Genetic Polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and the Risk of Idiopathic Recurrent Pregnancy Loss

Round 1

Reviewer 1 Report

Title: Genetic polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and risk of idiopathic

recurrent pregnancy loss.

Authors: Sung Hwan Cho, Ji Hyang Kim, Hui Jeong An, Jung Oh Kim, Jung Jae Ko, Nam Keun Kim

 

General opinion:

Thank you for the opportunity to re-revise the manuscript  “Genetic polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and risk of idiopathic recurrent pregnancy loss” by Suang Hwan Cho et.al. Since I have an impression that the present submission (ijms-1235002) resembles the previous one (ijms-783917) a lot, I would like to ask the authors to provide a point by point response to my previous remarks as all of them are uptodate.

Author Response

Title: Genetic polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and risk of idiopathic recurrent pregnancy loss” by Suang Hwan Cho et.al. Since I have an impression that the present submission (ijms-1235002) resembles the previous one (ijms-783917)

Reviewer 1

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	(x)	( )	( )	( )
Are the results clearly presented?	(x)	( )	( )	( )
Are the conclusions supported by the results?	( )	(x)	( )	( )

Comments and Suggestions for Authors

Title: Genetic polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and risk of idiopathic recurrent pregnancy loss.

Authors: Sung Hwan Cho, Ji Hyang Kim, Hui Jeong An, Jung Oh Kim, Jung Jae Ko, Nam Keun Kim

 General opinion:

Thank you for the opportunity to re-revise the manuscript  “Genetic polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and risk of idiopathic recurrent pregnancy loss” by Suang Hwan Cho et.al. Since I have an impression that the present submission (ijms-1235002) resembles the previous one (ijms-783917) a lot, I would like to ask the authors to provide a point by point response to my previous remarks as all of them are uptodate.

⇒ Thank you for kind comments.

Invaluable comments from Reviewer 1 have been confirmed to be the same for the current review (ijms-1235002) and the previous review (ijms-783917). As we understand, there was a first revision, perhaps at the request of an academic editor.

Comments and Suggestions for Authors

This article evaluates polymorphisms in five microRNAs that are associated with risk of idiopathic recurrent pregnancy loss. This study is being performed in vivo in a large patients group and relating the clinical parameter as observed, and in vitro study. The topic is interesting and the manuscript well written, however, there are some major points to be addressed. It is not clear the criteria of selection of the microRNAs evaluated in the study. It is not clear how the authors identify the miRNAs specifically.

In the "Introduction" there are other studies already about MicroRNAs related risk of idiopathic recurrent pregnancy loss that are not mentioned. The references reported in the manuscript should be updated. The authors perform the RFLP- PCR to evaluate the expression of SNPs MicroRNA, it is a technique outdated, it is better the Taqman microRNA assay.

■ It is not clear the criteria of selection of the microRNAs evaluated in the study. It is not clear how the authors identify the miRNAs specifically.

 

⇒ Thank you for kind comments. We focused on microRNAs targeting female reproduction-related genes.

 

We revised sentences in the Introduction as follows:

Transforming growth factor (TGF-β) superfamily members, including TGF-β and its associated regulatory miRNAs, miR-938 and miR-631 [30], exert critical functions in female reproductive physiology [31]. Another miRNA, miR-604, putatively binds targets related to placenta retention [32]. The miR-608C>G (rs4919510) polymorphism has been associated with the regulation of interleukin (IL)-6 expression [33], and expression of this cytokine was higher in mice who experienced recurrent spontaneous abortion than in those with a normal murine pregnancy [34]. Lastly, the miR-1302 family, which is derived from MER53 elements, comprises placental-specific miRNAs [35].

 

■ In the "Introduction" there are other studies already about MicroRNAs related risk of idiopathic recurrent pregnancy loss that are not mentioned. The references reported in the manuscript should be updated.

