Characterisation and comparison of semen microbiota and sperm function in men with infertility, recurrent miscarriage, or proven fertility

Reviewer #1 (Public Review):

Summary:

The authors analyzed the bacterial colonization of human sperm using 16S rRNA profiling. Patterns of microbiota colonization were subsequently correlated with clinical data, such as spermiogram analysis, the presence of reactive oxygen species (ROS), and DNA fragmentation. The authors identified three main clusters dominated by Streptococcus, Prevotella, and Lactobacillus & Gardnerella, respectively, which aligns with previous observations. Specific associations were observed for certain bacterial genera, such as Flavobacterium and semen quality. Overall, it is a well-conducted study that further supports the importance of the seminal microbiota.

Strengths:

- The authors performed the analysis on 223 samples, which is the largest dataset in semen microbiota analysis so far.
- Inclusion of negative controls to control contaminations.
- Inclusion of a positive control group consisting of men with proven fertility.

Weaknesses:
- The manuscript needs comprehensive proofreading for language and formatting. In many instances, spaces are missing or not required.
- Could the authors explore correlation network analyses to get additional insights into the structure of different clusters?
- The GitHub link is not correct.
- It is not possible to access the dataset on ENA.
- Add the graphs obtained with decontam analysis as a supplementary figure.
- 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?
- While correctly stated in the title, the term microbiota should be used throughout the manuscript instead of "microbiome"

Reviewer #2 (Public Review):

Summary:

The study by Mowla et al analysed seminal microbiome together with semen quality parameters in fertile men and men from infertile couples with different infertility diagnoses. The study is of potential interest, with solid study design and methodology, nevertheless, the statistical analysis approach is not fully justified.

-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.

-Were any covariables included in the statistical analyses, e.g. age, BMI, smoking, time of sexual abstinence, etc?

-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?

-Were the analyses of bacterial genera and species abundances with seminal quality parameters controlled for diagnosis and other confounders?

Strengths:

The cohort of participants seems to be homogenous in the sense of ethnicity and location.

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.

Weaknesses:

There is a lack of paired seminal/urinal samples.