Tryptophan is an essential amino acid that plays a vital role in the daily diet. There are three primary metabolic pathways for tryptophan in the gastrointestinal tract: indole derivatives, kynurenine pathway, and 5-hydroxytryptamine (5-HT) pathway. Among these pathways, the kynurenine pathway is regulated by the major rate-limiting enzyme Indoleamine2,3-dioxygenase1 (IDO1), whose synthesis can be expedited by the gut microbiota. In the 5-HT pathway, tryptophan hydroxylase 1 (TpH1) is the key enzyme responsible for 5-HT synthesis by colonic enterochromaffin cells (ECs) (Agus et al., 2018). Gut microbiota is of great significance in this process, specifically for indigenous spore-forming bacteria (Yano et al., 2015). For example, Clostridium ramosum (Mandić et al., 2019) and Blautia (Golubeva et al., 2017) were proved to promote 5-HT secretion from ECs. This specific mechanism may be related to the increased expression levels of Tph1, Sert, and Ido1. Tph activity determines the amount of 5-HT produced and released, whereas Sert controls the rate of 5-HT reuptake and breakdown (Mawe and Hoffman, 2013). Although the precise mechanism remains unclear, it is certain that the gut microbiota has a close affinity for tryptophan metabolism and the 5-HT pathway, which may be due to its metabolite SCFAs (Reigstad et al., 2015).

Furthermore, 5-HT exists in the sperm and is involved in sperm physiology. It can be observed in the mid-segment of human sperm (Jiménez-Trejo et al., 2012). A similar pattern for 5-HT selective reuptake transporter (SERT) was found in horse sperm (Gao et al., 2018). 5-HT can activate transmembrane adenylate cyclase (tmAC) and open membrane Ca²⁺ channels (CatSper), resulting in an influx of Ca²⁺ and the activation of protein kinase A (PKA) (Alasmari et al., 2013), which is involved in regulating tyrosine phosphorylation and sperm protein Ser/Thr phosphorylation (Kwon et al., 2014; Bae et al., 2020; Kushawaha et al., 2020). In addition, related to both CatSper and Ca²⁺ stores, the spermatozoon is hyperactivated, which is essential for sperm motility and fertilization (Alasmari et al., 2013). This range of Ca²⁺ influx can lead to improved sperm quality.

Although it has been proven that gut microbiota can improve tryptophan in serum and 5-HT in the central nervous system (Clarke et al., 2013), studies on whether 5-HT secreted with the help of gut microbiota can enter the testis through the blood circulation and play a role directly have yet to be conducted. Therefore, we supposed 5-HT secreted by the synergy of ECs and gut microbiota as a key factor that can enter the testis via blood circulation and maintain an effective concentration, which affects sperm motility partly by regulating Ca²⁺ (Figure 1).

Unsaturated fatty acids (UFAs) are fatty acids with double bonds in the hydrocarbon chains. They can be divided into monounsaturated fatty acids (MUFAs) and PUFAs. MUFAs do not have the potential adverse effects of PUFAs, such as lipid peroxidation, and can be used as precursors for PUFAs synthesis. PUFAs, especially long-chain polyunsaturated fatty acids, such as α-linoleic acid (ALA), arachidonic acid (ARA), and docosahexaenoic acid (DHA), are considered as essential fatty acids in the human body. Studies have found that PUFAs have important biological effects: (1) anti-inflammatory effects by interfering with the TLR4/MyD88 pathway and the GPR120/NF-κB pathway; (2) anti-oxidation effects by increasing the expression and activity of catalase and superoxide dismutase (SOD) to reduce reactive oxygen species (ROS) (Korbecki et al., 2019); and (3) increased stability and fluidity of lipid rafts (Kotlyarov and Kotlyarova, 2021) to ensure sperm activity (Castellini et al., 2022).

Interestingly, the gut microbiota can change the content and proportion of PUFAs in the body (Albouery et al., 2019). One of the reasons for this is the biotransformation by microorganisms. For example, Bacillus proteus and Lactobacillus plantarum reduce gut PUFAs levels by converting linoleic acid (LA) and ALA into stearic acid (Blanchard et al., 2013). Another reason may be that intestinal bacteria can regulate lipid droplet accumulation (Danielli et al., 2023) to affect the absorption of PUFAs.

