Author response:
The following is the authors’ response to the original reviews.
Reviewer 1:
- The manuscript needs comprehensive proofreading for language and formatting. In many instances, spaces are missing or not required.
Thank you for your comments. The manuscript has been thoroughly proofread for errors in language and formatting.
- Could the authors explore correlation network analyses to get additional insights into the structure of different clusters?
We have added a co-occurrence analysis (at species taxonomic level) based on SparCC to the manuscript (Figure 2).
This is described on Page 9 line 141-148
- The GitHub link is not correct.
The github repository has now been made public.
- It is not possible to access the dataset on ENA.
We have changed the ENA study PRJEB57401 status to open.
- Add the graphs obtained with decontam analysis as a supplementary figure.
We have added the outputs of decontam (.csv files with feature lists of ASVs that were filtered based on the prevalence and frequency tests) to the github repository.
- There is nothing about the RPL group in the results section, while the authors discuss this issue in the introduction. What about the controls with proven fertility?
Thank you. We have amended the manuscript to compare characteristics between the RPL, unexplained subfertility and controls groups.
Line 1279-130 page 8:
“The study group represented 85% of samples with high sperm DNA fragmentation, 85% of samples with elevated ROS and 79% of samples with oligospermia. Rates of abnormal seminal parameters including low sperm concentration, reduced progressive motility and ROS concentrations were found to be highest in the MFI group (Supplementary Figure 1). Baseline characteristics between the RPL, unexplained subfertility and controls groups were similar.
Line 150-154 Page 9:
“Bacterial richness, diversity and load were similar between all patient groups examined in the study (Supplementary Figure 4).
- While correctly stated in the title, the term microbiota should be used throughout the manuscript instead of "microbiome"
Thank you. This misnomer has been amended throughout the manuscript.
Minor corrections:
Line 25: provoke is not a good term here.
Thank you. The term ‘provoke’ has been removed
Line 26: why does semen culture have a limited scope?
Thank you. Line 40-41 Page 3 has been amended:
“It is therefore plausible that asymptomatic seminal infections may be associated with impaired reproductive function in some men. Since semen culture has a limited scope for studying the seminal microbiota due to its inability to identify all present microbiota next generation sequencing (NGS) approaches have been reported recently by a growing number of investigators (13, 14, 15, 16, 17, 18, 19)”.
Line 68: write μl correctly
Thank you. This has been corrected
Line 131: several organisms at the genus level.
Thank you. This has been corrected
Line 136: what are the relative abundances of these genera? Is this relevant?
The mean relative abundances for the key taxa mention in each cluster are all above 20%. This information has been added to the manuscript text on page 9, line 153.
Line 173: Molina et al.
Thank you. This has been corrected
Line 173: the contaminations are referred to the low biomass nature of testicular samples. If present, bacteria of accessory gland secretions are an integral part of the seminal microbiota itself. Please review these sentences.
Thank you. This had been reworked to highlight the important of urethral contamination, which you later allude to as a limitation of our study is the failure to provide paired urine and semen samples.
Page 11 line 194-196
“Molina et al report that 50%-70% of detected bacterial reads may be environmental contaminants in a sample from extracted testicular spermatozoa (35); with the addition of passage along the urethra it is likely that contamination of ejaculated semen would be much higher.”
Table 1: remove results interpretation from table caption.
Thank you this has been acted upon.
Table 1: why in some cases, like in DNA fragmentation index, the total is not equal to n=223?
This is due to missing data/ analysis not possible for some men due to the requirement of a minimum number of sperm in the ejaculate to perform DNA fragmentation testing.
Table 1: "frag" is not defined.
Thank you, this has been amended
Tables 2, 3 & 4: bacterial genera in italics.
Thank you, this has been amended
Figure 1A: add the fertility status information above the cluster colors.
Thank you, this has been amended in Figure 1.
Figure 1C: the color code is confusing. Use different colors for each cluster.
Figure 1 legend: bacterial genera in italics.
