Отправить статью по E-mail Оценка связи состава просветной и тканевой микробиоты с интраэпителиальными изменениями шейки матки
- Авторы: Чечко С.М.1,2, Лямин А.В.1, Казакова А.В.1, Янченко А.В.1, Сапожкова Н.В.2, Каторкина Е.С.3, Столбова М.Е.4
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Учреждения:
- ФГБОУ ВО Самарский государственный медицинский университет Минздрава РФ
- ГБУЗ СО Самарская городская клиническая больница № 2 имени Н.А. Семашко
- ФГБОУ ВО Самарский государственный медицинский университет Минздрава России
- ГБУЗ Самарский областной медицинский центр «Династия»
- Выпуск: Том 13, № 4 (2023)
- Страницы: 761-766
- Раздел: КРАТКИЕ СООБЩЕНИЯ
- Дата подачи: 20.05.2023
- Дата принятия к публикации: 28.08.2023
- Дата публикации: 24.10.2023
- URL: https://iimmun.ru/iimm/article/view/11258
- DOI: https://doi.org/10.15789/2220-7619-AAR-11258
- ID: 11258
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Аннотация
Заболеваемость раком шейки матки (РШМ) в России по-прежнему остается на высоком уровне и занимает четвертое место среди всех онкологических заболеваний. РШМ предшествует цервикальная интраэпителиальная неоплазия легкой, умеренной и тяжелой степени. Известно, что вирус папилломы человека (ВПЧ) служит основной причиной развития данной патологии и ответственен за 99% случаев развития рака. Несмотря на инфицирование ВПЧ онкологический процесс возникает только при наличии определенных условий. Существуют факторы риска, опосредованно влияющие на течение и возникновении дисплазии шейки матки. В последние годы активно обсуждается роль цервико-вагинального микробиома в возникновении и прогрессировании данной патологии. Многие исследования свидетельствуют о том, что дисбиоз, при наличии цервикальной интраэпителиальной дисплазии связан с повышенной вирусной нагрузкой ВПЧ, также уточняется роль конкретных микроорганизмов. Несмотря на быстро накапливающиеся знания о характере вагинального микробиома, при наличии предраковых процессов шейки матки, уровень его влияния на течение заболевания до конца не изучен и представляет большой интерес. Выявление микроорганизмов влияющих на возникновение и прогрессирование данной патологии позволит профилактировать и избирательно подходить к лечению дисбиоза. Целью нашего исследования было выявить связь между тканевым и просветным культуромом шейки матки и цервикальной интраэпителиальной неоплазией тяжелой степени. В исследовании оценивалась микробиота биоптата шейки матки и слизистой оболочки цервикального канала у пациентов с цервикальными интраэпителиальными изменениями. Согласно результатам исследования представители рода Streptococcus достоверно чаще встречались в биоптате шейки матки у пациенток с дисплазией тяжелой степени по сравнению с женщинами без интраэпителиальных изменений шейки матки, тогда как в посеве из цервикального канала эти результаты были сопоставимы, а бактерии из рода Corynebacterium обнаруживалась в биоптате шейки матки в 3 раза чаще, чем в мазке со слизистой оболочки цервикального канала. Микробиота биоптата шейки матки и цервикального канала качественно и количественно отличаются, в частности встречаемость представителей Streptococcus spp. отмечается чаще у пациентов с наличием дисплазии шейки матки, а Corynebacterium spp. — в биоптате шейки матки по сравнению с мазком из цервикального канала. Таким образом большое влияние на результат имеет способ сбора материала.
