Microbiota dysbiosis is associated with HPV‑induced cervical carcinogenesis
- Authors:
- Published online on: September 26, 2018 https://doi.org/10.3892/ol.2018.9509
- Pages: 7035-7047
Abstract
Introduction
It has been reported that the female cervix maintains communities of microbial species, which have a symbiotic relationship with the host (1). It has been demonstrated that the female cervix is colonized by diverse microbiota, which serve a crucial role in cervicovaginal health (1). However, it has been indicated that the type of organisms present is dependent on the prevailing environmental conditions and host factors (1,2). Over the last decade, it has been indicated that the development and introduction of molecular-based technology has provided novel information regarding the composition of the vaginal-cervical flora, as well as the abnormal colonization of the genital tract by pathogens (2). The aforementioned findings may help to elucidate the microbiome of the genital tract in healthy women and in human papilloma virus (HPV)-dependent carcinogenesis.
The majority of previous studies have indicated that the cervical/vaginal microbial flora has a prevalence of Lactobacillus species, which produce lactic acids that maintain an acidic environment and may inhibit pathogenic growth (2–8). Specifically, lactic acid and other related acidic compounds have been reported to inhibit bacterial growth associated with bacterial vaginosis (BV), as well as viral infections (3). In addition, lactic acid has been recognized as a component of the immune defence system, as it has been demonstrated to potentiate the production of protective proinflammatory cytokines by vaginal epithelial cells, to promote the activation of T helper 17 lymphocytes, to stimulate dendritic cell maturation and induce interferon production (1). Over 120 Lactobacillus species have been identified and over 20 species have been identified in the vagina (2).
The dominant microorganisms in the vagina of a healthy woman during puberty have been reported to be from the Lactobacillus genus, including L. acidophilus, L. fermentum, L. plantarum, L. brevis, L. jenseni, L. casei, L. catenaforme, L. delbrueckii and L. salivarius (1,2). It is considered that in the majority of cases, vaginal inflammation is not caused by novel microorganisms introduced from the outside, but instead by a disturbance to the proportion and number of microorganisms already existing in the vagina (4). Each bacterium has been reported as a potential etiological factor of inflammation, including Lactobacillus (4). It has been reported that anaerobic bacteria have a significantly increased pathogenic potential compared with aerobic bacteria (5,6). Factors, which have been reported to potentially disturb vaginal biocenosis include the following hormonal changes: Pregnancy, puberty, menopause and hormonal contraception, particularly using low doses of estrogen; vaginal sterilization following chemo- or antibiotic therapy; surgical conditions such as vaginoplasty, erosions, poor genitalia hygiene, including the vulva and the vagina, and lack of a regimented sex life, i.e. frequent, unprotected, lack of a regular sex life (4–6). Large variation in the number and composition of bacteria has been reported among women, and among time intervals for one woman (5,6). Therefore, attempting to evaluate the vaginal microbiome is extremely difficult.
A healthy cervicovaginal microenvironment has been reported to be characterised by high levels of different species of Lactobacillus, including the predominant L. crispatus, L. iners, L. jensenii and L. gasseri. Other species may occur occasionally (2,6). In rare cases, it has been reported that the cervix can be colonized by the same two or four Lactobacillus species (4). The aforementioned cases have been demonstrated to be dependent on a number of genetic and environmental factors, including nationality, diet and age (2,6). A number of previous studies have indicated that, in a significant proportion of healthy women, the Lactobacillus in the vagina may be replaced by other lactic acid-producing bacteria, including Atopobium vaginae, Megaspharea and Leptotrichia species (3). It has been indicated that an abnormal vaginal microenvironment may be caused by sexually transmitted infection (3). It has been reported that trichomoniasis may be caused by colonization with a microorganism not commonly identified within vaginal colonies, including Streptococcus pneumoniae, Haemophilus influenzae and Listeria monocytogenes (6,7). In addition, it has been reported that an abnormal microenvironment may be caused by an invasion of an alternative organism, which is a component of the normal vaginal flora, including Escherichia coli (3). Bacterial vaginosis has been reported as a disorder characterised by a decrease in the quality or quantity of Lactobacillus and the growth of Mycoplasma hominis, Gardnerella vaginalis, Mobiluncus species, Neisseria gonorrhea, Trichomonas vaginalis, Chlamydia trachomatis and Prevotella species (4,5). A previous meta-analysis reported a positive association between cervical HPV infection and BV (6,7). The reverse phenomenon of HPV as a risk factor for BV invasion has also been described (8).
