Oxidative Stress, Sperm DNA Fragmentation, or Both? Optimizing Test Selection in Male Infertility Evaluation

Round 1

Reviewer 1 Report

The manuscript provides a clear and useful overview of the relationship between oxidative stress (OS) and sperm DNA fragmentation (SDF), including a comparison of the main assessment methods (ROS chemiluminescence, ORP/MiOXSYS, TAC/FRAP, 8-OHdG, TBARS; and SCSA, TUNEL, Comet, SCD) and their indications in clinical practice. A notable strength of the manuscript is the parallel consideration of leukocyte assessment, as the OS signal may originate from both spermatozoa and leukocytes.

However, a significant gap is that the manuscript only marginally addresses single-cell fluorescence-based approaches (e.g., DCFDA, DHE) and does not provide a practical framework for modern cytometric or fluorescence-based quantification of ROS and mitochondrial dysfunction in spermatozoa (e.g., CellROX, MitoSOX, and related markers), nor does it sufficiently address fluorescence microscopy and issues of signal specificity in the context of sperm cells.

Major comments

Line 171 – The authors are encouraged to include additional markers, such as MitoSOX Red for the detection of mitochondrial superoxide (mito-O₂•⁻) and CellROX dyes (Green/Deep Red/Orange) as indicators of overall cellular oxidative status.

The manuscript would further benefit from the inclusion of a basic flow cytometry gating strategy, particularly since it already highlights the need to account for leukocytes and the potential confounding effect of leukocytospermia on OS measurements. In addition, the use of appropriate positive controls (e.g., H₂O₂-induced oxidative stress) should be described. Complementary fluorescence microscopy could also be suggested to help exclude non-specific fluorescence signals.

A minimal gating strategy could include:

• FSC/SSC gating to identify spermatozoa and exclude debris,
• doublet discrimination (FSC-A vs. FSC-H),
• viability gating (e.g., PI, 7-AAD, or DRAQ7),
• optional leukocyte identification using markers such as CD45.

Finally, the authors are encouraged to add a Future Perspectives section outlining potential strategies for mitigating the effects of oxidative stress on spermatozoa.

The manuscript provides a clear and useful overview of the relationship between oxidative stress (OS) and sperm DNA fragmentation (SDF), including a comparison of the main assessment methods (ROS chemiluminescence, ORP/MiOXSYS, TAC/FRAP, 8-OHdG, TBARS; and SCSA, TUNEL, Comet, SCD) and their indications in clinical practice. A notable strength of the manuscript is the parallel consideration of leukocyte assessment, as the OS signal may originate from both spermatozoa and leukocytes.

However, a significant gap is that the manuscript only marginally addresses single-cell fluorescence-based approaches (e.g., DCFDA, DHE) and does not provide a practical framework for modern cytometric or fluorescence-based quantification of ROS and mitochondrial dysfunction in spermatozoa (e.g., CellROX, MitoSOX, and related markers), nor does it sufficiently address fluorescence microscopy and issues of signal specificity in the context of sperm cells.

Major comments

Line 171 – The authors are encouraged to include additional markers, such as MitoSOX Red for the detection of mitochondrial superoxide (mito-O₂•⁻) and CellROX dyes (Green/Deep Red/Orange) as indicators of overall cellular oxidative status.

The manuscript would further benefit from the inclusion of a basic flow cytometry gating strategy, particularly since it already highlights the need to account for leukocytes and the potential confounding effect of leukocytospermia on OS measurements. In addition, the use of appropriate positive controls (e.g., H₂O₂-induced oxidative stress) should be described. Complementary fluorescence microscopy could also be suggested to help exclude non-specific fluorescence signals.

A minimal gating strategy could include:

• FSC/SSC gating to identify spermatozoa and exclude debris,
• doublet discrimination (FSC-A vs. FSC-H),
• viability gating (e.g., PI, 7-AAD, or DRAQ7),
• optional leukocyte identification using markers such as CD45.

Finally, the authors are encouraged to add a Future Perspectives section outlining potential strategies for mitigating the effects of oxidative stress on spermatozoa.

Author Response

Response to Reviewer 1

Comment 1 (Line1. Single-cell fluorescence ROS approaches; add CellROX and MitoSOX; improve framework)

The manuscript only marginally addresses single-cell fluorescence-based approaches (e.g., DCFDA, DHE) and lacks a practical framework for modern cytometric/fluorescence-based quantification of ROS and mitochondrial dysfunction in spermatozoa. Please include MitoSOX Red and CellROX dyes, and address signal specificity in sperm cells.

