Sperm Microbiota and Its Potential Impact on Male Fertility: A Systematic Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

My major concerns are as follows:

1. A space should be existed between “potential and impact” in title.
2. Conclusion in abstract was not proper and some contents (from Further investigations……) should be deleted and appeared in discussion part.
3. The framework for the paper was not proper and should be coordinated. The paper was a systematic review based on the title, but major contents seemed like a study. In addition to that, many data in the paper can be further analyzed. Therefore, “Systematic Reviews and Meta-Analyses” might be the proper characteristics or attribute for the paper.
4. Only descriptive introduction for the microbiota (tale 2) was not enough and made the paper simple and lack of comprehensive information. The studies should compare and analyze the bacteria most frequently found in normal semen and abnormal semen deeply, because this was the most important role. Some bacteria might exist at both the normal semen and abnormal semen, but the other bacteria might have different survival status or overlapping. In addition to that, different research might have different expression style. Detailed Meta-Analyses might provide the paper more value.
5. Actually the paper was a primary study and the precise mechanisms through which it influences on male fertility had not yet be elucidated. If only providing with simple results and without suggested normal or abnormal semen related seminal microbiota, the importance for the study was low.
6. More discussion contents in the results part (contents from line 135 to line 163) were not proper, and should be changed into discussion part.
7. The microbiota most frequently found at semen does not certainly imply its pathogenesis, and may be healthy carrier, such as “Ureaplasma urealyticum”, and related controversy existed extensively. Comprehensive explanation/analysis should be supplemented in discussion part.

Comments on the Quality of English Language

Language should be checked carefully. Some expressions were not logically reasonable, and might be verified, such as contents in line170 “For many years now”.

Bacteria are not equal to microbiota, and should not be used at random, such as “bacteria” in line 166, and in line 179.

Author Response

Thank you very much for taking the time to review our manuscript. Please find below our detailed responses, as well as the corresponding revisions and corrections.

Comments 1 : A space should be existed between “potential and impact” in title.

Response 1 : the title has been changed

Comments 2 : Conclusion in abstract was not proper and some contents (from Further investigations……) should be deleted and appeared in discussion part.

Response 2 : the sentence has been deleted

Comments 3 : The framework for the paper was not proper and should be coordinated. The paper was a systematic review based on the title, but major contents seemed like a study. In addition to that, many data in the paper can be further analyzed. Therefore, “Systematic Reviews and Meta-Analyses” might be the proper characteristics or attribute for the paper.

Response 3 : This is a systematic literature review, deliberately conducted without statistical analysis, aimed at providing an overview of published studies on the sperm microbiota and its impact on sperm quality. No quantitative analysis was performed because the data across studies were too heterogeneous to allow for rigorous comparison and the generation of statistically meaningful results. Attempting quantitative synthesis under such conditions would risk producing misleading or spurious conclusions due to the high clinical and methodological heterogeneity.

The included publications vary widely in terms of:
• Sampling protocols (e.g., semen collection methods, processing techniques, storage conditions),
• Microbiological assessment tools (e.g., culture-based methods vs. 16S rRNA gene sequencing),
• Study populations (e.g., fertile vs. infertile men, geographical differences, and the presence of confounding factors such as urogenital infections or lifestyle variables).

Nonetheless, by systematically identifying, appraising, and synthesizing the available evidence, this review highlights recurring microbial signatures. This synthesis reveals useful trends that can guide the interpretation of sperm microbiota findings. Moreover, these qualitative insights provide a crucial foundation for future research, helping refine hypotheses regarding the role of the sperm microbiome in male reproductive health.

Comments 4 : Only descriptive introduction for the microbiota (tale 2) was not enough and made the paper simple and lack of comprehensive information. The studies should compare and analyze the bacteria most frequently found in normal semen and abnormal semen deeply, because this was the most important role. Some bacteria might exist at both the normal semen and abnormal semen, but the other bacteria might have different survival status or overlapping. In addition to that, different research might have different expression style. Detailed Meta-Analyses might provide the paper more value.

Response 4 : The table provides a synthesis of the bacterial species identified in each patient or experimental group across the studies included in this review. It does not present original microbiological findings but rather collates and organizes data reported in previously published articles. The relationships and patterns observed—such as the association between specific bacterial profiles and the sperm quality are further interpreted and contextualized in the Discussion section.

