Association Study of Hyaluronan-Binding Protein 2 (HABP2) Gene Polymorphisms in Idiopathic Recurrent Pregnancy Loss (RPL) in Korean Women

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Association study of hyaluronan-binding protein 2 (HABP2) gene polymorphisms in idiopathic recurrent pregnancy loss (RPL). Lee et al.,

 

This study analyzes the association between Recurrent Pregnancy Loss (RPL) and HABP2 gene polymorphisms in Korean women. The authors conducted a large cohort analysis (388 RPL vs. 377 controls) of six SNPs (rs3832698, rs10885478, rs932650, rs7923349, rs1157916, rs2240879) in a large cohort (388 RPL vs 377 controls), reporting that the rs2240879 CC genotype is significantly associated with increased RPL risk. They also suggest that rs3832698 and rs1157916 are associated with changes in prothrombin time (PT). In conclusion, their study demonstrates that HABP2 can be utilized as a biomarker for RPL susceptibility.

This paper is clinically significant in proposing biomarkers for RPL. However, limitations include the absence of mechanistic validation and the analysis being restricted to Koreans.

[Major points]

• A description of how the polymorphisms identified by the authors could be associated with biological mechanisms is required. While the coagulation-related function of HABP2 (FSAP) is well described, presenting a specific model of how it directly contributes to RPL would greatly aid readers in understanding this paper.
• Although this study included only Korean women, the emphasis on ethnic limitations is mentioned only very briefly toward the end of the Discussion. The necessity for comparative studies in other ethnic groups should be strongly emphasized.

[Minor point]

• In Table 1, “FSH 7.8 ± 11.6” has an abnormally large standard deviation. A re-examination of the accuracy of this number is required.

Author Response

[Major point]

A description of how the polymorphisms identified by the authors could be associated with biological mechanisms is required.

While the coagulation-related function of HABP2 (FSAP) is well described, presenting a specific model of how it directly contributes to RPL would greatly aid readers in understanding this paper.

Although this study included only Korean women, the emphasis on ethnic limitations is mentioned only very briefly toward the end of the Discussion.

The necessity for comparative studies in other ethnic groups should be strongly emphasized.

 

Thank you for your comments and we added more information in ___228_____

There is limitation in our research. First, case-control studies were conducted only in the Korean population and conduct in other population will be required.

Second, our study sample size is small that is not represented all participants including recurrent pregnancy loss patients.

Third, relationship of HABP2 genotype and recurrent pregnancy loss is unclear, further study for molecular work is required.

[Minor point]

In Table 1, “FSH 7.8 ± 11.6” has an abnormally large standard deviation. A re-examination of the accuracy of this number is required.

 

Thank you for your comments. We are re-calculate FSH by R, and shows same result. Mean 7.8 standard deviation 11.6147

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This article analyzes polymorphisms in the HBP2 gene and their association with pregnancy loss, findings that resulted from previous work by the same research group. They examined six HABP2 variants (rs3832698 A>del, rs10885478 G>A, rs932650 T>C, rs7923349 G>T, rs1157916 G>A, and rs2240879 T>C) in order to clarify their association with pregnant loss risk. The manuscript is well-structured, with an introduction that clearly outlines the issues and provides all necessary background information. There are few studies regarding the action of the HBP2 gene variants in pregnancy loss, and concerning the association with polymorphisms, there are no reports.  This highlight the importance of analyzing polymorphisms in the HBP2 gene. 

Therefore, it is highlighted that the study has scientific potential and interest.

 The methodology is thoroughly detailed and encompasses all proposed steps, making it suitable for achieving the study's results and reproducible by other researchers.Regarding the sample size, it may not represent the population of women with pregnancy loss, as highlighted by the authors. However, the data demonstrate the association of two polymorphisms with pregnancy loss in this sample. Since the sample only represents women from South Korea, it would be interesting to emphasize this information, including  the article's title.

The manuscript contains eight detailed, and self-explanatory tables that clearly demonstrate the analyses and results obtained. 

