As a worldwide human health problem, infertility is widely reported to impact over 186 million individuals globally (Inhorn and Patrizio, 2015). There are numerous theories about the etiology of infertility, however, the specific mechanisms are not yet known. Up to 50% of infertility is due to male factors (Minhas et al., 2021). In recent decades, in both developed and developing countries, male fertility rates have declined, raising concerns among academics and society at large (Gunes et al., 2018). To our knowledge, there are no known molecular biomarkers for male infertility. An important risk factor for male infertility is poor sperm quality. In addition to overweight (Salas-Huetos et al., 2021), substance abuse (Gundersen et al., 2015), and testicular inflammation (Bryan et al., 2020), poor lifestyle habits such as smoking (Sharma et al., 2016), alcohol consumption (Li et al., 2022), and consumption of sugary beverages (Nassan et al., 2021) can affect sperm quality.

The gut microbiota, often referred to as the second human-genome, as a vast bacterial community, is essential for maintaining balance between the external and internal environments of the host. Gut microbiota has been linked to many diseases, including psychiatric disorders (Mangiola et al., 2016), hematological disorders (Yang et al., 2022), and orthopedic disorders (Liu et al., 2019). Studies suggest that there may be a correlation between gut microbiota and male fertility (Zhao et al., 2021). Confounding factors, such as environment, lifestyle, and dietary habits, often influence traditional observational studies and greatly interfere with the inference concerning the relationship of causality of risk factors and endpoint outcomes. In Mendelian randomization (MR), single nucleotide polymorphisms (SNPs), which are instrumental variables (IVs), are used to infer cause-and-effect correlations between exposure factors and outcomes (Greenland, 2000; Bowden and Holmes, 2019). In order to understand the causal relationship between gut microbiota and disease, MR methods are extensively used, and there are no studies on the causality between gut microbiota and male infertility risk. In this study, we will evaluate the existence of cause-and-effect correlation between the gut microbiota and male infertility through the use of two-sample MR.

Infertility, which is a worldwide public health problem (Inhorn and Patrizio, 2015), has been closely associated with the reduction of newborns and increased population aging, and could have a serious potential negative influence on social development. Throughout the human digestive tract, mucosal surfaces are colonized by a host of symbiotic microbes. Gut microbiota affect host systems such as digestion (Franzosa et al., 2019), immunity (Thaiss et al., 2016), and the nervous system (Sharon et al., 2016). In our current study, we aimed to discover whether male infertility is influenced by gut microbiota. It is the first two-sample Mendelian randomization study to examine the cause-and-effect links between gut microbiota and male infertility risk using the European cohort, to our knowledge.

Through the use of the MR method, 119 gut microbiota and male infertility were causally studied at the genetic level, effectively avoiding the confounding variables that can interfere with an observational study. In our study, we found a positive and direct genetic causality between human gut microbiota and male infertility, with Allisonella, Anaerotruncus, Barnesiella, Intestinibacter, and Lactococcus all showing to have an effect. Furthermore, Bacteroides Romboutsia, Ruminococcaceae (NK4A214group), and Ruminococcaceae (UCG011) were found to prevent male infertility. Combined with our study and a pubmed search, Allisonella and Intestinibacter were not reported to be associated with male infertility, which provided a new possible way to investigate male infertility and contributes positively to the genetic study of male infertility.

Several observational studies reported the association of gut microbiota and reproductive capacity in mammals. A potential mechanism for alteration of the reproductive system in male rats was found through the disruption of the gut microbiota by Ditridecyl-phthalate (Zhao et al., 2020a). Gut microbiota could also affect testicular secretion function of mice (Al-Asmakh et al., 2014). Translocation of the gut microbiota, through a multiple immunological mechanism, causes inflammation of the testicles and epididymis, which affects spermatogenesis (Wang and Xie, 2022). Self-reported stress was negatively associated with semen quality in a cross-sectional study (Nordkap et al., 2016). Gut flora is closely associated with the hypothalamic-pituitary-adrenal (HPA) axis, and the HPA regulates a series of physical processes to respond to external stresses (Foster and McVey Neufeld, 2013; Farzi et al., 2018). One animal experiment has reported the increased abundance of Anaerotruncus in prenatal stress (PNS) animal models (Golubeva et al., 2015), which suggests Anaerotruncus as a possible biomarker of stress. It has been shown that diabetes has negative effects on male fertility (Chen et al., 2021; Lotti and Maggi, 2023). The abundance of Anaerotruncus was significantly increased in mice that had testicular injury caused by diabetes, however, its abundance was significantly reduced after treatment with cornuside (Cor), which suggested that Anaerotruncus could be a potential biomarker of testicular injury (Liu et al., 2021); this is in line with our findings. Vitamin A has a critical effect on the process of spermatogenesis (Hogarth and Griswold, 2010). Mice with metabolic syndrome (Mets) have abnormal testicular spermatogenesis due to impaired vitamin A absorption in the intestine, which may be associated with the significantly reduced abundance of Ruminococcaceae (NK4A214group) (Zhang et al., 2022). As an androgen found in epididymal cells, dihydrotestosterone (DHT) regulates sperm maturation (Robaire and Henderson, 2006), and a large-scale animal study suggested that an abundance of Ruminococcaceae was positively correlated with DHT (Chen et al., 2023). Regulation of the gut microbiota to improve the symptoms of drug-induced male infertility was demonstrated by several studies (Zhao et al., 2020b; Sun et al., 2022). Triptolide -induced testicular injury mouse model results in an increase in relative abundance of Firmicutes and a decrease in relative abundance of Parabacteroides, causing the reduction of spermine synthesis. By transferring normal microbiota into mice, testicular dysfunction can be reversed (Zhao et al., 2021). Busulfan chemotherapy is an effective treatment for leukemia (Allan, 1989), although a side effect of this is male infertility. In the intestinal tract, Busulfan will increase the abundance of Firmicutes and Proteobacteria (Zhao et al., 2020b). Chestnut polysaccharides can restore the imbalance of gut microbiota and thus restore the damaged spermatogenesis caused by Busulfan (Sun et al., 2022). Alginate oligosaccharides (AOS) improve male infertility after Busulfan application with the improvement of gut microbiota (Zhao et al., 2020b). Research has demonstrated that AOS may improve sperm quality by increasing Bacteroides' abundance and mitigating Mucispirillum abundance in the gut microbiota (Hao et al., 2022).

