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

Abstract

The human body harbours trillions of microbes, and their influence on human health has been explored in many parts of the human body, including the male reproductive system. From routine culturing to polymerise chain reaction (PCR) and high throughput DNA sequencing, several studies have identified bacteria in the male reproductive system. In this review, we discuss the past and current literature surrounding the testicular and semen microbiome in correlation with male infertility. We further highlight the potential benefits of probiotics as an alternative therapeutic option for male infertility. Although not conclusive, emerging data are indicating potential implications of certain bacterial members on male fertility. There is a general agreement on the negative impact of some pathogenic bacterial species on semen parameters, including sperm counts, motility, morphology, and DNA integrity. On the other hand, Lactobacillus, known as a human-friendly bacteria, has shown protective effects on semen parameters, which makes it a potentially good probiotic. In order to confirm the findings of previous studies, more clinical studies with larger sample sizes and the right controls are needed.

Keywords:

• Microbiome
• male infertility
• semen microbiome
• testicular microbiome
• probiotic
• microbiome in ART

Introduction

Infertility is a common condition, affecting around 15% of couples around the world. Males contribute to almost half of the cases, either as a single factor or combined with other female issues (Agarwal et al., 2015). Male infertility can arise from chromosomal abnormalities, gene defects, hormonal, environmental, physical, or psychological issues. However, in several cases, the main cause of male infertility remains unknown. Infection in the male genitourinary (GU) tract is another infertility factor that is reported in about 15% of infertile men. Infections and subsequent inflammations within the GU tract can interfere with the male reproductive functions in several ways. The ascending of infectious agents from the lower GU tract can result in orchitis, prostatitis, epididymitis, or obstruction of the deferent ducts (Motrich et al., 2006; Pellati et al., 2008). Similarly, semen contamination by urinary tract microorganisms have been shown to be associated with impaired spermatogenesis and poor semen quality, including reduced sperm motility, abnormal mitochondrial and acrosomal functions, and loss of DNA integrity (Diemer et al., 2000; La Vignera et al., 2011; Pellati et al., 2008). Change in semen viscosity is another defect that is commonly reported in men who are diagnosed with infections (Elia et al., 2009). The level of leukocytes in semen (leukocytospermia) may increase during or after infection, causing elevated levels of reactive oxygen species (ROS) (Andrade-Rocha, 2009; Domes et al., 2012; Ho et al., 2022; Yao et al., 2022). Prolonged ROS exposure may subsequently deteriorate sperm functions and chromatin integrity (Agarwal et al., 2014; Ho et al., 2022).

Bacteriospermia was believed to always result from infections. However, the advances in high-throughput DNA sequencing methods contradicted that thought. By targeting the 16S rRNA gene, scientists revealed that semen harbours a wide diversity of microbes, even in healthy men. Understanding the complex microbial community within the male reproductive system may help to identify microbes associated with male infertility or discover healthy microbes that can be used in form of probiotics to improve male fertility. Herein, we provided a historical overview of the literature exploring semen and testicular microbiome regarding male infertility, with emphasis on findings of recent pilot metagenomics studies. In addition, we discussed the potential therapeutic role of probiotics, challenges of current data, and scope for improvements in future studies.

