Potential relationship of the gut microbiome with testosterone level in men: a systematic review

Introduction

A microbiome is a collection of all the taxa that constitute the microbial community in a particular organ or system. Bacteria constitute approximately 99% of all microbiomes in the human body, whereas other microorganisms, such as viruses, archaea, protozoa, and fungi, constitute the remaining 1% (Marchesi et al., 2016). Several studies have demonstrated the influence of the microbiome in predicting and improving the clinical outcomes of acne vulgaris (Ruchiatan et al., 2023), atopic dermatitis (Menul Ayu Umborowati et al., 2022), pemphigus vulgaris (Satriyo & Vidyani, 2024), neurodevelopment delay (Fadlyana et al., 2022), tuberculosis (Wiqoyah et al., 2021), and sexual dysfunction (Darmadi et al., 2024). The human gut microbiome has the highest density and number of microorganisms, with the small intestine and colon differing in their relative abundances (greater density at the distal end than at the proximal end) (Marchesi et al., 2016; Schroeder & Bäckhed, 2016). Trillions of microbes inhabit the gut of healthy individuals, with different parts of the gut harboring different microorganisms. The stomach and duodenum contain approximately 10¹–10² CFU/mL of the gut microbiome, the jejunum and ileum contain 10⁴–10⁸ CFU/mL of gut microbes, and the colon with 10¹⁰–10¹² CFU/mL is the most abundant in gut microbes (Cresci & Bawden, 2015). The majority of the gut microbiome comprises only five phyla (Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Verrucomicrobia). However, the numbers and species diversity varies considerably. Most of the gut microbiome is comprised of anaerobic bacteria with a mass of 1.5–2 kg (Schroeder & Bäckhed, 2016). The remaining 1% of the microorganisms in the human body are found in the respiratory, digestive, integumentary, and reproductive systems (Kim, 2022; Koedooder et al., 2019).

The gut microbiome is established early in life and remains relatively constant throughout adulthood. It is influenced by several factors such as genotype, body mass index (BMI), lifestyle, physical activity, and dietary and cultural habits (Koliada et al., 2021). Metagenomic research has demonstrated that ethnicity, age, stress, psychological factors, antibiotics, health of the pregnant mother, and delivery method influence the gut microbiome. The host and the microbiome communicate bidirectionally and affect each other’s functions (Kim, 2022). The gut microbiome influences health and disease physiology by affecting the metabolic functions and immune system, protecting against pathogens, and directly or indirectly contributing to various physiological processes (Shreiner, Kao & Young, 2015). It also communicates with the brain through the gut-brain axis (Ranuh et al., 2019). The gut microbiome is involved in the development, maturation, modulation, and stimulation of the host (Vemuri et al., 2019). Furthermore, the gut microbiota regulates metabolism by producing vitamins and short-chain fatty acids (SCFAs). The gut microbiome interacts with the intestine locally; it also interacts with and affects distant organs such as the adipose tissues, liver, pancreas, cardiovascular system, brain, lungs, and reproductive system. Each of these organs forms a network with the intestines, and the gut microbiome influences almost every organ of the human body (Li et al., 2022).