Importantly, this manuscript is a literature review; it involves no primary data collection, no patient recruitment, no laboratory interventions, and no original experimental procedures. All conclusions are drawn from critical analysis and synthesis of existing peer-reviewed publications.

Comments 5 : Actually the paper was a primary study and the precise mechanisms through which it influences on male fertility had not yet be elucidated. If only providing with simple results and without suggested normal or abnormal semen related seminal microbiota, the importance for the study was low.

Response 5 : The relationship between the seminal microbiota and sperm parameters—including the underlying biological mechanisms—has been thoroughly discussed in the Discussion section of this article. Specifically, the analysis distinguishes between bacterial taxa associated with normozoospermia (i.e., normal semen parameters) and those linked to pathological semen profiles (such as oligozoospermia, asthenozoospermia, or teratozoospermia). For each group, the potential roles of specific microorganisms—whether commensal, beneficial, or potentially pathogenic—are examined in light of current evidence, and plausible mechanisms by which these bacteria may influence sperm concentration, motility, morphology, or overall semen quality are outlined.

Comments 6 : More discussion contents in the results part (contents from line 135 to line 163) were not proper, and should be changed into discussion part.

Response 6 : It has been changed

Comments 7 : The microbiota most frequently found at semen does not certainly imply its pathogenesis, and may be healthy carrier, such as “Ureaplasma urealyticum”, and related controversy existed extensively. Comprehensive explanation/analysis should be supplemented in discussion part.

Response 7 : we have revised the paragraph on line 169 and clarified that certain bacteria are known to be pathogenic above a predefined threshold

Comments on the Quality of English Language : Language should be checked carefully. Some expressions were not logically reasonable, and might be verified,

Response : all the manuscript have been revised

Reviewer 2 Report

Comments and Suggestions for Authors

The main question addressed by the authors is whether certain characteristics of the sperm microbiota influence semen quality and fertility outcomes, including the effects of assisted reproductive technologies (ART). Therefore, the study aims to map the general microbial profiles of fertile versus infertile men, summarizing the body of research, particularly from the last 10 years. This is also a topic of current scientific interest. The conventional view of semen as a sterile, fixed fluid is slowly being subverted, and the contribution of indigenous microbial communities to sperm function, oxidative stability, and DNA quality has emerged as a new area of ​​research. The authors collect and interpret data from 20 studies, compiled between 2013 and 2024, thus presenting a practical synthesis in a field characterized by high methodological variability and fragmented evidence. In relation to previous reviews, the manuscript contributes to the literature by highlighting the characteristics of the taxon. Furthermore, the consistent presence of Lactobacillus and Bifidobacterium in semen samples with physiological values ​​is evident and described, compared to the overproduction of Prevotella, Pseudomonas, and Escherichia coli in pathological cases. A further advantage of this study is the addition of a practical table to quantify and represent microbial load thresholds for assisted reproductive technology (ART), as it provides clinicians with a reference for laboratory use. The section on pathogenic mechanisms—oxidative stress, mitochondrial dysfunction, and inflammatory cascades—is also well-conceived and educationally useful. However, the review's methodological framework needs to be strengthened before the article can be submitted for preliminary review and ready for publication. Authors should expand their literature search beyond PubMed and ScienceDirect to include sites such as Scopus, Web of Science, and Embase, and make search strings fully reproducible. For example, protocol registration (e.g., PROSPERO) and the PRISMA list could be used to increase transparency. The lack of a risk of bias assessment is a significant limitation; the use of standardized tools such as those from Joanna Briggs or the Cochrane Collaboration would better reflect the robustness of the synthesis. Since sequencing varies depending on the type of procedure, it is also desirable to present the targeted 16S hypervariable regions, extraction kits and control sets used, as well as the criteria for normal and abnormal semen parameters in the main comparative table. Such details are essential for interpreting inconsistencies between studies. A more systematic assessment of confounding variables, particularly the partner's microbiota, antibiotic exposure, and sampling techniques, would further enhance the discussion. There is consistent agreement with the evidence presented in the conclusions. Indeed, the authors rightly emphasize that, although multiple relationships exist between microbial signatures and sperm quality, causality has not been established and further standardized research is needed. This approach is carefully presented, with the prospect of integrating microbiome sequencing into the diagnostic workup for idiopathic infertility prudently positioned as a future direction rather than as an ongoing recommendation. Most of the relevant publications were written by 2024, so the cited sources were recent and in the appropriate place. Minor editorial changes in the margins would be helpful, though—a uniform DOI formatting would also be helpful—and perhaps we could consider including some methodological references to contamination control and low-biomass analysis methods in the citation font. Regarding visual content, Tables 1 and 2 are informative; Tables 1 and 2 contain information, but could be enhanced with columns indicating the sequencing regions, contamination controls, and diagnostic criteria. The table presenting bacterial thresholds for assisted reproduction (ART) is particularly useful, and a brief explanatory appendix indicating whether the thresholds are correct for pure or processed semen samples would be very helpful. The graphical features in Figures 2 and 3 are also well-designed, but indicating both sample sizes and study counts by taxon would further explain and contextualize the graphical data.

