Establishment of a Rapid Detection Method for Microorganisms in the Lower Genital Tract Using MALDI-TOF MS and Its Clinical Application in Infertility
Conditions: Infertility, Female
Sponsor: Shenzhen Second People's Hospital

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Establishment of a Rapid Detection Method for
Microorganisms in the Lower Genital Tract Using
MALDI-TOF MS and Its Clinical Application in
Infertility
Project source: Hospital-level clinical research at Shenzhen
Second People's Hospital
Research Unit: Shenzhen Second People's Hospital
Research leader: Zhiying Yu
Version number: V3.0
Version date: June 15, 2022
1.
Program Summary
Research Title
Establishment of a Rapid Detection Method for Microorganisms in the
Lower Genital Tract Using MALDI-TOF MS and Its Clinical Application
in Infertility
Purpose of the
study
1) Development and robustness evaluation of a rapid detection method
based on Matrix-Assisted Laser Desorption/Ionization Time of Flight
Mass Spectrometry (MALDI-TOF MS) for the simultaneous detection of
multiple pathogens and CST in the lower genital tract
2) Assessment of the correlation between lower genital tract pathogenic
infections, CST and infertility using the newly developed assay
Research
Hypothesis
Possible correlation between CST, pathogens, and infertility in the lower
genital tract
Study Design
Part I: Diagnostic tests; Part II: Case-control

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Case group
Part I: MALDI-TOF MS test; Part II: Infertile individuals
Control group
Part I: Microbiological culture; Part II: Clinically proven to be
spontaneous pregnancy
Sample size
Part I: 77 cases in each group; Part II: 114 cases in each group
Selection
Criteria
The first part of all test samples are acquired standards with no inclusion
criteria; the second part:
Inclusion Criteria:
Case group:
1) Female aged 18-40
2) Regular menstrual cycle
2) Conform to the clinical diagnosis of infertility
3) No sexual life 3 days before the examination
control group:
1) Female aged 18-40
2) Regular menstrual cycle
3) Patients who did pre-pregnancy consultation in our hospital, pregnant
at the time of 3-month follow-up
4) No sexual life 3 days before the examination
-
Exclusion Criteria:
Case group:
1) Infertility due to malformation of reproductive tract
2) Infertility caused by endocrine factors such as polycystic ovary
syndrome, premature ovarian failure and severe hyperthyroidism
3) Infertility due to male factors
4) Patients with malignant tumors
5) Patients with autoimmune diseases
6) Patients with severe heart, kidney and other basic diseases
7) Patients who consumed antibiotics 3 days before the examination

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8) Have the habit of smoking and drinking
Control group:
1) Pregnancy by various assisted reproductive means
2) Patients with malignant tumors
3) Patients with autoimmune diseases
4) Patients with severe heart, kidney and other basic diseases
5) Patients who consumed antibiotics 3 days before the examination
6) Have the habit of smoking and drinking
Exclusion
Criteria
All samples tested in Part I were acquired standards with no inclusion
criteria; Part II:
Case group:
1) Infertility due to malformation of reproductive tract
2) Infertility caused by endocrine factors such as polycystic ovary
syndrome, premature ovarian failure and severe hyperthyroidism
3) Infertility due to male factors
4) Patients with malignant tumors
5) Patients with autoimmune diseases
6) Patients with severe heart, kidney and other basic diseases
7) Patients who consumed antibiotics 3 days before the examination
8) Have the habit of smoking and drinking
Control group:
1) Pregnancy by various assisted reproductive means
2) Patients with malignant tumors
3) Patients with autoimmune diseases
4) Patients with severe heart, kidney and other basic diseases
5) Patients who consumed antibiotics 3 days before the examination
6) Have the habit of smoking and drinking
Ending
evaluation
Main indicators: microbiological test results, are recorded as negative,
positive