The interaction between gut microbiome and reproductive hormones was first introduced by Flak, Neves & Blumberg (2013) under the term “microgenderome”. In the testicles, Leydig cells produce testosterone, the main sex steroid, at a secretion rate of 7 mg/day (Hohl, 2023). The adrenal gland also produces approximately 5% of total testosterone. Testosterone can bind to sex hormone-binding globulin (SHBG) and albumin or circulate freely in plasma. The fundamental mechanism that influences testosterone production is the hypothalamic-pituitary-testicular axis. The gonadotropin-releasing hormone secreted by the hypothalamus stimulates the pituitary gland to release luteinizing hormone, which then stimulates the testes to produce testosterone (Hohl, 2023). Testosterone is essential for masculinization, normal sexual function, and spermatogenesis (Brinkmann, 2011; MacLeod et al., 2010). It targets the brain, skin, muscle, bone, hair follicles, and hematopoietic system. This vast biological effect demonstrates that testosterone is important for the overall health of men, including mood, energy levels, and cognitive function (Li et al., 2022). Studies have suggested a possible relationship between the gut microbiome and androgen levels in men. Androgens can profoundly alter gut flora via a complex pathway (Harada et al., 2016). Yan et al. (2024) reported a causal effect of SHBG on gut microbiota. Higher SHBG levels in men were associated with Dorea and Clostridiales. Genome-wide association studies have shown that Alphaproteobacteria are associated with high levels of SHBG, a carrier of sex hormones (Yan et al., 2024). The gut microbiome may also influence testosterone production and metabolism through various mechanisms (Colldén et al., 2019). Emerging evidence suggests that the gut microbiome influences testosterone production through various mechanisms. One proposed pathway involves the modulation of the hypothalamic-pituitary-gonadal (HPG) axis, where the gut microbiota may affect the release of gonadotropin-releasing hormone (GnRH) and, consequently, luteinizing hormone (LH), which stimulates testosterone synthesis in the testes (Chen et al., 2024). Additionally, certain gut microbes possess steroid-processing enzymes that can directly affect androgen metabolism, contributing to testosterone levels in circulation (Tang et al., 2024; Zou et al., 2024). In contrast, testosterone and other hormones influence the gut microbiome. Diet, lifestyle, toxins, and drugs, among other factors, can influence the gut microbiome (Li et al., 2022). Presently, no conclusive evidence is available supporting a direct link between the microbiome and testosterone levels, and the microgenome theory remains unproven. The gut microbiome may also affect sexual and reproductive problems in men, including infertility.

Notably, researchers have conducted a comprehensive analysis to evaluate the association between the gut microbiome and sex hormone levels in women (D’Afflitto et al., 2022); however, a similar analysis has not been conducted for men. In this systematic study, we aimed to determine the significance of microgenderomes and the probable relationship between the gut microbiome and testosterone levels in men.

Materials and Methods

Search strategy, study selection, and data extraction

Three authors (CP, GL, and MCH) performed keyword searches of the specified databases. All studies that matched the keyword findings were exported to the Mendeley reference manager. Duplicate screening was performed using Mendeley, followed by screening of titles and types of articles; two authors independently evaluated the articles using abstracts. In case of disagreement during screening/data extraction, the authors discussed it internally until an agreement was reached. For example, the design of the study included and the results. The authors also asked experts who were not included in the group for consideration. The PRISMA flowchart shows the study exclusion process and related reasons (Fig. 1).

Data were extracted from all eligible studies (CP, GL, and MCH). The first author’s name, publication year, research type, inclusion/exclusion criteria, sample size, dominant microbiome type, and study outcomes were recorded in an Excel spreadsheet. This information was obtained from the individual studies. The authors were contacted to obtain the missing information, if any. Statistical significance of the results was determined using appropriate measures of association, such as relative risk (RR), odds ratio (OR), or correlation coefficient (r), along with corresponding p-values. All authors collectively examined all the findings including those that were challenging to interpret. We performed qualitative analyses and comparisons without conducting quantitative analyses because statistical analyses and presentations differed across the studies.

Results

Discussion

Ten studies reported the relationship between the gut microbiome and testosterone levels. Wilmanski et al. (2019) discovered that the gut microbiome positively correlated with a testosterone metabolite (5α-androstane-3β-17α-diol disulfate), and Bacteroides were anti-correlated with this metabolite. Shin et al. (2019) reported that Acinetobacter (r = 0.3782, p = 0.0359), Dorea (r = 0.3976, p = 0.0268), Megammonas (r = 0.4161, p = 0.0199), and Ruminococcus (r = 0.4589, p = 0.0094) positively correlated with testosterone levels in men. Jie et al. (2021) found that the gut microbiome had the greatest predictive power for metabolites and plasma hormones. Matsushita et al. (2022b) reported an association between the gut microbiome and testosterone levels in a Japanese population, with the specific finding that the number of Firmicutes in the gut correlated with testosterone levels (r = 0.3323, p = 0.0141).