Table 1 compares baseline characterisitics between cases and controls where a difference is observed in the TSH, LH and ES parameters. There is no mention in the discussion regarding these parameters, which may be associated with pregnancy loss. To strengthen the study, it would be interesting to discuss these parameters, where the results indicate differences between the case and control groups. Could pregnancy losses be more strongly associated with these parameters than with polymorphisms in the HBP2 gene?

Tables 2 to 4 describe the genotyping analyses of the polymorphisms. Table 5 describes synergistic effect of HABP2 polymorphisms with clinical risk factor. Tables 6 to 8 summarize of one-way analysis of variance between clinical parameter and HABP2 polymorphisms. All tables are well described and reflect the results obtained, which are pointed out in the discussion.

The figures presented are quite illustrative and represent the findings observed in the study.

The discussion presents a correlation between polymorphisms in the HBP2 gene and pregnancy loss, highlighting the rs2240879 polymorphism, which has been shown to be associated with the risk of pregnancy loss, as well as the rs3832698 and rs10885478 polymorphisms, which have been associated with prothrombin time levels. The authors' conclusions align with the observations in this study, indicating that one polymorphism is associated with an increased risk of pregnancy loss, and another is associated with coagulation parameters. They also point out the study's limitations, highlighting the origin and size of the sample and the fact that the effect of HBP2 is unclear.

Based on the above, there are only a few considerations to be made:

1. Since the sample is restricted to women from South Korea, highlight this information.

2. Include in the title that it is a population from South Korea.

3. Discuss the difference between FSH and TSH between the groups. Could pregnancy losses be more strongly associated with these parameters than with polymorphisms in the HBP2 gene?

Author Response

1. Since the sample is restricted to women from South Korea, highlight this information.

Thank you for your comments and we added more information line _228_

We have a little limitation in our research. First, case-control studies were conducted only in the Korean population and conduct in other population will be required. Second, our study sample size is small that is not represented all participants including recurrent pregnancy loss patients. Third, relationship of HABP2 genotype and recurrent pregnancy loss is unclear, further study for molecular work is required.

2. Include in the title that it is a population from South Korea.

Thank you for your comment we have revised the title. _Line 4, in Korean women

3. Discuss the difference between FSH and TSH between the groups.

Could pregnancy losses be more strongly associated with these parameters than with polymorphisms in the HABP2 gene?

Thank you for your comment. We add information in line _221_

In Table 1, Hormone levels are significantly different between control and case. TSH levels are higher in RPL patients. Fumarola et al. reported that pregnancy rates is related in women with TSH levels, they discover high TSH level (> 2.5 mIU/L) result low pregnancy rate.(https://doi.org/10.1111/aji.12113).  Zhou et al also report that high TSH level (> 3 mIU/L) groups result low pregnancy rate. But by ROC curve, they also report that not enough as a biomarker for pregnancy rate (10.21037/tp-22-79). Seema et al. report that early pregnancy loss patients are high FSH level compared with control groups (fertility have taken). (10.22159/ajpcr.2023v16i5.47027)

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript investigates the association between polymorphisms in the HABP2 gene and idiopathic recurrent pregnancy loss (RPL) through comprehensive genetic and clinical analyses.

The study benefits from a well-defined cohort with age-matched controls and employs a broad array of genetic analyses, including SNP genotyping, haplotype analysis, and gene–clinical factor interactions. The statistical methods are thorough, supporting the identification of significant genetic variants linked to RPL susceptibility. The integration of clinical coagulation parameters enhances the biological relevance of the findings, but it seems not strongly linked to the HABP2 genotypes, so it is difficult to give a relevant meaning to these correlations.

The manuscript would benefit from clearer organization and presentation, particularly in the Results section, to improve readability.