Полный текст
Microbiota plays a key role in human physiology and maintenance of homeostasis. In recent years, knowledge about the microbiome has changed significantly [4]. It is known that the vaginal ecosystem is a metabolically and microbiologically complex environment. In most women, the vaginal microbiota is dominated by numerous varieties of Lactobacillus spp., which form the colonization resistance of the mucous membranes. Thus, the loss of dominance of Lactobacillus spp. promotes colonization by anaerobic bacteria and an increase in microbial diversity, which in some cases contributes to the progression of cervical intraepithelial neoplasia. The development of cervical cancer is known to be associated with persistent human papillomavirus (HPV) infection [3]. Most often, HPV infection is transient [5], but long-term persistence of HPV is associated with an increased risk of cervical intraepithelial neoplasia (CIN) and cervical cancer [5]. Dysbiotic changes in the vaginal microflora are a risk factor for the persistence of HPV infection [2, 7]. Recent studies show a relationship between cervico-vaginal microflora and the progression of CIN. With the advent of modern methods for diagnosing the state of microbiome and rapidly accumulating knowledge in this area, many issues require further study and systematization.
The purpose of the study was to reveal the connection of tissue and luminal culture of the cervix with severe cervical intraepithelial neoplasia.
The study was conducted on the basis of Samara State Medical University. The study involved 29 women of reproductive age (from 18 to 45 years). All patients were divided into two groups. The main group included women with severe intraepithelial lesions (10 people). The comparison group included women with no intraepithelial changes in the cervix (19 people).
The exclusion criteria for both groups were: pregnancy, HIV infection, hepatitis B, C, patients who received antibiotics within 15 days prior to taking a sample or who had intercourse/douching within 48 hours prior to sampling.
To study the cervical microbiota in women of both groups, a microbiological examination of cultures of the mucous membrane of the cervical canal and biopsy specimen of the cervix was carried out. The method of seeding the biopsy specimen of the cervix was conducted according to the author’s method (patent for invention No. 2784053). Collection of material from cervical canal of the cervix was carried out with a sterile swab, biopsy of the cervix with a gynecological conchotome with endovideo control (patent for utility model No. 213605). After collection, the samples were placed in liquid Amies transport medium and delivered to the laboratory within 2 hours under isothermal conditions. In the laboratory, the material was placed on an expanded set of solid nutrient media: 5% blood agar (HiMedia, India), anaerobic agar (HiMedia, India), veillonella isolation agar (HiMedia, India), clostridium isolation agar (HiMedia, India), bifidobacteria isolation agar (HiMedia, India), lactobacilli isolation agar (HiMedia, India), universal chromogenic medium (Bio-Rad, USA). The cultures were incubated for 5 days at 37°C under aerobic and anaerobic conditions. Then, using MALDI-ToF mass spectrometry (Microflex LT, Bruker), all isolated microorganisms were identified.
The present study evaluated the qualitative and quantitative composition of tissue and luminal microbiota in the smear from the cervical canal and biopsy specimen of the cervix, as well as the association of microbiota with cervical intraepithelial changes in the cervix.
Statistical analysis was carried out using the StatTech v. 2.8.7 (developer — Stattech LLC, Russia). Comparison of percentages in the analysis of the four-field contingency tables was performed using Pearson’s chi-square test, Fisher’s exact test. The link between the signs was regarded as statistically significant at a significance level of p < 0.05.
Representatives of the following genera of bacteria were isolated from the obtained material (Table 1).