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indicators
Safety
evaluation
indicators
Pain indicators: pain score; bleeding indicators: bleeding volume
assessment (blood staining area method), blood routine; cervical and
vaginal infection: white belt routine
2.
Research Background
1.1 Research significance
The cervical canal and vagina (collectively referred to as the lower genital tract)
are important components of the female reproductive tract, and infections of the
lower genital tract are also the most common gynecologic conditions. Infections
such as Neisseria gonorrhoeae (NG), Mycoplasma urealyticum (UU) and Chlamydia
trachomatis (CT) not only cause discomfort, but may also lead to infertility, ectopic
pregnancy, miscarriage and premature birth. These infections not only cause
uncomfortable symptoms, but also may lead to infertility, ectopic pregnancy,
miscarriage, premature birth and other adverse pregnancy outcomes, which seriously
affect women's physical and mental health and family harmony.1-2 The infection may
lead to infertility, ectopic pregnancy, miscarriage, preterm delivery and other adverse
pregnancy outcomes, which can seriously affect women's physical and mental health
and family harmony. Disruption of the ecosystem of the lower genital tract can lead
to overgrowth of pathogens, resulting in vaginal infections and sexually transmitted
infections (STIs).3 The disruption of the lower reproductive tract ecosystem can lead
to an overgrowth of pathogens that can cause vaginal infections and sexually
transmitted infections (STIs). The community state type (CST) of cervical and
vaginal colonizing bacteria is the most important component of the lower genital
tract microecosystem and has an important impact on the health of the lower genital
tract.4 It has an important impact on the health of the lower reproductive tract.
Recent studies have shown that abnormal CST is strongly associated with infertility
and poor pregnancy outcomes.4-5 . Therefore, rapid, accurate and cost-effective
detection of common pathogens and CST in the reproductive tract is a technique that
gynecologists are eager to achieve, not only to comprehensively analyze the etiology

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of infertility and poor pregnancy outcomes in patients, but also to further investigate
the correlation between dynamic changes in CST and various pathogenic infections
and diseases in the lower reproductive tract.
Currently, although inflammatory vaginal diseases such as mycosis fungoides,
bacterial vaginosis, and trichomoniasis can be detected rapidly (30-60 minutes) by
simultaneous routine leukocyte or vaginal microecological evaluation. However, in
order to assess the infection status of the lower genital tract, sampling, culture and
testing of Neisseria gonorrhoeae, mycoplasma and chlamydia need to be performed
separately, which takes a long time (ranging from 2-5 days) and increases the
patient's pain and may lead to an increase in the false-negative rate of the later
specimens due to repeated sampling, and CST analysis is costly and time-consuming
due to the use of 16SrRNA and high-throughput sequencing. Currently, CST analysis
is only used in scientific research and lacks practical significance for clinical
diagnosis and treatment of patients.
The emerging Matrix-Assisted Laser Desorption/Ionization Time of Flight
Mass Spectrometry (MALDI-TOF MS) detection system can detect multiple
pathogenic microorganisms and subtypes simultaneously than traditional detection
techniques such as fluorescent PCR technology, with high detection stability and
accuracy, and lower cost and faster analysis than second-generation sequencing, and
has been gradually used in recent years. in clinical testing6 The project team has
previously used MALDI to detect a variety of pathogenic microorganisms. The
project team has previously established a cervical fluid-based cell PAX1 methylation
detection platform using MALDI-TOF MS (Patent No. CN202010753067.8., paper
submitted), and has extensive experience in the development of MALDI-TOF MS
platform for clinical testing. This study will use the MALDI-TOF MS platform to
establish a combined detection system for common pathogens in the lower genital
tract and CST to fill this technical gap and address the practical needs of clinicians.
1.2 Current status of domestic and international research.
Sexually transmitted infections (STIs) are a major global health problem, with

