Peculiarities of microbiocenosis of the urogenital tract of women with HPV-associated squat cellular intraepitelial lesions of the uterine cervix

N.A. Shmakova¹, G.N. Chistjakova¹, I.V. Lavrent’eva²,

I.N. Kononova³, I.I. Remizova¹

 

¹ FGBU «Ural research Institute for maternal and infant health», Yekaterinburg, Russian Federation;

² «Ural State Medical University», Yekaterinburg, Russian Federation;

³ LLC interregional center of additional professional education, Yekaterinburg, Russian Federation

 

Abstract. Objective: To reveal the features of the microbiocenosis of the cervical canal with the species identification of lactobacilli in women with HPV-associated squamous intraepithelial lesions of the cervix. Materials and methods: A survey of 80 patients of reproductive age with HPV-associated squamous intraepithelial lesions of the cervix (SIL), who had not received local and systemic antibacterial drugs at the time of the examination, was carried out. According to the results of morphohistological examination of cervical biopsies, this cohort of patients was divided into two groups. Group 1 included 40 patients with CIN II and CIN III, which corresponds to Нigh-grade squamous intraepithelial lesions (HSIL), group 2 — 40 patients with CIN I, which corresponds to Low-grade squamous intraepithelial lesions (LSIL). The comparison group (3rd) consisted of 30 clinically healthy women. All patients underwent a cytological study by the «liquid method» BD SurePath technology, genotyping of the human papillomavirus by the polymerase chain reaction method (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), cultural studies of the microbiocenosis of the cervical canal (by the MALDI method — TOF mass spectrometry, in the VITEKMS analyzer), extended colposcopy. Conclusion. It was found that in all women with HPV-associated squamous intraepithelial lesions of the cervix, the microflora of the cervical canal is characterized by the presence of Gardnerella vaginalis, as well as a decrease in the detection rate and number of lactobacilli, with the presence of the facultative anaerobe Lactobacillus iners. In the structure of opportunistic microflora, the predominance of Enterococcus faecalis and Escherichia coli was demonstrated in HSIL, while for LSIL, representatives of the enterococcus group.

 

Keywords: intraepithelial neoplasia of the cervix, papillomavirus infection, dysbiosis

 

There is no conflict of interest.

Contact details of the corresponding author:

Shmakova Nadezhda Aleksandrovna

nadyushka.ru@yandex.ru

Received 25.09.2020

For citation:

Shmakova N.A., Chistjakova G.N., Lavrent’eva I.V., Kononova I.N., Remizov I.I.

Peculiarities of microbiocenosis of the urogenital tract of women with HPV-associated squat cellular intraepitelial lesions of the uterine cervix. Url: http://vestnikural.ru/article/1141. [Online] Vestn. Ural. Med. Akad. Nauki. = Journal of Ural Medical Academic Science. 2020, Vol. 17, no. 3, pp. 221–232. DOI: 10.22138/2500-0918-2020-17-3-221-232 (In Russ)

 

For more than one decade, squamous intraepithelial lesions of the cervix have been a fairly common pathology, occupying up to 38.8% in the structure of gynecological diseases [1, 2, 3], while according to official data, the frequency of squamous intraepithelial lesions of a low degree ranges from 1, 5-7.7%, high — 0.4-1.5%, depending on the population [3, 4, 5].

The undeniable fact is the high risk of SIL progression with the development of cervical cancer (CC), the incidence of which in the Russian Federation as of 2018. is 124 cases per 100 thousand women in the structure of oncopathology. At the same time, the average annual growth rate over the last decade is 2.06%, while the mortality rate is 8.13 per 100 thousand women per year, with the maximum rates recorded at the age of 15-39 years [6]. Thus, at present, the peak incidence of not only SIL, but also cervical cancer occurs at reproductive age, showing a tendency towards “rejuvenation”.

The accumulated data suggest that the main role in the development of SIL and further their oncotransformation belongs to the human papillomavirus (HPV) [7, 8, 9]. At the same time, world statistics show that 9% of all new cases of cervical cancer in women are associated with HPV infection [10, 11].

