Identification and Validation of miR-222-3p and miR-409-3p as Plasma Biomarkers in Gestational Diabetes Mellitus Sharing Validated Target Genes Involved in Metabolic Homeostasis

Round 1

Reviewer 1 Report

This manuscript aims to identify plasma biomarkers in gestational diabetes mellitus (GDM) patients. By purifying extracellular vesicles from plasm and profiling the RNAs with NanoString nCounter Human v3 microRNA 87 expression assay, they performed validation analysis in a larger cohort of GDM and NGT allowed to validate two upregulated TP MiR‐222‐3p and miR‐409‐3p. The overview of literature data and of the expression of miR‐222‐3p and miR‐409‐3p together with their validated targets allowed to hypothesize the putative role of these miRNAs in the pathophysiology of GDM by targeting to MGMT and PPP2R2A. How do miR-222-3p and 409-3p regulate MGMT and PPP2R2A remain under speculation without experimental data. However, the present data were sufficient to show the significance of these two miRNAs function as plasma biomarkers. Thus, I appreciate this study and suggest this manuscript is warrant for publication by IJMS.

Author Response

We thank the reviewer for the positive comments

Reviewer 2 Report

In this study, the authors have performed miRNA expression profiling in plasma and extracellular vesicles (EVs) secreted from gestational diabetes patients as well as normal glucose tolerant women, at third trimester of gestation. Authors have identified a set of differentially expressed miRNAs, which are linked to type 2 diabetes and gestational diabetes. It is proposed that miR‑222‑3p and miR‑409‑3p identified in the circulation (plasm and EVs) of validation cohort could serve as classification biomarkers for gestational diabetes.

The study is interesting and holds a scope, however, there are certain points which authors need to address. My comments are appended below.

(1). Introduce extracellular vesicles (EVs) in the abstract, their content and functions and applications, in one or two lines.

(2). Introduction needs more literature, both for pathophysiology of gestational diabetes mellitus, its diagnosis, and treatment options. Especially, related to scope of the current study, the diagnosis classification needs literature. Additionally, the role of dysregulated miRNAs in gestational diabetes mellitus needs more studies to be referred in the introduction section.

(3). The EVs biology has not been described in the introduction, for example, what are EVs, what content they carry and transport (especially noncoding RNAs, miRNAs), what are the functions of EVs in gestational diabetes, and glucose tolerance. Finally, EV associated miRNAs in gestational diabetes should be included.

(4). In the discovery cohort only 3 samples are included, and conclusions are drawn from comparing miRNAs from n = 3 of discovery cohort with miRNAs of n = 12 of validation cohort. How authors justify this discrepancy in terms of population size of the current study?.  

(5). In the figure legends, mention the number of replicates (n = ?) with each panel, and indicate the type of analysis/tools where applicable, and how data was presented e.g., SD ± x of n number of replicates.

(6).  In the methods, mention the ethical approval statement, ethical approval number, and the year of approval.

Other specific comments:  

(i). Gestational diabetes (GDM). What does letter M stand for? Either write Mellitus, or remove M.  

(ii). In the abstract, when first time mentioned in the text, abbreviate microRNA (miRNA, miR), and later use the abbreviation only.

(iii). Same applies to manuscript body, i.e, when first time mentioned in the introduction, abbreviate microRNA (miRNA, miR), and later use the abbreviation only.

(iv). The aim of this study was to evaluate microRNA expression in total plasma (TP) and extracellular vesicles (EVs) in GDM patients and normal glucose tolerance (NGT) women, at third trimester of gestation, to find new microRNAs that could improve GDM classification and molecular characterization. The phrase is too long to read.

(v). to evaluate microRNA expression…….. to find new microRNAs that could improve GDM classification. Perhaps better to write these aims together.

(vi). Perhaps better to avoid unnecessary abbreviations like total plasma (TP). Instead, use the word total plasma. 

Author Response

Reviewer 2

Comments and Suggestions for Authors

In this study, the authors have performed miRNA expression profiling in plasma and extracellular vesicles (EVs) secreted from gestational diabetes patients as well as normal glucose tolerant women, at third trimester of gestation. Authors have identified a set of differentially expressed miRNAs, which are linked to type 2 diabetes and gestational diabetes. It is proposed that miR‑222‑3p and miR‑409‑3p identified in the circulation (plasm and EVs) of validation cohort could serve as classification biomarkers for gestational diabetes.