Early studies on the semen microbiome and male infertility

Historically, culture-based approaches were the standard for microbial identification and diversity characterisation. However, some microbes are difficult to isolate and require strict conditions to grow in a lab. Conversely, some microbes are easy to culture, which can overcrowd or inhibit the growth of more fastidious bacteria. Accordingly, it is difficult to interpret results based on microbial culture methods as some species could be under-or over-represented. Despite these limitations, culture-based techniques have isolated various bacterial species from semen including Escherichia spp, Staphylococcus spp, Streptococcus spp, Enterococcus spp, and Ureaplasma spp (Mashaly et al., 2016; Nasrallah et al., 2018; Vilvanathan et al., 2016; Zeyad, Hamad, et al., 2018). The prevalence of bacteriospermia in sub-fertile and infertile men varied largely between studies, ranging from 6% to 68% (Bussen et al., 1997; Domes et al., 2012; Eggert-Kruse et al., 1995; Moretti et al., 2009; Vilvanathan et al., 2016). Most culture-based studies found significant reductions in sperm concentration and the percentage of progressively motile spermatozoa in men with bacteriospermia (Mashaly et al., 2016; Ricci et al., 2018; Zeyad et al., 2018; Zeyad, Hamad, Amor, et al., 2018). Sperm morphology, however, seems to be the least affected semen parameter in infected semen (Nasrallah et al., 2018). Still, those findings were challenged by other groups. Bacteriospermia was presented in normozoospermic men in similar proportions to that reported in infertile men (Bukharin et al., 2022; Cottell et al., 2000; Ivanov et al., 2009; Rehewy et al., 1979; Rodin et al., 2003). In addition, some studies have showed that the absence or presence of bacteriospermia does not influence sperm quality (Nasrallah et al., 2018; Vilvanathan et al., 2016). According to a large retrospective study performed by Domes et al. (2012), bacteriospermia was associated only with poor DNA integrity, and the decrease in sperm quality reported in their study was rather associated with the elevated levels of leukocytes than with bacteriospermia. However, a recent meta-analysis supported the negative impact of bacteriospermia on sperm counts, motility, and sperm DNA fragmentation (Farahani et al., 2021).

Ureaplasma urealyticum, Mycoplasma hominis and Chlamydia trachomatis are the most common pathogens causing sexually transmitted diseases (STDs) in developed countries (Gerbase et al., 1998; Yoshida et al., 2002). In 1977, Taylor-Robinson et al. (1977) demonstrated that U. urealyticum can cause nongonococcal urethritis. Since then, many studies have linked the presence of U. urealyticum in semen with male infertility (Debata et al., 1999; Ma & Gao, 2017; Wang et al., 2001, 2005). Compared to fertile men, infertility patients showed a significantly higher prevalence of the U. urealyticum in semen, and that was associated with decreased semen quality, including progressive motility, vitality, and DNA integrity (De Francesco et al., 2011; Lee et al., 2013; Ma & Gao, 2017; Moretti et al., 2009; Wang et al., 2005). Similarly, M. hominis was linked with deleterious impacts on semen quality, even higher than those observed in the presence of U. urealyticum (Gdoura et al., 2007; Rybar et al., 2012). Chlamydial infections can cause obstructive azoospermia if undiagnosed or left untreated (Paavonen & Eggert-Kruse, 1999). However, C. trachomatis has never been identified in semen using culturing methods (Farahani et al., 2021). In an in vitro study, coculturing of human spermatozoa with the microbe resulted in a significant reduction in sperm motility and increased damage to the spermatozoa (Hosseinzadeh et al., 2001).

The introduction of polymerase chain reaction (PCR) technology has enabled more sensitive detection and identification of specific microbial species. The technology has identified bacteria that have never been isolated from semen or found at very low levels using routine culturing methods (Gdoura et al., 2001; Jarvi et al., 1996; López-Hurtado et al., 2018; Motamedifar et al., 2020; Sellami et al., 2014). One of the earliest reports on this topic was published by Jarvi et al. (1996). In general, PCR data agreed with the negative impact of bacteriospermia on semen quality observed using culture-based methods. A recent study found bacteriospermia at a similar prevalence to that reported using culturing. Interestingly, bacteriospermia was more common in men who were infertile than healthy donors (Motamedifar et al., 2020).

