Dear Editor,

This manuscript introduces a unique concept in an area that is both intriguing and important for medical research. The topic is timely and is likely to engage the journal's readers. Additionally, the manuscript is well-written, presenting ideas clearly and logically.

Dear Authors,

You are doing excellent work, and this manuscript reflects a great deal of effort and thoughtful research. The study is promising and contributes meaningfully to the field. However, there are a few important points that require further clarification and revision to enhance the overall quality and impact of the manuscript.

The introduction provides a comprehensive overview of PCOS and the hypothesised role of the KISS1 gene; however, the specific knowledge gap addressed by the current study should be stated more explicitly. While conflicting evidence regarding serum kisspeptin levels and KISS1 polymorphisms is mentioned, the introduction would benefit from a clearer explanation of why existing studies are insufficient and how the present study advances the field.

It is preferred to use the following reference to support your updated citations:   (refer to 1)

  The reported prevalence of PCOS (6%–10%) is appropriate; however, the diagnostic criteria used in cited studies (e.g., NIH, Rotterdam, or AES) are not specified. Given that PCOS prevalence varies considerably depending on diagnostic criteria, the authors should clarify this to improve interpretability and contextual accuracy.

Although the introduction discusses the physiological role of kisspeptin in GnRH and gonadotropin regulation, the mechanistic pathway linking KISS1 SNPs to PCOS pathogenesis remains somewhat speculative. A more detailed explanation of how specific polymorphisms may alter kisspeptin expression or function would strengthen the biological plausibility of the study hypothesis.

It is preferred to use the following reference to support your updated citations: (refer to 2)

The discussion of KNDy neurons is relevant but underdeveloped. The authors should expand on how alterations in neurokinin B, dynorphin, and kisspeptin signaling specifically contribute to PCOS-related neuroendocrine dysfunction and how this supports investigating KISS1 gene variants.

The final sentence states that the study determined the impact of KISS1 gene polymorphisms and haplotypes on PCOS development. This objective would benefit from greater specificity, including which polymorphisms or haplotypes were analyzed and whether the focus is on association, causation, or functional prediction.

It is preferred to use the following reference to support your updated citations: •   (refer to 3)

The manuscript states that 80 women with PCOS and 76 controls were initially recruited, but only 60 participants per group met the inclusion criteria. The specific reasons for exclusion (e.g., incomplete data, failure to meet diagnostic criteria, withdrawal of consent) should be explicitly described to enhance transparency and reproducibility.

Oligo- or anovulation was determined solely based on self-reported menstrual cycle frequency, which may be subject to recall bias. The authors should clarify whether menstrual history was corroborated with clinical records or supported by biochemical markers (e.g., mid-luteal progesterone), particularly given the diagnostic importance of ovulatory dysfunction in PCOS.

Although hyperandrogenism was defined using Ferriman–Gallwey scores and serum total testosterone levels, the manuscript lacks detail regarding:

The assessor training or blinding for Ferriman–Gallwey scoring, and

The assay method, timing, and reference ranges for testosterone measurement.

These details are essential for ensuring diagnostic consistency and comparability with other studies.

Participants were described as “self-reported as having Indonesian ethnic origin.” While this helps reduce population stratification bias, the authors should acknowledge the limitations of self-reported ethnicity, particularly in genetic association studies, and consider discussing whether sub-ethnic heterogeneity was assessed or controlled.

The Results section refers to comparisons between “lean and obese groups” and mentions “four groups”; however, this stratification is not clearly introduced or defined in the Methods or at the beginning of the Results. The authors should explicitly describe how subjects were categorized (e.g., BMI cutoffs) and ensure consistency between Methods, Tables, and Results.

The manuscript reports median ages and median kisspeptin levels but does not consistently indicate whether other variables are presented as means ± SE or medians (IQR). This inconsistency makes interpretation difficult. The authors should clarify which variables were normally distributed, justify the choice of statistical descriptors, and ensure alignment with the statistical tests used.

Although no statistically significant difference in kisspeptin levels was observed, the Results section still emphasizes the lower median value in the PCOS group. This observation should be reported cautiously and without implying biological relevance unless supported by statistical significance or effect size analysis.

There appears to be confusion in allele nomenclature, particularly for rs5780218 (e.g., reference to a “CT allele”). Alleles should be clearly distinguished from genotypes. The authors must revise this section to ensure correct and unambiguous genetic terminology.

