Contrary to earlier perceptions that primarily portrayed bacteria as pathogenic adversaries, contemporary insights reveal a fascinating truth: the human body is teeming with more bacteria than human cells (1). This revelation aligns with the recognition that nearly all organs and systems host a companion microbiota composed of bacteria, fungi, and viruses that coexist harmoniously with human hosts (2). These organisms do not lead solitary lives; instead, they play a pivotal role in regulating bodily functions and maintaining hemostasis. Perturbations in the microbiota, termed dysbiosis, which can encompass imbalances in microbial community composition, loss of beneficial symbionts, proliferation of pathobionts or opportunistic organisms, and disruptions in inter-microbial competition and diversity, have been implicated in the onset or exacerbation of various diseases (3).

The term ‘microbiome’ refers to diverse microorganisms inhabiting specific organs, systems, or biofluids. Next-generation sequencing (NGS) technology has ushered in a new era of understanding the human microbiome, enabling the detection of previously unknown commensal and pathogenic microorganisms (4). Leveraging this high-throughput technique, the ‘Human Microbiome Project’ has characterized microbiomes in various bodily organs and has reported that the urogenital tract microbiome constitutes approximately 9% of the total human microbiota (5, 6). Notably, dysbiosis of the female reproductive microbiome has been associated with reduced pregnancy rates and adverse pregnancy outcomes (7).

Despite considerable progress in elucidating the human microbiome, the characterization of the male genital tract microbiome remains in its early stages. Most studies concerning the male reproductive microbiota center on the seminal microbiome. Semen comprises secretions from the testicles, epididymis, prostate, seminal vesicles, bulbourethral glands, and periurethral glands, providing a conducive environment for microbial growth due to its nutrient content (8, 9). Therefore, the seminal microbiome serves as a representative of the entire male genital system.

A male factor is identified in up to 50% of infertile couples, and urogenital tract infection represents a potential etiological contributor (10, 11). Additionally, as many as 25% of men with abnormal semen analysis results are categorized as having idiopathic infertility due to the absence of discernible causes using current diagnostic tools (7, 12). Beyond the conventional mechanisms by which pathogenic bacteria can adversely affect male fertility, such as impairing sperm motility and capacitation and inducing oxidative stress and apoptosis (13–21), some researchers propose that dysbiosis of the seminal microbiome may also exert adverse effects on male fertility through as-yet-unclear pathways (2).

To consolidate the evidence concerning the seminal microbiome and its association with male infertility, we conducted a comprehensive narrative review of all studies about the male reproductive tract microbiome in humans from 1980 to 2023. Our review encompassed research that employed various methodologies, including culture-based, polymerase chain reaction (PCR)-based, and NGS-based techniques to investigate the microbiome in human semen or testicular tissue samples. We systematically collected information on sample types (semen or testicular tissue), study designs, participant demographics, employed methodologies, and key findings. To assess the quality of the included studies, we utilized the ‘Study Quality Assessment Tool for Before-After (Pre-Post) Studies with no Control Group’ developed by the National Heart, Lung, and Blood Institute. The eligible studies were categorized into three tiers based on their quality: high, medium, or low (www.nhlbi.nih.gov/health-topics/study-quality-assessment-tools). Our review also explores the influence of environmental factors on the seminal microbiome. Finally, we examine the evolving clinical practices stemming from this emerging knowledge and recommend topics for future research to address the existing knowledge gaps.

The initial investigations into the bacterial content of semen relied on culture-based techniques primarily targeting well-known pathogenic bacteria, like Staphylococcus, Enterococcus, Escherichia, and Ureaplasma (22–24). Consequently, these earlier endeavors yielded limited insights into the resident seminal microbiota, particularly concerning anaerobes and fastidious bacteria, which are challenging to cultivate (5). Subsequently, PCR-based studies made strides in identifying a broader spectrum of bacteria genera. However, they still failed to provide a comprehensive overview of the seminal microbiome (25). This limitation arose from the requirement to predetermine the genera of bacteria under investigation, rendering the technique less effective for polymicrobial specimens and frequently resulting in data that were challenging to interpret (26).

