The semen microbiome and its impact on sperm function and male fertility: A systematic review and meta‐analysis

Male factor is attributable in up to 50% of cases of infertility. In vitro studies demonstrate that bacteria can negatively impact sperm function. The use of next‐generation sequencing techniques has provided a better understanding of the human microbiome, and dysbiosis has been reported to impact health. Evidence regarding the impact of the semen microbiome on sperm function and fertility remains conflicting.


| INTRODUC TI ON
Infertility affects 8%-12% of couples worldwide 1 and is defined as the failure to conceive after 12 months of regular unprotected intercourse. 2. Male factor infertility is attributable in up to 50% of cases, 3 and potential causes include urogenital tract infections (eg prostatitis, epididymitis). 4 In vitro studies have highlighted the mechanisms through which bacteria affect sperm function, including agglutination of motile sperm, induction of apoptosis, production of immobilization factors and impairment of the acrosome reaction. [5][6][7][8][9] However, the evidence for using empiric antibiotics in the clinical setting is controversial, 10 as there are conflicting data as to whether such pathogens cause abnormalities in semen parameters in vivo and whether treatment leads to an improvement in semen parameters and reproductive potential. Leucocytospermia has been posited as the pathogenesis for male factor infertility, and has been associated with elevated reactive oxygen species (ROS), 11 which are associated with DNA damage of the spermatozoa. 12 Sperm DNA damage is associated with adverse reproductive outcomes. [13][14][15] However, there are also conflicting data on the significance of leucocytospermia, with some studies reporting an association between bacteriospermia and leucocytospermia, 16,17 whilst others have found no such association. [18][19][20] The human microbiome is composed of the genetic material of the microbial community (eg bacteria, fungi and viruses) and is more complex than the human genome. The advent of next-generation sequencing (NGS), which uses the 16s ribosomal RNA region of the bacterial genome to identify bacteria, 21,22 has enabled more accurate characterization of the human microbiome, and large-scale microbial genome sequences can now be analysed.
Previously undetectable pathogens have been discovered using this novel technique. 23 The human microbiome project 24 has characterized the microbiome of the airway, skin, oral cavity, gut and vagina. Metagenomic research has increased our understanding of the microbiome and how dysbiosis plays a role in conditions, such as mental health disorders and cancers. [25][26][27] Research on the vaginal microbiome has identified over 100 bacterial species, and its impact on pregnancy, premature birth, infertility and gynaecological cancer has been studied. [28][29][30][31][32] Further evidence suggests that male and female interactions may influence the composition of the microbiome, 33 and further research is needed to understand how this may impact on reproductive health and pregnancy, and whether novel therapies targeting the seminal microbiome improve outcomes. 34 The objectives of this systematic review and meta-analysis were as follows: 1. To describe the species and communities present in semen 2. Assess the prevalence of bacteriospermia and the association with male infertility 3. Assess the association between bacterial species and semen quality 4. Provide a contemporary understanding of the seminal microbiome and its potential effects on reproductive health.

| E VIDEN CE ACQUIS ITION
A systematic search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement 35 (Table 1). The systematic review was registered with PROSPERO (ID Number CRD42019124483).
The databases MEDLINE, OVID and PubMed were searched to identify studies related to the identification of bacteria in the semen of infertile men, between 1 January 1992 and 1 September 2019. This time frame was chosen to facilitate the identification of studies using the 3rd to 5th editions of the World Health Organization (WHO) laboratory manual for the examination and processing of human semen. 36 The following terms were used in the search: "sperm OR semen OR seminal," AND "microbiome OR microbiota OR bacteria OR microorganisms," AND "fertility OR fertile OR infertility OR infertile" AND "man OR men OR male OR Objectives 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS).

Protocol and registration
5 Indicate if a review protocol exists, if and where it can be accessed (eg, Web address), and, if available, provide registration information including registration number.

5
Eligibility criteria 6 Specify study characteristics (eg, PICOS, length of follow-up) and report characteristics (eg, years considered, language, publication status) used as criteria for eligibility, giving rationale.

5
Information sources 7 Describe all information sources (eg, databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched.

5
Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.