Figures 1 & 2: the authors should use similar chart formatting in the two tables.
Thank you, this has been amended
Reviewer 2:
(1) The patient groups have different diagnoses and should be handled as different groups, and not fused into one 'patient' group in analyses.
Why are the data in tables presented as controls and cases? I would consider men from couples with recurrent pregnancy loss, unexplained infertility, and male factor infertility to have different seminal parameters (not to fuse them into one group). This means, that the statistical analyses should be performed considering each group separately, and not to fuse 3 different infertility diagnoses into one patient group.
We have conducted detailed analyses, requested by the reviewer, comparing seminal DNA, ROS and microbiota characteristics between each individual patient groups (Supplimental figures 1 and 4). No specific taxa (at either genera or species-level) were found to differ in relative abundance between the diagnostic groups. However, we expect associations between parameters such as reactive oxygen species, or DNA fragmentation, and relative abundance of bacterial species, to be general and not restricted to or specific to each diagnostic group. Therefore, we also conducted further analyses aggregating data from all patient groups to investigate relationships common to these different forms of male reproductive dysfunction.
(2) Were any covariables included in the statistical analyses, e.g. age, BMI, smoking, time of sexual abstinence, etc?
Covariates were not included in the statistical analyses. This has been added in the manuscript to the limitations.
Page 14 line 267-268
“Additionally, we did not have other covariables such as smoking status with which to include in further analyses”.
(3) Furthermore, it is known that 16S rRNA gene analysis does not provide sensitive enough detection of bacteria on the species level. How much do the authors trust their results on the species level?
The limitations of taxonomic assignment using 16S rRNA gene metataxonomics are well documented. However, the capacity to assign sequence amplicons at species level depends on the sequence variability of the 16S rRNA gene for each of the taxa reported and the specific gene region chosen. In this study, amplification of the V1-V2 region was performed using a mixed 28f primer set (see methods for details) that enables resolution and assignment of several bacterial species highly relevant to the reproductive tract including Lactobacillus spp., such as L. crispatus and L. iners, (e.g. https://doi.org/10.3389/fcell.2021.641921, https://doi.org/10.1128/msystems.01039-23, https://doi.org/10.1186/s12915-023-01702-2). In this study, we report the presence of L. iners, but not L. crispatus in semen samples, and we have also identified a specific association/co-occurrence between Gardnerella vaginalis and Lactobacillus iners, similar to that observed in vaginal bacterial communities.
(4) Were the analyses of bacterial genera and species abundances with seminal quality parameters controlled for diagnosis and other confounders?
As stated in point 2, no adjustment was made for co-variates. No differences in microbiome composition were observed among the three diagnostic groups, so no adjustments were made to our analysis.
(5) The authors stress that their study is the biggest on the microbiome in semen. However, when considering that the study consists of 4 groups (with n=46-63), it does not stand out from previous studies.
Our study is overall the largest investigating interactions between the seminal microbiome and male reproductive dysfunction. Other studies have included greater numbers of men with infertility.
(6) Weaknesses: There is a lack of paired seminal/urinal samples.
Thank you. This limitation has been added.
Page 14 line 266-267
“A further limitation of this study, and others, is the lack of reciprocal genital tract microbiota testing of the female partners, or paired seminal and urinary samples from male participants”.
Recommendation for authors to consider:
Including previous classical reviews in the introduction: DOI:10.1097/MOU.0000000000000742
DOI: 10.1038/s41585-019-0250-y
Thank you. This has been added.
Mentioning in the M&M section that there is a supplementary text with a more detailed M&M part.
Thank you. This has been added. Further methodological detail can be found in supplementary text.
Revising the use of 'microbiota' and 'microbiome', they are not synonyms. When talking of 16S rRNA gene analysis, we consider 'microbiome' analysis.
Thank you. This misnomer has been amended throughout the manuscript.
Revising the text, there are several erratas (e.g. verb missing, etc).
Thank you for your comments. The manuscript has been thoroughly proofread for errors in language and formatting.