Response:
Thank you. We expanded the discussion on single-cell fluorescence-based ROS assessment to better reflect modern practice and clearly position these assays as complementary to bulk chemiluminescence. We now explicitly include CellROX dyes (overall cellular oxidative status) and MitoSOX Red (mitochondrial superoxide), and we added a concise note on signal specificity challenges in sperm (autofluorescence and dead-cell signal inflation), emphasizing the need for viability gating and controls.

Changes in the text:
Page 9, Lines 179–188:
“Single-cell fluorescence approaches (flow cytometry and/or fluorescence microscopy) complement bulk chemiluminescence… Common probes include… DCFDA… DHE… CellROX dyes… and MitoSOX Red… However, probe chemistry, sperm autofluorescence, and dead-cell signal inflation necessitate strict viability gating and appropriate analytical controls…”

Comment 2 (R1. Add a basic flow cytometry gating strategy + positive controls; suggest microscopy to exclude nonspecific signals)

Please include a minimal flow cytometry gating strategy (FSC/SSC, doublet discrimination, viability gating, optional CD45 for leukocytes), describe appropriate positive controls (e.g., H₂O₂-induced OS), and suggest complementary fluorescence microscopy to exclude nonspecific fluorescence.

Response:
We agree and have added a practical, minimal gating workflow aligned with semen-specific analytical pitfalls, particularly leukocyte confounding. We also added a brief description of session controls (unstained/single-stained controls, oxidative challenge) and explicitly recommend fluorescence microscopy as a complementary approach to support signal specificity and localization.

Changes in the text:
Page 14, Lines 330–337:
“Where flow cytometry is employed… a minimal and reproducible gating workflow includes (i) FSC/SSC… (ii) doublet discrimination… (iii) viability gating… (iv) optional CD45… For assay control… include a positive oxidative challenge (e.g., H₂O₂ exposure; antimycin A when interrogating mitochondrial ROS with MitoSOX)… Complementary fluorescence microscopy can further support signal specificity…”

Comment 3 (R1. Add a Future Perspectives section on strategies to mitigate oxidative stress effects on spermatozoa)

Please add a Future Perspectives section outlining potential strategies for mitigating OS effects on spermatozoa.

Response:
Thank you. We added a dedicated Future Perspectives subsection that outlines forward-looking, clinically translatable mitigation directions, including (i) risk-factor/etiologic mitigation, (ii) precision redox-targeted therapeutics, (iii) ART/laboratory mitigation, and (iv) monitoring/endpoints/implementation to support prospective pathway validation.

Changes in the text:
Page 36, Lines 993–1024 and Page 37, Lines 1025–1043: New subsection “7.10. Future Perspectives: Mitigation Strategies to Reduce Oxidative Stress–Related Sperm Damage” with subheadings 7.10.1–7.10.4.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

This manuscript provides a timely and clinically relevant synthesis of oxidative stress (OS) and sperm DNA fragmentation (SDF) as complementary contributors to male infertility. It clearly distinguishes OS as a marker of disrupted seminal redox balance and SDF as a measure of downstream genomic damage, offering a coherent pathophysiological framework aligned with current knowledge. The structured overview of laboratory assays, including ROS measurement, oxidation reduction potential, antioxidant capacity, lipid peroxidation markers, and major SDF platforms, enhances its translational value. The alignment of testing strategies with guideline-based clinical indications is a significant strength.

The manuscript entitled “Oxidative Stress, Sperm DNA Fragmentation, or Both? Optimizing Test Selection in Male Infertility Evaluation” is a comprehensive, well structured, and clinically relevant review. However, I would like to suggest a few points that may further strengthen the manuscript:

 

Section 3.5. Biomarkers of Oxidative Damage: 8-OHdG and Lipid Peroxidation (MDA/TBARS)

In the section discussing TBARS as a marker of lipid peroxidation, it would be valuable to mention that, in addition to this technique, C11-BODIPY581/591 is a fluorescent probe that has been successfully used as a reliable measurement of lipid peroxidation, particularly in mammalian spermatozoa across different species. This probe allows for a more specific and dynamic assessment of membrane lipid oxidation and could enrich the discussion on modern and more sensitive biomarkers of oxidative damage in sperm cells.

