Review for 10.1186/s12876-020-01510-w (2020)

Reviewer’s report
Title: 
Characterization of Gut Microbiota Associated with Clinical Parameters in
Intrahepatic Cholestasis of Pregnancy
Version: 
0 
Date: 
08 Feb 2020
Reviewer: 
Valerio Iebba
Reviewer's report:
-page 2, line 4: please number the pages of your manuscript, following a
continuous line numbering (not starting page by page ex novo), and with a
proper line-aligned numbers.
-page 3, line 22: please add the region you amplified.
-page 3, line 40: please use Bray-Curtis distance instead of Unifrac, and redo the
overall PCoA.
-page 3, line 42: avoid subjective expression, refer only to statistical evidence.
-page 3, line 50: I would suggest to do a KEGG gene analysys, not only at
pathway level.
-page 3, line 54: erase "phylogenetic", it adds nothing to the sentence.
-page 3, line 57: pay attention to the term "composition": it means a general term
for beta-diversity. Thus, you should show a clear correlation of the beta-diversity
measure (e.g., I advice using Bray-Curtis distance) with the clinical parameters.
Please add the correlation analysis (a clustermap) of alfa- (Shannon and OTU
number) and beta-diversity (Bray-Curtis distance) measurement with clinical
parameters to Mat&Met and Result sections.
-page 3, line 59: maybe "related to" should replace "associated with"
-page 3, line 2: actually no results of progression were presented, only ICP status
vs controls. please acknowledge this aspect in your analyses, or provide a time-
course analysis.
-page 6, line 19: replace "roled" with "roles"
-page 6, line 21: the reference seems to be not appropriated (refers to worm
C.elegans which has only an innate immune system dissimalr to vertebrates). I
suggest to replace it with the following ones:
i) PMID: 23298474 DOI: 10.1177/039463201202500401
ii) PMID: 20656465 DOI: 10.1016/j.coi.2010.06.008
-page 6, line 31: the reference from Guarner, even if excellent, is somewhat
outdated. I would suggest to add these references to it:
- PMID: 26922981, New Microbiol. 2016 Jan;39(1):1-12
- PMID: 26210164 DOI: 10.1016/j.tibtech.2015.06.011
- PMID: 28626231 DOI: 10.1038/nrmicro.2017.58
-page 7, line 1-4: an important liver pathology, liver cirrhosis, wasn't mentioned
(especially for the interrelation biliary acids - portal blood - gut microbiota). I
would like Authors to add these references:
- 
PMID: 23851335 PMCID: PMC3839982 DOI: 10.4161/gmic.25723
- PMID: 29844325 PMCID: PMC5974022 DOI: 10.1038/s41598-018-26509-y
-page 7,, line 8: please add the actual number of women enrolled in the two
categories.
-page 7, line 13: specify the 16S region.
-page 7, line 36: provide the protocol number of EC approval for the present
study.
-page 7, line 45: please provide also the fecal BA quantitation (see as reference
Lipids Health Dis. 2018; 17: 121.
Published online 2018 May 23. doi: 10.1186/s12944-018-0774-9).
-page 7, line 53: did the patients enrolled had an antibiotic treatment within the
previous two months? That would be of extreme importance: please report ant
ATB treatment/history record for each patient and put this information (among
the others) intyo a Demographic Table describing patients' characteristics.
-page 7, line 59: please report the Bristol Score for each stool sample, and
provide a summary distribution pewrcentage within the Demographics Table.
-page 8, line 29: software or pipeline? Please explain deeply the characteristics
of your software/pipeline if not published yet (stay within general terms).
-page 8, line 33; tags or contigs?
-page 8, line 33: please provide the output of DADA2 pipeline, which is more
robust than OTU-based analysis (OTUs output rely on sample number, DADA2
not), and redo all the analysis to confirm and (possibly) strenghten your results.
-page 8, line 40: please provide also the taxonomical annotations made with
BLASTn using the 16S NCBI Database, which is updated every 2 days.
-page 8, line 48: please use only Bray-Curtis distance for all of your results and
figures. Rewrite results, figure legends, figure composition according only to
Bray-Curtis distance.
- page 9, line 1: insted of using LEfSe (which is known to enhance species that
are not prevalent within the cohorts, even in its advanced form LEfSe2), I
suggest to implement a PLS-DA analysis with a VIP plot, or to do a Random
Forest (RF) analysis. Please provide a VIP and a RF plot, and find the
similarities among them. Exclude LEfSe from your analysis.
-page 9, line 2: please redo the analysis using Picrust2, which is more affordable.
-page 9, line 15: please specify the percentage of FDR and if it was one-stage or
two-stages.
-page 9, line 17: I would suggest to not use Spearman's correlation (which is
based on median): instead use Pearson's correlation (which is based on mean) on
your normalized AND standardized 16S raw data. Please report if you made
these important steps of normalization (NOT logarythmization) and
standardization and repeat your analyses of alfa- and beta- diversity.