It has been demonstrated that HPV is considered a principal factor responsible for the development of cervical cancer (8). However, it has been suggested that HPV infection alone does not cause cervical carcinogenesis and that other factors, such as prolonged oral contraceptive or smoking, may be involved in the disease progression (8). To determine whether the vaginal microflora is affected by one of these factors, the present study investigated the association between cervical and vaginal infections, and pre-cancerous lesions of the cervix.
Materials and methods
Patient samples
Women included in the present study were recruited from cervical cancer screening in the Department of Gynaecological Oncology and Gynaecology, Medical University of Lublin in Poland between February 2003 and August 2015. The study group consisted of 250 women. Patients whose molecular analysis indicated that they were HPV-positive [HPV(+)] were included within the analysed group (n=180), whereas the healthy women [HPV(−)] were included within the control group (n=70).
Cytology and HPV status were used to classify the patients into the following 3 groups: Control group [n=70); HPV(−)]; women with low-grade squamous intraepithelial lesions [LSIL; n=95; HPV(−)], and women with high-grade squamous intraepithelial lesions [HSIL; n=85; HPV(+)]. The mean ages of patients with LSIL and HSIL, and the control group, did not significantly differ: 35 years (range, 25–48) and 37 years (range, 29–48) compared with 37 years (range 21–48), respectively (P>0.05). The present study was approved by the Ethics Committee of the Medical University of Lublin (Lublin, Poland). Written informed consent was obtained from all participants in the present study and the research was performed in accordance with the Declaration of Helsinki.
The patient inclusion criteria for the present study for the research and control groups were as follows: i) Cytological diagnosis of LSIL or HSIL or patients with a normal cytological swab; ii) HPV (+) or (−) status; iii) no use of oral and/or vaginal probiotics for 30 days prior to the start of the present study; iv) absence of genital tract infection during the 30 days prior to the classification of patients in the study, and v) a maximum of one sexual partner for 30 days prior to qualification testing.
The exclusion criteria for participating in the present study included the following: i) Vaginal bleeding of unknown etiology; ii) pregnancy; iii) oral contraceptive use or hormone replacement therapy; iv) cigarette smoking; v) history of other types of cancer; vi) systemic diseases; vii) diabetes, and viii) thyroid or other endocrine diseases.
Cervical swabs were collected from patients and to rule out experimental bias or random error. pooling was performed in 3 subgroups as follows: in the control group 23, 23, 24 swabs were pooled; in the LSIL group 31, 32, 32 were pooled, and in the HSIL group 28, 28, 29 swabs were pooled. The pooled cervical swabs were stored immediately at −80°C for a maximum 12 months. The cervical cytological findings were classified according to the Bethesda system (9).
DNA isolation from the swabs
Total DNA was extracted from cells using a DNA isolation kit (QIAamp DNA kit; cat. no. 51306; Qiagen GmbH, Hilden, Germany), according to the manufacturer's protocols.
Identification of HPV DNA
Identification of HPV-derived DNA was performed by polymerase chain reaction (PCR) amplification of HPV gene sequences. The MY09, MY11, LC1 and LC2 primers (Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland) that were complementary to the genomic sequence of the predominantly diagnosed HPV types were used, as previously described (10).
Typing of 16S ribosomal DNA (16S rRNA) by PCR-amplification and next generation sequencing
The V4 hypervariable regions of 16S rRNA in bacterial genes were detected by PCR, as previously described (11). Sequencing and sequence analysis was performed at Genomed S.A, Warszawa (Poland).