 

Thank you for kind comments. We revised sentences in the Introduction as follows:

⇒ Associations between several miRNAs, including miRNA machinery genes, and the risk of RPL have been reported [21-23]. In particular, gene polymorphisms miR-146aC>G (rs2910164), miR-149T>C (rs2292832), miR-196a2T>C (rs11614913), and miR-499A>G (rs3746444) are present in RPL patients [24]. MiR-146a regulates FAS expression by binding to FAS mRNA, thereby reducing apoptosis [25].

 

We added references as follows:

 

[21]. Kim YR, Ryu CS, Kim JO, An HJ, Cho SH, Ahn EH, Kim JH, Lee WS, Kim NK. Association study of AGO1 and AGO2 genes polymorphisms with recurrent pregnancy loss. Sci Rep. 2019 Oct 30;9(1):15591. doi: 10.1038/s41598-019-52073-0

[22]. Alipour M, Abtin M, Hosseinzadeh A, Maleki M. Association between miR-146a C > G, miR-149 T > C, miR-196a2 T > C, and miR-499 A > G polymorphisms and susceptibility to idiopathic recurrent pregnancy loss. J Assist Reprod Genet. 2019 Nov;36(11):2237-2244. doi: 10.1007/s10815-019-01573-z.

[23]. Du E, Cao Y, Feng C, Lu J, Yang H, Zhang Y. The Possible Involvement of miR-371a-5p Regulating XIAP in the Pathogenesis of Recurrent Pregnancy Loss. Reprod Sci. 2019 Nov;26(11):1468-1475. doi: 10.1177/1933719119828051.

[24]. Parveen F, Agrawal S. Recurrent miscarriage and micro-RNA among north Indian women. Reprod Sci. 2015;22:410–415.

[25]. Fluhr H, Wenig H, Spratte J, Heidrich S, Ehrhardt J, Zygmunt M. Non-apoptotic fas-induced regulation of cytokines in undifferentiated and decidualized human endometrial stromal cells depends on caspase-activity. Mol Hum Reprod. 2011;17:127–134.

 

■ The authors perform the RFLP- PCR to evaluate the expression of SNPs MicroRNA, it is a technique outdated, it is better the Taqman microRNA assay.

 

⇒ I agree with your point of view. All samples were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. As you mentioned above, it is better the Taqman assay. RFLP analysis is one of the oldest, most convenient and least expensive methods of genotyping, but is limited by the availability of restriction endonuclease sites. Although The PCR-RFLP method is convenient, validation was performed by sequencing when the RFLP genotyping result was uncertain.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this paper, the authors examined the polymorphisms in five microRNAs 12 (miRNAs), miR-604A>G, miR-608C>G, 631I/D, miR-938G>A, and miR-1302-3C>T, and their association with risk of idiopathic recurrent pregnancy loss (RPL).

Their results are of some interest, but there are some limitations in regards to the design of the study reducing the importance of the work. In fact, it is not clear why 5 microRNA ,were considered when it would have been necessary, and common practice, to use a signature in order to identify the specific microRNAs which are related to the mechanism of reproductive disorders.

 

Major Concerns:

In the results part is missing a figure that rapresents the expression of miR-938G>A. levels detected by miRNA reverse transcriptase quantitative PCR (qRT-PCR) in cells transfected

Expression of of miR-938 were detected by miRNA reverse transcriptase quantitative PCR (qRT-PCR) in cells transfected. Is missing also a figure that reports the results of flow cytometric analysis of CD56+ peripheral NK cells.

In the supplementary materials are missing all figures, there are only tables.The authors should represent the results of the study with Figures describing better the various end points examined.  

Comments for author File: Comments.pdf

Author Response

please see the attachment!

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors investigated the association between five genetic variants located in microRNAs and the risk of idiopathic recurrent pregnancy loss. The authors also conducted functional assays to evaluate whether allelic differences in regulatory activity observed for miR-604 and miR-938 could be attributed to differential binding to targets. The topic is relevant and of interest. The article is well written and the sample size is adequate.  The main limitation is the candidate-gene design.

