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Журнал микробиологии, эпидемиологии и иммунобиологии. 2020; 97: 26-31

Состав микробиоты репродуктивного тракта женщин при бесплодии

Годовалов А. П., Карпунина Т. И.

https://doi.org/10.36233/0372-9311-2020-97-1-26-31

Аннотация

Введение. В настоящее время вопрос об участии комменсальной, в том числе условно-патогенной, микрофлоры в развитии бесплодия остается дискутабельным. Для решения этого вопроса активно внедряются молекулярно-генетические методы исследования, которые превосходят рутинные методики культивирования по ряду позиций.

Цель исследования — оценить таксономическое разнообразие микроорганизмов в вагинальном биотопе при бесплодии.

Материал и методы. Для исследования использовали образцы вагинального содержимого, полученного из заднего свода влагалища 15 женщин, состоящих в бесплодном браке. Метагеномное исследование 16S рибосомной РНК образцов осуществлено на платформе Illumina MiSeq с использованием набора MiSeq Reagent Kits v3 (600-Cycle Kit) согласно рекомендациям производителя. Библиотеки для секвенирования участков V3-V4 гена 16S рибосомной РНК были приготовлены согласно 16S Metagenomic Sequencing Library Preparation Illumina. При биоинформационной оценке применяли программное обеспечение для метагеномного анализа — Kraken Metagenomics version 2.0.0 (классификатор ридов — коротких нуклеотидных последовательностей), используя стандартную базу данных.

Результаты. Показано, что встречаемость представителей семейства Lactobacillaceae в вагинальном биотопе варьирует от 12 до 84%. Ведущее положение среди представителей семейства занимал род Lactobacillus с доминированием L. jensenii, L. delbrueckii и L. amylolyticus. Во всех пробах обнаружены в большом количестве Moraxella spp., причем M. osloensis лидировали среди представителей всего сообщества. В половине случаев выявлено совместное присутствие M. osloensis и G. vaginalis.

Заключение. Установлено, что при бесплодии в вагинальной микробиоте существенно снижается количество представителей рода Lactobacillus, а также наблюдается смена лидирующего вида на L. jensenii, функциональная активность которых не обеспечивает в полной мере колонизационную резистентность вагинального биотопа, допуская избыточное размножение условно-патогенных микроорганизмов, в частности M. osloensis.

Список литературы

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4. Шишкова Ю.С., Долгушина В.Ф., Графова Е.Д., Завьялова С.А., Курносенко И.В., Евстигнеев а Н.П. и др. Взаимосвязь функционального статуса нейтрофилов цервикального секрета у беременных женщин с видовым составом лактофлоры. Журнал микробиологии, эпидемиологии и иммунобиологии. 2018; 95(4): 51-6. DOI: http://doi.org/10.36233/0372-9311-2018-4-51-56

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7. Sirota I., Zarek S.M., Segars J.H. Potential influence of the microbiome on infertility and assisted reproductive technology. Semin. Reprod. Med. 2014; 32(1): 35-42. DOI: http://doi.org/10.1055/s-0033-1361821

8. Franasiak J.M., Werner M.D., Juneau C.R., Tao X., Landis J., Zhan Y., et al. Endometrial microbiome at the time of embryo transfer: next-generation sequencing of the 16S ribosomal subunit. J. Assist. Reprod. Genet. 2016; 33(1): 129-36. DOI: http://doi.org/10.1007/s10815-015-0614-z

9. Campisciano G., Florian F., D’Eustacchio A., Stanković D., Ricci G., De Seta F., et al. Subclinical alteration of the cervical-vaginal microbiome in women with idiopathic infertility. J. Cell Physiol. 2017; 232(7): 1681-8. DOI: http://doi.org/10.1002/jcp.25806

10. van Oostrum N., De Sutter P., Meys J., Verstraelen H. Risks associated with bacterial vaginosis in infertility patients: a systematic review and meta-analysis. Hum. Reprod. 2013; 28(7): 1809-15. DOI: http://doi.org/10.1093/humrep/det096

11. Михайлова Н.А., Воеводин Д.А., Поддубиков А.В. Коррекция дисбиоза – основа регенеративной медицины. Журнал микробиологии, эпидемиологии и иммунобиологии. 2018; 95(5): 107-13. DOI: http://doi.org/10.36233/0372-9311-2018-5-107-113

12. Brotman R.M. Vaginal microbiome and sexually transmitted infections: an epidemiologic perspective. J. Clin. Invest. 2011; 121(12): 4610-7. DOI: http://doi.org/10.1172/JCI57172

13. Mastromarino P., Hemalatha R., Barbonetti A., Cinque B., Cifone M.G., Tammaro F., et al. Biological control of vaginosis to improve reproductive health. Indian J. Med. Res. 2014; 140(Suppl.): S91-7.