It should be borne in mind that the oncogenic potential of HPV is realized only in the presence of certain conditions in the body. In this case, both somatic and immunological predictors that contribute to changes in the cellular structure of the cervical epithelium can act as confounding [12].

However, there is evidence in the literature that, in addition to HPV, the development of precancer and cervical cancer is associated with hyperplastic processes in the cervical epithelium, inhibition or decreased activity of apoptosis processes, epigenetic mutations and imbalance of estrogen metabolites [13, 14].

 In this regard, literature data are important, indicating a close relationship between chronic inflammation of the cervix, regardless of viral or bacterial nature, with the development and oncotransformation of SIL [15, 16, 17, 18]. As a result, local tissue hypoxia occurs, leading to the formation of oxidative stress, thereby triggering the triggering of epigenetic mutations [19]. Ultimately, this provokes proliferative processes in the cervical epithelium with a high risk of outcome in the neoplastic and certainly determines the feasibility of assessing the composition of the microflora of the cervical canal in HPV-associated cervical intraepithelial neoplasias.

 However, despite the fairly broad knowledge in understanding the nuances of the mechanism of development of this process, there is insufficient effectiveness of the therapy used, which leads to the progression or recurrence of squamous intraepithelial lesions of the cervix after treatment [13, 20].

Thus, understanding the epidemiological situation, the high risk of oncotransformation of intraepithelial lesions of the cervix, one cannot but take into account other factors involved in the formation of this pathology, which in turn actualizes the study of the features of the microbiocenosis of the urogenital tract.

 

Purpose of the study

To reveal the peculiarities of the microbiocenosis of the cervical canal with the species identification of lactobacilli in women with HPV-associated squamous intraepithelial lesions of the cervix.

 

Material and methods

A survey of 80 patients with HPV-associated squamous intraepithelial lesions of the cervix was performed, which, depending on the degree of the neoplastic process, were divided into two groups. Group 1 included 40 patients with CIN II and CIN III, which corresponds to high-grade squamous intraepithelial lesions (HSIL), group 2 — 40 patients with CIN I, which corresponds to low-grade squamous intraepithelial lesions (LSIL). The comparison group (3rd) consisted of 30 conventionally healthy women. The average age of patients in the 1st, 2nd and 3rd groups was comparable — 30.3±0.97, 28.7±0.87 and in the third 29.3±1.16 years, respectively (p>0.05 in all cases). The inclusion criteria for the study were: the presence of HPV-associated squamous cell intraepithelial lesions of the cervix, the reproductive age of women (18-45 years), the absence of the use of local and systemic antibacterial drugs within 1 month before the study, written informed consent of the patients to participate in the study.

 All patients underwent a cytological study by the «liquid method» using the BD SurePath technology, genotyping of the human papillomavirus by the polymerase chain reaction method (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), microbiological (cultural) study of the microbiocenosis of the cervical canal for aerobic and facultative anaerobic microorganisms with the determination of sensitivity to an extended spectrum of antibiotics and bacteriophages, as well as an assessment of the species composition of lactobacilli (by the MALDI method — TOF mass spectrometry, in the VITEKMS analyzer), extended … The material was taken from the cervical canal with a swab probe and placed in an eSwab container, with a total storage and transportation time to the laboratory of no more than 24 hours. The result was expressed in CFU / sample.

 The data obtained as a result of the study were processed using a Microsoft Excel 2010 spreadsheet and standard software packages (SPSS 23.0, STATISTICA for Windows 10). The analysis of the statistical significance of differences in qualitative and quantitative features was carried out using the Pearson χ² test with Yates’ correction. Differences were considered statistically significant at p≤0.05. To assess the relationship between the indicators, the Spearman’s rank correlation coefficient was determined. The correlation ratios of features were taken into account, corresponding only to the average (0.7≥r≥0.5) and high (r≥0.7) degrees of connection at a confidence level of p ≤ 0.05 — 0.00.

