The microbiome and male infertility: looking into the past to move forward

Figures & data

Table 1. Summary of studies explored seminal fluid and testicular microbiome profiles utilising Next-generation sequencing of the 16S rRNA.

Author	Ethnicity	Patients grouping	Intervention	Control	Detection method	Detected bacteria	Main outcomes
Hou et al. (2013)	Chinese	Semen parameters (WHO 4th edition)	58 infertility patients: 10 AS 23 OS 25 AZ or sever OS	19 donors with normal semen parameters	QIAamp DNA Mini Kit V1-V2 16S rRNA Roche 454 GS-FLX	Dominant Phyla: NA Common genera: Streptococcus, Corynebacterium Finegoldia, Veillonella, Lactobacillus, Prevotella, Staphylococcus, Anaerococcus, Peptoniphilus, Ralstonia, Incertae_Sedis, Porphyromonas, Clostridiales.	No significant difference in semen microbiome between donors and infertility patients. Identified six bacteria clusters. Anaerococcus was linked with poor semen quality.
Weng et al. (2014)	Taiwanese	Semen parameters (WHO 5th edition)	60 infertility patients with abnormal semen parameters	36 infertility patients with normal semen parameters	QIAamp DNA Blood Mini Kit V4 16S rRNA Illumina Miseq	Dominant Phyla: NA Common genera:Lactobacillus, Pseudomonas, Prevotella, Gardnerella, Rhodanobacter, Streptococcus, Finegoldia, Haemophilus.	Identified three bacteria clusters (Pseudomonas, Lactobacillus, and Prevotella). Lactobacillus was associated with improved semen quality. Prevotella showed a negative influence on semen quality.
Monteiro et al. (2018)	Portuguese	Semen parameters (WHO 4th & 5th editions)	89 infertility patients: 27 AT 35 OAT 27 Hyperviscosity (group samples)	29 infertility patients with normal semen parameters (group samples)	QIAamp DNA Mini Kit V3-V6 16S rRNA Ion PGM	Dominant Phyla: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, Common genera: Enterococcus, Staphylococcus, Anaerococcus, Peptoniphilus, Aggregatibacter, Haemophilus, Klebsiella, Morganella, Actinomycetaceae, Corynebacterium, Propionibacterium, Flavobacteriaceae.	Lactobacillus was at low abundance in all groups, and at a significantly lower level in hyperviscosity group compared to the control. Increased abundance of Pseudomonas, Klebsiella, Neisseria, Aerococcus, and Actinobaculum negatively influence sperm count, motility, and viscosity.
Chen et al. (2018)	Chinese	Semen parameters (WHO 4th edition)	12 AZ men: 6 iOA 6 iNOA	5 donors with normal semen parameters	TIANamp Genomic DNA Kit V4 16S rRNA Illumina HiSeq	Dominant Phyla: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria. Common genera: Lactobacillus, Prevotella, Proteus, Pseudomonas, Veillonella, Corynebacterium, Rhodococcus, Staphylococcus, Bacillus.	Men with azoospermia had lower bacterial diversity and higher abundance of Firmicutes and Bacteroidetes phyla compared to the control. Disputed semen microbiome might increase the risk of metabolic, infectious, and immune disorders.
Alfano et al. (2018)	European–Caucasian	SSR results	10 AZ men 5 iNOA + ve SSR 5 iNOA − ve SSR	5 normozoospermic men	QIAamp DNA Kit V3-V5 16S rRNA Roche 454 GS-J	Dominant Phyla: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes. Common class: Bacilli, Alphaproteobacterial, Actinobacteria, clostridia, Gamaproteobacterial, Betaproteobacterial, Bacteroidi, Saprospirae.	Human testicular tissues are not microbe-free. iNOA men had higher bacterial DNA in their testes compared to normozoospermic men. iNOA lack of Bacteroidetes and Proteobacteria iNOA individuals with failed sperm retrieval had decreased amount of Clostridia.
Amato et al. (2020)	Caucasian	IUI results	23 Idiopathic infertile couples	16 Retrospective normozoospermic men	QIAamp BiOstic Bacteraemia DNA kit V1–V2 16S rRNA Illumina MiSeq	Dominant Phyla: NA Common families: Tissierellaceae, Lactobacillaceae, Streptococcaceae, Prevotellaceae, Corynebacteriaceae.	No differences in semen microbiome composition and diversity between IUI failure and IUI success groups.