The advantage of the PCR technology over culturing was more evident in detecting pathogens associated with STDs. In contrast to culture methods, several PCR studies have successfully detected C. trachomatis in semen (Gdoura et al., 2001, 2008; López-Hurtado et al., 2018; Sellami et al., 2014). Unexpectedly, C. trachomatis was found in about 40% of infertile men, according to a cross-sectional study by Gdoura et al. (2008). However, the prevalence of C. trachomatis in this study was not significantly different between fertile and infertile men (Gdoura et al., 2008). PCR methods revealed that U. Parvum, U. urealyticum, M. genitalium, and M. hominis were also commonly prevalent in semen (Gdoura et al., 2007, 2008; Ho et al., 2022; Huang et al., 2016; Lee et al., 2013; Liu et al., 2014).

Overall, PCR data supported the findings of culturing studies. The presence of pathogenic bacteria in semen was associated with declined sperm counts, motility, morphology, DNA integrity, and in vitro fertilisation (IVF) outcomes (Gdoura et al., 2007, 2008; Lee et al., 2013; Liu et al., 2014; Ricci et al., 2018). Bacterial identification using routine culturing methods, or the combination of culturing and PCR is clinically valuable, especially in detecting pathogenic agents. However, those techniques cannot provide detailed information about the whole community in semen samples.

Recent metagenomics studies

Since the initiation of the Human Microbiome Project (HMP), several metagenomics studies have explored different parts of the human body using high-throughput sequencing technologies such as next-generation sequencing (NGS) (NIH HMP Working Group, Peterson et al., 2009). The role of the microbiome in human reproductive health has largely been deduced from studies of the microbiota within the female reproductive system. For example, dysbiosis of the vaginal and uterine microbiomes have been widely explored and linked with adverse pregnancy rates and assisted reproductive technology (ART) outcomes (Franasiak & Scott, 2015). On the other hand, little is known about the role of the microbiome in male infertility. Here we discussed the current literature on this topic.

Semen microbiome

Semen was believed to be a sterile fluid in healthy conditions, and the presence of microbial agents within the semen was thought to be caused by infections. However, the use of advanced high-throughput sequencing methods revealed the opposite. The earliest investigation of human semen using NGS was carried out by Hou et al. (2013). The study included 56 infertile men, with at least one semen abnormality (World Health Organization, 1999), and 19 normozoospermic donors. The group utilised primers targeting two hypervariable regions (V1–V2) of the bacterial 16S rRNA. Sequenced reads were classified into different bacterial taxa with the use of the Ribosomal Database Project (RDP) Naïve Bayesian Classifier (Hou et al., 2013). Semen was then clustered into six groups based on similarities in bacterial compositions. However, the results showed no significant difference in semen parameters between the six groups, suggesting no association with semen quality (Hou et al., 2013). Upon further analysis of individual bacterial taxa, the only bacterial genus that showed a negative impact on semen quality was Anaerococcus (Hou et al., 2013). The major limitation of this observation is the significant microbial quantity present in each sample, suggesting potential inappropriate sample handling. However, the findings of this study have aroused interest in further investigation.

Since then, a few pilot metagenomic studies have explored the semen microbiome in correlation with infertility in men (Baud et al., 2019; Monteiro et al., 2018; Weng et al., 2014; Yang et al., 2020; Yao et al., 2022). Three studies have identified two to three predominant bacterial genera in the semen (Baud et al., 2019; Weng et al., 2014; Yao et al., 2022), where Lactobacillus was a common predominant group among those studies. The presence of Lactobacillus in semen was associated with improved semen quality, including sperm counts, motility, normal morphology, viscosity, and leukocytospermia (Baud et al., 2019; Monteiro et al., 2018; Weng et al., 2014; Yao et al., 2022). Those observations highlight the potential beneficial effects of the genus Lactobacillus on the spermatogenesis process. However, two recent studies contradicted the previous reports (Chen et al., 2018; Yang et al., 2020). The first study compared the microbiome in semen obtained from azoospermic men to that from normozoospermic donors (Chen et al., 2018). Interestingly, healthy donors showed an overall higher bacterial diversity, but a lower abundance of Lactobacillus compared to the azoospermic group. However, the sample size for this study was too small to be conclusive. The other study included 159 infertility patients clustered into five groups based on semen characteristics (Yang et al., 2020). The study found that Lactobacillus was more common in asthenozoospermia and oligoasthenozoospermia than normal semen (Yang et al., 2020).

