PONE-D-24-36829Human placental mesenchymal stem cells ameliorates premature ovarian insufficiency via modulating gut microbiota and suppressing the inflammation in ratsPLOS ONE

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Partly

Reviewer #2: No

Reviewer #3: Partly

Reviewer #4: Partly

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

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

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5. Review Comments to the Author

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Reviewer #1: The action mechanism of placental mesenchymal stem cells on premature ovarian insufficiency induced by cisplatin was discussed. There are the following problems to be solved:

1. When first using an abbreviation, provide the full term, such as in the Materials and Methods section when first mentioning SD rats, it should be noted as SD (Sprague Dawley).

2. Figures 2, 5, and 6 contain pathological images, which should include a scale bar on the images. The specific information of the scale bar should also be indicated in the Figure legends section. Additionally, the images, particularly those in the gut microbiota section, are not clear enough, which affects readability.

3. In the discussion section, "In addition, the flora disturbance of POI rats at the phylum level was also reflected in the significantly increased levels of actinomyces ...". The initial letter of “actinomyces” should be capitalized, please check the rest of the article to ensure adherence to spelling standards.

4.In the discussion section, "the PMSC group showed significant differences in the abundance of major component bacteria," should be "relative abundance."

5.In the discussion section, the changes in the gut microbiota do not adequately explain their role in improving premature ovarian insufficiency. For example, "Lachnospiraceae, which mainly exists in the intestinal flora of humans and mammals, belongs to the anaerobic bacteria, which can prevent the occurrence of colorectal cancer by producing butyric acid," "Ruminococcus, a vital cellulose-degrading bacterium, plays an irreplaceable role in maintaining intestinal microecological balance and promoting the overall health of the host." It is suggested that the authors focus their discussion on premature ovarian insufficiency rather than expending excessive narrative on the beneficial effects of the gut microbiota and its metabolic products on the host.

6.In this study investigating premature ovarian insufficiency, why were fecal samples collected to examine changes in the gut microbiota instead of scraping vaginal secretions to explore changes in the vaginal flora?

7.Compared to the targeted short-chain fatty acid detection used in this article, non-targeted metabolomics detection might provide more meaningful results.

8.If conditions permit, it is recommended to supplement a proteomic analysis of the ovaries, which could better infer the potential mechanisms of PMSC treatment for premature ovarian insufficiency. Moreover, the results of the proteomic analysis could be jointly analyzed with the 16S rDNA sequencing results.

9.premature ovarian insufficiency primarily affects fertility. If possible in the future, it could be considered to observe the effects of PMSC on the fertility of a premature ovarian insufficiency rat model, as well as the impact on the offspring of the premature ovarian insufficiency rat model.

Reviewer #2: Effectiveness of PMSCs on Ovarian Function: The results indicate that PMSC intervention improved ovarian weight and index in POI rats. How do these improvements compare quantitatively with other potential treatments for POI, such as hormone replacement therapy or other stem cell-based therapies?

Estrous Cycle Restoration: The study mentions that PMSCs helped restore the estrous cycle in POI rats. What mechanisms are proposed for PMSCs influencing the regularization of the estrous cycle, and were there any observed side effects during this restoration process?

Inflammatory Markers: The results show that PMSCs reduced the levels of pro-inflammatory cytokines such as IL-1β and IL-6 in plasma and ovarian tissue. Were these reductions sustained over time, and how do they correlate with long-term improvements in ovarian function?

Microbiota Changes: The study reports significant changes in gut microbiota composition following PMSC intervention. Which specific bacterial changes are hypothesized to be directly linked to ovarian recovery, and are these changes expected to persist long-term without continuous treatment?

SCFA Levels: Short-chain fatty acids (SCFAs) like caproic acid were significantly altered by PMSC intervention. How are these changes in SCFA levels mechanistically linked to improved ovarian function, and could SCFAs be potential biomarkers for therapeutic success?

LPS Levels and Endotoxemia: The reduction in plasma LPS levels suggests improved intestinal barrier function. Is there any evidence to support a direct relationship between the reduction in LPS levels and improvements in ovarian histopathology, or is this effect secondary?

Comparison with Control Groups: The results show that PMSC treatment had a more significant impact on ovarian function and inflammatory markers than hormone therapy (MED group). What specific aspects of PMSC treatment are believed to account for its superior efficacy compared to conventional hormone therapy?

Hormonal Regulation: While E2 levels were improved by PMSC intervention, they did not reach statistical significance compared to the control. What might explain the relatively modest impact on estrogen levels, and could different dosing or timing of PMSC administration enhance this effect?

Long-Term Efficacy: Has the study assessed the long-term effects of PMSCs on ovarian function, inflammatory markers, and gut microbiota, particularly after treatment cessation? How durable are the therapeutic benefits observed in this study?