For male reproductive function, PUFAs mainly affect sperm membrane stability, sperm motility, acrosome reaction, and sex hormone synthesis and increase the anti-inflammatory and antioxidant capacity of testicular cells (Please see Table 1 for details.).

Testicular tissue is a hypoxic organ that primarily uses the lactate produced by glycolysis within Sertoli cells as an energy substrate. Glucose transporters (GLUT) in Sertoli cells transport glucose from the interstitial fluid of seminiferous tubules to the cytoplasm of Sertoli cells for glycolysis. The resulting lactate is transported out of the cells through the monocarboxylate transporter (MCT) 4 on Sertoli cells, and then transported into spermatogenic cells by MCT2 for energy supply (Rato et al., 2012). Even under aerobic conditions, the head and principal piece (tail) of the spermatozoon are capacitated mainly by glycolysis (du Plessis et al., 2015).

Gut microbiota and its metabolites can promote glycolysis to maintain sperm energy supply and enhance sperm motility. For example, leucine supplementation can improve the average curve speed of sperm in boars (Lin et al., 2022). 5-HT can keep tryptophan away from the melatonin pathway through the kynurenine pathway to reduce circulating melatonin levels (Laborda-Illanes et al., 2021), which play a vital role in male reproduction, including promoting the production of lactate by Sertoli cells, activating some glycolysis-related enzymes (Rocha et al., 2014), and increasing the energy supply of sperm (Rato et al., 2012). In addition, Bacteroides, Streptococcaceae, Akkermansia are thought to improve the energy supply for spermatid cells by improving glycolysis (Zhu et al., 2021). Previous studies have found that some drugs, such as Pioglitazone (Meneses et al., 2016), Metformin (Alves et al., 2014), Spermidine (Wang et al., 2022) and Nicotinamide mononucleotide (Ma et al., 2022a), can support spermatogenesis by benefitting gut microbiota and improving the glycolysis of Sertoli cells.

In addition, the gut microbiota can regulate blood glucose levels, which is also an important substrate for supporting testicular glycolysis. For example, bile salt hydrolase (BSH) producing bacteria, Barnesiella and Clostridium XlVa, promote the production of unconjugated cholic acid (CA), chenodeoxycholic acid (CDCA), and the secondary bile acid deoxycholic acid (DCA) to inhibit hepatic gluconeogenesis via the FXR-SHP-FOXO1 pathway, thereby reducing blood glucose levels (Zhuang et al., 2021). And in one study, metformin was used to alter folate and methionine metabolism to inhibit the growth of B. fragilis, reduce BSH activity, thereby inhibiting intestinal FXR signaling (Sun et al., 2018) which can induce endoplasmic reticulum (ER) oxidative stress to significantly attenuate mitochondrial citrate synthase activity, triggering an increase in hepatic mitochondrial acetyl-CoA levels and pyruvic carboxylase (PC) activity (Xie et al., 2017). Furthermore, September Numata et al. also found a molecular basis to support our argument: not only SCs express GLUT, but sperm can also express a Na+-dependent sodium-glucose cotransporter (SGLT), whose deletion decreases glucose uptake, glycolytic activity, and ATP production (Numata et al., 2022). Therefore, we hypothesized that gut microbiota may alter sperm motility by regulating blood glucose levels through metabolites that affect SCs and sperm glycolysis. All in all, gut microbiota and its metabolites may act on testicular glycolysis and regulate blood glucose to affect germ cell energy supply and altering sperm motility. Metabolites of Gut Microbiota Participate in Spermatogenesis.

Insulin-like growth factor type I (IGF-1) is a key factor in maintaining the pluripotency of mouse spermatogonial stem cells (Huang et al., 2009). Gut microbiota metabolites such as branched chain amino acids (BCAAs) can also improve the secretion of IGF-1 (Pedrosa et al., 2013; Habibi et al., 2022). Serum IGF-1 levels can be increased by supplementation with Bacillus amyloliquefaciens C-1 and Bacillus subtilis (Du et al., 2018). IGF-1 can activate the Ras/MAPK, Ras/ERK (Shen et al., 2014) and PI3K/AKT (Pitetti et al., 2013) pathway to promote cell proliferation, cell differentiation, and cell survival, which can accelerate the differentiation of spermatogonia to primary spermatocytes (Hakuno and Takahashi, 2018; Józefiak et al., 2021). The insulin/IGF signaling pathway are involved in follicle-stimulating hormone-mediated (FSH-mediated) proliferation of immature Sertoli and Leydig cells. Without this, the proliferation and development speed of Sertoli and Leydig cells would slow, leading to testicular shrinkage and reduced sperm production (Pitetti et al., 2013; Neirijnck et al., 2018).