14. Donati L., Di Vico A., Nucci M., Quagliozzi L., Spagnuolo T., Labianca A., et al. Vaginal microbial flora and outcome of pregnancy. Arch. Gynecol. Obstet. 2010; 281(4): 589-600. DOI: http://doi.org/10.1007/s00404-009-1318-3

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16. Onderdonk A.B., Delaney M.L., Fichorova R.N. The human microbiome during bacterial vaginosis. Clin. Microbiol. Rev. 2016; 29(2): 223-38. DOI: http://doi.org/10.1128/CMR.00075-15

17. Mendes-Soares H., Krishnan V., Settles M.L., Ravel J., Brown C.J., Forney L.J. Fine-scale analysis of 16S rRNA se quences reveals a high level of taxonomic diversity among vaginal Atopobium spp. Pathog. Dis. 2015; 73(4): pii: ftv020. DOI: http://doi.org.10.1093/femspd/ftv020

18. Copeland A., Sikorski J., Lapidus A., Nolan M., Del Rio T.G., Lucas S., et al. Complete genome sequ ence of Atopobium parvulum type strain (IPP 1246). Stand. Genomic Sci. 2009; 1(2): 166-73. DOI: http://doi.org/10.4056/sigs.29547

19. Lewis F.M., Bernstein K.T., Aral S.O. Vaginal microbiome and its relationship to behavior, sexual health, and sexually transmitted diseases. Obstet. Gynecol. 2017; 129(4): 643-54. DOI: http://doi.org/10.1097/AOG.0000000000001932

20. Gómez-Camarasa C., Fernández-Parra J., Navarro-Marí J.M., Gutiérrez-Fernández J. Moraxella osloensis emerging infection. Visiting to genital infection. Rev. Esp. Quimioter. 2018; 31(2): 178-81. (in Spanish)

21. Goto T., Hirakawa H., Morita Y., Tomida J., Sato J., Matsumura Y., et al. Complete genome sequence of Moraxella osloensis strain KMC41, a producer of 4-Methyl-3-Hexenoic acid, a major malodor compound in laundry. Genome Announc. 2016; 4(4): e00705-16. DOI: http://doi.org/10.1128/genomeA.00705-16

22. Tan L., Grewal P.S. Pathogenicity of Moraxella osloensis, a bac terium associated with the nematode Phasmarhabditis hermaphrodita, to the slug Deroceras reticulatum. Appl. Environ. Microbiol. 2001; 67(11): 5010-6. DOI: http://doi.org/10.1128/AEM.67.11.5010-5016.2001

23. Morais I.M.C., Cordeiro A.L., Teixeira G.S., Domingues V.S., Nardi R.M.D., Monteiro A.S., et al. Biological and physico chemical properties of biosurfactants produced by Lactobacillus jen senii P6A and Lactobacillus gasseri P65. Microb. Cell Fact. 2017; 16(1): 155. DOI: http://doi.org/10.1186/s12934-017-0769-7

24. Martin R., Suarez J.E. Biosynthesis and degradation of H2O2 by vaginal lactobacilli. Appl. Environ. Microbiol. 2010; 76(2): 400-5. DOI: http://doi.org/10.1128/AEM.01631-09

25. O’Hanlon D.E., Moench T.R., Cone R.A. In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide. BMC Infect. Dis. 2011; 11: 200. DOI: http://doi.org/10.1186/1471-2334-11-200

26. Kim S., Gu S.A., Kim Y.H., Kim K.J. Crystal structure and thermodynamic properties of d-lactate dehydrogenase from Lactobacillus jensenii. Int. J. Biol. Macromol. 2014; 68: 151-7. DOI: http://doi.org/10.1016/j.ijbiomac.2014.04.048

27. Tsukida K., Takahashi T., Iida H., Kanmani P., Suda Y., Nochi T., et al. Immunoregulatory effects triggered by immunobiotic Lactobacillus jensenii TL2937 strain involve efficient phagocytosis in porcine antigen presenting cells. BMC Immunol. 2016; 17(1): 21. DOI: http://doi.org/10.1186/s12865-016-0160-1

28. Patnaik S., Davila C.D., Chennupati A., Rubin A. Endocarditis of the native aortic valve caused by Lactobacillus jensenii. BMJ Case Rep. 2015; 2015: bcr2014206288. DOI: http://doi.org/10.1136/bcr-2014-206288

Journal of microbiology, epidemiology and immunobiology. 2020; 97: 26-31

The Microbiota Continuum along the Reproductive Tract in Women with Infertility

Godovalov A. P., Karpunina T. I.

https://doi.org/10.36233/0372-9311-2020-97-1-26-31

Abstract

Introduction. At present, the question of commensal, including opportunistic, microflora participation in  infertility development remains debatable. In a number of studies, the translocation of the vaginal microflora into the endometrial tissue is considered as a factor contributing to inflammation development. In addition, the connection of some reproductive losses with the persistence of certain conditionally pathogenic microorganisms is shown. Today, to solve this issue, molecular genetic research methods are being actively introduced that surpass the routine cultivation techniques in a number of positions.

The aim of the study was to assess the taxonomic diversity of microorganisms in the vaginal biotope with infertility.