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127 million and 27 million adults worldwide having Chlamydia trachomatis and
gonococcal infections in 2012, according to statistics.7 . Both infections are mainly
asymptomatic and curable, but if untreated or ineffective, they can lead to pelvic
inflammatory disease, infertility and ectopic pregnancy, and Chlamydia trachomatis
infection is also associated with a variety of adverse pregnancy outcomes, such as
chorioamnionitis,
preterm
delivery
and
low
birth
weight1
.
Reproductive
mycoplasma infections are as high as 2.0%-7.3% and are thought to be associated
with UTIs, cervicitis, pelvic inflammatory disease, and infertility8-9 .
The vast majority of patients with lower genital tract pathogens are only
transiently infected and can be cleared by their own or pharmacological treatment,
while only a minority of patients have serious adverse outcomes, suggesting that
other factors influence the pathogenic evolution of these pathogens. Previous studies
have shown that the type and abundance of long-term parasitic flora in the human
cavity are closely related to important aspects of disease development such as
immune regulation, inflammatory response, cytogenetic mutation, angiogenesis, and
tissue remodeling.10-11 . Therefore, in recent years, microecology has received a lot of
attention from many fields such as immunity and metabolism. Since the discovery of
Lactobacillus vaginalis by Albert Doderlein in 1982, a number of studies have been
conducted to demonstrate that a decrease in the absence of Lactobacillus vaginalis is
a major cause of inflammatory vaginal diseases such as mycobacteria and bacterial
vaginitis.12 However, because early studies on genital tract pathogens were mainly
performed by microbiological microscopy, staining, and culture, it was not possible
to subtype Lactobacillus, and the correlation between lower genital tract CST and
obstetrical and gynecological diseases has only recently been emphasized. Recently,
many scholars have studied the relationship between vaginal CST and obstetric and
gynecological diseases, and such studies have suggested that abnormal CST in the
lower genital tract can lead to pelvic inflammatory disease13-15 infertility15-17 and
adverse pregnancy outcomes.15, 18 However, does abnormal CST promote gonorrhea?
However, it is unclear whether CST abnormalities promote infection and persistence
of infertility-causing pathogens such as Neisseria gonorrhoeae, Mycoplasma

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urealyticum and Chlamydia trachomatis.
Currently, most clinical tests for Neisseria gonorrhoeae, mycoplasma and
chlamydia are performed using microbial cultures, and vaginal CST analysis is
performed
using
16SrRNA
or
macrogenomic
sequencing.19-21
.
Therefore,
performing multiple analyses on a patient at the same time is not only expensive, but
also requires multiple sampling, which affects the accuracy of the results and makes
it difficult to be widely implemented in the clinic. To the best of our knowledge,
there is no accurate and cost-effective method for simultaneous detection of common
pathogenic microorganisms in the lower genital tract. Therefore, this study proposes
to establish a combined detection system for common pathogens and CST in the
lower genital tract based on the MALDI-TOF MS testing platform, which will
enable clinicians to accurately detect pathogens and common microorganisms in the
lower genital tract with a single sample at a reasonable testing cost, and provide
more accurate information and evidence for patient disease status assessment and
treatment guidance. In addition, the project team will evaluate and compare the
lower genital tract microecology of infertile and healthy control women using the
constructed assay, and initially explore the correlation between lower genital tract
microecology and infertility, and the correlation between various pathogenic
infections and lower genital tract CST.
1.3 References
1.
Tsevat, D. G.; Wiesenfeld, H. C.; Parks, C.; Peipert, J. F., Sexually transmitted diseases and
infertility. Am J Obstet Gynecol 2017, 216 (1), 1-9.
2.
Carey, A. J.; Beagley, K. W., Chlamydia trachomatis, a hidden epidemic: effects on female
reproduction and options for treatment. Am J Reprod Immunol 2010, 63 (6), 576-86.
3.
Chee, W. J. Y.; Chew, S. Y.; Than, L. T. L., Vaginal microbiota and the potential of
Lactobacillus derivatives in maintaining vaginal health. Microb Cell Fact 2020, 19 (1), 203.
4.
Smith, S. B.; Ravel, J., The vaginal microbiota, host defence and reproductive physiology. J
Physiol 2017, 595 (2), 451-463.
5.
Dunlop, A. L.; Satten, G. A.; Hu, Y. J.; Knight, A. K.; Hill, C. C.; Wright, M. L.; Smith, A. K.;
Read, T. D.; Pearce, B. D.; Corwin, E. J., Vaginal Microbiome Composition in Early Pregnancy and
Risk of Spontaneous Preterm and Early Term Birth Among African American Women. Front Cell
Infect Microbiol 2021, 11, 641005.
6.
Tsuchida, S.; Umemura, H.; Nakayama, T., Current Status of Matrix-Assisted Laser
Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) in Clinical Diagnostic