 

Results and discussion

During extended colposcopy, analysis of the type of transformation zone in the examined patients revealed a number of features. Significantly less often type 1 substitution therapy was observed in HSIL patients (67.5%, 27 women) compared to the same indicator in LSIL (87.5%, 35 women). The revealed indicators indicated a deep lesion of the cervix with insignificant visible changes or practically unchanged exocervix, which confirms its auxiliary value.

When assessing the prevalence of types of HPV infection in patients with SIL included in the study, the prevalence of genotype 16 was revealed. In second place, 33 types were found, which does not contradict well-known data. More than 10% of patients had human papillomavirus genotypes: 18, 31, 39, 45, 52. The frequency of detection of other types was less than 10% (35, 51, 56, 58, 59 types).

When conducting a microbiological study in the structure of the microbiocenosis of the cervical canal with SIL, opportunistic microorganisms predominated in 82.5% and 80.0% in the 1st and 2nd groups, in comparison with 10% of women in the 3rd group (χ²=33, 2 at p=0.001, χ²=30.9 at p=0.001, respectively). In almost half of the patients, the presence of opportunistic microflora (UPM) was combined with lactobacilli in 17 (42.5%) patients with HSIL and 20 (50%) with LSIL. However, the complete absence of any microorganisms was diagnosed in the 1st group in 7 (17.5%) and the 2nd in 8 (20%) women.

The structure of opportunistic microflora was represented by the following spectrum of microorganisms — Enterococcus faecalis, Escherichia coli, Streptococcus agalactiae, Klebsiella pneumoniae, Streptococcus epidermidis, Candida albicans (Fig. 1)

Fig. 1. Frequency of occurrence of opportunistic representatives of microbiocenosis of the cervical canal in the examined patients

 

The characteristic of opportunistic bacteria in terms of their frequency of occurrence demonstrated the predominance of Enterococcus faecalis, both in squamous intraepithelial lesions of high 24 (60%) (4.9±0.16 lg CFU / sample) and low degree 15 (37.5%) (4.7±0.15 lg CFU / sample), statistically significant relative to the comparison group (χ²=21.6 at p=0.001, χ²=9.5 at p=0.003, respectively). Escherichia coli (4.9±0.16 lg CFU / sample) was detected in 12 (30%) patients with HSIL, which was significant relative to healthy women, where this microorganism was absent (χ²=8.9 at p=0.003). In the contingent of patients with low-grade squamous intraepithelial lesions, this representative of the UPM was found in every seventh woman 6 (15.0%), however, there were significant differences both with the comparison group (p=0.074) and with respect to HSIL (p=0.18) not found. Streptococcus agalactiae was recorded in 7 (17.5%) LSIL patients (3.4±0.36 lg CFU / sample) and 3 (7.5%) women with HSIL (2.3±0.15 lg CFU / sample), (p=0.14 with LSIL and p=0.82 with HSIL). A variant of the association between the growth of Enterococcus faecalis and Escherichia coli was observed in high-grade squamous intraepithelial lesions in 11 (27.5%) and low-grade in 5 (12.5%) patients (p1-2>0.05). Klebsiella pneumonia (3.7±0.34 lg CFU / sample) was present only in patients with LSIL 3 (7.5%), and Streptococcus epidermidis in one (3.3%) woman from the comparison group.

Detection of yeast-like fungi of the genus Candida albicans was noted only in patients with squamous intraepithelial lesions, regardless of the degree: in group 1 — 4.5±0.22 lg CFU / sample and in group 2 — 4.6±0.24 lg CFU / sample, with a detection frequency of 15% and 12.5% of cases, versus 0% in the comparison group.

