Table 2.
Characteristics of studies investigating semen/sperm microbiome in infertile men.
| Author, year, (country) | Design | Patients | Sample type | Technique | Main Phyla/Genera | Other findings |
Study quality |
|---|---|---|---|---|---|---|---|
| Veneruso et al., 2023 (30) (Italy) |
Cross-sectional | 13 men with abnormal SA | Semen | Sequencing V4 – V6 hypervariable region of 16S rRNA gene | Proteobacteria were the most abundant phylum; Lactobacillus, Escherichia, Shigella, and Serratia were the most abundant genera |
The genera Mannheimia, Escherichia_Shigella, and Varibaculum
were significantly increased in men with abnormal SA when compared to men with normal SA |
High |
| Yao et al., 2022 (31) (China) |
Cross-sectional | 13 men with asthenozoospermia, 22 men with leukocytospermia, and 32 men with asthenozoospermia and leukocytospermia; |
Semen | Sequencing V3 - V4 hypervariable region of 16S rRNA gene | Overall: Streptococcus, Lactobacillus, Burkholderia-Caballeronia-Paraburkholderia, Staphylococcus, and Gardnerella;
Lactobacillus-enriched group predominated in men with asthenozoospermia, whereas Streptococcus-enriched group predominated in men with leukocytospermia |
Diversity increased in men with leukocytospermia; Bacteroides were increased in men with leukocytospermia |
High |
| Bukharin et al., 2022 (32) (Russia) |
Cross-sectional | 42 infertile men with abnormal SA | Semen | Culture + Sequencing of 16S rRNA gene | Staphylococcus, Corynebacterium, Enterococcus, Streptococcus, and Escherichia | Fair | |
| Molina et al., 2021 (53) (Spain) |
Cross-sectional | 7 azoospermic men (13 samples), 3 men with high SDF (9 samples), and 1 man with severe OAT (2 samples) |
Testicular tissue | Sequencing V3 and V4 hypervariable regions of 16S rRNA gene | Blautia, Cellulosibacter, Clostridium XIVa, Clostridium XIVb, Clostridium XVIII, Collinsella, Prevotella, Prolixibacter, Robinsoniella, and Wandonia. | 50-70% contamination | High |
| Lundy et al., 2021 (33) (USA) |
Cross-sectional | 25 men with primary idiopathic infertility | Semen, Urine, and Rectal swab |
Sequencing V3 - V4 hypervariable region of 16S rRNA gene | Infertile group: Increased Aerococcus, Prevotella, Pseudomonas, and decreased Collinsella
Infertile group + varicocele: Bacteroids, Peptoniphilus |
Rectum of infertile men: decreased Anaerococcus and increased Lachnospiraceae, Collinsella, and Coprococcus;
Urine of infertile men: increased Anaerococcus; SAM cycle strongly over-represented in the urine and semen of infertile men |
High |
| Pagliuca et al., 2021 (16) (Italy) |
Cross-sectional | 37 men with abnormal SA | Semen | Culture positive if concentration > 10³cfu/m and PCR |
Staphylococcus coagulase negative, Haemophilus haemolyticus, Enterococcus faecalis, Haemophilus parainfluenzae, Gardnerella vaginalis | Bacteria were found more frequently in men with abnormal SA when compared to those with normal SA (70% vs 31%) | High |
| Okwelogu et al., 2021 (34) (Nigeria) |
Cohort | 36 male partners of infertile couples: 7 men with oligozoospermia, 7 men with azoospermia, 10 men with asthenozoospermia, and 1 man with teratozoospermia | Semen | Sequencing V4 hypervariable region of 16S rRNA gene | Oligozoospermia: Prevotella, Escherichia, Lactobacillus, Shuttleworthia, Serratia, Megasphaera, Gardnerella, Sneathia, Porphyromonas;
Azoospermia: Lactobacillus, Enterococcus, Corynebacterium, Veillonella, Gardnerella, Ureaplasma, and Prevotella |
Leukocytospermia: Increased Bacteroides and Prevotella;
Decreased Lactobacillus reuteri group, and Faecalibacterium |
High |
| Campisciano et al., 2020 (54) (Italy) |
Cohort | 47 male partners of infertile couples: 22 men with explained infertility, and 25 with unexplained infertility | Semen | Sequencing V3 hypervariable region of 16S rRNA gene | Overall: Prevotella
Explained Infertility group: Increased Prevotella (p. bivia and Staphylococcus); Unexplained Infertility group: Increased Lactobacillus gasseri |