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Microbiology. Molecules 2020, 25 (20).
7.
Newman, L.; Rowley, J.; Vander Hoorn, S.; Wijesooriya, N. S.; Unemo, M.; Low, N.; Stevens,
G.; Gottlieb, S.; Kiarie, J.; Temmerman, M., Global Estimates of the Prevalence and Incidence of
Four Curable Sexually Transmitted Infections in 2012 Based on Systematic Review and Global
Reporting. PLoS One 2015, 10 (12), e0143304.
8.
McGowin, C. L.; Anderson-Smits, C., Mycoplasma genitalium: an emerging cause of sexually
transmitted disease in women. PLoS Pathog 2011, 7 (5), e1001324.
9.
Lis, R.; Rowhani-Rahbar, A.; Manhart, L. E., Mycoplasma genitalium infection and female
reproductive tract disease: a meta-analysis. Clin Infect Dis 2015, 61 (3), 418-26.
10.
Man, A. W. C.; Li, H.; Xia, N., Resveratrol and the Interaction between Gut Microbiota and
Arterial Remodelling. Nutrients 2020, 12 (1).
11.
Karki, R.; Kanneganti, T. D., Diverging inflammasome signals in tumorigenesis and potential
targeting. Nat Rev Cancer 2019, 19 (4), 197-214.
12.
Mitra, A.; MacIntyre, D. A.; Marchesi, J. R.; Lee, Y. S.; Bennett, P. R.; Kyrgiou, M., The
vaginal microbiota, human papillomavirus infection and cervical intraepithelial neoplasia: what do we
know and where are we going next? Microbiome 2016, 4 (1), 58.
13.
Wang, J.; Li, Z.; Ma, X.; Du, L.; Jia, Z.; Cui, X.; Yu, L.; Yang, J.; Xiao, L.; Zhang, B.; Fan, H.;
Zhao, F., Translocation of vaginal microbiota is involved in impairment and protection of uterine
health. Nat Commun 2021, 12 (1), 4191.
14.
Sharma, H.; Tal, R.; Clark, N. A.; Segars, J. H., Microbiota and pelvic inflammatory disease.
Semin Reprod Med 2014, 32 (1), 43-9.
15.
Chen, C.; Song, X.; Wei, W.; Zhong, H.; Dai, J.; Lan, Z.; Li, F.; Yu, X.; Feng, Q.; Wang, Z.; Xie,
H.; Chen, X.; Zeng, C.; Wen, B.; Zeng, L.; Du, H.; Tang, H.; Xu, C.; Xia, Y.; Xia, H.; Yang, H.; Wang,
J.; Wang, J.; Madsen, L.; Brix, S.; Kristiansen, K.; Xu, X.; Li, J.; Wu, R.; Jia, H., The microbiota
continuum along the female reproductive tract and its relation to uterine-related diseases. Nat
Commun 2017, 8 (1), 875.
16.
Salliss, M. E.; Farland, L. V.; Mahnert, N. D.; Herbst-Kralovetz, M. M., The role of gut and
genital microbiota and the estrobolome in endometriosis, infertility and chronic pelvic pain. Hum
Reprod Update 2021, 28 (1), 92-131.
17.
Hong, X.; Ma, J.; Yin, J.; Fang, S.; Geng, J.; Zhao, H.; Zhu, M.; Ye, M.; Zhu, X.; Xuan, Y.;
Wang, B., The association between vaginal microbiota and female infertility: a systematic review and
meta-analysis. Arch Gynecol Obstet 2020, 302 (3), 569-578.
18.
Vergaro, P.; Tiscornia, G.; Barragan, M.; Garcia, D.; Rodriguez, A.; Santalo, J.; Vassena, R.,
Vaginal microbiota profile at the time of embryo transfer does not affect live birth rate in IVF cycles
with donated oocytes. Reprod Biomed Online 2019, 38 (6), 883-891.
19.
Louis, P.; Hold, G. L.; Flint, H. J., The gut microbiota, bacterial metabolites and colorectal
cancer. Nat Rev Microbiol 2014, 12 (10), 661-72.
20.
Velmurugan, G.; Ramprasath, T.; Gilles, M.; Swaminathan, K.; Ramasamy, S., Gut Microbiota,
Endocrine-Disrupting Chemicals, and the Diabetes Epidemic. Trends Endocrinol Metab 2017, 28 (8),
612-625.
21.
Brotman, R. M.; Shardell, M. D.; Gajer, P.; Tracy, J. K.; Zenilman, J. M.; Ravel, J.; Gravitt, P.
E., Interplay between the temporal dynamics of the vaginal microbiota and human papillomavirus
detection. J Infect Dis 2014, 210 (11), 1723-33.