The emergence of NGS technology marked a remarkable breakthrough in exploring the human microbiome. This method directly sequences microbial DNA or RNA within samples, eliminating the reliance on traditional culture-based approaches (4). Two primary NGS techniques employed for microbiome characterization are amplicon sequencing and shotgun metagenomic sequencing (27).

Amplicon sequencing involves amplifying a specific region of DNA through PCR and then sequencing the resultant product. Typically, this entails targeting one or more hypervariable regions of the bacterial 16S ribosomal RNA (rRNA) gene (4). The hypervariable regions, being highly conserved and ubiquitous among bacteria, offer a suitable basis for analysis (28). Nevertheless, due to practical constraints related to time and cost, only a subset of these variable regions is generally chosen for sequencing. This approach introduces potential bias since no single region effectively distinguishes all bacteria species, and sequencing specific hypervariable regions may yield varying results.

In contrast, shotgun metagenomic sequencing (SMS) comprehensively assesses all the DNA within a given sample. This method involves DNA extraction and random fragmentation, followed by the ligation of barcodes and adapters to each fragment, facilitating sample identification and DNA sequencing. Subsequently, the obtaining reads are meticulously cleaned and aligned with a reference database to identify taxa and assess functional potential (28). Unlike amplicon sequencing, SMS metagenomic sequencing enables the detection of fungi, parasites, and DNA viruses (29). Furthermore, SMS has superior resolution and sensitivity in detecting species-level changes and predicting functional potential (28).

A limited number of studies employing NGS have investigated the seminal microbiome of healthy men, often including them as part of a control group ( Table 1 ). Notably, two distinct features have emerged regarding the seminal microbiome: a wide variation in species composition among individuals and the formation of microbial communities dominated by particular species (2, 22).

In one of the initial NGS-based studies, Hou et al. sequenced the V1-V2 regions of 16S rRNA genes, revealing that even in healthy sperm donors, semen harbors a more diverse bacteria population than sperm itself (43). Their findings highlighted Ralstonia, Lactobacillus, Corynebacterium, Streptococcus, and Staphylococcus as the most prevalent bacteria in seminal fluid; these bacteria are organized into six distinct communities based on species composition and structure.

Weng et al. employed sequencing of the V4 hypervariable region of the 16S rRNA gene to examine 36 semen samples with normal basic semen analysis parameters. Their study identified Lactobacillus, Pseudomonas, Gardnerella, Prevotella, and Rhodanobacter as the most common genera (27). Additionally, the bacterial communities formed three main clusters: Pseudomonas-predominant, Lactobacillus-predominant, and Prevotella-predominant, with Lactobacillus-predominant group being the most frequent in the normal samples.

Similarly, Baud et al., utilizing sequencing of the V1 and V2 hypervariable regions of the 16S rRNA gene, examined 26 samples from men undergoing fertility evaluation who had normal basic semen analysis parameters. Their findings revealed three distinct microbiota communities: a Lactobacillus-predominant group, a Prevotella-predominant group, and a polymicrobial group (36). Staphylococcus was associated with normal semen analysis parameters, while the Lactobacillus genus was enriched in samples with normal morphology.

Another study reported that Lactobacillus, Gardnerella, Veillonella, Corynebacterium, and Escherichia were the most prevalent genera in the semen of men with normal basic semen analysis results (34). Bukharin et al. analyzed the seminal microbiome composition in 30 healthy men, identifying Staphylococcus, Corynebacterium, Enterococcus, Neisseria, and Veillonella as the most prevalent genera (32). Moreover, Yao et al. examined semen samples of 20 men with normal basic semen analysis parameters, revealing the main genera as Streptococcus, Lactobacillus, Burkholderia-Caballeronia-Paraburkholderia, Staphylococcus and Gardnerella (31). Interestingly, a Lactobacillus-enriched group predominated among these men.