Identifying the specific microbes that predominantly affect testosterone levels could be valuable. Several microbiome types were consistently reported across studies, such as the phylums Bacteroidetes and Firmicutes (Jie et al., 2021; Koliada et al., 2021; Matsushita et al., 2022b; Mayneris-Perxachs et al., 2020; Shin et al., 2019; Wilmanski et al., 2019). In contrast to the other study findings, Mayneris-Perxachs et al. (2020) used an orthogonal partial least squares model to predict circulating testosterone levels in men and found that the gut microbiome could not predict circulating testosterone levels. A quantitative analysis of these study findings would be valuable; however, the data presentation across these studies differed significantly. Furthermore, some studies have reported only phyla rather than specific microbiome species (Jie et al., 2021; Matsushita et al., 2022b; Shin et al., 2019).

Thus, the gut microbiome influences testosterone levels, which may also control gut microbiome diversity. This is consistent with the findings of Matsushita et al. (2022b), who discovered a more significant number of gut microbes in the high-testosterone group than in the low-testosterone group. Androgens maintain intestinal homeostasis by balancing bone morphogenetic proteins and wingless/transforming growth factor-beta signaling in stromal cells (Yu et al., 2020). Therefore, the role of androgens in regulating the intestinal barrier and microenvironment may affect and govern the gut microbiome.

Sex hormones, such as estrogen and testosterone, influence gut microbiome diversity (D’Afflitto et al., 2022; Ma & Li, 2019). High estrogen levels are associated with higher Bacteroidetes levels and lower levels of Firmicutes and the Ruminococcaceae family (D’Afflitto et al., 2022). Progesterone boosts Bacteroides and Prevotella intermedius growth in mice (He et al., 2021). The gut microbiome composition of female mice was similar to that of castrated male mice (He et al., 2021). Males with high testosterone levels had higher gut microbiome diversity. After puberty, males have relatively lower α gut microbiome diversity than females (Wang et al., 2019); however, a Japanese study reported different results (Matsushita et al., 2022b). The gut microbiome diversity did not differ significantly between males and females (Takagi et al., 2019).

Shin et al. (2019) observed that Acitenobacter, Dorea, Ruminococcus, and Megamonas were prevalent in males with high testosterone levels, whereas Atopobium was less common. Of the gut microbiomes, Ruminoccus was the genus most significantly associated with testosterone levels. Other studies conducted under different conditions and locations have revealed significant differences in the gut microbiomes (Fransen et al., 2017; Haro et al., 2016; Markle et al., 2013). Firmicutes and Actinobacteria were more prevalent in men than in women, whereas Bacteroidetes were less prevalent (Koliada et al., 2021). Koliada et al. (2021) reported similar findings, in which men and women had different types and numbers of specific microbiomes. Sex-specific differences in the gut microbiome may be associated with the differences in hormone metabolism, sexual function, and health profiles between men and women.

Shin et al. (2019) reported that Acinetobacter, Dorea, Megammonas, and Ruminococcus were positively correlated with serum testosterone levels in Korean men. However, Mayneris-Perxachs et al. (2020) reported that Actinobacteria, Proteobacteria, Firmicutes, and Verrucomicrobia were negatively associated with testosterone levels in Spanish men, whereas Prevotellaceae, Cytophagaceae, Fibrobacteriaceae, Sphingobacteriaceae, and Idiomarinaceae were positively associated with testosterone levels. Matsushita et al. (2022b) also reported that Firmicutes were positively correlated with testosterone levels in a Japanese male population. In addition, Wilmanski et al. (2019) reported that microbiome diversity is positively associated with androstane-3-17-diol disulfate (a testosterone metabolite). The strongest positive predictor, 5-androstan-3-17 diol disulfate, also correlated with the Bacteroides genus, suggesting that the dominance of this taxon was linked to a decline in Shannon diversity. Diet, drugs/pharmaceuticals, geography, birth process, infant feeding methods, stress (exercise, metabolic, and psychological), lifestyle, physical activity, genotype, BMI, and cultural practices determine gut microbiome diversity (Cresci & Bawden, 2015; Koliada et al., 2021).

