Infertility is a social burden for the population, a difficult ordeal for couples to overcome. Its treatment can be stressful, invasive and costly. In some cases, it is the cause of marital discord or divorce. For a long time, women were helplessly blamed for a couple’s infertility. The World Health Organization (WHO) defines infertility as the inability of a couple to conceive a child after one year or more of regular, unprotected sexual intercourse at the right time [1] .

Infertility is a global public health problem affecting 10% - 20% [2] of couples of childbearing ages worldwide. Male infertility is present in 30% to 50% of cases [3] . It can be attributed to the male if there is an alteration in one or more sperm parameters, i.e. sperm concentration and/or motility and/or morphology [4] .

Uropathogenic infections can lead to infertility and bacteria are typically involved in infection and inflammation leading to bacteriospermia.

Excessive leukocyte infiltration in the urogenital tract in response to bacterial load results in oxidative stress (OS). By interacting directly with sperm or producing reactive oxygen species (ROS), bacteria can cause infertility by affecting sperm characteristics such as motility, volume, capacitation, and hyperactivation [5] [6] . Sperm analysis is essential in the search for the cause of male infertility, but it cannot diagnose or fully determine the primary causes of infertility [7] . For example, in around 30% of cases, the etiology of abnormal semen analysis parameters is not found. Thus, investigations involving the semen microbiome may bridge this gap [8] . The microbiome of semen is a field of increasing scientific interest, in recent years scientific evidence-based data from several studies demonstrated the existence of semen microbiome comprising different taxa, genus, and species of microorganisms originating from the urogenital tract [8] ; also, it is now known that in addition to the pathogenic microorganisms (some of which are sexually transmitted) that can cause male infertility, the sperm microbiota can influence male fertility [8] [9] . Even the hypothesis that microbiota may be involved in male infertility is short-explored because this microbial niche is currently understudied compared with other areas of microbiome research. With the development and accessibility of Next-Generation-Sequencing technologies, there are more and more studies in this avenue [8] .

Most previous studies have focused on the infected organs of the male genital tract, and few studies address the direct effect of microorganisms and their mechanisms of action on sperm.

The pathogens can induce various forms of damage to spermatozoa, such as DNA fragmentation, cell membrane peroxidation, and alteration of the acrosome [10] . This damage may be mediated by microorganisms’ toxins and metabolites, or by their direct binding to spermatozoa and subsequent activation of signaling pathways linked to oxidative stress, apoptosis, and inflammation [10] .

These induced changes can alter sperm parameters and subsequently lead to infertility. It appears that the effect of microorganisms on sperm function depends on the species and their concentration [10] .

In Burkina Faso, studies on this issue are still inadequate, and national statistics are poorly publicized. Furthermore, in Burkina Faso, clinical laboratories, and diagnostic algorithms, did not perform appropriate tests for male infertility etiology investigations. Clinical laboratories just run traditional culture methods for current bacteria detection and identification. Also, infertility is a taboo subject. Couples in this situation are often led to adopt isolation strategies, which only serve to distance them from the real solutions [11] . According to data from Burkina Faso 2010 demographic and health surveys (DHS), infertility affected 1.1% of the general population (DHS, 2010). This survey focused solely on the social, economic, and environmental factors of infertility. The present study is the first of its kind, and its overall aim is to explore specifically targeted pathogenic microorganisms in sperm that may be involved in male infertility, using conventional bacteriology techniques and real-time PCR.

The mean age of our study population was 36.15 years. The younger age bracket of 30 to 40 years was in the majority. These results are similar to Mohammed Frikh et al.’s study which found an average age of 35.35 in Morocco in 2021. This result could be explained by the fact that this age bracket corresponds to the period when men were much more interested in reproduction in their married life. Another hypothesis could be that this age bracket corresponds to the period of late awareness on the part of the man to establish his share of responsibility for the couple’s infertility. The distribution of patients according to profession showed that shopkeepers and civil servants were the majority in this study. These results show that infertility is not a problem of a single social stratum, but affects all social categories.