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below 

Comments 1 : Authors should expand their literature search beyond PubMed and ScienceDirect to include sites such as Scopus, Web of Science, and Embase, and make search strings fully reproducible

Response 1 : PubMed and ScienceDirect—the two databases used in this review—are among the largest and most comprehensive sources for peer-reviewed scientific literature. Extending the search to additional databases such as Embase and Web of Science did not yield any further relevant studies on the human seminal microbiota beyond those already identified through PubMed and ScienceDirect.

Comments 2 : The lack of a risk of bias assessment is a significant limitation; the use of standardized tools such as those from Joanna Briggs or the Cochrane Collaboration would better reflect the robustness of the synthesis

Response 2 : This is a systematic literature review, deliberately conducted without statistical analysis, aimed at providing an overview of published studies on the sperm microbiota and its impact on sperm quality. No quantitative analysis was performed, as the data from the articles were too heterogeneous to allow for rigorous comparison and the generation of statistically significant power. Attempting quantitative synthesis under such conditions would risk producing misleading or spurious conclusions due to high clinical and methodological heterogeneity.

The included publications vary widely in terms of:

• Sampling protocols (e.g., semen collection methods, processing techniques, storage conditions),
• Microbiological assessment tools (e.g., culture  vs. 16S rRNA gene sequencing),
• Study populations (e.g., fertile vs. infertile men, geographical differences, inclusion of confounding factors such as urogenital infections or lifestyle variables).

Nonetheless, by systematically identifying, appraising, and synthesizing current evidence, this review enables the identification of recurring microbial signatures. This synthesis has revealed some useful trends to guide the analysis of the sperm microbiota. Also,  these qualitative insights provide a crucial foundation for future research, helping to refine hypotheses into the role of the sperm microbiome in male reproductive health.

Comments 3 : Since sequencing varies depending on the type of procedure, it is also desirable to present the targeted 16S hypervariable regions, extraction kits and control sets used, as well as the criteria for normal and abnormal semen parameters in the main comparative table. Such details are essential for interpreting inconsistencies between studies.

Response 3 : we added the hypervariable regions and the extraction kits in Table 2 ; the criteria defining the sperm parameters are not specified in all the articles.

Comments 4 :  A more systematic assessment of confounding variables, particularly the partner's microbiota, antibiotic exposure, and sampling techniques, would further enhance the discussion.

Response 4 : we added it in the discussion page 16

Comments 5 : The table presenting bacterial thresholds for assisted reproduction (ART) is particularly useful, and a brief explanatory appendix indicating whether the thresholds are correct for pure or processed semen samples would be very helpful.