Endotoxemia refers to systemic inflammation caused by a poorly regulated host response to LPS (Płóciennikowska et al., 2015). When intestinal barrier permeability changes, microbial-related molecular patterns (MAMPs), such as LPS, lipoproteins, peptidoglycans, and lipoproteins, can cross the intestinal barrier and enter the circulation, then arrive at the testicles through the testicular artery and bind to pattern recognition receptors (PRRs) on the cell membrane of testicular cells, eventually causing cellular oxidative stress, local inflammation and destruction of the testicular structure (Wiest and Garcia-Tsao, 2005). The most typical and main-affecting MAMPs are LPS. The entry of LPS into circulation is believed to be a frequent cause of idiopathic male infertility due to systemic inflammation and oxidative stress (Agarwal et al., 2017).

Studies suggest that the classical inflammatory pathway, the Toll-like receptor (TLR) pathway, is involved in the response to orchitis induced by endotoxemia (Palladino et al., 2018). The TLR4/MyD88 pathway can activate IKK to induce orchitis via NF-kB (Saad et al., 2016), elevate the level of inflammation and oxidative stress, and finally result in direct damage to Leydig cell function and sperm DNA damage (Pearce et al., 2019). In addition, LPS-mediated TLRs activation can inhibit MAPK, p38, ERK, and JNK signaling, leading to autophagic dysfunction (Wang et al., 2021). This disrupts the ability of testicular cells to resist inflammation and oxidative stress, which leads to testicular injury in orchitis.

BTB is formed by the cellular junction of adjacent Sertoli cells at the base of the seminiferous tubule. It is composed of capillary endothelium, basement membrane, connective tissue, and multiple types of cell junctions from Sertoli cells, mainly consisting of tight junctions, basic electrophysiological specializations, gap junctions, and cell-like junctions (Neto et al., 2016; Lustig et al., 2020). Regulators of cell connections in the testis include hormones [such as testosterone (Yan et al., 2008) and androgens (Xia et al., 2005)], cell factors [IL-1a (Sarkar et al., 2008) and TNF-α (Li et al., 2006; Xia et al., 2009)], growth factors [HGF (Catizone et al., 2012), NO (Lee and Cheng, 2008) and TGF-β3 (Xia et al., 2009)], and gut microbiota.

The detrimental impact of the gut microbiota on BTB can be partially ascribed to inflammation and oxidation, mainly caused by LPS. A study found that LPS treatment can lead to orchitis and significantly decrease the expression of cell junctions, such as testicular intercellular adhesion molecule 1, tight junction protein 1, and gap junction α-1 protein (Shen et al., 2022). Furthermore, IL-6, induced by gut microbiota, can damage the tight junctions of Sertoli cells by disrupting the ERK-MAPK signaling pathway and changing the localization and number of BTB component proteins (Zhang et al., 2014). This change in BTB permeability can be restored to the levels of cell adhesion proteins by supplementation Clostridium butyricum, a high-level butyric acid secretor (Al-Asmakh et al., 2014). In summary, elevated LPS and systemic endotoxemia can damage the normal structure of the BTB by causing inflammatory damage and disrupting cell junctions (Figure 2).

The HPT axis means that the hypothalamus releases gonadotrophin releasing hormone (GnRH) to promote pituitary release ICSH and FSH, and the two can respectively regulate the release of testosterone from Leydig cells and anti-Mullerian (AMH) from Sertoli cells in the testis, also there exists a corresponding negative feedback mechanism (Aleksic et al., 2022). The gut microbiota can participate in the regulation of gonadal development and affect each level of the HPT axis (Shen et al., 2022). This includes both the supportive role of probiotics and damaging role of pathogenic bacteria.

