PONE-D-23-19156A comprehensive metabolic insight into recurrent spontaneous abortion based on UHPLC-MS/MS for oxidative stress and dysimmunityPLOS ONE

Dear Dr. Tuo,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: It is important to determine gene expression and discuss the RSA at the molecular level.

I suggest joining these findings with the abnormal expression of some specific immunoregulatory genes involved in T-cell activation and differentiation. According to psychophysiological interaction (PPI) analysis, TLR2, CXCL8, IFNG, IL2RA, and ITGAX were among the top five immunoregulatory hub genes of differentially expressed genes in unexplained recurrent spontaneous abortion (URSA). Among the identified differentially expressed genes (DEGs) in URSA, IFNG may play a key role in regulating maternal immune response. AS the key genes and functional pathways identified will provide new insights into the molecular mechanisms involved in RSA pathogenesis and provide potential diagnostic and therapeutic targets.

Reviewer #2: The research titled’’ A comprehensive metabolic insight into recurrent spontaneous abortion based on UHPLC-MS/MS for oxidative stress and dysimmunity ‘’ by Wu et al, investigated the underlying mechanisms contributing to the onset and progression of RSA by identifying potential metabolites as an initial step. This direct clinical study used eleven Serum samples from early pregnancy and 17 from recurrent spontaneous abortion were subjected to liquid chromatography/mass spectrometry (LC-MS) analysis.in addition to measurement of seven oxidative stress products, namely superoxide dismutase , catalase , malonaldehyde , glutathione , glutathione peroxidase , oxidized glutathione , heme oxygenase , was carried out using ELISA.

The current format could be improved by considering the following points:

1-Title: What does the term "dysimmunity" mean?Is it activation or suppression? It is redundant, in my opinion, and needs to be clarified.

2- Abstract:

In conclusion part : Our investigation furnished a more comprehensive analytical framework encompassing metabolites and oxykinase. what do you mean by oxykinase?

3-Introduction:

Both non-volatile targeted metabolites and volatile non-targeted metabolites have been employed on an MS platform to differentiate the RSA and healthy groups (6). The LC platform’s extensive coverage and board dynamic range of metabolites are its primary advantages

Please explain what these terms' abbreviation means.

4-How did you calculate the sample size for this work?

5- What criteria must the patients meet in order to be selected? In terms of age, race, and chronic illnesses like diabetes or other immunological dysfunctions? Metabolomics may be impacted by the factors listed above.

6- Data pretreatment and statistical analysis

To confirm the statistical significance of the differential metabolites, a student’s t-test was conducted between groups, with a p-value less than 0.05 based on OPLS-DA.

Please explain what these terms' abbreviation means.

7- Metabolites identification

AI database ?? Please explain what these terms' abbreviation means.

8- Study participants:

You mentioned that:

‘’The limited sample size precluded the statistical analysis of adverse factors to determine their association with disease’’

Please elaborate. because a greater sample size provides more useful information and lowers odds ratios.

Also, RSA is a prevalent situation that aids in gathering big sample sizes, hence further explanation of the causes of small sample sizes is required.

9- Identification of significantly altered metabolites and pathways

‘’Notably, approximately 200 of these metabolites were associated with drug treatment, likely stemming from prior interventions for pregnant women with RSA. As our investigation did not account for the influence of pharmaceuticals, these metabolites were excluded from our analysis’’

Clarify that please

10- Discussion

- you mentioned that RSA, an autoimmune disease with multifaceted etiology

I disagree that RSA is a disease because it is a disruption of a natural physiological function, possibly caused by an autoimmune condition, during pregnancy.

-The sample size of the cohort (17 patients and 11 healthy controls) was conducted, dynamic changes were captured, resulting in the extraction of dependable e differential information

I think this is a mistake

-Additionally, research has demonstrated that lipid mediators are not only a consequence of oxidative stress but also an inducement for modulating processes (17)

Old information and reference

- The biosynthesis of leukotrienes (LTs), which are derived from AA, involves a two-step process mediated by 5-lipoxygenase. LTs serve important roles in immune regulation, self-defense, and the maintenance of homeostasis in living systems.

-Old data with no reference

-of change in response to the perturbation of RSA. Notably, glutamine assumes a crucial role in energy metabolism by replenishing TCA cycle intermediates through a mitochondrial metabolic pathway known as “glutaminolysis”. In the presence of glutaminase (GIs), glutamine undergoes conversion to glutamate, which subsequently yields aketoglutarate (aKG) that enters the TCA cycle and undergoes catabolism to lactate. The onset of RSA elicits body's oxidative stress response, which promptly triggers biochemical reaction and amplifies the energy demand

where is the references for all of that?

-which stimulates fatty acid oxidation. M1 macrophages, characterized by classical activation, exhibit reduced susceptibility to glutamate metabolism. Upon activation, these macrophages promptly initiate a proinflammatory response to the occurrence of reproductive system associated events by detecting microbial components, including signaling lipoprotein, and damaged-associated molecular patterns that are released from shed abortive material resulting from sudden symptoms of miscarriage.

References??

11- Could you elaborate on the clinical implications of your findings and how it can assist in treating people who have RSA.

12-What is the limitations of your work in frank words

13-Refrences:

-No 2 please check

-Many old references e.g:7,8,9,10 references should be updated

I believe that those comments should be reviewed and corrected before any publication decisions are made.

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Reviewer #1: Yes: Entsar Rashad Abd-Allah

Reviewer #2: Yes: Fatma EL Zharaa Abdelhakam

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Attachments

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Submitted filename: Review.docx

Untargeted metabolomics analysis reveals the metabolic disturbances and exacerbation of oxidative stress in recurrent spontaneous abortion

PONE-D-23-19156R1

Dear Dr. Tuo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Mohammed Hamdy Farouk, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: the article can be accepted , but i hope to complete this study involving immunoregulatory genetic parameters.

Reviewer #2: The authors develop a unique theoretical framework, and I believe that they should highlight their originality much more.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Entsar Rashad Abd-Allah

Reviewer #2: Yes: Fatma EL Zahraa Abd EL Hakam

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Attachments

Attachment

Submitted filename: Review 2.docx