Frequency of Gene Polymorphisms in Admixed Venezuelan Women with Recurrent Pregnancy Loss: Microsomal Epoxy Hydroxylase (rs1051740) and Enos (rs1799983)

Round 1

Reviewer 1 Report

The article type “communication” entitled “Frequency of Gene Polymorphisms in Admixed Venezuelan Women with Recurrent Pregnancy Loss: Microsomal Epoxy Hydroxylase (RS1051740) and Enos (RS1799983).” (cimb-2964445). It is presented to de Section “Biochemistry, Molecular and Cellular Biology” in the Special Issue “Molecular Research in Reproductive Biology, 2nd Edition”.

The topic of this article aligns with the section in which it is presented.

The study aimed to evaluate certain aspects of oxidative metabolism in patients with frequent miscarriages by determining genetic polymorphisms in microsomal epoxy hydroxylase (mEPH or EPHX1, T612C, rs1051740) and nitric oxide synthase 3 (eNOS. G894T, rs1799983) in admixed Venezuelan women with primary Recurrent Pregnancy Loss (RPL).

Comments:

 The summary should indicate the design used.

In the introduction, the importance of the topic is discussed, but it is not informed why certain polymorphisms are used as suspected associations with RPL. It would be interesting to propose a reasoned hypothesis for why these polymorphisms are suspected in relation to RPL.

In the materials and methods section, the design used should be indicated; apparently, it appears to be a case-control design, but it is unbalanced. Normally, when the sample is small, double the number of cases than controls is used to compensate for the sample. Additionally, an appropriate sample size calculation should have been proposed to justify the sample used. Regarding the methodology used in determining the polymorphisms, I suggest reviewing the description of the procedure and structuring it if possible. In relation to the program used, the version should be stated. The statistical methodology should be thoroughly described.

Regarding the results, Table 1 should be included in the results section; it currently appears in the introduction. In the discussion, the contributions of the work are usually presented first, followed by a discussion of these results with the literature.

The discussion should be tailored to the topic at hand, which is these polymorphisms. Strengths and weaknesses of the study conducted should be added.

I recommend reviewing the text.

Author Response

We want to thank the reviewer for the useful comments.

In the introduction, the importance of the topic is discussed, but it is not informed why certain polymorphisms are used as suspected associations with RPL. It would be interesting to propose a reasoned hypothesis for why these polymorphisms are suspected in relation to RPL.

Response

1.- We added a paragraph specifying the hypothesis.

In the materials and methods section, the design used should be indicated; apparently, it appears to be a case-control design, but it is unbalanced. Normally, when the sample is small, double the number of cases than controls is used to compensate for the sample. Additionally, an appropriate sample size calculation should have been proposed to justify the sample used. Regarding the methodology used in determining the polymorphisms, I suggest reviewing the description of the procedure and structuring it if possible. In relation to the program used, the version should be stated. The statistical methodology should be thoroughly described.

2. The study's definition was added to the Material and Methods section. The number of controls was increased to 50, and the statistical analysis was added. The minimum number for the analysis was set at 45 based on the frequency of the mutated allele of microsomal Epoxy Hydrolase in the normal population. Now, the total number of controls is 50, and of patients, 63.

Regarding the results, Table 1 should be included in the results section; it currently appears in the introduction. In the discussion, the contributions of the work are usually presented first, followed by a discussion of these results with the literature.

3.- Table 1 was changed as proposed.

The discussion should be tailored to the topic at hand, which is these polymorphisms. The strengths and weaknesses of the study conducted should be added.

4.- A paragraph was added to the discussion. The manuscript's strength is the strict genetic analysis of the admixed population and the criteria of primary, recurrent pregnancy loss; unfortunately, this criterion decreases the number of available samples. Despite the low number significance in one SNP of epoxy hydrolase that was never assessed in recurrent pregnancy, the loss was significant along with the other from NOS3.

Reviewer 2 Report

 _{The purpose of the present study was to assess the frequency of two SNP in different enzymes involved in medical conditions observed during pregnancy. Microsomal epoxy hydrolase (mEPH) is involved in detoxifying xenobiotics. It is present in ovaries, and it is hormonally regulated. The endothelial nitric oxide synthase forms nitric oxide from arginine and is involved in vascular tone. As a results, authors suggested that genetic studies could provide crucial information on the aetiology of primary RPL.}

_{1) What is this study's hypotheses? Could you show this more exactly?} 

_{2) Authors mentioned that the number of samples was the main limitation. RPL samples were from patients whose medical history and follow-up were for over two years. Most of the RPL patients were attended by the fertility clinic without success. The controlsXXX as a limitation. Are there any other study limitations here?}

_{3) Please double check how to cite the literature.}

NA

Author Response

We want to thank the reviewer for the useful comments.

1) What is this study's hypotheses? Could you show this more exactly?

A paragraph was added

2) Authors mentioned that the number of samples was the main limitation. RPL samples were from patients whose medical history and follow-up were for over two years. Most of the RPL patients were attended by the fertility clinic without success. The controlsXXX as a limitation. Are there any other study limitations here?

The number of controls was increased. There was no other limitation.

3) Please double check how to cite the literature.

The references were checked.

Round 2

Reviewer 1 Report

I have thoroughly reviewed the latest version of the manuscript “Frequency of Gene Polymorphisms in Admixed Venezuelan Women with Recurrent Pregnancy Loss: Microsomal Epoxy Hydroxylase (RS1051740) and Enos (RS1799983)” (cimb-2964445)., along with the reviewers' response to our suggestions.

Regarding the Materials and Methods section, it is indicated in lines 170-171 the test used for comparison. I suggest specifying that both absolute frequency and relative frequency have been utilized, and significance has been attributed to a p-value less than 0.05.

Author Response

We would like to express our gratitude to the reviewer for the valuable comments which is incorporated in the text of the new version, as suggested