Conversely, Monteiro et al. reported a low prevalence of Lactobacillus and a high prevalence of Enterococcus in semen samples of men with normal basic semen analysis parameters, employing sequencing of the V3 to V6 hypervariable regions of the 16S rRNA gene (40). However, it is essential to note that all the samples used in this study were derived from leftovers of assisted reproduction procedures, making it possible that some cases might have involved male factor infertility. Correspondingly, Yang et al., using sequencing of the V1 and V2 hypervariable regions of the 16S rRNA gene, demonstrated that Pseudomonas, Propionibacterium, Boseagenosp, Bosea, and Afipia were the most prevalent genera in healthy men with normal basic semen analysis parameters (35). Intriguingly, these authors observed an increased abundance of Lactobacillus in men with abnormal semen analysis results.

Given the diverse microfluidic components of semen and the microbiome’s complexity, it is estimated that approximately 30% of microorganisms in the semen originate from the urethra microbiome (35). Furthermore, specific genera, such as Pseudomonas, Pseudoxanthomonas, and Acidovorax, are overrepresented in the seminal microbiome compared to the urethral microbiome, suggesting their origin from upstream anatomic compartments (33). Thus, the seminal microbiome represents a composite of the microbiomes of the testicular, epididymal, prostatic, vesicular, and urethral regions (33) ( Figure 1 ). Higher microbiota diversity in the gut, skin, and oral cavity is often considered beneficial for human health (45). Interestingly, data from studies regarding the male genital tract microbiome is heterogeneous. Some authors have suggested that greater microbiota diversity harms sperm health (32, 46, 47), while others have found that reduced seminal biodiversity is associated with poor semen quality (30, 39).

Most culture-based studies examining cohorts of infertile couples have failed to establish a conclusive link between the presence of bacteria in semen and abnormal semen analysis parameters (33, 41, 48–51). However, Ricci et al. reported reduced sperm motility in samples testing positive for microorganisms compared to negative samples (52). They also observed a negative association between E. faecalis and semen quality. Likewise, Zeyad et al. identified a negative impact of bacterial presence on sperm concentration and motility (38). Along these lines, Pagliuca et al. showed a significant correlation between infected status assessed by culture and PCR with semen volume, sperm concentration, and motility (16). Below, we summarize findings from studies using NGS to explore the microbiome of men with abnormal semen analysis parameters ( Figure 2 ; Table 2 ).

Evaluating the epididymal or testicular microbiome requires harvesting biofluids or tissue samples from these organs. In this context, Alfano et al. conducted a study using testicular samples from men with idiopathic non-obstructive azoospermia (iNOA) and normozoospermic men who underwent orchiectomy (37). Employing NGS to sequence the V3 to V5 hypervariable regions of the 16S rRNA gene with NGS, the authors made a groundbreaking discovery, revealing that the testicular compartment is not sterile. In the testicular tissue of men with normal spermatogenesis, they identified the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. However, despite the increased presence of bacterial DNA in testicular samples from men with iNOA, only the phyla Actinobacteria and Firmicutes were identified in these samples.

Furthermore, the testicular microbiome of NOA men with complete germline cell aplasia exhibited reduced bacterial richness and diversity, with a dominance of the Actinobacteria phylum and the absence of Clostridia. Similarly, Molina et al. utilized testicular samples from sperm retrieval procedures in men with azoospermia, severe oligoasthenozoospermia, or high DNA fragmentation to study the testicular microbiome (53). The authors also observed low levels of bacteria and identified ten genera specific to the testicles, including Blautia (phylum Firmicutes), Cellulosibacter (Firmicutes), Clostridium XIVa (Firmicutes), Clostridium XIVb (Firmicutes), Clostridium XVIII (Firmicutes), Collinsella (Actinobacteria), Prevotella (Bacteroidetes), Prolixibacter (Bacteroidetes), Robinsoniella (Firmicutes), and Wandonia (Bacteroidetes). Notably, despite stringent antiseptic measures, contamination accounted for 50–70% of all detected bacterial reads, suggesting that sperm retrieval from the testes is not performed under sterile conditions (53).