Statistical analysis
The difference in the mean age of the female patients was tested using the Kruskal-Wallis test and was statistically significant if the calculated P>0.05. Using the 16S rRNA gene sequencing data, the frequency of bacteria occurrences were calculated by multiplying the total number of present bacteria with the percentage of bacterial concentration in the cervical swabs. The statistical analysis was performed using k-means cluster analysis (k-means clustering algorithm) and Statistica 12.0 software (StatSoft Inc., Cracow, Poland).
Results
Identification and cluster analysis of bacteria in the control, LSIL HPV(+) and HSIL HPV(+) groups
16S rRNA sequence-based methods were used to identify healthy cervical microbial colonies, as well as those associated with HPV-dependent carcinogenesis. The identified microflora were grouped into CSTs as follows: Control, HPV(−); LSIL, HPV(+), and HSIL HPV(+). A total of 6,466 bacteria species, belonging to 74 classes, were identified from the vaginal/cervical swabs of the patients. The major classes observed are presented in Table I.
Identification and cluster analysis of bacterial classes in the control, LSIL HPV(+) and HSIL HPV(+) groups
Frequency analysis of bacterial classes in the CST healthy women, as well as in patients with a diagnosis of LSIL HPV(+) or HSIL HPV(+) revealed the presence of three major clusters. The results of the bacterial classes cluster analysis are presented in Table II. The bacterial classes, which were distinct from the other classes regardless of the type of diagnosis, included Bacilli, Actinobacteria and Gammaproteobacteria (Table II). Analysis of the frequencies of the individual classes demonstrated that the CST composition in the healthy women, as well as the women diagnosed with LSIL was formed predominantly of the Bacilli bacteria. In CST patients with cancer, the Gammaproteobacteria class was additionally detectable. Further cluster analysis, from classes to species, was performed to determine the bacterial species making up the three selected bacteria classes.
 | Table II.Three major clusters of bacteria classes in volunteers and patients with LSIL and HSIL diagnosis. |
Identification and cluster analysis of bacterial species in the control, LSIL HPV(+) and HSIL HPV(+) groups
Analysis of the bacteria species in each of the three classes of bacteria revealed no cluster formation within the Gammaproteobacteria class. The further analysis subsequently focussed only on the Actinobacterium and Bacilli classes of bacterial species. Regardless of the type of diagnosis, the bacterial species within the analysed classes formed two clusters. The results are presented in Tables III and IV (data repository).
| Table III.The bacteria species of the class Actinobacterium forming clusters in volunteers and patients with L-SIL or H-SIL histopathological diagnosis. Gr.- cluster number, Dist.- Euclidean distance. |
| Table IV.The bacteria species of the class Bacilli forming clusters in volunteers and patients with L-SIL or H-SIL histopathological diagnosis. Gr.- cluster number, Dist.- Euclidean distance. |
The Actinobacterium class included Gardnerella vaginalis and Propionibacterium acnes identified in the CST healthy women; Gardnerella vaginalis and Actinomyces turicensis in the CST women diagnosed with LSIL, and Gardnerella vaginalis, Corynebacterium glaucum, Corynebacterium matruchotii, Propionibacterium acnes and Propionibacterium humerusii in women diagnosed with HSIL, which formed clusters separate from the other bacterial species (Table III, data repository).
The cluster analysis of the Bacilli bacterial species revealed the presence of a CST subpopulation, which in the healthy women consisted of Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis. In women diagnosed with LSIL it included Lactobacillus iners and Lactobacillus acidophilus, and in women diagnosed with HSIL it included Lactobacillus iners, Lactobacillus acidophilus and Lactobacillus crispatus (Table IV, data repository). For further analysis, the levels of bacterial characteristics were determined in each of the analysed patient groups (Table V).
| Table V.Analysis of the selected bacteria species frequency in control, LSIL and HSIL diagnosed patients. Frequencies >10 have been highlighted. |
The analysis of the bacterial classes indicated that CST cervical swabs of the female patients were colonised by Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis, however, Gardnerella vaginalis and Lactobacillus acidophilus were almost not identified. In the CST patients diagnosed with LSIL, the predominant types of bacteria were Lactobacillus acidophilus and Lactobacillus iners, while Lactobacillus crispatus frequency was lower than in the control group. In CST patients diagnosed with HSIL, high abundance of Gardnerella vaginalis, Lactobacillus acidophilus was identified, however, Lactobacillus taiwanensis, Lactobacillus iners and Lactobacillus crispatus frequencies were lower than in the control group. The level of Lactobacillus acidophilus in the CST patients diagnosed with LSIL swabs were compared with swabs taken from women with HSIL.