Host and gut microbiomes communicate bidirectionally, indicating that they influence each other’s functions (Kim, 2022). A well-known function of the gut microbiome is to modulate the immune system (particularly the innate immune system) which influences gut-specific diseases and conditions. Obesity-related diseases, liver diseases, inflammatory bowel diseases, and colitis-associated malignancies are associated with the gut microbiome (Marchesi et al., 2016). Folates, indoles, secondary bile acids, trimethylamine-N-oxide, neurotransmitters (serotonin and gamma-aminobutyric acid), and short-chain fatty acids (SCFA) are vital metabolites in the gut microbiome. Previous study findings show that these microbial metabolites affect the host metabolism by binding to specific nuclear receptors or membranes. SCFA are the most widely investigated metabolites because they are recognized by G-protein-coupled receptors (GPR-41 and GPR-43). Stimulation of these receptors by short-chain fatty acids (SCFA) activates the secretion of peptide-1, such as glucagon or peptide YY, which plays a role in glucose metabolism. The gut microbiome can also influence testosterone levels through the gut-brain axis. Alterations in the composition of the gut microbiome and its active metabolites, such as trimethylamine N-oxide and lipopolysaccharides, cause an imbalance in gastrointestinal homeostasis, resulting in an inflammatory state, glycometabolic disorders, and insulin resistance (Tanase et al., 2020). Lachnospirales were identified as the cause of substantial increases in plasma glucose and reduced serum insulin in colonized germ-free mice; however, no study has reported the direct effect of this species on the synthesis or metabolism of testosterone in type 2 diabetes mellitus patients (Liu et al., 2022). In addition, gut-derived bacterial endotoxins may trigger testosterone deficiency in men with obesity by impairing testosterone production in the Leydig cells (Tremellen, 2016).

Notably, germ-free male mice have a distinct transcriptional profile in the hippocampus compared to conventionally raised mice (Zhou et al., 2020). The most interesting difference was in the transcription factor associated with gonadotropin-releasing hormone signaling, suggesting that the gut microbiota may influence androgen synthesis by interacting with the hypothalamic-pituitary-gonadal axis (Seminara & Crowley, 2008; Zhou et al., 2020). Microbes act on different organs by activating the enteroendocrine cells to produce hormones (Cani, 2018). This shows the versatility of the gut microbiome to affect distant organs such as the testicles (Li et al., 2022).

The gut microbiome is a key regulator of androgen metabolism, and (Colldén et al., 2019; Yoon & Kim, 2021) testosterone and dihydrotestosterone can be conjugated by glucuronidation, which increases their water solubility. These glucuronidated androgens are excreted in urine or through bile into the small intestine. An in vitro study showed that Clostridium scindens has the potential to cleave the side chains of host glucocorticoids, converting them into androgens, and (Ridlon et al., 2013) Colldén et al. (2019) reported the involvement of the gut microbiome in androgen metabolism and glucuronidation. Gut flora may efficiently deglucuronidate the intestinal glucuronidated testosterone and dihydrotestosterone excreted by the liver (Colldén et al., 2019). According to a previous study, germ-free mice have higher glucuronidated testosterone and dihydrotestosterone levels in their intestines than their counterparts (He et al., 2021; Markle et al., 2013). In contrast, free dihydrotestosterone levels increase in the blood, highlighting the importance of the gut microbiome in preventing excretion and sustaining testosterone levels. Microbiome transplantation reverses this effect (Aguilera, Gálvez-Ontiveros & Rivas, 2020; He et al., 2021). In addition to their deglucuronidation activity, some specific microbial taxa express steroid-processing enzymes that enable them to directly metabolize steroid hormones (Li et al., 2022). Widely distributed environmental bacteria may feed on testosterone and other steroids to eliminate contamination (Chen et al., 2016). The endobolome involves the gut microbiota genes, pathways, and enzymes involved in steroid hormone metabolism (Aguilera, Gálvez-Ontiveros & Rivas, 2020). The gut microbiome expresses classic steroid-metabolizing enzymes such as 17,20-desmolase, 20a-HSDH, 3a-HSDH, 20b-HSDH, and 5b-reductase (Aguilera, Gálvez-Ontiveros & Rivas, 2020; He et al., 2021). Diviccaro et al. (2020) observed that dihydrotestosterone, 3a-diol, and 17b-E, testosterone metabolites, were substantially higher in the colon than in the plasma. Specific bacteria such as Butyricicoccus desmolans and Clostridiu scindens, possess steroid-metabolizing enzymes that can convert and use sex steroids (Ly et al., 2020; Maffei et al., 2022; Ridlon et al., 2013).