My basic consideration is that when studying the correlation between a polymorphism and a given phenomenon, it is essential to report the frequency of the polymorphism in the study population. The frequency data are crucial to determine whether the polymorphism is truly associated with the observed phenomenon or if its presence might be due to chance. Including this comparison strengthens the validity and interpretability of the study's findings and is a standard practice in genetic association research.

Moreover, in the Introduction is reported that “In this study, we analyzed relationships between HABP2 polymorphisms and RPL. Six polymorphisms (rs3832698 A>del, rs10885478 G>A, rs932650 T>C, rs7923349 G>T, rs1157916 G>A, and rs2240879 T>C) located in the promoter region, introns, and 3′ untranslated region (UTR) were selected” no other explanation is present. But why were these polymorphisms chosen? They are present in non-coding regions such as promoters, introns, and UTRs, so can they have regulatory roles influencing gene expression? Which biological pathways are involved that can be related to the RPL phenomena?

In light of this, the discussion could be strengthened by a deeper exploration of the functional implications of the identified polymorphisms and their mechanistic role in RPL pathophysiology. Limitations regarding population specificity and the absence of experimental validation should be more explicitly addressed. 

Comments on the Quality of English Language

Language improvements for clarity and conciseness are also recommended.

Author Response

Reviewer 3

 

The manuscript investigates the association between polymorphisms in the HABP2 gene and idiopathic recurrent pregnancy loss (RPL) through comprehensive genetic and clinical analyses.

The study benefits from a well-defined cohort with age-matched controls and employs a broad array of genetic analyses, including SNP genotyping, haplotype analysis, and gene–clinical factor interactions. The statistical methods are thorough, supporting the identification of significant genetic variants linked to RPL susceptibility. The integration of clinical coagulation parameters enhances the biological relevance of the findings, but it seems not strongly linked to the HABP2 genotypes, so it is difficult to give a relevant meaning to these correlations.

The manuscript would benefit from clearer organization and presentation, particularly in the Results section, to improve readability.

My basic consideration is that when studying the correlation between a polymorphism and a given phenomenon, it is essential to report the frequency of the polymorphism in the study population. The frequency data are crucial to determine whether the polymorphism is truly associated with the observed phenomenon or if its presence might be due to chance. Including this comparison strengthens the validity and interpretability of the study's findings and is a standard practice in genetic association research.

Moreover, in the Introduction is reported that “In this study, we analyzed relationships between HABP2 polymorphisms and RPL. Six polymorphisms (rs3832698 A>del, rs10885478 G>A, rs932650 T>C, rs7923349 G>T, rs1157916 G>A, and rs2240879 T>C) located in the promoter region, introns, and 3′ untranslated region (UTR) were selected” no other explanation is present. But why were these polymorphisms chosen? They are present in non-coding regions such as promoters, introns, and UTRs, so can they have regulatory roles influencing gene expression? Which biological pathways are involved that can be related to the RPL phenomena?

In light of this, the discussion could be strengthened by a deeper exploration of the functional implications of the identified polymorphisms and their mechanistic role in RPL pathophysiology. Limitations regarding population specificity and the absence of experimental validation should be more explicitly addressed. 

 

1. About population frequencies of each SNP.

Thank you for your comment. We add more information in line _161_

Each polymorphisms are well established and matched with Korean minor allele frequency such as KRGDB and Korea 1K. And all SNPs are satisfied with Hardy Weinberg equilibrium in control group. In contrast, several SNPs showed deviation from HWE in patient groups. As HWE disruption in affected individuas may reflect disease associated or selection of selection pressure. Therefore, the HWE deviation in patients does not undermine the allele frequency comparison or the conclusions of the study

 

2. About regulatory roles of introns such as 3’UTR and promoter and could possible to influencing gene expression.

Thank you for your comment. Each polymorphism are located in non-coding regions such as promoters and UTR region. However there are lots of reports that UTR and promoters are possible to change gene expression level. So we add more information in line _70_

however, even non-coding regions possible to change gene expression. Therefore we examined there frequencies in Korean women with RPL and in healthy controls.

Author Response File: Author Response.pdf