Baud et al. (2019)	Estonian	Semen parameters (WHO 5th edition)	68 infertility patients with abnormal semen parameters	26 infertility patients with normal semen parameters	QIAamp DNA Kit V1–V2 16S rRNA Illumina MiSeq	Dominant Phyla: Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria Common genera: Corynebacterium, Burkholderia, Finegoldia, Haemophilus, Lactobacillus, Prevotella Planococcaceae, Streptococcus, Staphylococcus.	Identified three bacteria clusters (Prevotella, Lactobacillus, and polymicrobial). Prevotella was linked with decreased sperm motility and increased bacterial load. Staphylococcus and Lactobacillus were positively associated with sperm motility and morphology, respectively.
Štšepetova et al. (2020)	Estonian	NA	50 IVF patients	NA	QIAamp DNA mini kit V2–V3 16S rRNA Roche 454 FLX NGS	Dominant Phyla: Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria. Common genera: Lactobacillus, Staphylococcus, Prevotella, Incertae_Sedis_X, Phyllobacterium, Corynebacterium, Peptoniphilus, Micrococcu.	Contamination of IVF culture medium was high (8%). Semen processing minimised the prevalence of contamination in semen but did not eliminate them. Staphylococcus spp and Alphaproteobacteria was linked with poor sperm parameters and embryo quality of IVF embryos.
Yang et al. (2020)	Chinese	Semen parameters (WHO 5th edition)	101 infertility patients: 22 OA 58 AS 8 AZ 13 OS	58 infertile patients with normal semen parameters	CTAB DNA Kit V1–V2 16S rRNA Illumina HiSeq	Dominant Phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Fusobacteria. Common genera: Lactobacillus, Corynebacterium, Acinetobacter, Prevotella, Enterococcus, Veillonella, Streptococcus, Porphyromonas, Sneathia, Pelomonas.	Lactobacillus presented at a high abundance in all semen, with a notably greater abundance in AS and OA compared to the control. Anaerococcus was linked with poor sperm motility. Proposed 4 bacterial biomarkers associated with AS and 6 bacterial biomarkers linked with OA. lipid metabolism was significantly different between the intervention groups.
Lundy et al. (2021)	Americans of different races	Fertility status	25 men with idiopathic primary infertility.	12 health men with proven paternity	QIAamp Microbiome Kit + Qiagen PowerFecal pro kit V3–V4 16S rRNA Illumina MiSeq	Dominant phyla: Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria Common genera: Gardnerella, Collinsella, Bacteroides, Veillonella, Enterococcus, Staphylococcus, Peptoniphilus, Pseudomonas, Aerococcus, Corynebacterium, and Pseudoxanthomonas.	Infertile men presented with a significantly higher alpha-diversity and distinct beta-diversity compared to fertile men. Aerococcus was inversely associated with both leukocytospermia and semen viscosity. Prevotella was linked to low sperm counts. Pseudomonas was directly associated with total motile sperm concentration.
Molina et al. (2021)	Spanish	Semen parameters (WHO 5th edition)	11 infertility patients attended for SSR: 3 High SDF 1 OAT 7 AZ	None	QIAamp cador Pathogen Kit V3–V4 16S rRNA Illumina MiSeq	Dominant phyla: Firmicutes, Bacteroidetes, Actinobacteria. Common genera: Blautia Cellulosibacter, Clostridium XIVa, Clostridium XIVb, Clostridium XVIII, Collinsella, Prevotella, Prolixibacter, Robinsoniella, Wandonia.	Testicle is a low microbial biomass site. Identified 10 microbial genera as testicular sperm specific microbes.
Yao et al. (2022)	Chinese	Semen parameters (WHO 5th edition)	67 infertility patients: 13 AS 22 Leu 32 LA	20 infertile patients with normal semen parameters	QIAamp DNA Kit V3–V4 16S rRNA Illumina MiSeq	Dominant Phyla: Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes Common genera: Streptococcus, Lactobacillus, Burkholderia-Caballeronia-Paraburkholderia, Staphylococcus, Gardnerella, Ralstonia, Corynebacterium, Veillonella, Acinetobacter, Rhodococcus, Finegoldia, Peptoniphilus, Enterococcus, Prevotella, Haemophilus	The proportion of Bacteroidetes was significantly different between the study groups. Most of the control and AS patients were clustered in the Lactobacillus-enriched. Leukocyteospermia was inversely linked with Streptococcus.