Probiotics can restore damage to the HPT axis. Gut microbiota can improve insulin-like growth factor receptor type I (IGF1R), possibly through SCFAs (Yan and Charles, 2018). Supplementation with Bacillus amyloliquefaciens C-1 and Bacillus subtilis increased the abundance of SCFA-producing bacteria and elevated serum IGF-1 concentrations (Du et al., 2018). IGF-1 appears to be involved in the proliferation and differentiation of Sertoli cells by mediating the effects of FSH through the PI3K/AKT pathway (Cannarella et al., 2018). Moreover, IGF-1 can participate in mitochondriogenesis in Leydig cells and affect steroid hormone synthesis (Radovic et al., 2019). In a mouse model lacking the insulin receptor (InsR) and IGF1R, the adrenal cortex and testis are significantly shrunk along with develop-inhibited Leydig cells, leading to low corticosterone and testosterone (Neirijnck et al., 2018).

However, the entry of LPS into circulation is hypothesized to be a key factor in triggering male hypogonadism (Tremellen, 2016). LPS, which is mainly produced by cytoderm lysis of gram-negative bacteria, can act on all levels of the HPT axis independently and eventually damages the male gonad. LPS treatment resulted in the activation of the medial preoptic area (mPOA) of the hypothalamus, increased FSH and ICSH release, and HPT axis disorder (Shen et al., 2022). Studies have shown that elevated LPS may directly result in compromised Leydig cells functionality (Tremellen et al., 2018). Even low doses of LPS can drive the body's inflammatory response independently of FSH and ICSH, damage Leydig cells, and lower serum testosterone (Tremellen et al., 2017, 2018). However, after 6 h of low-dose LPS injection, treatment with RAPID (reduced temperature, acidification, protease inhibition, isotope exogenous control, and dilution) effectively increased corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH) levels (Goebel et al., 2011). This provides a methodological basis and valuable opportunity for us to deal with acute endotoxemia and save male reproductive function.

Ever since the first evidence of cortisol synthesis by microbiota was found (Nabarro et al., 1957), the human gut microbiome (HGM) has gradually been recognized to play a more important role in steroid modification than the host, which is critical for estrous cycles, testosterone levels, and reproductive function (Cross et al., 2018).

Most bacteria located in the intestinal and urogenital tracts encode cortisol-inducible operons. These include steroid-17,20-desmolase, synthesized by HGM, which can inactivate cortisol by cutting the side chain (Devendran et al., 2018). Typical representatives, such as Clostridium scindens and Propionimicrobium lymphophilum, have extremely high activities against glucocorticoids (Ly et al., 2020).

Numerous studies have shown that the gut microbiota can produce HSDH and impact male reproductive health. Here, we list some common examples: Mycobacterium neoaurum can degrade serum testosterone with the help of 3β-HSDH (Li et al., 2022a). Compared to wild-type Comamonas testosterone, the 7α-HSDH knockout mutant showed reduced degradation of testosterone, estradiol, and cholesterol (Ji et al., 2014). As an important glucocorticoid metabolizing enzyme in Leydig cells, 11β-HSDH is involved in regulating steroidogenic gene expression and testosterone production in Leydig cells (Wang et al., 2019). Butyricicoccus desmolans and Clostridium cadaveris have previously been reported to demonstrate steroid-17,20-desmolase and 20β-HSD activities, which are responsible for the formation of androstanes from cortisol (Devendran et al., 2017).

Notably, the conversion of dehydroepiandrosterone, androstenedione, and testosterone to dihydrotestosterone (DHT) is an essential function of 17β-HSDH enzymes (Bélanger et al., 2003). Some attempts have been made to introduce gene fragments of 17β-HSDH producing bacteria into Mycobacterium smegmatis by genetic engineering technology, and natural sterols have been bioconverted to produce testosterone successfully (Fernández-Cabezón et al., 2017). In human patients, 17β-HSDH deficiency caused by related gene mutations also results in low serum testosterone (Ben Rhouma et al., 2017; Yang et al., 2017). Perhaps the usage of the bacterial-derived HSDH production pathway will become a new dawn for patients with HSDH-related diseases, but whether it has the same effects as endogenous HSDH remains to be studied. Although the role of microbial HSDH has not been fully elucidated, it has therapeutic potential as a steroid pool modulator or drug targets in the future (Doden and Ridlon, 2021).

Overall, the expression of HSDH by gut microbiota forms the physiological basis for intestinal bacteria to affect the host's sex hormones.