Prevotella had a higher relative abundance in HPV-positive semen samples (25% vs. 17%) | High |
| Yang et al., 2020 (35) (China) |
Cross-sectional | 8 men with azoospermia, 58 men with asthenozoospermia, and 22 men with oligoasthenozoospermia |
Semen | Sequencing V1 and V2 hypervariable region of 16S rRNA gene | Men with asthenozoospermia had increased abundance of Sneathia, Ralstonia, Ureaplasma, Bacteroides, and Chryseobacterium Men with oligoasthenozoospermia had an increased abundance of Ralstonia, Oscillospira, Parabacteroides, Lachnospira, and Phascolarctobacterium |
Fair | |
| Baud et al., 2019 (36) (Switzerland) |
Cross-sectional | 68 men with abnormal SA | Semen | Sequencing V1 and V2 hypervariable regions of 16S rRNA gene | Prevotella genus was significantly enriched in the abnormal SA group | Three broad microbiota profiles identified: Prevotella-dominant, Lactobacillus-dominant, and Polymicrobial |
High |
| Ndiokwere et al., 2019 (55) (Nigeria) |
Cross-sectional | 22 semen samples from men undergoing fertility evaluation | Semen | Sequencing V4 hypervariable region of 16S rRNA gene | Serratia, Lactobacillus, Corynebacterium, Staphylococcus, and Prevotella | Species: Serratia marcescens Lactobacillus iners, Serratia entomophila, Haemophilus parainfluenzae, and Corynebacterium tuberculostearicum | Fair |
| Zeyad et al., 2018 (38) (Germany) |
Cross-sectional | 29 men with bacteriospermia | Semen | Culture: positive if concentration > 10³cfu/ml |
S. aureus (9%), E. coli (7%), S. epidermidis (6%), S. haemolyticus (5%), E. faecalis (5%), and S. agalactiae (2%) |
Bacteriospermia 34.5% of samples; Bacteriospermia associated with reduced sperm concentration and motility; Bacteriospermia not associated with increased DFI; Bacteriospermia associated with decreased fertilization |
High |
| Ricci et al., 2018 (52) (Italy) | Cross-sectional | 285 male partners of infertile couples | Semen | Culture positive if concentration > 10³cfu/m | Bacteriospermia in 29.1% of specimens; Staphylococcus aureus (0.7%), Enterococcus fecalis (11.6%), Streptococcus agalactiae (4.6%), Escherichia coli (6.7%), Streptococcus anginosus (0.3%), S. haemolyticus (2%), and U. urealyiticum (2%) |
Bacteriospermia associated with a decrease in total motility and progressive motility; Enterococcus fecalis associated with reduced sperm motility and morphology |
High |
| Chen et al., 2018 (39) (China) |
Cross | 6 men with OA; 6 men with iNOA |
Semen | Sequencing V4 hypervariable regions of 16S rRNA gene | Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria were the predominant phyla Lactobacillus, Prevotella, Proteus, Pseudomonas, and Veillonella were the dominant genera |
Solibacillus, Campylobacter, Campyiobacteraceae and Plesiomonas were reduced in the OA group; Sneathia and Lysobacter were reduced in iNOA group |
High |
| Alfano et al., 2018 (37) (Italy) |
Cross-sectional | 10 men with iNOA: 5 with positive sperm retrieval, and 5 with negative sperm retrieval |
Testicular tissue | Sequencing V3 to V5 hypervariable regions of 16S rRNA gene |
Actinobacteria
and Firmicutes |
Increased number of bacteria in the testis of iNOA men; Positive sperm retrievals: Actinobacteria and Firmicutes Negative sperm retrievals: Actinobacteria |
High |
| Zeyad et al., 2017 (56) (Germany) |
Cross-sectional | 36 men with bacteriospermia | Semen | Culture: positive if concentration > 10³cfu/ml |
Staphylococcus sp (15%; aureus, epidermidis, haemolyticus, xylosus); Escherichia coli (5%); Streptococcus spp (6%: agalactie, pneumoniae); Enterococcus faecalis (4%), and Klebsiella pneumoniae (1.6%) |
Bacteriospermia associated with reduced sperm concentration and motility; Neither morphology nor DFI was significantly impacted by bacteriospermia |
High |
| Monteiro et al., 2017 (40) (Portugal) |
Cross-sectional | 27 men with AT, 35 men with OAT, And 27 men with hyperviscosity |
Semen (pooled by subgroups) |