A Westernized diet (a diet high in high saturated fatty acids and refined sugar refined) has been reported as a significant factor in the long-term decline in human sperm concentrations (Wu et al., 2011). Research indicates that the different types of diet patterns based on saturated, monounsaturated, or polyunsaturated fatty acids may induce alterations in gut microbiota composition (Patterson et al., 2014). The fatty acids level (Bunay et al., 2021), especially DHA, are associated with spermatogenesis (Hale et al., 2019). A high-fat diet (HFD) induces disruption of the gut microbiome, as reflected by decreases in Bacteroides and increases in Firmicutes and Aspergillus in mice models (Hildebrandt et al., 2009; Graham et al., 2015). Moreover, HFD also leads to alterations in the mucosal epithelium of the intestine, which results in intestinal barrier dysfunction (Moreira et al., 2012), higher permeability (Ji et al., 2011), and more diffusion of endotoxins into the bloodstream (Ji et al., 2011). In mice, excess endotoxin activates the immune system, resulting in an increase in proinflammatory factors in the epididymis, and decreasing sperm viability (Ding et al., 2020).

The major metabolism products of the gut microbiota are short-chain fatty acids (SCFA), which are essential for intestinal immunity and health of the host (Martin-Gallausiaux et al., 2021). Research shows that butyric acid may have a facilitative effect on spermatogenesis (Yan et al., 2022). For our study, the bacteria Ruminococcaceae (NK4A214group) (Verhoeven et al., 2021), Ruminococcaceae (UCG011) (Radjabzadeh et al., 2022), and Romboutsia (Qin et al., 2021) were able to produce butyric acid. Major sources of gastrointestinal epithelium are SCFAs (Martin-Gallausiaux et al., 2021), which are closely associated with maintaining the mucosal barrier function of the intestinal tract. An animal study reported that the dietary fiber supplements improve spermatogenesis and semen quality by promoting SCFA production through improved gut microbiota (Lin et al., 2022). A metabolically healthier gut microbiota may be achieved by a diet that is supplemented with low animal fat, low animal protein, and high fiber (Fan and Pedersen, 2021), to prevent endotoxin diffusion into blood; this might improve infertility in males in some cases. However, this requires a further randomized controlled experiment to demonstrate.

Our study contains several strengths. (1) The data on gut microbiota and male infertility were obtained from a European population, effectively avoiding bias caused by differences in ethnicity; this can better reflect the cause-and-effect correlation between gut microbiota and male infertility. (2) Gut microbiota inherited variants from the largest usable GWAS metadata, which guarantees power of the IVs for the analysis of MR. (3) Genetic variants found in gut microbiota were derived from the maximum amount of GWAS metadata available, ensuring IV power in MR analyses. (4) Our study met all three assumptions of the MR analysis. To elaborate, we screened SNPs that were closely related with the gut microbiota (p < 1 × 10⁻⁵) and passed the F-statistic checks (F > 10), which satisfied the first assumption. The screened SNPs were evaluated by LD analysis, which fulfilled the second assumption. We conducted a study in which we used MR-Egger regression and MRPRESSO analyses to exclude SNPs with horizontal pleiotropy, and the last acquired SNPs satisfied the third hypothesis. (5) There may be some reduction in interference from confounding factors and reverse causation with MR methods. However, it is also important to note that our study has some limitations. Firstly, the results of the analysis, which was performed only for European populations, were not sufficiently well representative of populations in other regions. Secondly, there may be a potential bias in the study as the number of cases in the GWAS data for male infertility is relatively small. And finally, GWAS of male infertility were not categorized, so our study was not well able to reveal the association of gut microbiota with specific types of male infertility. Future studies and randomized controlled trials are required to better reveal the cause-and-effect correlation between gut microbiota and male infertility.

In summary, the MR analysis performed using data of a large sample of GWAS analysis showed a cause-and-effect relationship between gut microbiota and male infertility. However, greater GWAS databases need to be established for the exploration of mechanisms between gut microbiota and male infertility. There are several gut bacteria identified that may reduce the prevalence of male infertility and could offer hope in treating and preventing male infertility.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was not required from the participants or the participants' legal guardians/next of kin in accordance with the national legislation and the institutional requirements.

Conceptualization and writing—original draft preparation: Z-dF and YW. Writing—review and editing: H-lY. All authors contributed to the article and approved the submitted version.