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3. Purpose of the study
1)
Development
and
robustness
of
a
rapid
detection
method
for
microorganisms in the lower genital tract based on time-of-flight mass spectrometry
(MALDI-TOF MS) for simultaneous detection of multiple pathogens and CST
2)
Assessment of the association between lower genital tract pathogenic
infections, CST and infertility using a newly developed assay
4. Study design
The first part of this study is a diagnostic test using using a standard pathogenic strain;
the second part is a prospective case-control study
5. Case selection
5.1 Entry Criteria
1. Subjects voluntarily enrolled in the study and signed an informed consent form
2. The inclusion criteria for the case and control groups were as follows.
Case group:
1) Female aged 18-40
2) Regular menstrual cycle
2) Conform to the clinical diagnosis of infertility
3) No sexual life 3 days before the examination
control group:
1) Female aged 18-40
2) Regular menstrual cycle
3) Patients who did pre-pregnancy consultation in our hospital, pregnant at the time of
3-month follow-up
4) No sexual life 3 days before the examination
5.2 Exclusion criteria.
Case group:
1) Infertility due to malformation of reproductive tract
2) Infertility caused by endocrine factors such as polycystic ovary syndrome,

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premature ovarian failure and severe hyperthyroidism
3) Infertility due to male factors
4) Patients with malignant tumors
5) Patients with autoimmune diseases
6) Patients with severe heart, kidney and other basic diseases
7) Patients who consumed antibiotics 3 days before the examination
8) Have the habit of smoking and drinking
Control group:
1) Pregnancy by various assisted reproductive means
2) Patients with malignant tumors
3) Patients with autoimmune diseases
4) Patients with severe heart, kidney and other basic diseases
5) Patients who consumed antibiotics 3 days before the examination
6) Have the habit of smoking and drinking
5.3 Exit Criteria
Case group.
1）Unsatisfactory sample nucleic acid extraction
2）Intraoperative
detection
of
developmental
malformations
or
other
non-colony related factors that can clearly cause infertility
Control group.
1)
Unsatisfactory sample nucleic acid extraction
2)
No spontaneous pregnancy at the time of follow-up
6. estimation of sample size and grouping method
6.1 Estimation of sample size
6.1.1 Part I: Comparison of sensitivity of dose information between the two groups
Single and mixed standard bacterial solutions and blank controls were 11 in total, so
the chance of the appearance of four microorganisms in various standard strain solutions
was 0.18, and different studies showed that the culture sensitivity of gonococci, lactobacilli,
mycoplasma, and chlamydia was 40-60% and specificity 95-98%; time-of-flight mass
spectrometry detection was >99% in different studies, and the parameters were brought into