Staphylococcus is another genus of the phylum Firmicutes that has been detected in semen (Hou et al., 2013; Lundy et al., 2021; Monteiro et al., 2018; Weng et al., 2014; Yang et al., 2020; Yao et al., 2022). A recent study has linked the presence of this genus in semen with improved sperm motility (Monteiro et al., 2018). This observation was in accordance with previous reports based on culturing methods (Ivanov et al., 2009; Mändar, 2013). A very recent study by Lundy et al. (2021) highlighted an interesting observation regarding the origin of Staphylococcus and other microbes, namely Collinsella in semen. The group hypothesised that Staphylococcus and Collinsella originated from the testes or the epididymis, as they were depleted in semen obtained from vasectomised men (Lundy et al., 2021). However, further investigations are needed to confirm this observation and enhance our understanding of microbes’ role in spermatogenesis. Gardnerella was also found to be at an increased abundance in normozoospermic men and was linked with improved semen parameters (Chen et al., 2018; Lundy et al., 2021; Weng et al., 2014). This challenged the negative influence of this microbe on semen that was concluded from findings of culture and PCR-based studies (Andrade-Rocha, 2009; De Francesco et al., 2011).

On the other hand, there was a general agreement on the negative impact of Prevotella on semen characteristics (Baud et al., 2019; Chen et al., 2018; Lundy et al., 2021; Weng et al., 2014). Two studies have assigned Prevotella as the predominant genus in the semen of infertile men. Interestingly, most abnormal semen samples were enriched with Prevotella (Baud et al., 2019; Weng et al., 2014). Another study detected Prevotella at higher proportions in men with azoospermia compared to healthy controls (Chen et al., 2018). This was supported by the findings of a recent study (Lundy et al., 2021) which reported an inverse association between the abundance of Prevotella in semen and sperm concentration (Lundy et al., 2021). Prevotella has also been shown to influence the overall diversity and compositions of the microbial community within the semen (Chen et al., 2018). A group of well-known pathogens, such as Pseudomonas, Klebsiella, Neisseria, Aerococcus, Streptococcus, Anaerococcus, Aggregatibacter, and Actinobaculum, were associated with poor sperm counts, decreased sperm motility, abnormal viscosity, and Leukocytospermia (Chen et al., 2018; Lundy et al., 2021; Monteiro et al., 2018; Weng et al., 2014; Yang et al., 2020; Yao et al., 2022).

Testicular microbiome

Until recently, testes were believed to be entirely sterile. However, this assumption could not remain valid with the current data. The first evidence of a testicular microbiome was reported by Alfano et al. (2018) using the NGS technology. The group extracted testicular samples from ten men affected with idiopathic nonobstructive azoospermia (iNOA) and five healthy individuals with normal spermatogenesis (Alfano et al., 2018). Surprisingly, bacterial DNA was found in significant quantities in both groups (Alfano et al., 2018). Moreover, azoospermic men showed significantly higher bacterial counts compared to healthy patients, with a predominance of Actinobacteria and a depletion of Bacteroidetes and Proteobacteria (Alfano et al., 2018). Further investigation of the azoospermia group revealed an absence of Clostridia in men with negative retrieval at microdissection testicular sperm extraction (micro-TESE) (Alfano et al., 2018). This observation was of potential clinical relevance since two genera of the class Clostridia have been previously linked with improved sperm motility and morphology (Hou et al., 2013; Weng et al., 2014). Although this was an innovative observation, the small sample size and the lack of environmental controls limited the strength of this report. A more recent study has supported the previous observation by following a stricter methodology (Molina et al., 2021). The group applied environmental controls throughout all the experimental steps to adjust for contaminants’ contributions to the overall detected microbes. Bacteria were found in all the testicular samples, and Prevotella was the most common genus among all patients (Molina et al., 2021). Prevotella was previously reported in semen and linked with reduced semen parameters (Baud et al., 2019; Weng et al., 2014). Therefore, its presence within the testicular tissues of infertile men could point to its negative impact on spermatogenesis. What is interesting about the previous two studies is that they both detected seminal microbiota. This highlights the possible contribution of the testes to the semen microbiome and encourages researchers to further investigate the testicular microbial environment.

