The female urinary microbiota in relation to the reproductive tract microbiota

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Human urine is traditionally considered to be sterile, and whether the urine harbours distinct microbial communities has been a matter of debate. Potential links between female urine and reproductive tract microbial communities is currently not clear. Here, we collected urine samples from 147 Chinese women of reproductive age and explored the nature of colonization by 16S rRNA gene amplicon sequencing, quantitative real-time PCR, and live bacteria culture. To demonstrate the utility of this approach, the intra-individual Spearman’s correlation was used to explore the relationship between urine and multiple sites of the female reproductive tract. PERMANOVA was also performed to explore potential correlations between the lifestyle and various clinical factors and urinary bacterial communities. Our data demonstrated distinct bacterial communities in urine, indicative of a non-sterile environment. Streptococcus-dominated, Lactobacillus-dominated, and diverse type were the three most common urinary bacterial community types in the cohort. Detailed comparison of the urinary microbiota with multiple sites of the female reproductive tract microbiota demonstrated that the urinary microbiota were more similar to the microbiota in the cervix and uterine cavity than to those of the vagina in the same women. Our data demonstrate the potential connectivity among microbiota in the female urogenital system and provide insight and resources for exploring diseases of the urethra and genital tract.

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  1. Now published in Gigabyte doi: 10.46471/gigabyte.9

    **Reviewer 2. Levi Waldron ** Chen et al. use 16S amplicon metagenomic sequencing to investigate urinary bacterial communities and their correlation to lifestyle and clinical factors, and reproductive tract (cervix, uterine cavity, vagina) microbiota in a cross-sectional study of 147 Chinese women of reproductive age. This is an important but challenging study, because of the threat of microbial contamination in low microbial biomass specimens such as the upper reproductive tract and urine.

    Checklist item 4.0

    The laboratory /center where laboratory work was done is not actually stated in lines 121-133.

    Negative controls and contamination

    Negative controls were generated for the 10 women undergoing surgery through as sterile saline collected through the urine catheter. I assume this was done after the catheter was used for urine collection, but this should be stated.

    No negative controls were used for the self-collected urine specimens. However it seems likely that mid-stream self-collection would be more prone to contamination than catheter sampling by a doctor during surgery. Some possibilities for negative controls in this setting exist, such as including a sample of sterile saline with the self-collection kit and asking participants to fill another vial with it immediately following urine collection. The lack of negative controls for self- collected specimens should be stated as a limitation.

    The authors identify the risk of contamination from vulvovaginal region (lines 192-193) but not of cross-contamination. Discussion of the risk of cross-contamination during collection and subsequent processing, steps to mitigate and identify it, and comparison of results to bacterial taxa identified as common contaminants (e.g. Eisenhofer et al, PMID 30497919), is warranted.

    Comparability of urine sampling methods

    Since no specimens were collected by both self-collection and catheter sampling during surgery, there is no way to directly assess the accuracy of self-collection using catheter as a “gold standard” This should be stated as a limitation.

    I could not find an analysis comparing the microbial composition of the catheter-collected and self-collected specimens. Some analysis comparing the two could help address the quality of self-collected specimens lacking negative controls.


    The authors do not include overall interpretation or limitations in the Discussion, saying under checklist items 12.0, 12.1, 13.0 “The discussion was suggested to focus on the potential uses according to the article format.” I think the editors should clarify to authors where these key discussion points belong. I think no article is complete without some discussion of limitations; see above for limitations noted of this study.

    Checklist item 13.2 Generalizability

    Authors state “The generalizability of the study is to women of reproductive age, and is shown in line 236-237” but on these lines I see description of statistical methods. This does deserve some discussion though, because the sample includes only women who underwent hysteroscopy and/or laparoscopy for conditions without infections, and has a number of exclusion criteria. This cannot be a representative cross-section of all women of reproductive age, so some discussion of how this sample may be different or similar to the population of all women of reproductive age is warranted. If the authors claim this sample should be generalizable to all women of reproductive age, that should be stated along with the intentional restrictions of the sampling and rationale of why these criteria are not expected to have any impact on the microbiota sampled.

    Clustering of patients

    Lines 212-213: cutting a hierarchical clustering into discrete groups can be done for any dataset, and without some analysis such as Prediction Strength (Tibshirani and Walther, J. Comput. Graph. Stat. 14, 511–528 (2005)) or another measure of cluster validation, this isn’t evidence of distinct patient groups and that should be stated clearly. It is OK to use the grouping to discuss general trends as long as care is made not to imply these are distinct patient subsets without further analysis. I am cautious about this because distinct subsets are intuitively appealing to many readers and the existence of distinct subsets can be harder to correct than to claim.


    Line 241 “As the large-scale cohort” -> As a large-scale cohort

  2. Abstract

    **Reviewer 1. Christopher Hunter ** Is the language of sufficient quality?


    Is the data all available and does it match the descriptions in the paper?


    Comment: line 96-97 "In this study, a total of 147 reproductive age women (age 22-48) were recruited by Peking University Shenzhen Hospital (Supplementary Table 1)." B utSup. table 1 has only 137 samples. Revise text to explain only 137 samples were used for the main analysis, with the 10 extra for validation. Line 103 -104 "None of the subjects received any hormone treatments, antibiotics or vaginal medications within a month of sampling." Sup Table 1 has a column for "Antibiotic use True/False", 41 samples have "T"? this needs explaining. Its possible the spreadsheet True is referring to a longer time period, but thats not explained anywhere. line 110-112 "The samples from an additional 10 women were collected for validation purposes by a doctor during the surgery in July 2017." Where are these metadata? they are not included in Sup table 1. The data presented and discussed in "additional-findings.docx" are not included in the data files (yet), these should either be removed (as not included in the main article), or expand upon the methods (to include negative control details) and add this to main text.

    Is the data and metadata consistent with relevant minimum information or reporting standards?


    Comment. The supplemental tables need some better legends/descriptions to help readers understand what data is in them.

    Is the data acquisition clear, complete and methodologically sound?


    Comment. The wet and bioinformatics methods could benefit from being included in

    Is there sufficient detail in the methods and data-processing steps to allow reproduction?


    Is there sufficient data validation and statistical analyses of data quality?


    Is the validation suitable for this type of data?


    Is there sufficient information for others to reuse this dataset or integrate it with other data?


    Any Additional Overall Comments to the Author


    The Figure appear to be mixed up, what’s displayed as Figure 1 in the manuscript appears to relate to the legend given for Figure 2, Figure 2 relates to legend of Figure 3, and Figure 3 relates to the legend of Fig 1!!! line 69 -Chen et al. no citation number link provided line 74 -Thomas-White et al. (2018) no citation number link provided line 79 -Gottschick et al. (2017) no citation number link provided line 246-248 "The initial results here indicate a close link between the urinary microbiota with the general and diseased physiological conditions,... " As this study is looking at "Healthy" individuals I do not believe there is sufficient evidence to back up this statement about the "diseased" physiological conditions. line 274-275 "The sequences of bacterial isolates have been deposited in the European Nucleotide Archive with the accession number PRJEB36743" this accession is not public so I am unable to see whats included here. If available we would like to see the Real-Time PCR Data from the experiments made available in Real-Time PCR Data Markup Language (RDML). The additional cohort of 10 women is almost a different study, it didn't have the same 16s RNA amplicon sequencing done, and was only a validation that some live bacteria can be cultured from urine in a small number of cases (3/10). If it is to be included table S5 should be updated to include the specific INSDC accessions for the submitted sequences. (title of Table S5 in file is currently saying Table 1).