5
Study selection 9 State the process for selecting studies (ie, screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis).

5-6
Data collection process 10 Describe method of data extraction from reports (eg, piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.

5-6
Data items 11 List and define all variables for which data were sought (eg, PICOS, funding sources) and any assumptions and simplifications made.

5-6
Risk of bias in individual studies

12
Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.

6
Summary measures 13 State the principal summary measures (eg, risk ratio, difference in means). 6

Synthesis of results 14
Describe the methods of handling data and combining results of studies, if done, including measures of consistency (eg, I 2 ) for each meta-analysis.

6
Risk of bias across studies

15
Specify any assessment of risk of bias that may affect the cumulative evidence (eg, publication bias, selective reporting within studies).

Additional analyses 16
Describe methods of additional analyses (eg, sensitivity or subgroup analyses, metaregression), if done, indicating which were pre-specified.

Study selection 17
Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. Study characteristics 18 For each study, present characteristics for which data were extracted (eg, study size, PICOS, follow-up period) and provide the citations. Tables 2-4 Risk of bias within studies

19
Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12).

| E VIDEN CE SYNTHE S IS
A total of 55 studies fulfilled the criteria for inclusion to this systematic review, with a total of 51 299 subjects. 24 studies were included in the meta-analysis, with a total of 29 358 subjects. The PRISMA flow chart describes the cases excluded from this review ( Figure 1). All the studies were observational, assessing the prevalence and impact of bacteriospermia in infertile men. Thirty-nine cross-sectional studies and 16 case-control studies were included (Tables 2-4). Risk of bias for each study was ascertained (Tables 5   and 6).

Section/topic # Checklist item
Reported on page #

Synthesis of results 21
Present results of each meta-analysis done, including confidence intervals and measures of consistency.

Figures 4-24
Risk of bias across studies

22
Present results of any assessment of risk of bias across studies (see Item 15).  This form of transformation is preferred for meta-analysis as it stabilizes the variance of the estimates, and studies with zero or one effect size can be included. 38,39 Heterogeneity within and between subgroups was assessed with the I 2 statistic. 40 Significance was set at the .05 level.

| Types of bacteria
Four studies used NGS to identify and quantify the bacterial species present in the semen. Two of the studies used the V1-V2 region of the gene, 41,42 one used V4, 43 and the fourth used the V3-V6 region. 17 The genera identified consisted of aerobic, facultative anaerobic and strictly anaerobic bacteria, and Significantly higher prevalence of UU in IF (P = .02) No difference in prevalence of MH (P = .32) UU associated with a significant reduction in total (P = .015) and progressive motility (P = .03) MH associated with a significant reduction in concentration (P = .01), total motility (0.001) and progressive motility (0.001)  and Mycoplasma genitalium (2.5%-28.8%).

| Fertility
All four NGS studies reported a seminal microbiome that was rich and diverse in both infertile and fertile men. One case-control study 42  higher in infertile men, 78 whereas another found it was significantly higher in fertile men (P = .02). 68 A meta-analysis conducted on these four studies found that there was no significant difference in the prevalence of this species between fertile and infertile men (OR: 1.34, 95% CI: 0.51-3.5, I 2 : 91.73%; Figure 6).