Discussion
It has been indicated that the introduction of Next-Generation Sequencing (NGS) into research allowed for the identification of specific ecological niches for microorganisms within living organisms. This was possible due to the amplification and parallel sequencing of gene fragments, which were highly conserved among microorganisms, the most common being the 16S rRNA subunit, as well as RAD51 recombinase and inhibin subunit a (12). Highly conserved regions of the 16S rRNA gene (V1-V9) have been reported to allow for phylogenetic and taxonomic characterisation of the analysed microbial communities (12). In the present study, the V4 hypervariable regions of 16S rRNA were used, which allowed for the identification of 3 CSTs of HSIL HPV(+), LSIL HPV(+) and the healthy group HPV(−).
In addition, >70 classes of bacteria were identified and the analysis of their frequencies demonstrated that cervical swabs of flora obtained from the volunteers and women diagnosed with LSIL HPV(+) were predominantly composed of the Bacilli class. The presence of Gammaproteobacteria and Actinobacterium classes in patients with HSIL HPV(+) were also detected. Further analysis revealed no cluster formation within the Gammaproteobacteria class and, therefore, this class was excluded from the present study.
Further analysis focussed only on the Actinobacterium and Bacilli classes of bacterial species. Regardless of the type of diagnosis, the bacterial species within the analysed classes only formed two clusters. It was observed that swabs from healthy women were characterised by an increased level of Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis. However, Gardnerella vaginalis and Lactobacillus acidophilus were absent. In the CST patients diagnosed with LSIL HPV(+) the predominant types of bacteria were Lactobacillus acidophilus and Lactobacillus iners, and there was no presence of Lactobacillus crispatus detected. The CST cervical swabs obtained from women with HSIL HPV(+) were rich in Gardnerella vaginalis and Lactobacillus acidophilus, while Lactobacillus taiwanensis, Lactobacillus iners and Lactobacillus crispatus were not detected. However, similar levels of Lactobacillus acidophilus were identified in the control and LSIL HPV(+) groups.
In the majority of healthy women in the present study, Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis were the dominant cervical microbiota. According to previous studies, these species were also reported to be dominant in the vaginal microbiota of Asian women (6,13). The women with the highest risk of HSIL HPV(+) indicated low levels of Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis. It has been reported that hydrogen peroxide-producing Lactobacilli are present in 96% of women with a normal vaginal bacterial community (4). In addition, species in the Lactobacillus genus have been reported to maintain a low pH by producing lactic acid (4). It has been reported that Lactobacillus crispatus was associated with a low vaginal pH compared with Lactobacillus iners, suggesting that these two species differ in ecological function (14). The cervical swabs obtained from healthy women consisted mainly of Lactobacillus bacteria. Due to their presence, the physiological pH value (3.6–4.5) was achieved by fermentation of epithelial glycogen. In addition, it has been reported that Lactobacillus bacteria have a high adherence potential, which allows them to adhere tightly to the vaginal epithelium and cover its surface, protecting the vagina from colonisation by pathogenic microorganisms (15–17). A number of Lactobacillus species can form biofilms and produce bacteriocins and hydrogen peroxide, which together have been reported to inhibit the development of undesirable anaerobes in the vagina (15,16). Lactobacillus iners has been previously reported to become a predominant part of the microbial community in the vaginal microbiota transition between abnormal and normal states (17). HPV infection can alter the mucosal metabolism and host immunity, and can induce changes in the vaginal microbiota (18).