Sequencing V3 to V6 hypervariable regions of 16S rRNA gene | Overall: Enterococcus, Staphylococcus, Anaerococcus, Corynebacterium, Peptoniphilus, and Propionibacterium; OAT and Hyperviscosity groups: Cyanobacteria and Fusobacteria |
Lower prevalence of Lactobacillus and Propionibacterium; Higher prevalence of Pseudomonas, Klebsiella, Aerococcus, Actinobaculum, and Neisseria in OAT and hyperviscosity groups |
High |
| Vilvanathan et al., 2016 (41) (India) |
Cross-sectional | 37 men with oligozoospermia and 1 individual with azoospermia |
Semen | Culture: positive if concentration > 10³cfu/ml | Bacteriospermia in 35% of specimens; Overall: E. faecalis (30%), Coagulase-negative Staphylococcus (23.3%), Staphylococcus aureus (20%), E. coli (10%), Klebsiella pneumoniae (6.6%), Proteus sp (6.6%), and Citrobacter sp (3.3%) |
Presence of asymptomatic bacteriospermia not associated with abnormal semen parameters; Altered semen quality among different bacterial species lacked significant associations |
Fair |
| Mashaly et al., 2016 (57) (Egypt) | Cross-sectional | 60 infertile men: 30 without leukocytospermia (G1), and 30 with leukocytospermia (G2) | Semen | Culture: positive if concentration > 10.000 cfu/ml | G1: Corynebacterium (26.7%), Corynebacterium + E. coli (3.3%), Staphylococcus aureus (13.3%), Haemolytic streptococci + E.coli (3.3%); G2: Corynebacterium (10%), Corynebacterium + E.coli (0%), Staphylococcus aureus (10%), Haemolytic streptococci + E.coli (0%) |
Bacteriospermia in 33% of specimens; 20% Corynebacteria;
Sperm motility considerably lower in positive culture with Corynebacteria; Nonsignificant difference in sperm concentration and morphology between patients with Corynebacteria positive or negative cultures |
High |
| Ruggeri et al., 2016 (58) (Italy) | Cross-sectional | 246 male partners of infertile couples: 212 negative semen culture; 15 positive semen culture; 19 mixed flora | Semen | Not specified |
Enterococcus faecalis most common in both men (2.8%) and women (3.6%); Escherichia coli: men (0.8%) vs. women (3.2%); Ureaplasma urealyticum: 3.2% (men) |
High | |
| Fraczek et al., 2016 (42) (Poland) |
Cross-sectional | 30 normozoospermic men with isolated bacteriospermia; 22 normozoospermic with bacteriospermia and leukocytospermia; 19 normozoospermic with isolated leukocytospermia; |
Semen | Culture: positive if concentration > 10⁴cfu/ml | Coagulase-negative: Staphylococcus (22.9%), Streptococcus spp (12.3%), Enterococcus spp (13.8%), Mycoplasma spp (4.6%), Gram+ aerobic (16.5%), Corynebacterium glucuronolyticum-seminale, C. striatum, and C. propinquum; Gram negative aerobic (3.7%): Escherichia coli, and Proteus mirabilis); Gram+ anaeroibic (6.4%): Propionibacterium acnes, P. propionicum, P. avidum, Bifidobacterium sp.) Gram negative anaerobic (13.8%): Bacteroides ureolyticum, Prevotella melaninogenica, P. intermedia, and Fusobacterium varium One sample: Candida albicans |
Reduced sperm concentration in all groups compared to the control group; Significant sdeterioration of motility in the isolated leucocytospermia group; Necrozoospermia significantly higher in the combined bacteriospermia + leucocytospermia group; Teratozoospermia significantly higher in the isolated bacteriospermia group |
Fair |
| Mändar et al., 2015 (59) (Estonia) |
Cross-sectional | 23 infertile men | Semen | Sequencing V6 hypervariable region of 16S rRNA gene |
Lactobacillus, Flavobacterium, Prevotela, Porphyromonas, and Gardnerella;
The mean proportion of proteobacteria was higher in leukocytospermic men |
After intercourse, the seminal microbiome shifted the vaginal microbiome | High |
| Weng et al., 2014 (27) (China) |
Cross-sectional | 10 men with abnormal semen volume, 13 men with oligozoospermia, 12 men with asthenozoospermia, 44 men with teratozoospermia, 10 men with antisperm antibodies, And 18 men with; leukocytospermia |