As part of the cervical microflora, 37 patients with squamous intraepithelial lesions and all of the comparison group had lactobacilli. Colonization with lactobacilli was 2.9±0.08 lg CFU / sample in group 1, 3.1±0.17 lg CFU / sample in group 2, and 6.3±0.09 lg CFU / sample in group 3. sample, having significant differences between themselves (p1-3, 2-3=0.00). The species composition of lactobacilli was presented: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners (Fig. 2). It was found that in patients with SIL, regardless of the degree, facultative anaerobic Lactobacillus iners was recorded in 13 (27.5%) with HSIL and 12 (30%) with LSIL, which was significant relative to the comparison group (χ²=9.9 at p=0.002, χ²=8.9 at p=0.003, respectively), with an increase of 6.1±0.09 lg CFU / sample and 5.1±0.24 lg CFU / sample, respectively. The incidence of Gardnerella vaginalis at a concentration of ≥104CFU / sample did not differ from L. iners. A strong positive correlation was determined between these microorganisms (r=+0.84 at p=0.001). Peroxide-producing lactobacilli were also present in the studied contingent of patients, however, significantly less frequently (10% and 20% in groups 1 and 2, р1-3, 2-3=0.001) and the degree of their colonization was noticeably lower (10²-10³ CFU / sample) relative to the comparison group (≥106 CFU / sample). Lactobacillus crispatus was found in all groups, with a predominance of growth in the comparison group (1st — 2.3±0.33 lg CFU / sample, 2nd — 2.5±0.5 lg CFU / sample, 3rd — 6±0 lg CFU / sample, p1-3, 2-3 <0.0001), while Lactobacillus jеnsenii (6.1±0.09 lg CFU / sample) and Lactobacillus acidophilus (5.3±0, 32 lg CFU / sample) only in healthy women. The data obtained were significant relative to the comparison group, where Lactobacillus iners was not diagnosed and the growth of peroxidizing lactobacilli was observed within the normative values (≥10⁶ CFU / sample).

Fig. 2. Assessment of the species composition of lactobacilli

 

Thus, it was found that in all women with squamous intraepithelial lesions of the cervix, dysbiotic shifts in the cervical microflora were observed, characterized by the predominance of the growth of opportunistic microorganisms, regardless of their quantitative content, which confirms the existing opinions about the progression of the cervical neoplastic process against the background of violations of the cervico-vaginal microbiocenosis [17, 24].

In the structure of opportunistic microflora, the predominance of Enterococcus faecalis and Escherichia coli in HSIL was demonstrated, while for LSIL, the growth of only a representative of the enterococcus group was statistically significant. The results obtained confirm the relationship of chronic inflammation of the cervix, regardless of the viral or bacterial nature, with the development and oncotransformation of SIL [15, 16, 17, 18]. However, statistics on the analysis of the UPM of this localization are not yet widely presented.

 For more than 10 years, research has been carried out in the world, the purpose of which is to find parallels between HPV-associated squamous cell intraepithelial lesions of the cervix and disorders of the microbiocenosis of the urogenital tract. To date, according to the results of numerous meta-analyzes, a fairly clear connection with anaerobic dysbiosis associated with Gardnerella vaginalis has been revealed. At the same time, the risk of not only infection, but also the persistence of PVI and the development of SIL has been proven [22, 23].

The results of our research on the species composition of lactobacilli are of particular interest. In addition to a decrease in the frequency of detection and the number of lactobacilli, a statistically significant presence of Lactobacillus iners was revealed. As a facultatively anaerobic rod-shaped bacterium, it exhibits aggressive properties, atypical for normal microflora, destroying the cells of the cervical epithelium, which in turn disrupts the activation of Toll-like receptors (TLR) on their surface. At the same time, it is known that one of the functions of lactoflora is the activation of receptors TLR-1, 2, 6, 7, 8 of epithelial cells that recognize both viruses and bacteria for the production of pro-inflammatory cytokines that control the reproduction of unwanted microflora [11].

 

Conclusions

1. Enterococcus faecalis and Escherichia coli predominate in the structure of the opportunistic microflora of the cervical canal in women with HPV-associated squamous intraepithelial lesions of the cervix.

2. A distinctive feature of the microbiocenosis of women with HPV-associated squamous intraepithelial lesions of the cervix is a decrease in the detection rate and the number of lactobacilli with the presence of the facultative anaerobe Lactobacillus iners.

Thus, the revealed violations of the cervical microbiocenosis determine the need for further more detailed study of this issue, including the identification of the species composition of lactobacilli in patients with SIL.