Semen microbiome and assisted reproductive technology

While most studies have focussed on variations in semen parameters, two recent studies have linked the semen microbiome with assisted reproduction technology (ART) outcomes (Amato et al., 2020; Štšepetova et al., 2020). Amato et al. (2020) studied the microbial composition of semen in a group of idiopathic infertility patients who underwent intrauterine insemination (IUI). The semen microbiome didn’t differ significantly in couples with failed IUI compared to those who achieved clinical pregnancy (Amato et al., 2020). On the other hand, the presence of specific bacterial species in semen seems to affect the quality of IVF embryos (Štšepetova et al., 2020). The Alphaproteobacteria and Gammaproteobacteria were inversely associated with poor embryo quality, whereas Enterobacteriaceae were directly linked with better-graded embryos. However, the study failed to adjust for other confounding factors such as the autecology of infertility, variations in stimulation protocols, and the number of collected oocytes.

Potential mechanisms

The exact mechanisms of how the microbiome can impact male fertility are not yet fully understood. However, metagenomics analysis revealed potential pathways that may explain the association between the microbiome and semen quality. Chen et al. (2018) found that azoospermic patients had increased expression of several genes associated with metabolic disorders, enzyme families, and glycan biosynthesis (Chen et al., 2018). Similar results were also reported in men with asthenozoospermia and oligoasthenospermia compared to normozoospermic donors (Yang et al., 2020). Cytokine levels in the semen of asthenozoospermic patients are shown to be at lower levels compared to healthy men (Chen et al., 2021). This could influence the microbial composition of the semen. However, the hypothesis has yet to be tested. Another metagenomic study reported that the metabolite S-adenosyl-L-methionine cycle (SAM) was strongly overrepresented in the semen of infertile patients compared to fertile men (Lundy et al., 2021). This metabolite has well-known roles in DNA methylation, oxidative stress modulation, and polyamines synthesis (Bottiglieri, 2002). Therefore, changes in the SAM pathway may play a critical role in sperm production and sperm quality (Lundy et al., 2021). In addition to SAM, a group of other pathways were differentially expressed between fertile and infertile men. Those were associated with the biosynthesis of L-threonine, guanosine ribonucleotides, 5-aminoimidazole ribonucleotides, and queuosine, as well as pyruvate fermentation and methylerythritol phosphate pathways (Lundy et al., 2021). The findings of the previous studies are encouraging, but extensive examinations should be done to explore how those mechanisms work in correlation with seminal microbiota and male infertility.

Taken together, the NGS technology identified bacterial species that have either not been identified in semen or found at a very low abundance using culturing and PCR-based methods. Findings of the previous metagenomics studies indicated that human semen contains a diverse range of bacteria, even in healthy men, and that changes in the seminal microbiota can influence semen characteristics such as sperm counts, motility, morphology, and DNA integrity. However, it should be noted that some of the NGS findings were not consistent across studies. Conflicting results between studies could be explained by the differences between ethnic groups or geographical populations, study designs, semen classification guidelines (WHO4 vs WHO5) (World Health Organization, 1999; World Health Organization, 2010), the use of different hypervariable regions of the 16S rRNA and different sequencing methods (Table 1).

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.

