The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases

ARTICLE DOI: 10.1038/s41467-017-00901-0 OPEN The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases Chen Chen1,2, Xiaolei Song1,3, Weixia Wei4,5, Huanzi Zhong 1,2,6, Juanjuan Dai4,5, Zhou Lan1, Fei Li1,2,3, Xinlei Yu1,2, Qiang Feng1,7, Zirong Wang1, Hailiang Xie1, Xiaomin Chen1, Chunwei Zeng1, Bo Wen1,2, Liping Zeng4,5, Hui Du4,5, Huiru Tang4,5, Changlu Xu1,8, Yan Xia1,3, Huihua Xia1,2,9, Huanming Yang1,10, Jian Wang1,10, Jun Wang1,11, Lise Madsen 1,6,12, Susanne Brix 13, Karsten Kristiansen1,6, Xun Xu1,2, Junhua Li 1,2,9,14, Ruifang Wu4,5 & Huijue Jia 1,2,9,11 Reports on bacteria detected in maternal fluids during pregnancy are typically associated with adverse consequences, and whether the female reproductive tract harbours distinct microbial communities beyond the vagina has been a matter of debate. Here we systematically sample the microbiota within the female reproductive tract in 110 women of reproductive age, and examine the nature of colonisation by 16S rRNA gene amplicon sequencing and cultivation. We find distinct microbial communities in cervical canal, uterus, fallopian tubes and peritoneal fluid, differing from that of the vagina. The results reflect a microbiota continuum along the female reproductive tract, indicative of a non-sterile environment. We also identify microbial taxa and potential functions that correlate with the menstrual cycle or are overrepresented in subjects with adenomyosis or infertility due to endometriosis. The study provides insight into the nature of the vagino-uterine microbiome, and suggests that surveying the vaginal or cervical microbiota might be useful for detection of common diseases in the upper reproductive tract. 1 BGI-Shenzhen, Shenzhen 518083, China. 2 China National Genebank, BGI-Shenzhen, Shenzhen 518120, China. 3 BGI Education Center, University of Chinese Academy of Sciences, Shenzhen 518083, China. 4 Peking University Shenzhen Hospital, Shenzhen 518036, China. 5 Shenzhen Key Laboratory on Technology for Early Diagnosis of Major Gynecological diseases, Shenzhen 518036, China. 6 Department of Biology, Laboratory of Genomics and Molecular Biomedicine, University of Copenhagen, Universitetsparken 13, 2100 Copenhagen, Denmark. 7 Shenzhen Engineering Laboratory of Detection and Intervention of human intestinal microbiome, BGI-Shenzhen, Shenzhen 518083, China. 8 Qingdao University-BGI Joint Innovation College, Qingdao University, Qingdao 266071, China. 9 Shenzhen Key Laboratory of Human Commensal Microorganisms and Health Research, BGI-Shenzhen, Shenzhen 518083, China. 10 James D. Watson Institute of Genome Sciences, Hangzhou 310000, China. 11 Macau University of Science and Technology, Taipa, Macau 999078, China. 12 National Institute of Nutrition and Seafood Research, (NIFES), Postboks 2029, Nordnes, N-5817 Bergen, Norway. 13 Department of Biotechnology and Biomedicine, Technical University of Denmark, Soltofts Plads, Building 221, 2800 Kongens Lyngby, Denmark. 14 School of Bioscience and Biotechnology, South China University of Technology, Guangzhou 510006, China. Chen Chen, Xiaolei Song, Weixia Wei, Huanzi Zhong, Juanjuan Dai and Zhou Lan contributed equally to this work. Correspondence and requests for materials should be addressed to J.L. (email: ) or to R.W. (email: ) or to H.J. (email: ) NATURE COMMUNICATIONS | 8: 875 | DOI: 10.1038/s41467-017-00901-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00901-0 I n marsupials and placental mammals, the female reproductive tract has developed unique structures such as the vagina and the uterus. During the reproductive cycle, mature oocytes from the ovaries enter the peritoneal cavity to be captured by fimbriae of the fallopian tubes. The oocytes are fertilised in the fallopian tubes, and the zygotes develop and translocate to the uterus for implantation. While the vagina is home for trillions of bacteria, the uterus and the fallopian tubes are generally believed to be sterile, which would require the cervix to be a perfect barrier. Mucins in the cervix, however, are known to change conformation, leading to aggregations dependent on pH variations during the menstrual cycle1. Such changes may in theory allow passage of bacteria during certain conditions. Lactobacilli are known as the keystone species of the vaginal microbiota in reproductive-age women2. Indeed, cultureindependent 16S rRNA gene amplicon sequencing studies from the United States have identified 5 community types of vaginal microbiota, 3 or 4 of which contain >90% Lactobacillus3–5. The lactic acid produced by the vaginal microbiota helps maintain a low pH of 3.5–4.5, a major factor in limiting the growth of potentially harmful bacteria. Alterations in the vaginal microbiota play a role in common conditions such as bacterial vaginosis, sexually transmitted diseases, urinary infections and preterm birth2, 6, 7. In contrast, the upper reproductive tract remains largely unexplored. Bacteria have mostly been studied in small sample sizes in the context of infection, especially in relation to preterm birth6. Although controversial, the placenta has recently been reported to harbour a microbiota8. It is as yet not clear what type of bacteria, if any, may exist in the upper reproductive tract of the vast majority of women who are not in periods of infection or pregnancy. Furthermore, it is not known if an upper reproductive tract microbiota could play a role in uterinerelated diseases such as hysteromyoma, adenomyosis and endometriosis. Here, we systematically sampled the microbiota at six sites within the female reproductive tract, from a large cohort of Chinese women of reproductive age. Bacteria were identified by using 16S rRNA gene amplicon sequencing, real-time qPCR, as well as conventional bacterial culturing. The results indicate a continuity of the vagino-uterine microbiome, with a distinct trend within the same individual. Potential bacterial markers for adenomyosis and endometriosis were also identified. Results Microbiota composition at six sites within the female reproductive tract. To explore the microbiota beyond the vagina, we collected samples from six locations (CL, lower third of vagina; CU, posterior fornix; CV, cervical mucus drawn from the cervical canal; ET, endometrium; FLL and FRL, left and right fallopian tubes; PF, peritoneal fluid from the pouch of Douglas) throughout the female reproductive tract from an initial study cohort of 95 Chinese women submitted to surgery for conditions not known to involve infections (Fig. 1). These conditions included hysteromyoma (also known as uterine fibroids), adenomyosis, endometriosis and salpingemphraxis, which are to our knowledge the best proxies for the upper reproductive tract in healthy young women (Supplementary Data 1). Samples from the vagina and the cervical mucus (CL, CU, CV) were taken upon visit to the clinic (without any prior disturbance (...truncated)