Proteome Analysis of Daily Urine Samples of Pregnant Rats Unveils Developmental Processes of Fetus as Well as Physiological Changes in Mother Rats

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript entitled “Proteome analysis of daily urine samples of pregnant rats unveils developmental processes of fetus as well as physiological changes of mother rats” presents a novel, high‑resolution proteomic investigation of daily urine samples collected throughout rat pregnancy. The work is timely, the methodology well‑described, and the dataset generated is rich in biological information. The study’s strengths include the daily sampling strategy, robust proteome coverage (> 2900 proteins per sample), and the use of both DIA and DDA modes for hybrid spectral library construction.

Nevertheless, several issues should be addressed to improve the scientific rigor and interpretability of the work:

1. Sample size limitations – The use of only four pregnant and four control animals limits statistical power and increases susceptibility to biological variation. This limitation should be explicitly acknowledged in the Discussion.
2. Translational relevance – While the study is performed in rats, the manuscript does not provide sufficient commentary on how these results might correlate with human pregnancy or potential challenges in clinical translation. Include a short but focused paragraph on this aspect.
3. Validation of candidate biomarkers – The identification of key differentially expressed proteins and biological processes is compelling, but none of these findings are confirmed via an orthogonal method (e.g., Western blot, ELISA). Even minimal experimental validation of a subset of proteins would greatly strengthen the conclusions.
4. Discussion depth – The Discussion currently lacks mechanistic detail, particularly regarding how observed proteomic changes map to known maternal and fetal physiological events in early, mid, and late gestation. Adding literature context and discussing possible biological mechanisms would enhance the manuscript’s impact.
5. Variability among animals – Consider adding analyses that quantify inter‑individual variation within each group, to show whether daily proteome trends are consistent across all subjects.
6. Data presentation – In several cases (e.g., DEPs across days), full numerical data are relegated to supplementary files. Summarizing key numbers in the main text tables or figures would improve accessibility for readers.
7. Limitations section – Clearly state any constraints in study design, technical procedures, or analysis that may affect reproducibility or interpretation.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

Comments and Suggestions for Authors

 

The current manuscript provides detailed information on daily changes in the urinary proteome during pregnancy in rats. The aim is to identify daily differences in the urinary proteome during pregnancy in rats through comparative analysis. The authors of this study identified 3,455 proteins in all samples. They showed that there were significant changes associated with blastocyst formation and cell division in the early stage of pregnancy. Moreover, they showed that there were the changes associated with embryonic development and organ morphogenesis and changes specific to the mother, such as lactation in late pregnancy. The authors have done a commendable job of substantiating their claims with appropriate methodology. The manuscript is well illustrated with clear, well-detailed methods. This study provides accurate information useful for confirming and monitoring gestation through urinary proteome analysis. The manuscript could be considered for publication after addressing the following shortcomings:

 

1. Line 58-59: Please delete the sentence “In this study, female Wistar rats were selected and urine samples were collected from 58 the 1st to the 18th day of pregnancy, and a non - pregnant control group was also included.” which should be in the materials and methods section and is already there.

 

2. Line 139, 142, 144: Please, put in lowercase the probability p.

 

3. Authors should refer to author the journal's guideline to write the References section.

Author Response

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Author Response File: Author Response.docx

Reviewer 3 Report

Comments and Suggestions for Authors

The research may be of interest to readers; however, some revisions are necessary before its final publication.

SIMPLE SUMMARY:
I recommend including this section.

ABSTRACT:
General comments: I recommend mentioning the most important findings of this study.
Line 15 – Please revise this phrase: “pregnancy, this study”.

INTRODUCTION
General comments: I recommend adding information related to urinary proteomics. In addition, please describe the most important endocrine events occurring from the 1st to the 18th day of pregnancy in female Wistar rats. For example, embryo implantation.

MATERIALS AND METHODS
Specific comments:
Line 66 – In the section “Rat caging,” I recommend specifying the selection criteria.

RESULTS AND DISCUSSION
Specific comments:
Line 148 – Please clarify this section: you first mention “3,455 proteins were identified in all samples,” but in line 159 you state that “3,201 proteins were retained in the subsequent analysis.” What was the data filtering criterion?
Lines 231–239 – “For example, events related to gastrula and placenta development, including gastrulation, mesodermal cell differentiation, cell migration involved in gastrulation, and labyrinthine layer blood vessel development. Other events related to morphogenesis of anatomical structure were also observed during pregnancy, including notochord formation, cochlea morphogenesis, embryonic cranial skeleton morphogenesis, embryonic limb morphogenesis, embryonic forelimb morphogenesis, embryonic skeletal system morphogenesis, and embryonic morphogenesis. Post-embryonic development occurred on day 16, whose specific outcome is the completion of embryonic development to the mature structure.” I recommend citing references for this information or integrating it with the following section.
Line 248 – In the section “Organ development of embryo,” I recommend supporting all statements with references and describing the possible implications of the described findings.

CONCLUSIONS
I recommend adjusting and expanding this section according to the most relevant results and implications.

Author Response

Please see the attachment.

Author Response File: Author Response.docx