Another proposed mechanism involving the gut microbiome and androgens is sex hormone-binding globulin (SHBG) metabolism. Yan et al. (2024) reported a causal effect of SHBG on the gut microbiota. Higher SHBG levels in men were associated with Dorea and Clostridiales (Yan et al., 2024). In contrast, the gut microbiome may also influence testosterone production and metabolism (Colldén et al., 2019). Genome-wide association studies have shown Alphaproteobacteria are associated with high levels of SHBG, a carrier of sex hormones (Yan et al., 2024). Microbiome modulation is an emerging therapeutic strategy that affects testosterone levels in men. Altering the composition of the gut microbiome may enhance microbial taxa associated with healthy testosterone levels. Probiotic supplementation, dietary interventions, prebiotic intake, and fecal microbiota transplantation (FMT) are under investigation for their potential to restore or optimize gut microbiota composition and could serve as novel treatment pathways for testosterone-related disorders.

This study has some limitations. First, this review included a small number of studies; only ten studies met the inclusion criteria. While the meta-analysis included a substantial sample size of 35,904 men, the limited number of studies involved may render the conclusions susceptible to modification by future research. Second, the included studies showed significant heterogeneity. The studies included in this review were conducted on six populations: Spanish, Japanese, Ukrainian, Australian, American, Chinese, and Korean. However, these findings may not apply to other ethnicities. Third, we could not perform quantitative analysis. A quantitative analysis with big data from different background, such as different races, would have provided a better and clearer picture of the biodiversity of the microbiomes and the relationship between the gut microbiomes and testosterone levels, limiting our conclusions to being qualitative. Future research should focus on identifying the microbial taxa that are most beneficial for testosterone production and investigating the effects of targeted microbiome interventions in clinical settings. Clinical trials evaluating the impact of specific probiotics, dietary adjustments, and FMT on testosterone levels could provide insights into effective strategies for managing low testosterone levels via microbiome modulation. Additionally, exploring personalized microbiome therapies could be a promising avenue for precision medicine, enabling interventions tailored to individual gut microbiome profiles.

Conclusion

In conclusion, most studies reported a significant relationship between the gut microbiome and testosterone levels. Of the ten studies included in this review, a significant relationship was observed between the gut microbiome and testosterone levels. The association between the gut microbiome and testosterone levels appears to be bidirectional, although its extent remains unclear. The gut microbiome has a qualitative relationship with testosterone levels; however, the exact quantitative relationship remains unknown. The findings regarding microbial diversity differed significantly across studies. It would be interesting to determine whether specific types of microorganisms are correlated with higher testosterone levels. This will be useful for designing microbiome transplants for therapeutic purposes. The gut-testicular and gut-brain axes could have significant effects on testosterone production, metabolism, and action. Nevertheless, the relationship between the microbiome and testosterone levels is not straightforward, as comorbidities, obesity, dietary patterns, and lifestyle may influence the gut microbiome. This field is still in its infancy and much work is required to determine the association between the gut microbiome and testosterone levels. Future research should focus on standardizing microbiome analysis techniques and identifying the microbial taxa most critical for testosterone production.

Supplemental Information