Using a different strategy, Lundy et al. demonstrated that Collinsella and Staphylococcus were prevalent in semen samples from healthy fertile men and depleted in samples from men who underwent vasectomy, implying that these two genera are constituents of the testicular or epididymal microbiome (33). To date, no studies have examined the epididymal microbiome.

The seminal microbiome influences the microflora of the female genital tract. A well-established example of such influence is the association between bacterial vaginosis, a dysbiotic condition, and frequent vaginal intercourse (83). A study examining risk factors for bacterial vaginosis in women with and without HIV infection demonstrated that the presence of spermatozoa in Gram-stained vaginal smear samples, which serves as a biological marker of recent exposure to semen, was the only common factor in both groups (84).

Furthermore, the production of H₂O₂ by certain vaginal lactobacilli is essential for maintaining a healthy vaginal environment (85). Having more than two sexual partners during the past year has been identified as a risk factor for the absence of H2O2-producing lactobacilli among women with bacterial vaginosis (86). Several studies that simultaneously assessed the seminal and vaginal microbiomes of sexual partners have confirmed this finding. For instance, Okwelogu et al. found that couples shared 56% of predominant genera, suggesting that the composition of the reproductive tract microbiota, whether healthy or dysbiotic, could influence the microbial composition of their sexual partners (34). Similarly, Campisciano et al. demonstrated that couples with infertility of known causes exhibited an overlap in the bacterial composition of their seminal and vaginal microbiomes, including an increased prevalence of Staphylococcus and Streptococcus genera (54). The authors also noted a higher abundance of Lactobacillus gasseri in the semen of couples with unexplained infertility than those with explained infertility. Using PCR and culture-based techniques, Borovkova et al. found that up to seven new species could be introduced, and the same number removed from vaginal microflora after intercourse (87). Additionally, using culture-based methods, Ricci et al. found that 61% of couples with positive cultures in both partners shared at least one genital pathogen (52).

The emerging field of seminal microbiota research has illuminated the intricate microbial communities residing in the male reproductive tract. While significant progress has been made in characterizing these microorganisms and their potential functions of these microorganisms, several critical areas detailed below merit further exploration (93).

The exploration of the seminal microbiome has opened a fascinating realm of research, shedding light on the intricate microbial communities residing within the male reproductive tract. This emerging field has revealed a complex interplay between these microorganisms and male fertility, semen quality, and their potential influence on female reproductive health. The evidence compiled from various studies using culture-based and NGS techniques has provided valuable insights into these microbial communities’ composition, dynamics, and potential functions. One of the key takeaways from this review is the pressing need for standardized protocols and best practices in sample collection, DNA extraction, and NGS analysis. By establishing rigorous methodologies, the scientific community can ensure the reliability and reproducibility of results, fostering more robust comparisons between studies and facilitating meta-analyses. Additionally, the choice of variable regions within the 16S rRNA gene for sequencing and the application of shotgun metagenomics for a comprehensive assessment of viruses and fungi within the seminal microbiota are vital considerations for future research. Prospective and longitudinal studies and investigations into the impacts of various male infertility causes and commonly prescribed drugs hold promise for unraveling the intricate relationships between the seminal microbiome and male reproductive outcomes. This knowledge enhances our understanding of male fertility and paves the way for personalized interventions and treatments tailored to individual patients. Exploring the seminal microbiome represents a dynamic and rapidly evolving field poised to advance our comprehension of male reproductive health and potentially revolutionize clinical approaches to male infertility and semen alterations.

FL: Writing – original draft. MV: Writing – original draft. FC: Funding acquisition, Writing – review & editing. AC: Funding acquisition, Writing – review & editing. CA: Funding acquisition, Writing – review & editing. SE: Conceptualization, Methodology, Project administration, Supervision, Writing – review & editing.