Response 5 : we added a comment on page 4. All the samples are fresh sperm.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear authors

I read your systematic review “Sperm microbiota and its potentialimpact on male fertility: A  systematic review”, the topic is surely of interest however there are some issues:

• Title: please divide the words “potential” and “impact”.
• Introduction: I would make a distinction between alteration in seminal microbiota and seminal infection. In fact, the first can be only qualitative and not quantitative not causing bacteriospermia and seminal infection. For these reasons, I would focus more on the possible role of alteration in seminal microbiota in differential diagnosis of idiopathic infertility instead of in the contest of seminal infections.
• Materials and Methods: I would not report the sentence: “This systematic review aims to highlight the link between sperm microbiota and male fertility” because it is the purpose of the study not the way you tried to reach that purpose. Figure 1: It is reported “articles included in the meta-analysis” but a systematic review without meta-analysis is described in the paper. I would report in a clearer way (e.g.: using a table) the subdivision of the studies considering different populations analyzed (e.g.: idiopathic infertility or unexplained infertility). It is important considering that infertility is a clinical definition not based on seminal parameters. That analysis could be helpful to evaluate possible alteration in seminal microbiota that could be useful in clinical practice. Table 3: what is the meaning of pathogenic thresholds? Is it the negative outcome of ART? Was a control group analyzed in these studies? These data should be reported in the table or the caption. The term pathogenic is usually related to the concept of causality, if studies were not able to demonstrate causality, I would not use the term pathogenic. Figure 2: it shows damages deriving from bacterial infection and leucocitospermia, we can’t be sure that the same type of damage is carried out also in qualitative alteration of seminal microbiota, for that reason I would delate that image.

• Discussion: In Line 406 the word “could” is repeated. Among limitations I would highlight the absence of studies with main outcome natural fertility rates that are the first desire of patients going through fertility evaluations.

Best regards

Author Response

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below :

Comments 1 : Title: please divide the words “potential” and “impact”.

Response 1 : it is done

Comments 2 : I would make a distinction between alteration in seminal microbiota and seminal infection. In fact, the first can be only qualitative and not quantitative not causing bacteriospermia and seminal infection. For these reasons, I would focus more on the possible role of alteration in seminal microbiota in differential diagnosis of idiopathic infertility instead of in the contest of seminal infections.

Response 2 : we add : In the context of male infertility, it is crucial to distinguish between seminal infection—typically defined by elevated bacterial counts (bacteriospermia), leukocytospermia, and clinical signs of inflammation—and alterations of the seminal microbiota, which may occur in the absence of overt infection. Indeed, dysbiosis of the seminal microbiome can be primarily qualitative, involving shifts in microbial composition or loss of beneficial taxa, without necessarily exceeding phatogenic thresholds or triggering an inflammatory response. Such subtle microbial imbalances may contribute to impaired sperm function even in men with otherwise unexplained (idiopathic) infertility.

Comments 3 :  I would not report the sentence: “This systematic review aims to highlight the link between sperm microbiota and male fertility” because it is the purpose of the study not the way you tried to reach that purpose

Response 3 : the sentence has been deleted

Comments 4 : Figure 1: It is reported “articles included in the meta-analysis” but a systematic review witho

Response 4 : it has been changed

Comments 5 : Table 3: what is the meaning of pathogenic thresholds? Is it the negative outcome of ART? Was a control group analyzed in these studies? These data should be reported in the table or the caption. The term pathogenic is usually related to the concept of causality, if studies were not able to demonstrate causality, I would not use the term pathogenic. 

Response 5 : the title has been changed : Table 3.Pathogenicity thresholds (CFU/mL) for major semen bacteria in ART, based on Boitrelle et al., indicating levels at which antimicrobial treatment is clinically recommended to improve sperm quality and enhance the likelihood of achieving pregnancy through assisted reproductive technology

Comments 6 : Figure 2: it shows damages deriving from bacterial infection and leucocitospermia, we can’t be sure that the same type of damage is carried out also in qualitative alteration of seminal microbiota, for that reason I would delate that image

Response 6 : the figure has been deleted

Comments 7 : . Among limitations I would highlight the absence of studies with main outcome natural fertility rates that are the first desire of patients going through fertility evaluations.

Response 7 : we add the sentence : Finally, among the limitations of this review, it is important to highlight the lack of studies in which the primary outcome is the rate of natural fertility, which remains a key goal for most patients undergoing fertility evaluation.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The paper was only an initial summary of related information, and more work can be done in the future. Hope the authors continuously focused on the topic.

Comments on the Quality of English Language

language should be checked carefully.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors, thanks for the corrections and integrations done, in my opinion the paper is now ready for publication.

Best regards