 

REFERENCES

  1.      Aksel E. M. Morbidity and mortality from malignant neoplasms of the female reproductive system in Russia. Oncogynecology=Onkoginekologiya. 2015, no.1, рр. 6–15, (In Russ.).
  2.      Davydov M. I., Maksimovich D.I., Zaridze D.G. Dynamics of morbidity and mortality from malignant neoplasms in Russia. Bulletin of the Russian Oncology Center N.N. Blokhin RAMS=Vestnik RONC zim. N.N.Bloxina RAMN. 2015, no.26, рр. 1–5, (In Russ.).
  3.      Benard V.B, Castle P.E, Jenison SA. For the New Mexico HPV Pap Registry Steering Committee Population-Based Incidence Rates of Cervical Intraepithelial Neoplasia in the Human Papillomavirus Vaccine Era. JAMA Oncol. 2017, Vol. 3(6), рр. 833-837.

DOI:10.1001/jamaoncol.2016.3609.

  1.      Sanad A.S., Kamel H.H., Hasan M.M.. Prevalence of cervical intraepithelial neoplasia (CIN) in patients attending Minia Maternity University Hospital. Arch Gynecol Obstet. 2014 Jun., Vol. 289(6), рр. 1211-1217.

DOI:10.1007/s00404-013-3109-0.

  1.      Shahida S.M., Lipi L.B., Rifat J.A.; et al. Prevalence of Cervical Intraepithelial Neoplasia in four Upazila of Dhaka Division. Mymensingh Med J. 2019 Jul., Vol. 28(3), рр. 655-656.
  2.      Kaprin A. D., Starinskij V.V., Petrova G.V. Malignant neoplasms in Russia in 2018 (morbidity and mortality). Moscow: MNIOI im. P.A. Gercena filial FGBU «NMIRC» Minzdrava Rossii; 2019, (In Russ.).
  3.      Gravitt, P.E., Winer R.L. Natural History of HPV Infection across the Lifespan: Role of Viral Latency. Viruses. 2017, Vol. 9(267), рр. 2-10.

DOI:10.3390/v9100267.

  1.      Bird Y., Obidiya O., Mahmood R.; et al. Human Papillomavirus Vaccination Uptake in Canada: A Systematic Review and Meta-analysis. International J Preventive Medicine. 2017, Vol. 14(8), рр. 71.
  2.      Vargas – Hernández V. M., Vargas – Aguilar V. M, J. M. Tovar — Rodríguez J. M.; et al. Primary cervical cancer screening. Cir Cir. 2015, Vol. 83(5), рр. 448 — 453.
  3.    Kaprina A.D., Starinskogo V.V., Petrovoj G.V. The state of cancer care to the population of Russia in 2016. Moscow: MNIOI im. P.A.Gercena – filial FGBU NMICR; 2017, (In Russ.).
  4.    Tihomirov A.L., Sarsanija S.I., Filatova G.A. Human papillomavirus: from an understanding of immunopathogenesis to rational management tactics. Gynecology=Ginekologija. 2018, no. 20(3), рр. 5–11. (In Russ.).

DOI:10.26442/2079-5696_2018.3.5-11.

  1.    Radzinskij V.E., Ordijanc I.M., Bebneva T.N. The fight against HPV: many boundaries of a single issue. Moscow: Redakcija zhurnala StatusPraesens. 2018, (In Russ.).
  2.    Ashrafjan L. A., Kiseljov V. I., Aleshikova O. I.; et al. Results of conservative therapy in patients with grade III cervical intraepithelial neoplasia (CIN I-II). Оbstetrics and gynecology=Akusherstvo i ginekologija. 2015, no.12, рр.103-109, (In Russ.).
  3.    Bebneva T.N., Mujzhnek E.L., Rogovskaja S.I.; et al. Pathogenetic treatment of neoplastic processes of the cervix: new approaches. DoktorRu. 2016, no. 3, рр.9-14, (In Russ.).
  4.    Belokrinickaja T.E., Frolova N.I., Tarbaeva D.A.; et al. Associations of genital infections and human papillomavirus as confounding factors of cervical intraepithelial neoplasia. DoctorRи=DoktorRu. 2015, no. 2(12), рр.14–1, (In Russ.).
  5.    Krasnopol’skij V.I., Zarochenceva N.V, Mikaeljan A.V.; et al. The role of human papillomavirus infection in the pathology of pregnancy and the outcome for the newborn (modern ideas). Russian Bulletin of Obstetrician Gynecologist=Rossijskij vestnik akushera-ginekologa. 2016, no.16(2), рр. 30 – 36, (In Russ.).

