly, which increased from 3.01% to 19.87%. Both
Lactobacillus and Gardnerella vaginalis became the major genera
of the vaginal flora in the HPV + group. Compared with the HPV-
group, the contents of Prevotella Shan and Collins, Atopobium,
Leptotrichia Trevisan, Peptostreptococcus, anaerobicbacteria, and
mycoplasma were all increased. Also, Brucella appeared. The bac-
terial genera of each group were shown in Fig. 5.
Analysis results of vaginal flora among groups
Two independent sample nonparametric tests were used for sta-
tistical analysis. Among all the samples, a huge gap in PD index
values existed. The PD index of group B was the lowest at 36.42,
followed by group A and group C, successively. The PD index of
group D was the highest at 93.44, indicating the high diversity
of vaginal microorganisms. The groups were significantly differ-
ent. There was a statistically significant difference in the PD index
between group A and group B (P = 0.002), while there was no sta-
tistically significant difference with group H (P = 0.520). There was
a statistically significant difference in PD index between group B
and group C (P = 0.001). The differences in species richness index
between group D and the other groups were statistically signifi-
cant (P = 0.000, P = 0.000, P = 0.001). The Shannon index, which was
also affected by the mean, was the lowest in group A. There was
no significant difference between group A and group B (P = 0.344).
The comparison between group A and group C was statistically sig-
nificant (P = 0.002). The comparison between group B and group
C was statistically significant (P = 0.004). Compared with the rest
of the groups, the differences in the Shannon index were sta-
tistically significant (P = 0.000, P = 0.000, P = 0.001), as shown in
Figs. 6 and 7. In general, with the occurrence and development of
Fig. 6. Comparison of PD index of vaginal flora between groups.
Fig. 7. Comparison of the Shannon index of vaginal flora between groups.
cervical lesions, the diversity of vaginal microorganisms increased,
the relative abundance of each flora changed, the community
structure of the vaginal flora changed significantly, and the vagi-
nal micro-ecological changes in the cervical cancer patient group
reached the maximum.
The 16SrRNA-V4 region sequences of vaginal microorganisms
in all collected samples were sequenced by Illumina, and a total of
22 bacteria were detected. Six kinds of bacteria had relatively large
abundance and changed greatly in the occurrence and development
of cervical lesions. These bacteria were Phylum Firmicutes, Acti-
nobacteria, Proteobacteria, Bacteroides, Fusobacteria, and Phylum
Tenericutes. In the vaginal flora of group A, the Phylum Firmicutes
occupied the highest proportion, accounting for 90.69%. Also, the
sum of the remaining 21 kinds of bacteria accounted for less than
10%. Compared with group A, the proportion of Phylum Firmicutes
in the vaginal flora of group B decreased significantly, accounting
for only 56.01%. In addition, the bacteria that underwent significant
changes in the relative abundance also included Actinobacteria and
Phylum Tenericutes, which accounted for only 3.95% and 0.49% in
group A and increased to 34.33% and 7.69% in group B. In the vaginal
flora of group C, the relative abundance of the Phylum Firmicutes
did not change significantly (54.53%), and the proportion of Acti-
nobacteria was slightly reduced (26.09%). The states of bacteria of
each group were shown in Fig. 8.
The characteristics and changes of the vaginal flora commu-
nity structure in each group were analyzed at the genera level.
In group A, Lactobacillus was the dominant genus, accounting for
81.54%. Other genera were also included, such as Gardnerella vagi-
nalis and streptococcus. However, the overall contents were less.
The structure of the vaginal flora community in group B changed
significantly at the genus level. The most prominent manifestation
 W. Cheng et al. / Journal of Infection and Public Health 13 (2020) 1961–1966
Fig. 8. Schematic diagram of vaginal bacteria in each group.
Fig. 9. Schematic diagram of the genus vaginalis in each group.
was the decrease in the content of Lactobacillus and the increase in
the proportion of Gardnerella vaginalis. Both of them constituted
the dominant bacteria of the vaginal flora. The relative abundance
of Lactobacillus in group B decreased to 50.60%, and the proportion
of Gardnerella vaginalis in the entire vaginal flora increased from
3.01% in group A to 32.35%. Meanwhile, compared with group A,
the relative abundance of bacteria in group B also included Ure-
aplasma and Mycoplasma, which increased from 0.48%, 0.01% to
5.48%, 2.22%, respectively. The species diversity of vaginal microor-
ganisms in group C increased, and the internal structure of the
vaginal flora was complicated. The contents of Lactobacillus and
Gardnerella vaginalis were slightly lower than those in Group B.