S. aureus was the most bacteria isolated by culture in this study with a rate of 5.55%. Our results are similar to those of Sanocka-Maciejewska et al. in Poland, who reported a rate of 7% [12] . Other species such as E. faecalis, S. agalactiae, S. hoemalyticus, and K. pneumoniae were isolated in low proportion in culture. Certain bacteria isolated by the culture-based method are part of the semen microbiome and their variation in quality or quantity are factors that influence male fertility. Some of the commensal opportunistic pathogenic bacteria can also influence male fertility. It is known that the culture of semen specimens from healthy individual men is positive in around 50% of cases. Recently, the use of high-throughput sequencing for semen microbiome studies has revealed the presence of bacteria such as Corynebacterium, Lactobacillus, Pseudomonas, Prevotella, Streptococcus, Gardnerella, Enterococcus, Veillonella, Porphyromonas, Sneathia and Pelomonas in semen samples from patients and healthy controls. Osadchiy et al. (2024) have found that bacteria in the Pseudomonas genus and Lactobacillus iners can play a role in male fertility [8] .

These studies have also found potentially pathogenic bacteria such as Finegoldia, Anaerococcus, and others [8] .

This study also revealed the lack of high sensitivity of culture-based, appropriate diagnostic algorithms in Burkina Faso for semen analysis in the exploration of male infertility.

Furthermore, this culture method with inappropriate culture media and diagnostic algorithm did not allow the isolation of fragile and demanding microorganisms such as Mycoplasmes spp, Ureaplasma spp, and C. trichomonas.

In contrast, qPCR showed in our study that U. urealyticum is the most common sexually transmitted pathogen in men, with a frequency of 21.05%. It was detected in the samples of some patients in common with certain sperm parameters such as leukospermia, asthenozoospermia, and necrozoospermia as reported by the study of Hannichi et al. in Tunisia in 2018 with a higher rate of 43.1% [13] [14] . Others have only reported a decrease in sperm count [15] . We also reported U. parvum infection at 18.42%. Our frequency results of these bacteria are corroborated by other studies, Gdoura et al. 2008 [11] [16] and Jalal et al. 2013 [17] .

Regarding the possibility of microbial colonization alterations in the sperm of varicocele patients, research has indicated that the colonization of U. urealyticum in varicocele patients is substantially more than in healthy men. Although there isn’t any concrete proof that U. urealyticum harms sperm, the oxidative stress that the parasite causes may hurt the quality of semen [18] .

Other pathogenic microorganisms such as C. trachomatis (7.89%), M. hominis (15.79%), M. genitalium (10.53%), and T. vaginalis (2.63%) were reported in this study by molecular detection using qPCR.

Pathogenic microorganisms are responsible for silent infections in patients and are not systematically detected by conventional bacteriology [19] - [23] .

Some studies have already demonstrated that this pathogen is responsible for male infertility. Roh et al. were able to show that T. vaginalis affects sperm fertilization capacity [24] . The germ is also responsible for sperm agglutination [25] .

Male infertility may also arise from the loss of seminal ocilium caused by the coinfection of C. trachomatis, U. urealyticum, and M. hominis with varicocele inflammation [26] - [29] .

These microorganisms, which cannot be isolated by culture in the absence of specific media, were detected by real-time PCR. As a result, real-time PCR can be used as a complement to culture for non-cultivable or difficult-to-cultivate microorganisms, making PCR the method of choice.

Infertile patients infected with pathogenic microorganisms such as pathogenic bacteria can regain their fertility if successfully treated with appropriate therapeutic management. There are some examples of infected infertile men by M. genitalium or M. Hominis, successfully treated with the improvement of the semen quality and wives of certain became pregnant 4 months after the treatment completion [19] [20] The aim of our study was not to perform a multivariate statistical association analysis to estimate the association of microorganisms with abnormal semen parameters but to demonstrate that in Burkina Faso there is a misdiagnosis of microorganisms involved in male infertility. There is therefore a need to improve the diagnosis of male infertility and the investigation of its causes, all of which would lead to good therapeutic management.

The results of this pilot study pave the way for a large and relevant study of microorganisms that could be involved in infertility in Burkina Faso. This study using routine diagnostic samples has some limitations, such as the size of the samples, the lack of suitable culture media for the different micro-organisms that can be routinely isolated, and the lack of reagents to identify other viral pathogens involved in male infertility.

Sperm analysis is an essential tool in the search for the cause of male infertility. In this study, several pathogenic microorganisms were identified in sperm by culture or qPCR. Sexually transmitted pathogens identified by qPCR were more prevalent. Given the limitations of conventional bacterial culture, such as time, workload, and low sensitivity, PCR-based diagnostic methods, which offer rapid and sensitive detection of microbiota, should be used as a complement in clinical laboratories in Burkina Faso to strengthen the management of infertile men. In perspective, we will design a new study that will allow us to determine the clinical significance of microorganisms in male infertility and to explore other potential factors contributing to male infertility, such as genetic or lifestyle factors.