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PASS according to α=0.1 Software calculation path: Tests for Two Independent Sensitivities,
N=44-72, taking the least integer multiple of 11 for >72, i.e., 77 cases in each group
6.1.2 Part 2: Comparison of independent sample rates between the two groups
Comparison of the independent sample rates of the two groups: the infection rate of at
least one pathogen in the whole population is about 0.01%, and the infection rate of patients
who visited our hospital for infertility is about 0.46%, and this data was brought into SPSS
software: Confidence Intervals for the Difference Between Two Proportions, n=114 cases,
so 114 cases were included in the experimental group and the control group each
6.2 Grouping method
Case group were patients with clinical diagnosis of infertility: sexually active,
uncontraceptive and infertile for 2 years
The control group was those with spontaneous pregnancy: those with spontaneous
pregnancy at the follow-up visit 3 months after consultation
7. Interventions
This study did not involve an intervention
8. Research steps
8.1 Part I. Development of a rapid detection method based on MALDI-TOF MS
for simultaneous detection of multiple pathogens and CST in the lower genital
tract and evaluation of diagnostic efficacy
8.1.1 Find the relatively conserved regions of virulence factor sequences of
each pathogen according to the database, intercept the specific sequences from the
conserved regions of each pathogen to design multiplex PCR amplification primers
and single base extension (SBE) primers for each strain to construct MALDI-TOF
MS assays.
8.1.2 Purchase standard pathogen strains, culture standard concentrations (i.e.,
high concentrations) of standard pathogens using the culture method to identify
standard pathogens and their activities
8.1.3 Formulation of single, mixed and negative control standards with standard
pathogens.

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8.1.4 Gold standard: The actual pathogen type added to the standard is the gold
standard.
8.1.5 Detection of standards at gradient dilution concentrations by incubation
method and MALDI-TOF MS, respectively.
8.1.6 Performing diagnostic effectiveness analysis
8.2 Part II Assessment of the association between lower genital tract pathogen
infection, CST and infertility using newly developed assays
8.2.1 Select infertile women of childbearing age who meet the criteria for nadir
to sign informed consent and be enrolled in the study.
8.2.2 Completion of an information collection form to obtain information on
confounding factors that may affect this study (age, weight, height, ethnicity,
ancestry, place of long residence, number of pregnancies and births, time of last
menstruation and menstrual cycle, duration of infertility, relevant diagnoses
leading to infertility, etc.), which is collected daily by the information
management specialist and kept.
8.2.3 Sample collection: take cervical canal and vaginal secretions during
routine
gynecological
examination
and
routine
leucorrhea
examination
secretions during routine consultation. ④Place the swab head in sterile storage
solution and tighten the cap ⑤Label the bottle with patient information ⑥
Store in 4℃refrigerator.
8.2.4 Isolation and extraction of pathogen DNA.
8.2.5 Detection of microorganisms in the lower genital tract by a constructed
MALDI-TOF MS assay.
8.2.5 Uniform disposal of remaining specimens in accordance with medical
waste after sample testing is completed.
8.2.6 Statistical analysis to assess the correlation between various pathogenic
infections and CST status and infertility separately
8.2.7 Statistical analysis to assess differences in CST status between infected
and uninfected individuals with various pathogens
8.3 Sample preservation and handling

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All specimens will be stored in a 4℃refrigerator on the same day and DNA will be
extracted within 24 hours. DNA specimens will be marked with specimen number and
sampling time as required and then stored in a -80℃refrigerator in the gynecology
department of the central laboratory, and the remainder will be disposed of in accordance
with the medical waste disposal process. After the DNA specimens are tested and the results
are analyzed, they will be disposed of according to the medical waste disposal process.
9. Research Flow Chart
Part I.
Part II.

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10. Ending evaluation indicators
10.1 Mycoplasma solium, Mycoplasma humanum, Chlamydia antigen, and gonococcus were
recorded as positive or negative according to the clinical report.
10.2 MALDI-TOF MS test results were recorded as: microbial type, positive, negative
11. Security evaluation
This study involves only 1 vaginal and cervical secretion collection, which is consistent
with HPV sampling during cervical screening in routine clinical testing and does not cause
additional collection points or additional consultation costs, and is safe. It may cause pain and a
little bleeding to the patient, neither of which will have any impact on the patient's health. Inform
the patient before the operation to avoid causing panic. If the patient has intercourse immediately
after the test or performs inappropriate transvaginal operations that may lead to infection, the
patient is advised to avoid intercourse, sitz baths and other transvaginal operations without
professional medical guidance for 3 days after sampling.
11.1 Vital signs observation and laboratory tests
Pain: WHO Pain Classification
Bleeding:
①Bleeding
volume
assessment:
(according
to
the
bleeding
area
assessment method in the Guidelines for the Diagnosis and Treatment of Abnormal
Uterine Bleeding); ②Blood count
Infections: white belt routine, blood count
11.2 Adverse Events
(List of possible adverse events, all possible adverse reactions in the study should be
fully considered, and should include possible adverse reactions in all aspects of laboratory
tests (e.g. blood collection, ECG, imaging, etc.), interventions (e.g. drug treatment, surgical
treatment, etc.) and other non-interventions (e.g. postural tests, scale tests, questionnaires,
etc.), and evaluate the severity of the adverse reactions . (The same should also be reflected
in the informed consent form, fully informed to the subject.)
Pain: pain can be graded (0-4), ≥1 noted as an adverse event
Grading: Grade 0: No pain.
Grade 1 (mild pain): painful but not severe, tolerable, sleep is not affected
Grade 2 (moderate pain): pain is obvious and unbearable, sleep is disturbed,

