Major comment 1: Specify whether the study is retrospective or prospective.

Response: We agree. The study is now explicitly described as a multicentre retrospective cross-sectional study based on routinely collected, anonymized clinical and laboratory records. We also clarified that there was no prospective intervention or additional specimen collection.

Locations: Abstract, Methods sentence; Section 2.1, Study Design and Setting, first paragraph; Section 2.3, Data Collection, opening sentence.

Major comment 2: Explain why 49 initially ASA-positive cases were reclassified as negative.

Response: We clarified that the discrepancy was a categorical coding error in the source spreadsheet. The numeric MAR percentages were not changed. Binary ASA status was recalculated uniformly from the recorded MAR percentage using the prespecified threshold: negative <50% and positive >=50%.

Location: Section 2.5, Statistical Analysis, final data-cleaning sentence; related clarification retained in the first paragraph of the Discussion.

Major comment 3: Describe the direct MAR technique in sufficient detail for replication.

Response: We added the reagent volumes, specimen condition, mixing sequence, microscope magnification, reading time, number and type of sperm counted, delayed rereading procedure, and interpretation threshold. The procedure follows the FertiPro SpermMar IgG instructions and contemporary methodological guidance.

Location: Section 2.3, Data Collection, new paragraphs immediately after the ASA measurement bullet. Citation added as reference 23.

Major comment 4: Reduced motility may prevent antibody binding and affect the results.

Response: We agree. We now state that direct MAR scoring depends on motile spermatozoa, that marked asthenozoospermia can reduce the number of scorable cells and potentially reduce sensitivity, and that this may bias comparisons toward higher apparent ASA prevalence among men with better-preserved motility. We recommend indirect MAR or immunobead testing in future studies when adequate motile cells cannot be scored.

Locations: Section 2.3, new methodological limitation paragraph; Discussion, Limitations paragraph.

Major comment 5: The statistical analysis needs to be reviewed.

Response: We reviewed the analysis and strengthened its interpretation. The 10-predictor logistic model is now explicitly identified as exploratory because 53 positive events correspond to approximately 5.3 events per parameter. We emphasize effect estimates, confidence intervals, discrimination, calibration, and held-out test performance rather than p-values or variable-importance rankings alone.

Location: Section 2.5, Statistical Analysis, paragraph beginning “The prevalence of ASA positivity...” and the existing model-performance discussion.

Minor comment 1: Specify the supplements received by patients.

Response: The source records captured supplement use only as yes/no and did not consistently contain product name, formulation, dose, or duration. We therefore did not invent categories. The manuscript now transparently states this limitation and retains supplement exposure as a binary variable.

Location: Section 2.3, Data Collection, Demographic and lifestyle bullet.

Minor comment 2: Avoid repeating results in both tables and figures; remove redundant presentations.

Response: We retained the tables because they provide exact values and removed the five redundant graphical presentations corresponding to former Figure 2 through Figure 6. Textual cross-references were revised accordingly.

Locations: Results Sections 3.2, 3.4, 3.5, 3.6, and 3.7; former Figures 2-6 deleted.

Minor comment 3: Update outdated references.

Response: Foundational references were retained only where historically or methodologically necessary. We added a broad contemporary review and global survey of ASA testing practices and used it to support the expanded methods and international context. The manuscript already includes recent evidence from 2022, 2024, 2025, and 2026.

Locations: Introduction, regional/international variability paragraph; Section 2.3, MAR method; References, new reference 23.

Consider broader formulations in the title and abstract because the cohort is characterized by semen and general-health parameters.

Response: We broadened the title to include semen-quality phenotypes and added a sentence in the Abstract noting heterogeneity in motility, morphology, and semen-profile clustering, while retaining ASA as the main outcome.

Locations: Manuscript title; Abstract, immediately before Conclusions.

The absence of a control group is a serious limitation.

Response: We agree and have strengthened this limitation. The revised text states that the study estimates prevalence only within a referred infertile cohort and cannot quantify excess ASA positivity relative to fertile men from the same population.

Location: Discussion, Limitations paragraph.

Figure 8 has overlapping points; use clearer colour intensity and consider an axis break.

Response: The PCA figure uses high-contrast cluster colours, distinct geometric symbols for ASA-positive and ASA-negative cases, and transparency so overlapping observations can be recognized. We clarified these encodings in the caption. We did not break the PCA axis because a discontinuity would distort Euclidean distances and the geometry of the ordination; a broken axis is more appropriate for univariate plots than for a PCA biplot.

Location: Figure 3 (previous Figure 8) caption.

Include more references from populations outside the region.

Response: We added a global review and survey covering clinical and laboratory practices across multiple continents, and incorporated it into the Introduction and methods discussion.

Locations: Introduction, regional/international variability paragraph; References, new reference 23, 24.