Semen | Sequencing V4 hypervariable region of 16S rRNA gene | Abnormal SA group: Lactobacillus, Prevotella, Pseudomonas, Haemophilus, Finegoldia, Rhodanobacter, Corynebacterium and Streptococcus | High | |
| Sellami et al., 2014 (24) (Tunisia) | Cross-sectional | 85 infertile men | Semen | Culture positive if concentration > 10⁴cfu/m, and PCR | Bacteriospermia in 7% of specimens; Culture: Group B Streptococcus (3.5%), Enterococcus spp (1.1%), Staphylococcus aureus (1.1%), and Corynebacterium spp (1.1%); PCR: C. trachomatis (15.2%), N gonorrhea (5.8%), U. urealyticum (5.8%), M. genitalium (5.8%), U. parvum (5.8%), and M. hominis (5.8%) |
C. trachomatis associated with decreased sperm quality and increased apoptosis | High |
| Hou et al., 2013 (43) (China) |
Cross-sectional | 10 men with asthenozoospermia, 23 men with oligoasthenozoospermia, and 25 with oligozoospermia or azoospermia |
Semen | Sequencing V1 and V2 hypervariable regions of 16S rRNA gene) | Overall: Ralstonia, Lactobacillus, Corynebacterium, Streptococcus, Staphylococcus, Prevotella, Finegoldia, and Anaerococcus; No differences among the groups |
Anaerococcus had a negative association with sperm quality | Fair |
| Aghazarian et al., 2013 (50) (Iran) |
Cross-sectional | 171 men undergoing infertility evaluation | Semen | Not specified | Bacteriospermia in 36.2% of specimens; Ureaplasma urealyticum + Gardnerella vaginalis (25.8%), Ureaplasma urealyticum (19.4%), G. vaginalis (16.1%), Enterococcus faecalis (9.7%), E. coli + E. faecalis (1.6%) |
No significant association between bacteriospermia and leukocytospermia; No significant differences in semen parameters in men with bacteriospermia |
High |
| Domes et al., 2012 (51) (Canada) |
Retrospective cohort | 4935 samples from infertile men | Semen | Culture positive if concentration > 10³cfu/m |
Bacteriospermia in 15% of specimens; Staphylococcus aureus (5%), Enterococcus fecalis (56%), Escherichia coli (16%), Group B streptococcus (13%), Klebsiella pneumoniae (2.2%), Proteus mirabilis (1.7%), Citrobacter koseri (1.5%), and Morganella morganii (1.3%) |
Bacteriospermia associated with an increase in DFI; Elevated seminal leukocytes dominant factor associated with deterioration in semen parameters |
High |
| Isaiah et al., 2011 (60) (Nigeria) | Cross-sectional | 140 infertile men | Semen | Culture | Bacteriospermia in 65.7% of specimens; Staphylococcus aureus (28.3%), Staphylococcus saprohyticus (13%), Pseudomonas aerouginosa (6.5%), Escherichia coli (19.6%), Proteus mirabilis (10.8%), Staphylococcus spp (10.8%), and Proteus vulgaris (10.8%) |
Staphylococcus saprohyticus and Escherichia coli associated with altered sperm motility and morphology; Significant (p<0.001) relationship between bacteriospermia, leukocytes, and total sperm count |
High |
| Moretti et al., 2009 (61) (Italy) | Cross-sectional | 236 men with bacteriospermia | Semen | Culture: positive if concentration > 10⁴cfu/ml if gram + and > 10⁵cfu/ml if gram |
E. faecalis Bacteriospermia in 33.2% of specimens; (32.1%), E.coli (20.3%), Streptococcus agalactiae (13.4%), U. urealyiticum (11.8%), Staphylococcus epidermidis (9.7%), Streptococcus anginosus (9.3%), and Morganella morganii (3.2%) |
Sperm concentration lower than in controls; progressive motility lower than controls except for samples positive for S. agalactiae and S. anginosus | High |
| Gdoura et al., 2008 (62) (Tunisia) |
Cross-sectional | 166 men undergoing infertility evaluation | Semen | Culture and PCR | Overall: Chlamydia trachomatis (41.4%), Ureaplasma urealyticum (15.5%), and Mycoplasma hominis (10.3%) Culture: E. coli (1.7%), Streptococcus agalactiae (0.9%), Citrobacter diversus (0.9%), Enterococcus faecalis (0.9%), and Gardnerella vaginalis (0.9%) |
Bacteriospermia 56.9%; bacteria in 56% of semen samples by PCR; Bacteria in 5.2% semen samples by culture |
High |
| Virecoulon et al., 2005 (45) (France) |