| Impact on semen parameters
All four NGS studies found an association between certain bacterial species and semen parameters. Lactobacillus was associated with improvements in sperm quality in two studies, and one study reported an abundance of Lactobacillus in samples with normal morphology. 42 Two studies reported that Prevotella appeared to exert a negative effect on sperm quality, as there was an increased abundance of this genus in specimens that had abnormal parameters. One study reported that Anaerococcus was negatively associated with semen quality (P = .0012). 42 Monteiro et al reported that oligoasthenospermia and hyperviscosity phenotypes were associated with a higher prevalence of Pseudomonas and Klebsiella.
Ten culture-based studies reported on the impact of bacteriospermia on semen parameters. 16,18,20,[44][45][46][47]56,58,59 Six studies reported on the impact of bacteriospermia on mean sperm concentration, and five of these reported a significant negative impact on concentration. A meta-analysis was conducted on these six studies  Figure 10).
Four studies reported on the impact of bacteriospermia on DNA fragmentation. Two studies reported a significant increase in the DFI, 18,58 whilst two others reported an increase in DFI that was not statistically significant. 16,46 Two of these studies assessed DNA fragmentation using the same technique (TUNEL assay) and reported results enabling a pooled analysis, which noted a significantly higher DNA fragmentation index in the presence of bacteriospermia (difference in means: 3.518, 95% CI: 0.907 to 6.129, P = .008, I 2 : 0.0%; Figure 11). Two studies found that there was a significant impact on protamine deficiency, reporting increased chromomycin levels (CMA3) in the presence of bacteria. 16 70.42%; Figure 12).  Figure 15). The impact of other bacterial species was also investigated in a number of other studies. Two studies found that there was a significant decrease in total motility in the presence of Enterococcus faecalis 47,56 with a pooled analysis concurring with these findings (difference in means: −11.034, 95% CI: −17.845 to −4.223, P = .001, I 2 : 99%; Figure 24). One study found a significant decrease in motility with Corynebacteria spp. 44 The majority of studies that reported on it found that there were no differences in semen parameters with S aureus, or E coli. 45

| S TRENG TH S AND LIMITATI ON S
We conducted a thorough systematic review and meta-analysis of the literature, using standardized tools for the assessment of study methodology.
When assessing the risk of bias, the majority of studies included were of fair quality; however, some were of poor quality.
In view of the low quality of the studies, it was impossible to control for confounding variables, particularly between the fertile and infertile populations. The majority of the included studies clearly stated the research objectives, and whilst patient selection was generally acceptable, a few studies did not clearly report the inclusion criteria. Additionally, it could not be determined for the majority of studies whether the outcome assessors were blinded to the exposure status. There was a great deal of methodological variability between studies (eg different hypervariable regions (NGS), different culture media), which may explain the differences in the reported prevalence of organisms. As the aim of this systematic review was to investigate the association of bacterial species in semen with fertility and semen parameters, studies that used other specimens for investigation (eg urethral swabs) were not included, and therefore, a possible limitation is that not all species in the male genital tract were identified.
A meta-analysis was conducted on various outcome data, and it should be noted that within these groups there was significant heterogeneity, and therefore, the results should be interpreted with caution.
There are currently no published core outcome sets for male fertility, which limits meaningful comparison of the data. 90 There are limited published data using NGS to characterize the seminal microbiome and its impact on fertility. Culture and PCR have their place in the clinical setting, but their inherent weakness is the inability to identify all bacterial species, leading to inaccuracies in the reported outcomes. Was exposure assessed more than once over time?    injury. There is little in vivo evidence to explain the mechanisms by which bacteria cause male infertility, with some studies implicating leucocytospermia, and others suggesting that bacteria act independently of leucocytes. The proposed mechanisms include lowering the mitochondrial membrane potential causing apoptosis, and increased protamine deficiency. A previously published meta-analysis concluded that protamine deficiency was significantly associated with sperm DNA damage, 93 and this study found that protamine deficiency and DNA fragmentation were increased in the presence of bacteriospermia.

| D ISCUSS I ON
Studies analysing the microbiome of other body sites suggest that there is a fine balance of the community, with dysbiosis leading to domination by opportunistic or occasional pathogens, causing infections and inflammatory responses, for example E coli and inflammatory bowel disease. 94 This would suggest that the microbiome is necessary for the normal functioning of the semen and sperm, rather than having a strictly deleterious effect, similar to the vaginal microbiome, which plays a role in host defence. 95 Two studies in this review found that only certain strains or clonal variants This study has not found any differences in the presence of CT,

S aureus or E coli.
Future research should therefore also focus on non-pathogenic organisms that may have a protective role and how these can be developed as therapeutic options (eg probiotics), and well-designed randomized-controlled studies should be conducted to assess the impact of these interventions.
Currently, for clinicians who are managing couples with male infertility, it would be prudent to investigate leucocytospermia and bacteriospermia and treat seminal infections according to speciality guidelines. Additionally, couples should be informed that given the limited evidence available, the impact of the semen microbiome on fertility is inconclusive, and further studies are required.

CO N FLI C T O F I NTE R E S T
None declared.