Lines 251-252

In the paragraph stating: “Centrifugation steps, especially high-speed or repeated centrifugation, can artificially increase ROS generation.”

I suggest adding that during sperm washing, the mechanical action of pipetting introduces several stressors that contribute to Reactive Oxygen Species (ROS) generation. This clarification is relevant because not only centrifugation but also repeated mechanical handling during sample processing may significantly contribute to artifactual oxidative stress.

Section 4.6. Ancillary and Emerging Approaches of SDF Testing

In the paragraph stating: “Histochemical stains such as aniline blue and toluidine blue index chromatin packaging defects rather than strand breaks and therefore serve as complementary markers of protamination status rather than primary measures of DNA fragmentation [81].”

It would be appropriate to include that Chromomycin A3 (CMA3) is also a useful tool for the detection of protamine deficiency in sperm chromatin, as it competes with protamines for GC-rich binding sites and enables the assessment of chromatin packaging defects related to incomplete protamination.

Including CMA3 would provide a more complete overview of indirect markers of chromatin integrity and protamination status.

Author Response

Response to Reviewer 2

Comment 1 (Section 3.5. Biomarkers of Oxidative Damage: 8-OHdG and Lipid Peroxidation (MDA/TBARS))

In addition to TBARS, please mention C11-BODIPY581/591 as a fluorescent probe used as a reliable measurement of lipid peroxidation in spermatozoa across species, allowing a more specific/dynamic assessment of membrane lipid oxidation.

Response:
Thank you for this valuable suggestion. We have added C11-BODIPY581/591 as a modern, cell-resolved lipid peroxidation probe and briefly described its advantages (ratiometric/spectral shift; single-cell readout by microscopy or flow cytometry), while noting the need for protocol standardization and lab-specific validation.

Changes in the text:
Page 13, Lines 304–310:
“To complement TBARS with a more cell-resolved assessment, C11-BODIPY581/591 has been employed as a ratiometric, spectral shift–based fluorescent probe of membrane lipid peroxidation that can be quantified by fluorescence microscopy or flow cytometry. This method captures lipid peroxidation at the single-cell level, including within viability-gated sperm populations, thereby improving interpretability compared with bulk TBARS measurements, while still requiring rigorous protocol standardization and laboratory-specific performance validation and thresholds.”

Comment 2 (Lines 251–252; pre-analytics/handling)

In addition to centrifugation increasing ROS, please add that mechanical pipetting during sperm washing introduces stressors that can contribute to ROS generation and artifactual oxidative stress.

Response:
We agree. We expanded the pre-analytical/handling section to explicitly include mechanical handling during processing, emphasizing that repeated pipetting/resuspension and wash steps can inflate oxidative readouts via shear stress and related artifacts, alongside centrifugation effects.

Changes in the text:
Page 13, Lines 321–330:
“Centrifugation steps, especially high-speed or repeated centrifugation, can artificially increase ROS generation and thereby distort results of both chemiluminescence and ORP assays. Mechanical handling represents an additional and frequently under-recognized source of analytical artifact. Repeated pipetting, vigorous resuspension, and multiple wash steps can increase oxidative readouts by introducing shear stress and triggering redox-sensitive processes, thereby inflating apparent ROS signals even in the absence of genuine biological change.”

Comment 3 (Section 4.6. Ancillary and Emerging Approaches of SDF Testing)

When listing indirect chromatin/protamination markers (aniline blue, toluidine blue), please also add Chromomycin A3 (CMA3) as a tool for protamine deficiency/chromatin packaging defects (competes with protamines for GC-rich sites).

Response:
Thank you. We have incorporated CMA3 into the list of indirect chromatin packaging/protamination markers and added a short mechanistic clarification (competitive binding at GC-rich sites; higher staining suggests incomplete protamination), while keeping the distinction clear that these stains do not measure strand breaks.

Changes in the text:
Page 17, Lines 465–471:
“Histochemical stains such as aniline blue, and toluidine blue and Chromomycin A3 (CMA3) index chromatin packaging defects rather than strand breaks and therefore serve as complementary markers of protamination status rather than primary measures of DNA fragmentation. CMA3 is a fluorochrome-based stain used as an indirect marker of protamine deficiency and abnormal chromatin packaging. It binds to GC-rich sites and competes with protamines, so increased CMA3 staining suggests incomplete protamination.”

 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

I have no futher concerns.

The authors have addressed my comments, and I have no further concerns.