Material and methods. For the study samples of vaginal contents obtained from the posterior vaginal fornix of 15 women, consisting of barren marriage, were used. A metagenomic study of 16S ribosomal RNA samples was carried out on the Illumina MiSeq platform, using the MiSeq Reagent Kits v3 kit (600-Cycle Kit), as recommended by the manufacturer. Libraries for sequencing plots of the V3-V4 gene of the 16S ribosomal RNA were prepared according to the 16S Metagenomic Sequencing Library Preparation Illumina. In bioinformatics assessment, Kraken Metagenomics version 2.0.0 software for metagenomic analysis (classifier of reads — short nucleotide sequences) was used using a standard database.

Results. It was shown that the occurrence of representatives of the Lactobacillaceae family in the vaginal biotope varies from 12 to 84%. The genus Lactobacillus with the dominance of L. jensenii, L. delbrueckii and L. amylolyticus occupied the leading position among the members of the family. In all samples, Moraxella spp. was found in large numbers, with M. osloensis leading among the representatives of the entire community. In half of the cases, the joint presence of M. osloensis and G. vaginalis was revealed.

Conclusion. It has been established that in infertility in the vaginal microbiota the number of representatives of the genus Lactobacillus is significantly reduced, and there is also a change in the leading species to L. jensenii, whose functional activity does not fully ensure the colonization resistance of the vaginal biotope, allowing for excessive reproduction of oppotrunistic microorganisms, in particular, M. osloensis.

References

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2. Stone L. Infection: Vaginal microbiota and infectious infertility. Nat. Rev. Urol. 2018; 15(3): 136. DOI: http://doi.org/10.1038/nrurol.2018.11

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17. Mendes-Soares H., Krishnan V., Settles M.L., Ravel J., Brown C.J., Forney L.J. Fine-scale analysis of 16S rRNA se quences reveals a high level of taxonomic diversity among vaginal Atopobium spp. Pathog. Dis. 2015; 73(4): pii: ftv020. DOI: http://doi.org.10.1093/femspd/ftv020

18. Copeland A., Sikorski J., Lapidus A., Nolan M., Del Rio T.G., Lucas S., et al. Complete genome sequ ence of Atopobium parvulum type strain (IPP 1246). Stand. Genomic Sci. 2009; 1(2): 166-73. DOI: http://doi.org/10.4056/sigs.29547

19. Lewis F.M., Bernstein K.T., Aral S.O. Vaginal microbiome and its relationship to behavior, sexual health, and sexually transmitted diseases. Obstet. Gynecol. 2017; 129(4): 643-54. DOI: http://doi.org/10.1097/AOG.0000000000001932

20. Gómez-Camarasa C., Fernández-Parra J., Navarro-Marí J.M., Gutiérrez-Fernández J. Moraxella osloensis emerging infection. Visiting to genital infection. Rev. Esp. Quimioter. 2018; 31(2): 178-81. (in Spanish)

21. Goto T., Hirakawa H., Morita Y., Tomida J., Sato J., Matsumura Y., et al. Complete genome sequence of Moraxella osloensis strain KMC41, a producer of 4-Methyl-3-Hexenoic acid, a major malodor compound in laundry. Genome Announc. 2016; 4(4): e00705-16. DOI: http://doi.org/10.1128/genomeA.00705-16

22. Tan L., Grewal P.S. Pathogenicity of Moraxella osloensis, a bac terium associated with the nematode Phasmarhabditis hermaphrodita, to the slug Deroceras reticulatum. Appl. Environ. Microbiol. 2001; 67(11): 5010-6. DOI: http://doi.org/10.1128/AEM.67.11.5010-5016.2001

23. Morais I.M.C., Cordeiro A.L., Teixeira G.S., Domingues V.S., Nardi R.M.D., Monteiro A.S., et al. Biological and physico chemical properties of biosurfactants produced by Lactobacillus jen senii P6A and Lactobacillus gasseri P65. Microb. Cell Fact. 2017; 16(1): 155. DOI: http://doi.org/10.1186/s12934-017-0769-7

24. Martin R., Suarez J.E. Biosynthesis and degradation of H2O2 by vaginal lactobacilli. Appl. Environ. Microbiol. 2010; 76(2): 400-5. DOI: http://doi.org/10.1128/AEM.01631-09

25. O’Hanlon D.E., Moench T.R., Cone R.A. In vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide. BMC Infect. Dis. 2011; 11: 200. DOI: http://doi.org/10.1186/1471-2334-11-200

26. Kim S., Gu S.A., Kim Y.H., Kim K.J. Crystal structure and thermodynamic properties of d-lactate dehydrogenase from Lactobacillus jensenii. Int. J. Biol. Macromol. 2014; 68: 151-7. DOI: http://doi.org/10.1016/j.ijbiomac.2014.04.048

27. Tsukida K., Takahashi T., Iida H., Kanmani P., Suda Y., Nochi T., et al. Immunoregulatory effects triggered by immunobiotic Lactobacillus jensenii TL2937 strain involve efficient phagocytosis in porcine antigen presenting cells. BMC Immunol. 2016; 17(1): 21. DOI: http://doi.org/10.1186/s12865-016-0160-1

28. Patnaik S., Davila C.D., Chennupati A., Rubin A. Endocarditis of the native aortic valve caused by Lactobacillus jensenii. BMJ Case Rep. 2015; 2015: bcr2014206288. DOI: http://doi.org/10.1136/bcr-2014-206288