Reviewer #3: Thanks for your exciting piece of work. I have listed some general and technical issues. However,

Due to the low resolution of figures 9-13 and the lack of line numbers, I couldn’t properly review the manuscript.

General Issue,

1- Define all abbreviations in the abstract

2- Show the positive or negative correlation of the mentioned phyla in the abstract and not just say: “remarkable difference.”

3- Mention the concentration of collagenase A and DNase for placenta cell isolation

4- Why didn’t you use the shaker incubator for digestion?

5- Mention the yield of cell isolation and the reference of the method you used

6- Figures 9-13 are of low quality, and I am not able to review the section of this manuscript that comprises them.

Technical issues

1- How come you used one fluorochrome (PE) for different markers (CD45, CD73, CD90, and CD105) and FITC for HLA-DR, CD34, and CD14? So, the flow cytometry analysis (Fig. 2E) is not understandable on the first page of your results.

2- I can not understand the meaning of Control cells in this sentence (At the same time, control cells continued to be added to the mesenchymal stem cells-specific culture medium.)

3- In the result section entitled:” MSCs intervention ameliorated the homeostasis of steroid hormones in rats with POI,” it seems that in the PMCS group, the level of LH is significantly higher than in MOD, which is not reflected in the graph. Can you check?

4- According to Figure 8, the LPS graph, all groups have the same LPS. Please change the information in the results accordingly

5- Please include the effect of PMCS regarding the microbial changes among these lines at page 22:

“Analysis results showed that the MOD group was enriched in

Rikenella, Micrococcaceae, Bacteroidales, and Desulfovibrionaceae, while the CON group enriched in Lachnospiraceae and Clostridia (Fig. 9E). These findings provided important clues for us to understand further the effects of PMSCs on POI by modulating the intestinal microbiota”.

6- The information you provided for this title:” The difference in intestinal flora in rats of each group at the phylum level,” has been repeated:” The difference in intestinal flora in rats of each group at the genus level.” You may need to omit from the second part.

Final Note: Despite having trouble interpreting the results for Figures 9-13, I believe that the discussion of gut microbiota, inflammatory cytokines, and SCFA was comprehensive and informative. Therefore, I asked the corresponding editor to send me the high-resolution figures and a version of the manuscript with the line number to finish my review.

Reviewer #4: This peer-reviewed manuscript is devoted to the current topic of research on gut microbiota composition in association with the different health status of the host. This study aimed to investigate the effects and potential mechanism of human placental mesenchymal stem cells (PMSCs) on improving early-onset ovarian dysfunction (POI) in a rat model.

Unfortunately, the authors did not use line numbers in the document; thus, it was difficult to specify the comments.

1. The first comment concerns terminology. I recommend replacing "intestinal" with "gut", since the work did not study the parietal microbiota, but the microbiota that passes through the tract and comes out in the form of feces.

2. The methods section "Intestinal microbiota analysis" needs to be further assessed. For example, I doubt whether PCR was actually performed in "15 mL of Phusion® hi-Fi PCR Master Mix", and what was the final volume of the reaction mixture so that the concentrations of other components could be adequately recalculated? What sequencing mode was used by the authors?

3. The next comment is also terminological and structural. Correct: Firmicutes, Bacteroides, Proteobacteria, and so on in the text. There are no such taxa in modern bacterial taxonomy. The authors describe microbial communities; therefore, they must use modern bacterial taxonomy. Therefore, the entire taxonomy of the article must be as follows: https://www.bacterio.net/ and Oren A, Garrity GM. Valid publication of the names of forty-two phyla of prokaryotes. Int J Syst Evol Microbiol 2021; 71:5056.

In the text of the article, the authors use three variants of names for some taxa. In the sections "PMSCs intervention modulated the difference of overall community structure of intestinal microbiota", "The difference in intestinal flora in rats of each group at the phylum level", and "The difference of intestinal flora in rats of each group at genus level", the names of all bacterial taxa must be strictly consistent with the version presented in the corresponding figures. The authors did not describe the community at the species level; therefore, it is necessary to either provide an additional description or exclude this mention from the text.

4. I cannot assess the quality and consistency of the results in Figure 12 because it is of very low resolution; in Fig. 12C, undeciphered marking appears. The drawing requires correction.

Overall, the article requires additional correction and technical revisions.

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Reviewer #1: Yes:  Zhitong Deng

Reviewer #2: No

Reviewer #3: Yes:  Fariba Ghiamati Yazdi

Reviewer #4: No

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Human placental mesenchymal stem cells ameliorates premature ovarian insufficiency via modulating gut microbiota and suppressing the inflammation in rats

PONE-D-24-36829R1

Dear Dr. Wang,

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