Probiotic supplementation and semen quality

Probiotics are live microbial food ingredients that exert health benefits on the consumer when ingested in sufficient amounts (Ashwell, 2002). The use of probiotics as a therapeutic option for many diseases has gained increasing interest in recent years, and their minimal side effects make them an attractive option for treatment in several areas of medicine. In reproductive health, probiotic administration has shown promising results in treating female genital tract infections, e.g., bacterial vaginosis (Mastromarino et al., 2009). However, little is known about the benefits of probiotic therapy in the case of male fertility. In an in vitro study, a combination of three strains of Lactobacilli: L. Brevis, L. Salivarius, and L. Plantarum, have shown a protective effect on sperm lipid peroxidation level, sperm motility, and viability (Barbonetti et al., 2011). The mechanisms underlying the positive effect of probiotics on semen are not clear yet. However, the group suggested an indirect role of probiotics in improving sperm quality by reducing oxidative stress on sperm cells (Barbonetti et al., 2011). This observation was later supported by Chen et al. (2013) in vivo studies, using animal models. In rats, probiotic supplementation has protected sperm quality from oxidative stress induced by a high-fat diet (Chen et al., 2013). The author reported a significant reduction in lipid peroxidation, and increases in all semen parameters, including sperm counts, motility, viability, and DNA integrity (Chen et al., 2013). A similar study by Dardmeh et al. (2017) also showed that probiotic supplementation has positive effects on sperm quality, testicular weight, and reproductive hormone profiles including testosterone, follicular stimulation hormone (FSH), and luteinizing hormone (LH) in male mice (Dardmeh et al., 2017). However, the authors of those studies did not measure the level of reactive oxygen species (ROS) before, during, or after probiotic ingestion.

In humans, three recent studies have reported a significant improvement in semen quality after probiotic administration (Table 2). Valcarce et al. (2017) recruited nine men with poor sperm motility (asthenozoospermia) to evaluate the effect of two selected antioxidant probiotic strains (L. rhamnosus CECT8361 and B. longum CECT7347) on semen quality (Valcarce et al., 2017). Semen evaluation was performed after 3 and 6 weeks of probiotic ingestion, and the results were promising (Valcarce et al., 2017). The group reported a 6-fold increase in sperm motility, and 1.2-fold and 3.5-fold reductions in DNA fragmentation and ROS levels, respectively, with no change in sperm counts, volume, pH, and viability (Valcarce et al., 2017). The small sample size, short duration of probiotics consumption, and lack of control patients were limitations to this observation. In another study, those limitations were overcome by randomising 41 infertile patients with idiopathic oligoasthenoteratospermia (iOAT) into two groups: placebo and probiotic (Flortec) (Maretti & Cavallini, 2017). The six-month course of probiotic ingestion showed significant improvements in semen parameters, including semen volume, sperm counts, motility, and morphology (Maretti & Cavallini, 2017). Besides, the levels of total testosterone, FSH, and LH were increased after treatment, while no change was observed in the levels of 17-beta-2-oestradiol (E2) and prolactin (PRL) (Maretti & Cavallini, 2017). On the other hand, there was no significant change in any of the semen parameters or reproductive hormone profiles in the placebo group (Maretti & Cavallini, 2017).

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.

A recent double-blind randomised controlled clinical trial has also reported increased semen quality after the probiotic supplementation (Helli et al., 2020). A total of 50 men with iOAT were randomly allocated to either a placebo or probiotic group (Helli et al., 2020). Participants in the placebo group received a capsule containing Maltodextrin only, while a combination of probiotics was given in capsule form to men in the intervention group (Helli et al., 2020). Semen evaluation after a 10-week course of probiotic intake showed a significant increase in semen volume, sperm counts, motility, and viability (Helli et al., 2020). Moreover, there was a decrease in the level of oxidative stress and inflammatory markers after treatment. The levels of testosterone, FSH, LH, and PRL didn’t significantly change, but slightly improved at the end of the study (Helli et al., 2020).