-page 9, line 25: equal or less than 0.05, not only less than 0.05. I would see a
Bonferroni correction for Mann-Whitney U test comparison among the two
cohorts (ICP vs controls) and not an FDR. FDR is usually used for multiple
comparison within correlograms, where you have tons of linear correlation
among the variables of dataset A (e.g., species) with the same variables of
dataset A (auto-correlation clustermap), or with a different dataset B (cross-
corelation). Please redo the Pvalues after Mann-Whitney U test with Bonferroni
adjustment and not FDR.
-page 9, line 31: please provide in a new figure a correlogram of bacterial
species having a prevalence >= 20% with the clinical/biochemical parameters,
only for the ICP cohort, and draw significant correlations in a red-to-blue fashion
for positive-to-negative Pearson correlations (naturally on normalized AND
standardized data!).
-page 10, line 3: please report the interquartile range, the mean value (with
SEM), and the test you used to calculate the p-value. Of note, be consistent
throughout your manuscript with italicizing the P (or the p) of P-value (or p-
value).
- page 10, line 32: again, tags or contigs? Maybe you mean contigs (the
superimposition of R1 and R2 reads provided within the same fastq file). Tags
are a different thing, please be precise in your descriptions.
-page 10, line 38: please provide the TOTAL number of OTUs you found for the
two cohorts: it would be nice to have a description of your rarefaction strategy in
Mat&Met (for example, if you split the OTUs ruling out those having a total
population of only 10 contigs, or if you normalized your samples taking the
same number of contigs based on the sample having the lowest number of them).
Please explain your rarefaction strategy in Mat&Met.
-page 10, line 44: again, evrytime the P (or p) values (not the numbers, only the
P or p) needs to be reported in italic.
-page 10, line 50: please avoid subjective affirmation and report objectively the
mean (and SEM) percentage of coverage for both your cohorts (IPC and
controls).
-page 11, line 4: please report and redo PCoA with only Bray-Curtis distance,
which better handle sparse matrices as always present in microbial relative
abundance tables.
-page 11, line 23: redo the analysis after normalization/standardization using
Pearson's correlation. Use FDR.
-page 13, line 23: I suggest to not show Figure 4.
-page 13, line 30: see my comments about LEfSe above.
-page 13, line 49: please shorten the Discussion + Conclusions section: this
section should not be longer than Results one (table excluded, in the present
manuscript Result section is two-pages).
- page 14, line 46: about the role of gut microbiota in liver cirrhosis please report
also these references:
- 
PMID: 23851335 PMCID: PMC3839982 DOI: 10.4161/gmic.25723
- PMID: 29844325 PMCID: PMC5974022 DOI: 10.1038/s41598-018-26509-y
- PMID: 26210164 DOI: 10.1016/j.tibtech.2015.06.011
-page 15, line 27-34: please report a reference for this speculative affirmation.
-page 15, line 40: typo in staphylococcus
-page 15, line 54: typo in Citrobacter rodentium
-page 17, line 47: please provide a working link to the Project Databse
CNP0000403.
-page 23, line 34: figure legends should be readable and auto-explanaible, thus
please write more infos in each of them, describing briefly the methods and the
depicted parts (if relevant) of each figure.
-page 25: Figure 1 should be represented not as violin plots, but boxplots
reporting all the single datapoints for each cohort.
-page 26: Figure 2 should report 95% CI ellipses for the two cohorts, in order to
ease the reader in understanding the putative separation among them. Again, p
values in italic (only the p, not the number). Please report only PCoA with Bray-
Curtis.
-page 27: Figure 3 should report asterisks (*, **, ***) for the different P values
thresholds, for each 2-by-2 comparison when significant. Again, replace barplots
with boxplots and draw all the single datapoints. Replace "abundant" with
"abundance", and add the unit of measurement (%).
-page 28: Figure 4 should be canceled.
-page 29: Figure 5 should report VIP and RF plots, along with species
similarities among the two different algorithms. Report also aside species names
the corresponding asterisks if those species are significantly and differently
distributed among the cohorts (only for relative abundance, not for prevalence).
Erase completely all LEfSe results.
Are the methods appropriate and well described?
If not, please specify what is required in your comments to the authors.
Yes
Does the work include the necessary controls?
If not, please specify which controls are required in your comments to the
authors.
Yes
Are the conclusions drawn adequately supported by the data shown?
If not, please explain in your comments to the authors.
No
Are you able to assess any statistics in the manuscript or would you
recommend an additional statistical review?
If an additional statistical review is recommended, please specify what aspects
require further assessment in your comments to the editors.
I am able to assess the statistics
Quality of written English
Please indicate the quality of language in the manuscript:
Needs some language corrections before being published
Declaration of competing interests
Please complete a declaration of competing interests, considering the following
questions:
1
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Do you hold or are you currently applying for any patents relating to the
content of the manuscript?
4
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organization that holds or has applied for patents relating to the content of
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5
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Do you have any other financial competing interests?
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paper?
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