Table 1. Inter-group qualitative characteristics of cervical and cervical canal biopsy microbiota
The genus of microorganisms | Cervical biopsy specimen (number of patients) | Cervical canal smear (number of patients) | P | ||
Comparison group (n = 19) | Main group (n = 10) | Comparison group (n = 19) | Main group (n = 10) | ||
Staphylococcus spp. | 14 (73.7) | 8 (80.0) | 9 (47.4) | 8 (80.0) | 0.155 |
Photobacterium spp. | 0 (0.0) | 0 (0.0) | 1 (5.3) | 1 (10.0) | 0.473 |
Escherichia spp. | 4 (21.1) | 1 (10.0) | 5 (26.3) | 1 (11.1) | 0.664 |
Enterococcus spp. | 9 (47.4) | 3 (30.0) | 10 (55.6) | 5 (50.0) | 0.632 |
Rothia spp. | 0 (0.0) | 0 (0.0) | 1 (5.3) | 1 (10.0) | 0.473 |
Haemophilus spp. | 1 (5.3) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0.554 |
Klebsiella spp. | 0 (0.0) | 0 (0.0) | 2 (10.5) | 0 (0.0) | 0.236 |
Morganella spp. | 0 (0.0) | 1 (10.0) | 1 (5.3) | 1 (10.0) | 0.575 |
Candida spp. | 2 (10.5) | 0 (0.0) | 1 (5.3) | 0 (0.0) | 0.532 |
Enterobacter spp. | 1 (5.3) | 1 (10.0) | 0 (0.0) | 0 (0.0) | 0.473 |
Microbacterium spp. | 1 (5.3) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0.554 |
Stenotrophomonas spp. | 0 (0.0) | 1 (10.0) | 0 (0.0) | 0 (0.0) | 0.180 |
Brevibacterium spp. | 1 (5.3) | 0 (0.0) | 1 (5.3) | 0 (0.0) | 0.779 |
Gardnerella spp. | 5 (26.3) | 1 (10.0) | 4 (21.1) | 3 (30.0) | 0.705 |
Cutibacterium spp. | 2 (10.5) | 0 (0.0) | 0 (0.0) | 1 (10.0) | 0.367 |
Actinomyces spp. | 0 (0.0) | 1 (10.0) | 1 (5.3) | 1 (10.0) | 0.575 |
Campylobacter spp. | 0 (0.0) | 1 (10.0) | 0 (0.0) | 0 (0.0) | 0.180 |
Fusobacterium spp. | 0 (0.0) | 1 (10.0) | 0 (0.0) | 0 (0.0) | 0.180 |
Peptostreptococcus spp. | 0 (0.0) | 1 (10.0) | 0 (0.0) | 0 (0.0) | 0.180 |
Kocuria spp. | 1 (5.3) | 0 (0.0) | 1 (5.3) | 0 (0.0) | 0.779 |
Aerococcus spp. | 0 (0.0) | 0 (0.0) | 1 (5.3) | 0 (0.0) | 0.554 |
Peptoniphilus spp. | 0 (0.0) | 1 (10.0) | 0 (0.0) | 0 (0.0) | 0.188 |
Bifidobacterium spp. | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (10.0) | 0.180 |
Acinetobacter spp. | 0 (0.0) | 0 (0.0) | 1 (5.3) | 0 (0.0) | 0.554 |
Dermabacter spp. | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (10.0) | 0.180 |
Metamycoplasma spp. | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (10.0) | 0.180 |
Dialister spp. | 0 (0.0) | 0 (0.0) | 0 (0.0) | 1 (10.0) | 0.180 |
Alloscardovia spp. | 0 (0.0) | 1 (10.0) | 0 (0.0) | 1 (10.0) | 0.268 |
Citrobacter spp. | 1 (5.3) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0.554 |
Micrococcus spp. | 2 (10.5) | 0 (0.0) | 0 (0.0) | 0 (0.0) | 0.236 |
Streptococcus spp. | 4 (21.1) | 7 (70.0) | 9 (47.4) | 5 (50.0) | 0.017* |
Corynebacterium spp. | 9 (47.4) | 6 (60.0) | 3 (15.8) | 2 (20.0) | 0.043* |
Note. *Significant differences (p < 0.05).
As a result of evaluating the frequency of isolation of Streptococcus spp. bacteria, depending on the presence of intraepithelial changes in the cervix, statistically significant differences were found (p = 0.017). In the biopsy specimen of the cervix with intraepithelial changes, bacteria of the genus Streptococcus were found in 70% of cases, whereas in the biopsy specimen without dysplasia — in 21.1% of cases.
The presence of Streptococcus spp. in the biopsy specimens in patients of the main group was detected 8.750 times more often than in the control group, the differences in chances were statistically significant (95% CI: 1.528–50.112).