It is preferred to use the following reference to support your updated citations:      (refer to 4 )

The Discussion appropriately acknowledges conflicting reports regarding serum kisspeptin levels in PCOS; however, the interpretation remains largely speculative. The authors should more clearly distinguish between central (hypothalamic) kisspeptin signalling and circulating kisspeptin levels, as serum measurements may not accurately reflect hypothalamic activity. This distinction is critical for reconciling discrepancies with studies reporting elevated kisspeptin expression in PCOS.

It is preferred to use the following reference to support your updated citations: (refer to 5)

Conclusion should be objective, with further perspective,e or should add at least a few sentences about future study/future perspective of .it

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

Yes

Are all the source data underlying the results available to ensure full reproducibility?

Yes

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Yes

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

Molecular Biology, Microbiology

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Congratulations on a great research report. It focuses on an upstream neuroendocrine gene (KISS1) with mechanistic plausibility, also including haplotype analysis,  Day 3–5 sampling attempt, and exclusion of recent HPG-altering medications with open data availability. 

Here are the issues identified:

Primary outcome specification. The manuscript does not pre-specify a single primary genetic model/outcome; multiple inheritance models are tested across two SNPs (± haplotypes).

Sample size and power: No a priori power calculation is provided; small genotype cell counts (e.g., rs4889 GG) inflate ORs and widen CIs. Need to add an a priori (or post-hoc, with caveats) power analysis indicating detectable OR at α (after correction) for your MAF. Interpret large ORs from rare genotypes cautiously.

Recruitment flow & attrition recruited 80 PCOS and 76 controls, but analyzed 60+60; reasons for exclusion are not detailed. Can you provide a CONSORT-style flow diagram with reasons for exclusion and any differences between included vs excluded? 

Group definition and stratification Results text refers to “four groups” (lean vs obese strata), but Methods define only cases/controls; BMI cut-offs not specified. Please clearly define BMI strata (cut-points, rationale), when this stratification was planned, and where those subgroup results appear (tables/appendix). Align wording (two vs four groups).

Normality, summary measures, and test. Text says data are “mean ± SE” and “independent t-test” used, yet tables present medians (ranges), and several variables are non-normal. Please reassess distributions; report appropriate central tendency (mean±SD for normal; median[IQR] for skewed) and use Mann-Whitney U where needed. Ensure test choice matches what’s reported in tables.

Genotyping quality control: Limited QC details for Sanger calls (call rate, duplicate concordance, blinded repeats). Can you report per-SNP call rate, sample call rate, replicate concordance, and any re-reads? Include HWE p-values for  controls (and for cases with caveat) and confirm all expected cell counts ≥5; use Fisher’s exact where violated.

Population structure “Indonesian ethnic origin” is self-reported in a genetically diverse setting. Please describe recruitment geography and, if available, adjust for population substructure (e.g., principal components). At a minimum, discuss the risk of stratification bias in limitations.

Confounding adjustment Logistic regression via SNPStats appears unadjusted for age, BMI, and insulin resistance. Please present adjusted models (age, BMI, HOMA-IR at a minimum). State covariate selection strategy and show both crude and adjusted ORs with 95% CIs.

Haplotype and LD reporting. Only D′ is reported; r² is not provided; haplotype inference power is limited with n=120.. Please add r² values, haplotype frequencies with CIs, and sensitivity analyses (e.g., EM convergence diagnostics). Discuss low-frequency haplotype instability. 

Kisspeptin assay standardization. Samples taken “day 3–5” may not be physiologically comparable in anovulatory PCOS; assay performance details (LoD, intra/inter-assay CVs, freeze-thaw cycles) are missing. Please provide preanalytical handling, kit performance metrics, and justify timing equivalence for PCOS vs controls; consider sensitivity analysis excluding uncertain cycle-phase samples.

Diagnostic criteria implementation: Hyperandrogenism definition mixes clinical (FG >8) and biochemical (T>60 ng/dL) using immunoassay (possible bias at low female T). Please clarify assay method limitations (vs LC-MS/MS), laboratory reference ranges, and how hirsutism scoring handles hair removal. Provide inter-rater reliability if available.

Causal language “Predisposing factor” overstates causality for a cross-sectional association. Please replace with “associated with increased odds of PCOS” throughout, including the title and conclusions. 

That's all. Thank you

Is the work clearly and accurately presented and does it cite the current literature?

Yes

If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Yes

Reviewer Expertise:

need to be a researcher working on the same field as this is too niche

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.