The bacteria in the group with increased contents also included
Streptococcus, Prevotella Shan and Collins, and Atopobium. Except
for changes in the proportion of some relatively abundant bacteria,
the content of low-abundance bacteria and even unknown bacte-
ria in the vaginal micro-ecology increased from 4.38% in group B to
7.70%. The species diversity of the vaginal flora in group D increased
further. The most significant change in the genus level of the vagi-
nal flora was the substantial increase in the relative abundance of
low-abundance bacteria or even unknown bacteria, accounting for
19.30% of the total vaginal flora, indicating the flora community was
disordered. In addition, the relative abundance of Lactobacillus and
Gardnerella vaginalis continued to decline. The bacterial genera of
each group were shown in Fig. 9.
Discussion
Scholars have tested the HPV virus, mycoplasma, and
Ureaplasma urealyticum by vaginal secretion simultaneously.
Researchers have found that when mycoplasma is positive, the
HPV infection rate doubles. Ureaplasma urealyticum increases the
HPV infection rate by 4.7 times. Therefore, Ureaplasma urealyticum
combined with HPV infection should attract sufficient attention
[12]. In addition, scholars have suggested that the co-infection
of Ureaplasma urealyticum and HPV has a synergistic effect on
the occurrence of cervical cancer. Ureaplasma urealyticum is a
1965
cofactor for cervical cancer caused by HPV infection [13]. The
researchers have tested both mycoplasma and HPV in patients diag-
nosed with low-grade intraepithelial neoplasia. The results have
indicated that the rate of combined infection was higher in the low-
grade intraepithelial neoplasia group, i.e., HR-HPV was combined
with mycoplasma infection or Ureaplasma urealyticum infection
[14,15]. The results of this experiment were consistent with the
above results. In this study, the proportion of mycoplasma in the
four groups of vaginal flora was 0.01%, 2.22%, 0.45%, and 1.37%. The
relative abundance of Ureaplasma urealyticum in the four groups
was 0.48%, 5.48%, 0.69%, and 1.28%. Both the proportion and the rel-
ative abundance of mycoplasma were the highest in the low-level
groups.
The results of this study indicated that mycoplasma and Ure-
aplasma urealyticum might play a synergistic role in the initial
stage of cervical lesions caused by HR-HPV infection. However,
there was no significant correlation with the further progression of
cervical lesions. The specific mechanism of cervical lesions caused
by mycoplasma and Ureaplasma urealyticum and HR-HPV infection
remained to be studied. Still, the results of this study suggested that
the prevention and treatment of mycoplasma and Ureaplasma ure-
alyticum should be valued in clinical practices to prevent HR-HPV
infections and cervical lesions.
Current research has confirmed that HR-HPV infection is a
necessary factor for the development of cervical cancer, but the
progression from HR-HPV infection to cervical cancer is a long
and reversible intermediate stage. The cause of persistent infection
remains unclear. Illumina high-throughput sequencing technol-
ogy is a new sequencing technology that uses a proprietary
reversible terminator chemistry method to sequence millions of
fragments on a large scale. It can detect low-abundance bacte-
ria or even unknown bacteria [16]. In this study, the technique
was used to detect vaginal flora in vaginal secretions, which
breaks through the drawbacks of traditional methods that are
difficult to cultivate, isolate, and identify bacteria, thereby obtain-
ing accurate, comprehensive, systematic, and structured vaginal
microbial flora information. However, the research results in
this study were still not comprehensive enough. For example,
the sample types were fewer, and experimental environmental
errors were not completely removed. In the subsequent study,
samples of different living conditions would be classified and ana-
lyzed.
Conclusion
In this study, the Illumina high-throughput sequencing technol-
ogy and BIPES, a new sequence research method, were used to show
the bacterial flora distribution of vaginal microorganisms in women
with HR-HPV infection and healthy women. Meanwhile, the species
diversity and flora structure of several groups of female vaginal
microorganisms were compared and analyzed. The results showed
that the structure of vaginal flora in healthy women was relatively
simple, with Lactobacillus as the dominant bacteria, accounting for
more than 80%. The structure of vaginal flora in women infected
with HR-HPV was significantly different from that in non-infected
women. The former had a significantly increased species diversity,
which was mainly manifested by a decrease in Lactobacillus and
an increase in Gardnerella vaginalis. Under normal circumstances,
Lactobacillus is dominant in the vaginal microorganisms, which
plays an essential role in maintaining the vaginal micro-ecological
balance, inhibiting the growth of pathogenic microorganisms, and
enhancing the local anti-infection and anti-tumor capabilities of
the vagina. In the future, the research methods would be further
extended to clinical applications, thereby improving the examina-
tion efficiency of vaginal microecology.
1966 W. Cheng et al. / Journal of Infection and Public Health 13 (2020) 1961–1966
Acknowledgment
This work was supported by the General Project Funds from
the Health Department of Zhejiang Province(2017KY199 and
2018KY240).
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