In general, active hormones bind to glucuronides in the liver to produce the main metabolites of testosterone (T), such as testosterone glucuronide (TG), androsterone glucuronide (AG), etiocholanolone glucuronide (EtioG), and dihydrotestosterone glucuronide (DHTG). These metabolites are transported via the bile duct, with one part entering the blood and the other part excreted through urine and feces (Auer et al., 2020). Notably, intestinal microorganisms can mediate the deglucuronidation of DHT and T during this process, releasing inactive free androgens for host re-absorption (Colldén et al., 2019). This may be related to Clostridia, which codes for β-glucuronidase and participates in the deglucuronidation of DHT and T (Gloux et al., 2011; Flores et al., 2012; Colldén et al., 2019). However, there is a lack of adequate research to prove that sex hormones deglucuronidated by gut microbiota can affect male reproductive function or sexual behavior, and further research efforts still need to be invested in order to explore this promising issue in greater depth.

Probiotics are live bacteria that offer a range of potential health benefits to the human body. Oral supplementary probiotics, Lactobacillus, Bifidobacterium, Enterococcus, Collinsella, and Blautia, can enhance sperm quality by alleviating sperm inflammatory response and oxidative stress (Helli et al., 2022; Cao et al., 2023; Zhang et al., 2023). These may be linked to probiotics can regulate the Nrf2-Keap1-ARE signaling pathway to increase antioxidant activity and enhance neutralization of reactive oxygen species, ultimately resulting in improved sperm concentration and motility in infertile men (Helli et al., 2022).

Above all, it is Lactobacillus that are widely believed to have potential benefit for male infertility (Doroftei et al., 2022). We have drawn Table 2 to give a superficial description of recent studies on the effects and mechanisms of lactic acid bacteria on the male reproductive system. Especially L. rhamnosus strain NCDC 610 and L. fermentum strain NCDC 400 are suggested as alternative options to pharmaceuticals because of their positive impact on weight reduction and their ability to enhance endogenous testosterone levels (Akram et al., 2022). The studies have demonstrated the efficacy of probiotics in addressing male infertility, and their considerable therapeutic potential and extensive development space should not be overlooked. Therefore, we anticipate additional research efforts to delve more deeply.

FMT is a valuable tool for demonstrating the vital role of gut microbiota in regulating host physiology (Antushevich, 2020), and conducts a potential to improve infertility. It is common to see to increase beneficial bacteria by drugs and transplant them through FMT to improve male infertility (Figure 3). For example, by transplanting fecal microbiota from AOS users (AOS-FMT), Zhang et al. showed that the gut microbiota can be used to improve spermatogenesis and treat busulfan-induced male infertility for the first time (Zhang et al., 2021a,b). And AOS-FMT are also used to treating male infertility induced by HFD (Hao et al., 2022a), T2DM (Yan et al., 2022) and T1DM (Hao et al., 2022b). What's more, FMT, from healthy microbiota benefitted by traditional Chinese medicine such as Guijiajiao (Sheng et al., 2023), Radix Rehmanniae and Cornus Officinalis (Chen et al., 2022b) to male patients with impaired fertility, can improve testicular damage and restore spermatogenesis. Even FMT from ordinary healthy mice was able to exert a lesser degree of beneficial effect on the alleviation of testicular damage (Hao et al., 2022b).

These mechanisms are mostly related to restoring the diversity of gut microbiota, changing the blood metabolome of the host (Yan et al., 2022), inhabiting epididymal inflammation (Sheng et al., 2023), and restoring testicular microenvironment (Yan et al., 2022) to improve spermatogenesis and male infertility. For example, AOS-FMT increased beneficial bacteria such as Bacteroidales, Lactobacillaceae, Bifidobacteria, Sphingomonadales and Campylobacterales, to improve spermatogenesis and semen quality by increasing DHA and EPA in blood metabolome and testis metabolome (Zhang et al., 2021a; Hao et al., 2022b). Parabacteroides distasonis transplantation increased polyamine levels in the testis and cecum, and improved testicular histology, testicular index, testicular testosterone levels, expression of genes involved in spermatogenic events, inflammatory factors, and oxidative stress (Zhao et al., 2021).

All in all, FMT may serve as a novel and promising therapeutic approach to improve semen quality and male fertility via the gut microbiota-testis axis.