It has been reported that microbe-induced inflammation can contribute to cervical cancer by stimulating the production of specific cytokines and chemokines, which promote proliferation and/or inhibit apoptosis (18,19). Microbiota have been demonstrated to increase, as well as decrease susceptibility to HPV infection (18,19). An association between vaginal microbiota and HPV infection was previously described by Gao et al (19). They observed a significant presence of Lactobacillus species, including Lactobacillus gallinarum, Lactobacillus iners and Lactobacillus gasseri in all women as well as Lactobacillus gasseri and Gardnerella vaginalis in HPV(+) women. The reduced population of the Lactobacillus species and the presence of Fusobacteria species in HPV(+) patients was also observed by Lee et al (6). The present study demonstrated that bacterial communities in the cervix are more complex than previously thought. The analyses suggest an association between HPV infection and decreased abundance of Lactobacillus species and increased abundance of Gammaproteobacteria anaerobes. The aforementioned results are similar to those reported by Lee et al (6) and Dareng et al (8). The changes to the ‘core microbiome’ may be associated with changes in human health and risk of exposure to HPV infection and cervical cancer development.
In summary, analysis of the association between the occurrence of bacterial species and the histopathological diagnosis in the analysed population revealed that the vaginal microbiota may be clustered into two groups. Cluster 1 was predominantly affected by Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis and Cluster 2 predominated by Gardnerella vaginalis. The frequency of Lactobacillus acidophilus was identical in the clustered groups. The proposed mechanism of a cervical cancer formation may start with a sexually transmitted carcinogenic HPV infection (Fig. 1).
The fastest rate of HPV clearance may occur in women with Cluster 1 bacteria as the dominant microbiota as >50% of all infections were cleared within a year. The lowest clearance rate may occur in women classified with Cluster 2 dominant microbiota. In the aforementioned patients the chance of HSIL(+) development gradually increases, representing the growth of a clonal high-grade lesion up to a number of years following HPV infection. It was recently demonstrated that Gardnerella vaginalis, a dominant Cluster 2 bacterium, was able to adhere to and displace precoated protective Lactobacilli from vaginal epithelial cells, while other BV-associated anaerobes, including Atopobium vaginae, were less virulent (20).
The findings of the present study demonstrated a possible interaction between bacterial flora and HPV infection, as well as an association between this interaction and clinical cervical neoplasia. It was observed that bacterial dysbiosis, characterized by a predominance of Gardnerella vaginalis and a concomitant paucity of Lactobacillus crispatus, Lactobacillus iners and Lactobacillus taiwanensis, may be an HPV-dependent cofactor for cervical neoplasia development. However, without continuous observation it is difficult to confirm that microbiota dysbiosis contributes to HPV infection and carcinogenesis. Future researches are required to confirm the results of 16S rRNA sequencing and determine that microflora dysbiosis may be associated with HPV-induced cervical carcinogenicity.
Acknowledgements
Not applicable.
Funding
The present study was funded by grants from the Medical University of Lublin (Lublin, Poland; grant nos., DS 120, DS 128 and MB 128).
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. The datasets presented in Tables III and IV can be found online at the following webpages: http://www.katbiolkom.ump.edu.pl/wp-content/uploads/2018/07/table_III.docx and http://www.katbiolkom.ump.edu.pl/wp-content/uploads/2018/07/table_IV.doc × (The repository of the University of Medical Sciences, Poznan, Poland).
Authors' contributions
WK conceived the study, collected samples, wrote the materials and methods and edited the manuscript. MWC analyzed the data and wrote the results and discussion sections. JK conceived the study, collected samples and contributed resources. DK performed laboratory assays. WW conceived the study, prepared figures and wrote background information. AK conceived the study, collected samples, contributed resources and approved the final draft. AGJ conceived the study, contributed resources, collected samples and approved the final draft.
Ethics approval and consent to participate
The investigations were approved by the Ethics Committee of the Medical University of Lublin (Lublin, Poland; Resolution of the Bioethics Committee; approval no: 0254/30/2002. Written informed consent was obtained from all individuals, and the research was performed by the principles of the Helsinki Declaration.
Patient consent for publication
Study participants provided written informed consent for the publication of any data and associated images.
Competing interests
The authors declare that they have no competing interests.
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