Title: Diagnostic gene biomarkers for predicting immune infiltration in endometriosis
Version: 1 Date: 11 Feb 2022
Reviewer's report:
• The current study uses data available in the GEO database to find new markers in
endometriosis. This approach has recently been actively used to study various conditions,
including endometriosis. The GEO datasets used in the current study have also been used as the
same set (PMID: 34232189) or in different combinations with data from other investigations.
the strength of the current study is that some findings have been experimentally proved and the
novel aspect is the use of the cell-type analysis.
• My main concern is the objective of the study. The study aims to determine the potential
diagnostic markers in endometriosis and concludes that AQP1 and ZWINT can be regarded
as diagnostic markers of endometriosis. However, there is no reasonable explanation how the
expression level of markers in endometriotic lesions and endometrial tissue could be used as
diagnostic markers.
•
• In addition:
• 1. The details of original publications are provided in GEO and should be referred to if
using the data generated in these studies: GSE7305, Hever et al (PMID:17640886), GSE25628,
Crispi et al (PMID: 23460397), GSE5108, Eyster et al (PMID: 17462640). Also, the authors
mention that because of the retrospective nature of their research, they were not able to obtain
the clinical information associated with the samples. All three original publications include
the characteristics of patients and samples. Moreover, all three studies have analysed different
types of endometriotic lesions: GSE7305 used ovarian lesions, GSE25628 used deep infiltrating
lesions and GSE5108 analysed peritoneal implants and endometriomas. The co-analysis of
different types of lesions may affect the results and this issue should at least be discussed.
2. For verification of the markers, endometrial tissues and endometriotic lesions were collected.
However, there are no characteristics of the patients included. How many patients, what kind
of lesions, the use of hormones etc. Was written informed consent obtained from each patient?
Furthermore, it is mentioned that all tissue samples were taken from tissues discarded during
surgery. Why was eutopic endometrial tissue removed from these patients? It is usually not
necessary during laparoscopy of endometriosis.
3. The methods used for verification should be better described. It won’t suffice to cite the
previous publication where different antibodies were used.
4. Results: 53 DEGs were obtained: 28 significantly upregulated and 25 significantly
downregulated genes (Fig.1). However, 69 genes are shown in Fig. 1. Are they all DEGs?
Furthermore, the profiles of two endometrial samples are very different from the rest of
endometria. If these samples are clear outliers, they should be excluded from the analysis. The
figure legend is too scarce and it is unclear in which tissue (eutopic/ectopic) the genes are up
or down-regulated.
5. Fig. 2A. It is not clear whether all shown correlations are statistically significant.
6. As already mentioned, the part of the manuscript describing the identification of diagnostic
markers remains unclear, as there is no justifications of how these genes could be used as
diagnostic markers. At best, this would allow a distinction to be made between endometrial and
lesion tissue biopsies.
7. As it is not mentioned how many samples were used for verification, it is not possible to assess
the reliability of the verification is. Also, no p-values are shown.
8. It is said that the proportions of M2 macrophages (P < 0.001), and activated mast cells (P =
0.001) were significantly lower in EMS tissues than in endometrial tissues. However, the results
shown in Fig. 7A show the opposite: the mean values of both cell-types appear to be higher
in lesions.
9. The discussion is largely a repetition of the results. instead, the authors should discuss how
to put these results into practice or what they add to our knowledge of the pathogenesis of the
disease.
Minor comments:
Abstract. The first sentence of Results says: Fifty-three genes were identified in cells from
benign neoplasms, polycystic ovary syndrome, gallbladder carcinomas, and adenomas. This is
unclear as endometriosis was investigated.
The introduction contains a lot of long-known knowledge that would rather fit in the textbook
and is not relevant in the context of the current manuscript.