I only have a few comments and observations.

• In the introduction, if such findings are available, I would add a sentence on studies supporting the hypothesis that idiopathic recurrent pregnancy loss might be a heritable or familial trait (thus further supporting the need to explore the potential contribution of genetic variants in this trait)
• In the last paragraph of the Introduction, the authors report that Investigated miRNAs were selected based on those previously implicated in the reproductive function. What about how were genetic variants chosen? Are these the only ones located in these miRNAs, or how were the variants selected?
• In my opinion the sample is very well characterized and statistical analyses are sound
• A reference for the protocol of the functional assay might be added

Author Response

Title: Genetic polymorphisms in miR-604A>G, miR-938G>A, miR-1302-3C>T and risk of idiopathic recurrent pregnancy loss” by Suang Hwan Cho et.al. Since I have an impression that the present submission (ijms-1235002) resembles the previous one (ijms-783917)

Reviewer 3

Open Review

(x) I would not like to sign my review report
( ) I would like to sign my review report

English language and style

( ) Extensive editing of English language and style required
( ) Moderate English changes required
(x) English language and style are fine/minor spell check required
( ) I don't feel qualified to judge about the English language and style

 

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	(x)	( )	( )	( )
Is the research design appropriate?	(x)	( )	( )	( )
Are the methods adequately described?	( )	(x)	( )	( )
Are the results clearly presented?	(x)	( )	( )	( )
Are the conclusions supported by the results?	(x)	( )	( )	( )

Comments and Suggestions for Authors

The authors investigated the association between five genetic variants located in microRNAs and the risk of idiopathic recurrent pregnancy loss. The authors also conducted functional assays to evaluate whether allelic differences in regulatory activity observed for miR-604 and miR-938 could be attributed to differential binding to targets. The topic is relevant and of interest. The article is well written and the sample size is adequate.  The main limitation is the candidate-gene design.

I only have a few comments and observations.

• In the introduction, if such findings are available, I would add a sentence on studies supporting the hypothesis that idiopathic recurrent pregnancy loss might be a heritable or familial trait (thus further supporting the need to explore the potential contribution of genetic variants in this trait)

⇒ Thank you for kind comments. We added sentences in the Introduction as follows: “Notably, miRNA regulatory effect is a heritable trait in humans and that a polymorphism of the miRNA and microRNA machinary gene contributes to the observed inter-individual differences” [28, 29].

References:

[28] Geeleher, P.; Huang, S.R.; Gamazon, E.R.; Golden, A.; Seoighe, C. The regulatory effect of miRNAs is a heritable genetic trait in humans. BMC Genomics. 2012; 13, 383.

[29] Ponsuksili, S.; Oster, M.; Reyer, H.; Hadlich, F.; Trakooljul, N.; Rodehutscord, M.; Camarinha-Silva, A.;  Bennewitz, J,; Wimmers, K. Genetic regulation and heritability of miRNA and mRNA expression link to phosphorus utilization and gut microbiome. Open Biol. 2021, 11(2), 200182.

 

• In the last paragraph of the Introduction, the authors report that Investigated miRNAs were selected based on those previously implicated in the reproductive function. What about how were genetic variants chosen? Are these the only ones located in these miRNAs, or how were the variants selected?

⇒ Thank you for kind comments.

A previous report showed that the miRNA miR-938 is associated with the transforming growth factor (TGF-β) signaling pathway [Butz et al]. TGF-β superfamily members exert critical functions in the female reproductive system [Night et al]. Given the roles of TGF-β and miRNAs in female reproductive physiology, and the miR-938–TGF-β association, we sought to delineate the role, if any, of miR-938 polymorphisms in RPL. Polymorphism of miR-938G>A (rs12416605) is located seed sequence of mature miRNA. There are several polymorphisms in miRNA. Among them, It has been reported that the presence of pre- and mature-miRNA polymorphisms affects miRNA expression [Mishira et al., Kontorovich et al].