Cross-sectional | 534 male partners of infertile couples | Semen | Culture: positive if concentration > 10³cfu/ml | Gardnerella vaginalis (26.1%), coagulase-negative staphylococci (15.7%), Streptococcus anginosus (14.2%), Ureaplasma urealyticum (15.5%), Enterobacteriaceae (E. coli, Proteus mirabilis), Corynebacterium spp, and Lactobacillus spp | Sterile in 28.8%; polymicrobial flora in 49.3%; No relationship between the bacterial flora and leukocytospermia; Low titers of U. urealyticum in semen were not associated with a disturbance of the ecosystem |
High |
| Levy et al., 1999 (24) (France) | Cross-sectional | 92 male partners of infertile couples | Semen | Culture positive if concentration > 10⁴cfu/m, and PCR | Culture: Ureaplama urealyticum (13%) PCR: Chlamydia trachomatis (11%) |
No relation between the presence of microorganisms in semen and serum antibodies | High |
| Debata et al., 1999 (63) (India) | Cross-sectional | 197 infertile men | Semen | Culture | Ureaplasma. urealyticum (43%), Mycoplasma hominis (17%) | No association between Ureaplasma and sperm count; Bacteriospermia associated with altered sperm morphology |
High |
| Kjaergaard et al., 1997 (44) (Denmark) |
Cross-sectional | 60 men with mild/moderate oligozoospermia and 26 men with severe oligozoospermia | Semen | Culture positive if concentration > 10³cfu/m, and PCR | Mild/moderate oligozoospermia: Commensals, Ureaplasm. Urealyticum, Gardnerella vaginalis, Enterococcus faecalis, Enterobacteriaceae, and Mycoplasma;
Severe oligozoospermia: Commensals, Ureaplasm. Urealyticum, Enterococcus faecalis, Gardnerella vaginalis, Enterobacteriaceae, and Mycoplasma |
No association between semen quality and microorganisms | High |
| Bussen et al., 1997 (49) (Italy) | Cross-sectional | 88 male partners of infertile couples Group 1: 28 negative culture + 14 positive culture for microorganisms that colonize skin (considered control group); Group 2: 46 positive cultures |
Semen | >100 colonies per plate | Bacteriospermia in 68% of specimens; S. epidermidis (33%): considered to be commensal S. aureus (9%); E. coli (8%); Enterobacter spp. (7%); Group B streptococcus (8%); Corynebacteria (8%) |
No differences in sperm concentration, count, sperm morphology, and fertilization rates between groups | High |
| Shalika et al., 1996 (64) (USA) | Cross-seccional | 342 male partners of infertile couples | Semen | Culture | Bacteriospermia in 32% of specimens; Culture: S. aureus (3%), Enterococcus spp (23%), Ureaplasma spp (11%), E. coli (3%), Proteus mirabillis (0.5%), and Streptococcus spp (2%) |
Enterococcus spp did not adversely affect IVF pregnancy rate; E. coli, S aureus, and Ureaplasma urealyticum potentially affecting IVF pregnancy rates |
High |
| Eggert-Kruse et al., 1995 (48) (Germany) |
Cross-sectional | 126 male partners of infertile couples | Semen | Culture: positive if concentration > 106cfu/ml |
Peptococcus sp (38.1%), Peptostreptococcus sp (32.5%), Veillonella spp (27.8%), Lactobacillus spp (20.6%), Bacterioides spp (7.9%: B. disiens, B.capillosus, B. ruminicola, B. bivius), Propionibacterium spp (7.1%), Fusobacterium spp (3.2%: F. varium, F. mortiferum, F. nucleatum), Gardnerella vaginalis (3.1%), and Actinomyces spp (1.6%: A. meyeri, A. viscosus); Gram-negative non-identified anaerobic rods (5.6%); Anaerobic bacteria not identified (11.9%) Mycoplasma hominis (6.1%); Ureaplasma urealyticum (21.2%) |
99% of samples colonized with anaerobic; 71% potentially pathogenic species; Potentially pathogenic aerobic microorganisms more frequent in oligozoospermia group; Bacteroides spp and Fusobacterium spp more frequent in the asthenozoospermia and teratozoospermia groups (not statistically significant) |
Fair |
AT, asthenoteratozoospermia; OA, Obstructive Azoospermia; OAT, oligoasthenoteratozoospermia; H, hyperviscosity; iNOA, idiopathic non-obstructive azoospermia; SA, semen analysis; SDF, Sperm DNA Fragmentation; DFI, sperm DNA fragmentation index; PCR, polymerase chain reaction; cfu, colony forming units; HPV, human papillomavirus; SAM, S-adenosyl-L-methionine.