The study is interesting and holds a scope, however, there are certain points which authors need to address. My comments are appended below.

(1). Introduce extracellular vesicles (EVs) in the abstract, their content and functions and applications, in one or two lines.

Authors’ Response: We thank the reviewer for this comment. We have modified the abstract, according to the reviewer suggestion (lines 25-27)

(2). Introduction needs more literature, both for pathophysiology of gestational diabetes mellitus, its diagnosis, and treatment options. Especially, related to scope of the current study, the diagnosis classification needs literature. Additionally, the role of dysregulated miRNAs in gestational diabetes mellitus needs more studies to be referred in the introduction section.

Authors’ Response: As suggested, the introduction section has been extensively revised and the requested information has been included (lines 59-78)

(3). The EVs biology has not been described in the introduction, for example, what are EVs, what content they carry and transport (especially noncoding RNAs, miRNAs), what are the functions of EVs in gestational diabetes, and glucose tolerance. Finally, EV associated miRNAs in gestational diabetes should be included.

Authors’ Response: As suggested, the introduction section has been revised and the requested information has been included (lines 83-97)

(4). In the discovery cohort only 3 samples are included, and conclusions are drawn from comparing miRNAs from n = 3 of discovery cohort with miRNAs of n = 12 of validation cohort. How authors justify this discrepancy in terms of population size of the current study?.  

Authors’ Response: We thank the reviewer for this question. The discovery cohort (n=3) was used to perform a pilot study that allowed us to define the miRNAs profile of GDM and NGT pregnant women by using the nCounter Sprint Profiler (Nanostring Inc.), a high-throughput screening assay that detects the 800 most biologically relevant human miRNAs. Then, in order to validate the most biological relevant GDM-related miRNAs and to corroborate the results obtained from the profiling analysis, we decided not only to extend the cohort of patients but also to use another detection method. Thus, we analysed the expression of the selected GDM-biologically relevant miRNAs on a wider and independent cohort of samples (n=12) by using RT-qPCR.

(5). In the figure legends, mention the number of replicates (n = ?) with each panel, and indicate the type of analysis/tools where applicable, and how data was presented e.g., SD ± x of n number of replicates.

Authors’ Response: As suggested by the reviewer, we modified figure legends by adding the requested information.

(6).  In the methods, mention the ethical approval statement, ethical approval number, and the year of approval.

Authors’ Response: the requested information has been added in the method section (lines 295-296) 

Other specific comments:

(i). Gestational diabetes (GDM). What does letter M stand for? Either write Mellitus, or remove M. 

Authors’ Response: Thank you for your observation. The word “mellitus” has been added in the abstract.

(ii). In the abstract, when first time mentioned in the text, abbreviate microRNA (miRNA, miR), and later use the abbreviation only.

Authors’ Response: Thank you for your observation, we modified as suggested.

(iii). Same applies to manuscript body, i.e, when first time mentioned in the introduction, abbreviate microRNA (miRNA, miR), and later use the abbreviation only.

Authors’ Response: As suggested by the reviewer, we abbreviated microRNA throughout the text by using miRNA.

(iv). The aim of this study was to evaluate microRNA expression in total plasma (TP) and extracellular vesicles (EVs) in GDM patients and normal glucose tolerance (NGT) women, at third trimester of gestation, to find new microRNAs that could improve GDM classification and molecular characterization. The phrase is too long to read.

Authors’ Response: Thank you for your comment. The sentence has been shortened and part of the information has been reported in the next line (lines 27-30).

(v). to evaluate microRNA expression…….. to find new microRNAs that could improve GDM classification. Perhaps better to write these aims together.

Authors’ Response: Thank you for your comment. The two aims have been merged and the sentence has been modified (lines 27-28)

(vi). Perhaps better to avoid unnecessary abbreviations like total plasma (TP). Instead, use the word total plasma. 

Authors’ Response: Thank you for this observation. We modified the text accordingly.

Round 2

Reviewer 2 Report

The authors have revised the manuscript in accordance with my comments, and have further improved their manuscript. 

I have no further comments and endorse the publication of this work.