One-carbon metabolism is associated with defects in blood coagulation factors, abortion, abnormal plasma urate, folate, and homocysteine levels, all of which are risk factors for vascular disease and RPL. MTHFR is at a critical metabolic branch point of one-carbon metabolism and is a putative binding target of miR-604. Polymorphism of miR-604A>G (rs2368393) is located in pre-form miR-604. In addition, the miR-1302 family, which is derived from MER53 elements, comprises placental-specific miRNAs. Polymorphism of miR-1302C>T (rs2368393) is located in pre-form miR-1302. Thus, we evaluated whether allele differences in miR-938 (rs12416605), miR-604 (rs2368393) and miR-1302 (rs2368393) in activity affect the regulation of target mRNA.

 

⇒ We revised sentence in the Introduction as follows:

“In this study, we investigated possible genetic causes of RPL by examining miRNA nucleotide polymorphisms in individuals suffering from this condition, focusing on those miRNAs previously implicated in reproductive diseases” [26, 27].

References:

Butz H., Likó I., Czirják S.> Igaz P., Korbonits M., Rácz K., Patócs A. MicroRNA profile indicates downregulation of the TGF pathway in sporadic non-functioning pituitary adenomas.Pituitary.2011,14,112–124.

Knight P.G., Glister C. TGF-β superfamily members and ovarian follicle development.Reproduction.2006,132,191–206.

Mishra PJ and Bertino JR. MicroRNA polymorphisms: the future of pharmacogenomics, molecular epidemiology and individualized medicine. Pharmacogenomics. 2009, 10, 399–416.

Kontorovich T, Levy A, Korostishevsky M , Nir U, Friedman E. Single nucleotide polymorphisms in miRNA binding sites and miRNA genes as breast/ovarian cancer risk modifiers in Jewish high-risk women. Int. J. Cancer, 2010, 127, 589–597

[26] Cho SH, Ahn EH, An HJ, Kim JH, Ko JJ, Kim YR, Lee WS, Kim NK. Association of miR-938G>A Polymorphisms with Primary Ovarian Insufficiency (POI)-Related Gene Expression. Int J Mol Sci. 2017,  18(6), 1255.

[27] Lee HA, Ahn EH, Jang HG, Kim JO, Kim JH, Lee YB, Lee WS, Kim NK Association Between miR-605A>G, miR-608G>C, miR-631I>D, miR-938C>T, and miR-1302-3C>T Polymorphisms and Risk of Recurrent Implantation Failure. Reprod Sci. 2019, 26, 469-475.

 

• In my opinion the sample is very well characterized and statistical analyses are sound

⇒ We really appreciate the reviewer's favorable comments.

 

• A reference for the protocol of the functional assay might be added

⇒ Thank you for kind comments. We added references in the materials & methods as follows:

4.5. Expression vectors construction

[64] Heibel SK, Lopez GY, Panglao M, Sodha S, Mariño-Ramírez L, Tuchman M, Caldovic L. Transcriptional regulation of N-acetylglutamate synthase. PLoS One. 2012;7(2):e29527.

[65] Thorne N, Inglese J, Auld DS. Illuminating Insights into Firefly Luciferase and Other Bioluminescent Reporters Used in Chemical Biology. Chemistry & Biology. 2010; 6, 646.

 

4.6. Cell transfection and luciferase assay

[66] Unal H. Luciferase reporter assay for unlocking ligand-mediated signaling of GPCRs. Methods Cell Biol. 2019, 149:19-30.

[67] Müller K, Ogris M, Sami H. Firefly Luciferase-Based Reporter Gene Assay for Investigating Nanoparticle-Mediated Nucleic Acid Delivery. Methods Mol Biol. 2019,1943:227-239

 

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

The authors have successfully addressed all the concerns that I raised in my first report. Thus, I have no further objections against the publication of the manuscript.