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analgesics are requested
Grade 3 (severe pain): severe pain, unbearable sleep severely disturbed,
analgesics needed
Bleeding: small amount 0.1-1ml, medium amount 1-5ml, large amount >5ml, medium
and large amount of bleeding recorded as adverse events
Infections.
Mild infection - increased cervical or vaginal discharge, white belt routine
indicating cleanliness grade III or leukocytes +++
Moderate - Large amount of discharge with abnormal characteristics, white belt
routine indicating cleanliness IV or white blood cells ++++ or BV positive
Severe - with pain or pressure and the presence of infection are recorded as
adverse events
11.3 Pre-assessment of relevant risks and disposal plans
Pain: ≥grade 1 may be given pain medication
Bleeding: Vaginal gauze is used to stop heavy bleeding
Infection: mild infection - vaginal douching; moderate - vaginal medication based on
routine
leucorrhoea
test
results;
severe
-
vaginal
medication
combined
with
oral
anti-infection therapy based on routine leucorrhoea test results
*The study sponsor will bear all the costs related to the above adverse events and
exempt the subjects from the costs related to the adverse events, including all the costs
related to the adverse events during and after the end of the trial.
12. Data management and statistical analysis
(The following points should be clarified.
a. Whether data collection, processing and analysis are managed by a dedicated person
and the manager has a GCP certificate.
b. the use of questionnaires, completed by patients with the assistance of the
investigator, to complete the questionnaires and collect data.
c. Using Empower statistical software, sensitivity (Se), specificity (Sp), positive
predictive value (PV+), and negative predictive value (PV-) were used for the first part of
the diagnostic efficacy assessment, and chi-square test, rank sum test, stratified analysis,

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and logistic regression correlation analysis were used for the second part.
d. Establish standard operating procedures and quality control procedures for each part
of the operational process, and the experimental test report form must be complete with all
items and all reports written using uniform standards to ensure accurate and valid data.
e. All data are collected and managed in a confidential account, and the paper files are
stored in the department's clinical research file safe, so that any non-data administrator
conducting data analysis cannot obtain patients' personal privacy information such as name,
ID number, telephone number, and address.
13. Confidentiality measures
The results of the research through this project may be published in medical journals, but
the investigators will keep the patients' information confidential as required by law and the
patients' personal information will not be disclosed. When necessary, the government
administration and the hospital ethics committee and its related personnel may have access to the
patient's information as required.
14. Ethical standards
The study protocol and informed consent for this study should be approved in writing by the
Clinical Research Ethics Committee of Shenzhen Second People's Hospital before proceeding.
All original informed consent forms signed and dated by the subject or their legal
representative and the person conducting the informed consent process should be kept in a
copy by the investigator.
The investigator or a person authorized by the investigator will be responsible for
explaining the benefits and risks of participation in the study to each patient, the patient's legal
representative or notary witness, if any, and shall obtain written informed consent prior to the
patient's entry into the study.
This study is a non-interventional study. Only one additional tube of cervical and vaginal
secretions was collected during routine gynecological examinations, and the sampling method
was the common clinical method for HPV DNA testing. All subjects participating in the study
were exempted from registration fees and gynecological examination fees (which are necessarily
incurred in the course of routine treatment) as financial subsidies for study-related visits and
follow-ups.