Although data is not enough to be conclusive, changes in the microbial community caused by microbiome drifting may have an impact male fertility status. Probiotic ingestion at certain amounts showed beneficial effects on semen quality, and that could be through several mechanisms, including changes in the level of inflammation, oxidative stress, and reproductive hormones (Helli et al., 2020; Maretti & Cavallini, 2017; Valcarce et al., 2017). Given the limited number of studies, variations in probiotic composition and administration protocols, and the lack of sperm function assessments or clinical outcomes after treatments, it is not yet clear if probiotics have any therapeutic value in treating male infertility. More investigations are needed to answer some pending questions. For example, do probiotics have dose-response effects? How long does the effect last? Furthermore, none of the previous studies has investigated the change in the semen microbiome before and after probiotic therapy. Therefore, there is still a need for high-quality evidence to address that and establish whether probiotics should be considered a therapeutic modality in the case of male infertility.

Challenges and perspectives for future microbiome studies

Molecular-based approaches provide a less biased representation of the semen microbiota. However, it cannot distinguish between live and dead microbes. Therefore, data from NGS studies may not just represent the bacterial levels at the time of sample collection but also the short-term history of the semen microbiota within the host environment. Furthermore, all the semen microbiome studies published thus far have explored semen samples at a single time point, providing no evidence on whether the microbiome is permanent or transient in semen. Although it might be costly, exploring that may offer insights into the dynamics of changes in the microbial community over time.

The majority of the NGS studies suggested a correlation between the microbiome profile in semen and male infertility. However, the data from those studies is not robust enough to be conclusive. One reason for this is that most studies used abnormal semen parameters as an indicator for male infertility. Although semen analysis is routinely used in male fertility assessment, the functional ability of spermatozoa to undergo pivotal processes required for fertilisation, such as acrosome reaction (AR) or zona pellucida (ZP) binding and oocyte penetration, cannot be precisely evaluated based on routine semen analysis (Wang & Swerdloff, 2014). Therefore, we encourage scientists to consider combining semen analysis with other sperm function tests in future studies. Including a paternity-proven cohort as a fertile control could be another way to challenge this limitation. In fact, only one of the NGS studies so far has involved a cohort of fertile participants (Lundy et al., 2021).

Positive and negative control samples are essential in all metagenomics studies. The use of positive controls can detect potential biases in the microbial community and minimise the risk of data misinterpretation (D’Amore et al., 2016). However, only a few studies have collected parallel samples, such as urine or gut samples, as a positive control (Lundy et al., 2021; Yao et al., 2022). On the other hand, the use of negative controls can detect contaminants, identify their sources, and exclude them from the subsequent microbiome analysis. That was supported by three recent studies (Baud et al., 2019; Lundy et al., 2021; Molina et al., 2021). Therefore, we strongly suggest including control samples in all future studies.

Conclusion

Most of our understanding of bacteriospermia has evolved from traditional culturing studies. However, the presence of bacteria at very low levels or uncultivable ones was the main challenge to the culturing-based studies. The introduction of PCR technology was beneficial in detecting pathogenic bacteria, especially Chlamydia spp. and Mycoplasma spp. Recently, there has been a huge interest in understanding the semen microbiome and how dysbiosis of the microbiome profile within semen can interfere with fertility. A few metagenomics studies have been published thus far in this regard and suggest a potential impact of a specific microbial species on semen quality. However, the results of those studies are not yet sufficient to draw a clear connection with male infertility. Routine sperm analysis does not fully capture fertility status; perhaps microbiome analysis can explain some idiopathic infertility, particularly in men with relatively normal semen parameters. Furthermore, studying the microbiome of the male reproductive system will increase our understanding of the effect of microbiome drifting on male fertility and help to identify bacteria with beneficial profiles that can be used in the form of probiotics to treat or improve male fertility. The result of this review emphasises the need for well-designed clinical studies with a larger sample size and appropriate controls to assure the previous observations.

Disclosure statement

The authors declare that there is no conflict of interest could be perceived as prejudicing the impartiality of this review.

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

This research was supported by the Health and Medical Research fund [Grant number 19181162].