Then a study of Streptococcus spp. in a smear from the mucous membrane of the cervical canal was conducted. In patients with cervical dysplasia, bacteria of this genus were detected in 50% of cases, without dysplasia in 47.4% of cases, while there were no statistically significant differences between the groups.
A study of the species composition of Streptococcus spp. in a biopsy specimen and in a smear from the cervical canal was also carried out (Table 2).
Table 2. Inter-group Streptococcus spp. species composition
Species of Streptococcus spp. | Biopsy specimen of the cervix (number of patients) | Smear from the cervical canal (number of patients) | ||
Comparison group (n = 19) | Main group (n = 10) | Comparison group (n = 19) | Main group (n = 10) | |
Streptococcus vestibularis | – | – | 1 | – |
Streptococcus oralis | 1 | 2 | 1 | – |
Streptococcus anginosus | 2 | 7 | 4 | 2 |
Streptococcus mitis | – | 1 | – | – |
Streptococcus sanguinis | – | 1 | – | – |
Streptococcus pseudopneumonia | – | – | – | 1 |
Streptococcus galloliticus | – | – | – | – |
Streptococcus agalactiae | – | 1 | 2 | 1 |
The data obtained suggest that the presence of Streptococcus spp. is a marker associated with the presence of cervical dysplasia. The greatest number and variety of strains of Streptococcus spp. was found in a biopsy specimen of the cervix in patients with dysplasia. The most common was S. anginosus, which, along with S. oralis, S. mitis, S. sanguinis, is part of the oropharyngeal microflora and these species cause oral pathology. The role of these microorganisms in the development of genital pathology is not fully understood. Their appearance in the vaginal microflora may be due to the prevalence of unprotected oral-genital contact. Thus, representatives of the genus Streptococcus are associated with the activation of many inflammatory cytokines and can affect the epithelial cells of the vagina and cervix, thereby contributing to the occurrence or progression of intraepithelial lesions of the cervix [6, 8, 12, 13, 14].
In comparing the qualitative characteristics of the microbiota of the biopsy specimen of the cervix and that of the cervical canal in the study groups, statistically significant differences were found for representatives of the genus Corynebacterium spp. These bacteria are able to adhere to vaginal cells and bind to extracellular matrix proteins such as fibronectin. When forming microbiocenosis in female reproductive tract, they affect the production of cytokines in the epithelial cells of the vagina. This allows them to compete with other microorganisms for adhesion sites and exist in the vaginal ecosystem both in health conditions and in various infectious diseases of the external and internal genital organs.
The data obtained by us indicate the ability of Corynebacterium spp. to invade, since they were significantly more common in the biopsy specimen of the cervix than in the smear from the cervical canal, which is an important result of the diagnostic search. However, there was no difference in the quantitative composition of Corynebacterium spp. in the biopsy specimen of the cervix of the main group (47.4%) and the comparison group (60%), as well as in the smear from the cervical canal — 15.8% and 20% respectively.
The species composition of Corynebacterium spp. is presented in Table 3.
Table 3. Inter-group Corynebacterium spp. species composition
Species of Corynebacterium spp. | Biopsy specimen of the cervix (number of patients) | Smear from the cervical canal (number of patients) | ||
Comparison group (n =19) | Main group (n = 10) | Comparison group (n =19) | Main group (n = 10) | |
Corynebacterium aurumucosum | 1 | 1 | 1 | 2 |
Corynebacterium amylocatum | 5 | 3 | 4 | 2 |
Corynebacterium tuberculostearicum | 1 | 2 | 2 | 2 |
Corynebacterium simulans | – | – | 1 | 1 |
Corynebacterium pyruviciproducens | – | – | 1 | – |
Corynebacterium mucifaciens | – | 1 | – | – |
Corynebacterium coyleae | – | 1 | 1 | 1 |
Corynebacterium riegelii | – | – | 1 | – |
Corynebacterium accolens | – | – | – | 1 |
The greatest variety of Corynebacterium spp. was found in the cervical canal of the cervix, which confirms the ability of this genus of bacteria to adhere. Colonizing the vaginal biotope, corynebacteria interact with various strains of microorganisms [1, 11]. Scientific publications show conflicting data about their role in the human body. C. aurimucosum causes urinary tract infection, C. accolens — pelvic osteomyelitis and granulomatous mastitis. In women with cervical cancer, C. amycolatum is found in the vaginal microbiome, but according to another study, this strain is considered as a probiotic, so the role of the Corynebacterium genus in the vaginal microbiome is not fully understood [1, 4, 9].