DOI:10.17116/rosakush201616230-36.

  1.    Pestrikova T.Ju., Panfilova Ju.O. Sensitivity of pathogenic microflora of the cervical canal to antibacterial drugs in patients with chronic cervicitis and HPV. Gynecology=Ginekologija. 2015, no. 7(1), рр.18–21, (In Russ.).

DOI:10.26442/2079-5831_17.1.18-21.

  1.    Reid G., Hons B.S. Modulating the Vaginal Microbiome: The Need for a Bridge between Science and Practice. Semin Reprod Med. 2014, Vol. 32(1), рр. 28–34.

DOI:10.1055/s-0033-1361820 .

  1.    Karaulov A.V., Afanas’ev S.S., Aljoshkin V.A. The role of microbiocenoses and innate immunity in mucosal protective reactions and the development of inflammation. Physiology and pathology of the immune system Immunofarmakogenomika. Russian Journal of Immunology=Rossijskij immunologicheskij zhurnal. 2013, no. 17(4), рр. 3-10, (In Russ.).
  2.    Rogovskaja S.I., Lipova E.V. Cervix, vagina, vulva. Physiology, pathology, colposcopy, aesthetic correction: a guide for practitioners. Moscow: izd-vo zhurnala StatusPraesens; 2014, (In Russ.).
  3.    Radzinskij V.E. Guidelines for outpatient care in obstetrics and gynecology. M.: GJeOTAR-Media; 2016, (In Russ.).
  4.    Budanov P.V., Churganova A.A, Bahtijarov K.R. Human papillomavirus infection: the effectiveness of the treatment with inosine pranobex. Gynecology=Ginekologija. 2015, no. 17(6), рр. 56–59, (In Russ.).

DOI:10.26442/2079-5696_17.6.56-59.

  1.    Radzinskij V.E. Relapses tank. vaginosis and lactoflora: from current ambiguity to practical solutions. Moscow: Status Praesens; 2013, (In Russ.).
  2.    Kawai T., Akira S. Toll-like receptors and their crosstalk with otherinnate receptors in infection and immunity. Immunity. 2011, Vol. 34, рр. 637–50.

 

Authors:

Nadezhda A. Shmakova

FSBI «Ural Research Institute of OMM» of the Ministry of Health of Russia

Postgraduate student, obstetrician-gynecologist of the first category

Rossijskaja Federacija, 620028, g. Ekaterinburg, ul. Repina,1

nadyushka.ru@yandex.ru

 

Guzel’ N. Chistjakova

FSBI «SRI OMM» of the Ministry of Health of the Russian Federation

Doctor of Medical Sciences, Professor, Head of the Department of Immunology and Clinical Microbiology

Rossijskaja Federacija, 620028, g. Ekaterinburg, ul. Repina,1

7@niiomm.ru

 

Inna V. Lavrentieva

Ural state medical university Yekaterinburg, Russian Federation

PHD, MD, Department of obstetrics and gynecology faculty of continuing education, assistant Professor

Rossijskaja Federacija, 620028, g. Ekaterinburg, ul. Repina,3

lavr6607@bk.ru

 

Irina N. Kononova

LLC «Interregional Center for Continuing Professional Education»

Doctor of Medical Sciences, Deputy Director

Rossijskaja Federacija, 620075, g. Ekaterinburg, ul. Lenina, 52 / 3A

irkonmed@mail.ru

 

Irina I. Remizova .

FSBI «SRI OMM» of the Ministry of Health of the Russian Federation

Candidate of Biological Sciences, Senior Researcher, Scientific Department of Immunology and Microbiology

Rossijskaja Federacija, 620028, g. Ekaterinburg, ul. Repina,1

RemizovaII@yandex.ru

 

 
 
 

Authorization