PONE-D-21-32980Proteomic analysis of the umbilical cord in fetal growth restriction and preeclampsiaPLOS ONE

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Additional Editor Comments:

Editors comments:

This is an important Ms. insofar as it demonstrates that in fetal growth restricted pregnancies and in preeclampsia, the proteome of the Wharton’s Jelly component of the umbilical cord is largely altered when compared to that of a normal control group. The affected proteins included those connected with extracellular matrix, cellular processes, inflammation and angiogenesis, and it is suggested that these data could ultimately contribute to an improved understanding of the etiology of fetal growth restriction and preeclampsia.

The reviewers have provided detailed suggestions for necessary improvements of the Ms. and these must be satisfactorily dealt with by the authors before this Ms. can be reconsidered for publication.

Some additional changes suggested for the text:

Should use min, sec, h throughout

Table 1: Maternal Age (years) FGR value incorrect? 31.6 (50)? Should this be: 31.6 (5.0)??

Fig. 1: Increase Y-axis legend font size as in Suppl. Fig.1: Area (mm2)??

Suppl. Fig. 1: IUGR??

P. 23, ¶2, line 2: immune response factors

P. 24, ¶3, line 5: the majority of proteins

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The work presented by Conrad et al. examined changes in the umbilical cord (UC) composition in pregnancies complicated by FGR and FGR with PreE by histologic and proteomic analysis for Wharton’s Jelly samples.

Introduction reads well and the study design is technically valid. But

My comments to the author are listed below:

• The first word in the title is incomplete (I think the author means Proteomic)

• In the last step of sample preparation for proteomics, why the supernatant was not desalted first before drying on vacufuge instead of drying then desalting and drying again.

• In Histological analysis of the umbilical cord and Wharton’s Jelly area please specify which groups were significantly different according to the post-hok results of the ANOVA. This can also be indicated in the legend of Figure 1. Please Add the statistical test to the legend of figure 1.

• The flow of the proteomics section in the results is difficult. The authors only summarize the results rather than describing it. A table that summarizes number of changed proteins using spectral count and peak intensity methods between different compared group might be added.

• According to the author “A strength of this study is the novel use of five protein search and inference engines for identification of proteins from the mass spectrometry data as well as using both spectral count and peak intensity methods. By using the overlap data of commonly identified proteins that were significantly changed, this increases the confidence that this is a true change”.

Any common proteins that were significantly changed using spectral count and peak intensity? Why authors used two methods if they end with different altered proteins for the same set of samples when using the two analytical methods? Were proteins significantly altered by the two methods involved in similar biochemical processes?

• In the discussion of the proteomics findings, the author only mentioned the pathways in which the proteins are involved without linking this to underlying mechanisms involved in preeclampsia or FGR. Please highlight how these proteins are related to the pathophysiology of the studied disorders

• How proteomic changes in the Wharton’s Jelly can be linked to other proteomic studies that investigated different sample types including placenta and blood.

• the small sample size (n=5) in each group is of concern and the authors mentioned this in the limitation section.

• The authors made 6 binary comparisons (using two analytical methods). I was wondering how they decided for specific comparisons to be presented in the text while others as supplementary

• Please be consistent (e.g., either PreE or preeclampsia)

Reviewer #2: Re: Proteomic analysis of the umbilical cord in fetal growth restriction and preeclampsia

Manuscript Number: PONE-D-21-32980

This is a proteomic analysis of the umbilical cord in a case control study of FGR, FGR with preeclampsia and control pregnancies. It is novel, well-organized and well-written.

Comments:

1. Introduction, fifth paragraph. The patients are described as “pre-eclamptic women.” Respectfully, they should not be defined by their disease and should be referred to as “women with preeclampsia.”

2. Introduction, last sentence. “Specifically, these changes will include decreased cross-sectional area of the umbilical cord and proteomic changes in extracellular matrix protein composition that contribute to formation of the Wharton’s Jelly.” The phrase “that contribute to the formation of Wharton’s Jelly” suggests that some property of Wharton’s Jelly will be enhanced (and is kind of non-specific). I do not believe the data support this. If so, one of two responses by the authors is appropriate. Either rephrase this hypothesis to suggest that nothing is “enhanced” or include a discussion (in the discussion section) as to why this part of the hypothesis was not confirmed.

3. Methods. Parity is not seen in the available data. Parity has a tremendous effect on many outcomes of pregnancy. If there are any data in the literature showing that parity has no effect on umbilical cord volume or other parameters, parity could be sefaly omitted. In the absence of any such data, parity should be included in the demographic data.

4. Methods, Sample preparation for proteomics. In this paragraph, the biologic tissue is separated into supernatant and pellet. The authors use the word “sample” numerous times. For clarity, I believe it is best to specify supernatant or pellet each time the word "sample" appears.

5. Methods, LC-MS/MS. This paragraph starts with, “Protein identification was performed . . .” For clarity, I believe is is best to specify the sample used (Supernatant or pellet? Wharton’s Jelly or vessel?)

6. Results, Histology. Regarding the use of the phrase “cesarean section,” a more respectful and contemporary phrase is “cesarean delivery.” “Cesarean section” only refers to the act of cutting, not the delivery.

7. Table 1. Apgars not normally distributed. Should properly be shown as median (range or IQR).

8. Figures. For clarity, whenever “PreE” appears, it should be “FGR with PreE.”

9. Figures. Figures should stand alone, thus, abbreviations should be explained in footnotes. (FGR, PreE, Cont)

10. Supplemental Figure 2. “IUGR” should read “FGR.”

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Reviewer #1: No

Reviewer #2: Yes: Daniel W Skupski, MD

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Proteomic analysis of the umbilical cord in fetal growth restriction and preeclampsia

PONE-D-21-32980R1

Dear Dr. Rood,

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Academic Editor

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