No connection between the remaining identified microorganisms and intraepithelial changes in the cervix was found.
In our study, we have identified an association between the presence of Streptococcus spp. in the biopsy specimen of the cervix and the presence of dysplasia. These results are consistent with the previous studies that have shown a correlation between impaired cervical microbiota and the development of cervical dysplasia [5, 14].
However, we found no statistically significant differences in the presence of Streptococcus spp. in a smear from the cervical canal in patients of both groups, which may indicate the ability of Streptococcus to adhere to the mucous membrane with further invasion. The conducted study clearly demonstrates the decisive importance of the method of collecting material. The resulting microflora qualitatively and quantitatively differs in the biopsy specimen of the cervix and the smear from the cervical canal. The data obtained confirm the need for further research in this area.
Об авторах
Светлана Михайловна Чечко
ФГБОУ ВО Самарский государственный медицинский университет Минздрава РФ; ГБУЗ СО Самарская городская клиническая больница № 2 имени Н.А. Семашко
Email: svetlana-chechko92@mail.ru
ORCID iD: 0000-0002-3890-9944
ассистент кафедры акушерства и гинекологии Института педиатрии, врач акушер-гинеколог отделения оперативной гинекологии
Россия, 443099, Самара, ул. Чапаевская, 89; СамараАртем Викторович Лямин
ФГБОУ ВО Самарский государственный медицинский университет Минздрава РФ
Email: avlyamin@rambler.ru
ORCID iD: 0000-0002-5905-1895
доктор медицинских наук, директор Научно-образовательного профессионального центра генетических и лабораторных технологий
Россия, 443099, Самара, ул. Чапаевская, 89Анна Владимировна Казакова
ФГБОУ ВО Самарский государственный медицинский университет Минздрава РФ
Email: a.v.kazakova@samsmu.ru
ORCID iD: 0000-0002-9483-8909
доктор медицинских наук, доцент, зав. кафедрой акушерства и гинекологии Института педиатрии
Россия, 443099, Самара, ул. Чапаевская, 89Анна Витальевна Янченко
ФГБОУ ВО Самарский государственный медицинский университет Минздрава РФ
Email: pystnica131902@gmail.com
ORCID iD: 0009-0005-7718-6266
специалист Научно-образовательного профессионального центра генетических и лабораторных технологий
Россия, 443099, Самара, ул. Чапаевская, 89Наталья Владимировна Сапожкова
ГБУЗ СО Самарская городская клиническая больница № 2 имени Н.А. Семашко
Email: Sapozhkova.natalya@bk.ru
ORCID iD: 0009-0001-8163-8772
зав. отделением оперативной гинекологии
Россия, СамараЕлена Сергеевна Каторкина
ФГБОУ ВО Самарский государственный медицинский университет Минздрава России
Автор, ответственный за переписку.
Email: katorkina2005@mail.ru
ORCID iD: 0009-0003-7190-4795
зав. отделением гинекологии в Клинике
Россия, 443099, Самара, ул. Чапаевская, 89Марина Евгеньевна Столбова
ГБУЗ Самарский областной медицинский центр «Династия»
Email: m-stolbova@mail.ru
ORCID iD: 0009-0009-3508-9379
врач акушер-гинеколог
Россия, СамараСписок литературы
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