Title: Does the uterine microbiota affect the reproductive outcomes in women with recurrent
implantation failures?
Version: 2 Date: 02 Jan 2022
Reviewer's report:
Thank you for the opportunity to evaluate this report. The paper is about the effect of uterine
microbiata on recurrent implantation failure  in IVF cycles. I have the following comments:
1. Please define the “recurrent implantation failure” in the methods section.
2. There are repetitive words in the sentence of  “Microorganisms of the
genera Lactobacillus, Tepidimonas, Streptococcus, Acinetobacter, Proteus, Acinetobacter,
Enterococcus, Pseudomonas, Veillonella were detected in these samples; such genera were also
detected in negative control samples.”
3. The sentences between the line 268 and 282 should be in the discussion section, not in the
results section. The results section should be include only the study outcomes. Comments on
the results should be in the discussion section.
4. In the results section, you say “We conducted a comparative analysis of the microbiota of the
uterine cavity and the cervical canal of 130 women.” and then “ The analysis showed that the
microbiota of the uterine cavity and the cervical canal was identical only in 14 (12.1%) of 116
women, and it was different in qualitative composition in 102 (87.9%) patients.” Why didn’t
you mention the results of the remaining 14 women?
5. Why did you carry out metagenomic study in only 30 samples, not in the all samples? Please
discuss this issue in the discussion section.
6. In discussion section, strenghts and limitations of study should be discussed.
7. There is not enough reference in the paragraph between the lines 358 and 377. Please add
references at the end of indicated sentences.
“A separate stage of the study was the metagenomic sequencing of the uterine microbiota (the
distal part of the embryo transfer catheter in the culture medium).  This method of studying
microbiota is described in the studies of some researchers [10, 13, 14]. In most cases, the
microorganism diversity in the uterine cavity was the same according to microbiological studies
and metagenomic analysis (reference). In some cases, there were bacterial genera that were
not identified with microbiological studies (reference). The results can be explained by the
presence of microorganisms in the sample; these microorganisms did not grow in the culture
media for various reasons, but their DNA was present in the sample. The obtained data may
be due to the presence of trace amounts of bacterial DNA in the reagents and indicate that
sequencing method is not sufficiently specific for analyzing samples with an extremely low
DNA concentration. It can be assumed that besides the presence of microorganisms in the
uterine cavity, a quantitative assessment of the composition of the microflora is important;
however, it can be problematic to carry out the assessment at the time of embryo transfer due
to insufficient amount of material obtained with an embryo transfer catheter. The condition
of the whole organism is also essential since the endometrium can be protected against the
destructive effect of the microbial factor. Microorganisms are known to be inactivated by cells
involved in innate and adaptive immunity responses under physiological conditions (reference).
In the early stages, this process is carried out by Toll-, NOD-, and RIG-like receptors which
are located on the surface of cells of the immune system, epithelial cells, endothelial cells, and
fibroblasts (reference). Microbial colonization of the endometrium is also restrained by Toll-
like receptors, which stimulate the mechanisms of innate antimicrobial resistance by interacting
with the structures of the microbial cell (reference).”
8. The conclusion section should be revised. There are sentences that are not supported by the
results of the research in this section. These sentences should be removed. The conclusion should
emphasize the main outcomes of the study.
9. The 9th and 2st references are the same article. Please revise the references.