iOA: idiopathic obstructive azoospermia; iNOA: idiopathic non-obstructive azoospermia; NA: Not Available; SSR: sperm surgical retrieval; IUI: Intrauterine insemination; IVF: in vitro fertilisation; AS: Asthenzoospermia; OS: Oligozoospermia; AZ: Azoospermia; AT: Asthenoteratozoospermia; OAT: Oligoasthenoteratozoospermia; OA: Oligoasthenozoospermia; SDF: sperm DNA fragmentationLeu: Leukocytospermia only; LA: Leukocytospermia and Asthenozoospermia.

Table 2. Summary of studies investigated the effect of probiotics use on semen quality.

	Valcarce et al. (2017)	Maretti et al. (2017)	Helli et al. (2020)
Sample size	9	41	50
Intervention	9	21	25
Control	0	20	25
Probiotics	L. Rhamnosus B. Longum	L. Paracasei	L. Casei L. Rhamnosus L. Bulgaricus L. Acidophilus B. Breve B. Longum S. Thermophilus
Duration of ingestion	6 weeks	6 months	10 weeks
Semen parameters
Volume	↔	↑	↑
Concentration	↔	↑	↑
Motility	↑	↑	↑
Morphology	NR	↑	↔
Viability	↔	NR	↑
DNA fragmentation	↓	NR	NR
ROS level	↓	NR	NR
Hormone profiles
FSH	NR	↑	↔
LH	NR	↑	↔
Testosterone	NR	↑	↔
Prolactin	NR	↔	↔
Oestradiol	NR	↔	NR

ROS: Reactive oxygen species; FSH: Follicular stimulation hormone; LH: Luteinizing hormone; NR: Not recorded.

Hou, D., Zhou, X., Zhong, X., Settles, M. L., Herring, J., Wang, L., Abdo, Z., Forney, L. J., & Xu, C. (2013). Microbiota of the seminal fluid from healthy and infertile men. Fertility and Sterility, 100(5), 1261–1269. https://doi.org/10.1016/j.fertnstert.2013.07.1991

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Weng, S. L., Chiu, C. M., Lin, F. M., Huang, W. C., Liang, C., Yang, T., Yang, T. L., Liu, C. Y., Wu, W. Y., Chang, Y. A., Chang, T. H., & Huang, H. D. (2014). Bacterial communities in semen from men of infertile couples: Metagenomic sequencing reveals relationships of seminal microbiota to semen quality. PloS One, 9(10), e110152. https://doi.org/10.1371/journal.pone.0110152

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Monteiro, C., Marques, P. I., Cavadas, B., Damião, I., Almeida, V., Barros, N., Barros, A., Carvalho, F., Gomes, S., & Seixas, S. (2018). Characterization of microbiota in male infertility cases uncovers differences in seminal hyperviscosity and oligoasthenoteratozoospermia possibly correlated with increased prevalence of infectious bacteria. American Journal of Reproductive Immunology (New York, N.Y. : 1989), 79(6), e12838. https://doi.org/10.1111/aji.12838

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Chen, H., Luo, T., Chen, T., & Wang, G. (2018). Seminal bacterial composition in patients with obstructive and non-obstructive azoospermia. Experimental and Therapeutic Medicine, 15(3), 2884–2890. https://doi.org/10.3892/etm.2018.5778

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Baud, D., Pattaroni, C., Vulliemoz, N., Castella, V., Marsland, B. J., & Stojanov, M. (2019). Sperm microbiota and its impact on semen parameters. Frontiers in Microbiology, 10, 234. https://doi.org/10.3389/fmicb.2019.00234

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Yang, H., Zhang, J., Xue, Z., Zhao, C., Lei, L., Wen, Y., Dong, Y., Yang, J., & Zhang, L. (2020). Potential pathogenic bacteria in seminal microbiota of patients with different types of dysspermatism. Scientific Reports, 10(1), 6876. https://doi.org/10.1038/s41598-020-63787-x

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Lundy, S. D., Sangwan, N., Parekh, N. V., Selvam, M., Gupta, S., McCaffrey, P., Bessoff, K., Vala, A., Agarwal, A., Sabanegh, E. S., Vij, S. C., & Eng, C. (2021). Functional and taxonomic dysbiosis of the gut, urine, and semen microbiomes in male infertility. European Urology, 79(6), 826–836. https://doi.org/10.1016/j.eururo.2021.01.014

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Valcarce, D., Genovés, S., Riesco, M., Martorell, P., Herráez, M., Ramón, D., & Robles, V. (2017). Probiotic administration improves sperm quality in asthenozoospermic human donors. Beneficial Microbes, 8(2), 193–206. https://doi.org/10.3920/BM2016.0122

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Helli, B., Kavianpour, M., Ghaedi, E., Dadfar, M., & Haghighian, H. K. (2020). Probiotic effects on sperm parameters, oxidative stress index, inflammatory factors and sex hormones in infertile men. Human Fertility, 1–9. Advance online publication. https://doi.org/10.1080/14647273.2020.1824080

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