Reviewer’s report
Title: Evaluation of apoptosis and angiogenesis in ectopic and eutopic stromal cells of patients with
endometriosis compared to non-endometriotic controls
Version: 1 Date: 01 Oct 2019
Reviewer: Sandra Cecconi
Reviewer's report:
In  this  study,  the  authors   investigated  the  differences  in  gene  expression  of    apoptotic  and
angiogenetic markers ( Bcl-2, Bcl-xL, Bax, caspase 3, VEGF-A) among  three types of stromal cells
isolated from biopsies of endometriotic patients (ectopic: EESCs and eutopic: EuESCs) and non-
endometriotic  patients  (CESCs).   They  found  higher  expression  of  Bcl-2,  Bcl-xL in  EESCs  in
comparison with EuESCs and CESCs, lower expression of caspase 3 in EuESCs in comparison with
EESCs and CESCs, higher expression of VEGF-A in both endometriotic samples  compared with
control.  Bax gene  was  similar  among  the  three  groups. The  authors   concluded  that  absence  of
apoptosis  and  the  increased  angiogenesis  potential  in  EESCs  could  contribute  to  explain   the
pathogenesis of  endometriosis.
Although of great interest, the following  major revisions are required before publication.
MAJOR REVISIONS:
1.On the basis of  literature data, the authors suggested the involvement of apoptotic proteins as
important  factors  in  the  etiology  and  pathophysiology  of  endometriosis  (Refs  6-8,  10,  11).  As
mentioned in these papers and considering the fact that endometriosis is notably an inflammatory
disease, a pivotal role is played by Fas/FasL and TNF activation, both activators of the extrinsic
apoptotic pathway. The partial  activation of this pathway  has been reported in a recent paper, highly
pertinent to this Ms, that is not cited in the text (PMID: 31416694).  Please add and discuss differences.
2.A large number of biomarkers are involved in the angiogenic mechanisms of endometriotic
ectopic/eutopic lesions (PMID: 27541444), as VEGFR2, HIF1A, HGF, and PDGFB.  It is important to
add the analysis of some of these biomarkers to the panel of angiogenic factors studied to support
conclusions. It is not correct to speculate about increased angiogenic potential by investigating only
VEGFA.
3.The  analysis  of  gene  expression  is  not  per  se  indicative  of   protein  expression  levels.
Immunolocalization of proteins and/or WB analysis should better  support results.
Materials and methods:
4.Paragraph "Sample collection": 
*Lines 118: "Finally, cells from 17 eutopic and 11 ectopic endometrial tissues of endometriotic
patients and 15 eutopic endometrial tissue from non-endometriotic patients were used in this study".
These numbers are in contrast with the total number of patients claimed in the beginning of "Patients"
section. The authors enrolled for the study 25 endometriotic women and 20 non-endometriotic women.
Here, they stated that they use 28 tissues of endometriotic patients. Did the AA take more than one
sample for some patients? Please, explain.
5.In this section the paragraph "Statistical analysis" is missing. The authors reported statistical
significance  without explaining anywhere  which test has been used. Additionally, it is not clear how
experimental  data were expressed. What is the "expression ratio"?  Usually,  value of each gene is
compared  with the related housekeeping (gene expression/beta-actin).
Results:
6.Line  146-48.  Since  the  authors  reported  immunofluorescence  staining  as  method  for  the
evaluation of the purity of all ESCs groups, this reviewer suggests adding representative images for
each ESCs group.
7.Figures: in all the legends the authors  claimed that:
*"Data are expressed as mean and error". Please clarify. 
*"Each bar represents levels of … gene expression in two different endometrial cells". Do you
mean two cell groups? If so, please correct accordingly.
Discussion:
8.Lines 195-202: The authors  reported the results of Bax as not significant without discussing
this result. Please, explain which could be the reason(s) of such Bax gene expression.
9.Lines 219-37: As stated before, since the authors evaluated  only VEGF-A gene expression as
marker of angiogenesis,  this result  is not sufficient   to support the effective increase in angiogenesis.
Please, add  experiments with at least one  angiogenic marker other than VEGFA (See point 2).
Alternatively,  the strength of this paragraph and conclusions needs to be modified. 
MINOR REVISIONS:
10.Line 55: Correct "Despite being a quite common among women" with "Despite being quite
common among women", or "Despite being a quite common pathology among women".
11.Line 162: Correct "as" with "is".
12.437-38: Put "value" as subscripts.
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 recommend additional statistical review
Quality of written English
Please indicate the quality of language in the manuscript:
Acceptable
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