Osteoporosis (OP) is a skeletal and systemic disorder characterized by progressive loss of bone mineral density (BMD), microarchitectural deterioration, and decreased bone strength, leading to risk of fractures (Ensrud and Crandall, 2024). Multiple factors influence bone health include nutritional factors, genetics, metabolic status, and environmental exposures. In recent years, increasing evidence has revealed that gut microbiota (GM) composition plays a pivotal role in the development and progression of OP.

GM promotes the absorption of minerals such as calcium, magnesium, and phosphorus, thereby increasing the BMD through their involvement in the bile acid metabolism and vitamin B and K biosynthesis (Rodríguez et al., 2013; Clarke et al., 2014). Clinical studies have identified distinct taxonomic shifts in osteoporotic patients compared to healthy controls (Xu et al., 2020; Qin et al., 2021; Wang et al., 2022, 2023a). Evidence demonstrates significant taxonomic differences between the GM of osteoporotic patients and healthy controls. For instance, a higher abundance of Enterobacteriaceae members (e.g., Klebsiella, Citrobacter), a reduced Firmicutes/Bacteroidetes ratio and depletion of beneficial taxa such as Bifidobacterium, Lachnospiraceae and Blautia have been associated with bone loss (Li et al., 2019b; Lyu et al., 2023).

Moreover, functional pathways prediction has revealed depletion of the short-chain fatty acid biosynthesis pathway and increased activity in bacterial peptidase pathway (Ling et al., 2021; Akinsuyi and Roesch, 2023). Several studies have demonstrated distinct amino acids (AAs) metabolic signatures associated with enhanced BMD and reduced fracture incidence (Su et al., 2019; Grahnemo et al., 2023). A Mendelian randomization study and monozygotic twin research have demonstrated that AAs exert osteogenic effects independently of genetic factors (Jennings et al., 2016). Despite these advances, most studies have been conducted in Chinese and European populations (Akinsuyi and Roesch, 2023), with limited data from Latin America. This evidence points out the need to understand the bidirectional nature of host-microbiota-AA interactions in bone homeostasis. Therefore, this study explores the interactions between GM composition and serum metabolites in Mexican postmenopausal women associated with BMD status.

This is the first large-scale study analyzing the influence of GM diversity, functional pathways, and metabolomics on bone metabolism in Mexican postmenopausal women with low BMD. Our findings revealed a higher abundance of the genera Bacteroides, Parabacteroides, Barnesiella, Odoribacter, Sutterella, Butyricimonas, Coprobacter, Angelakisiella, and Oscillibacter associated with low-BMD. Consistent with our findings, previous studies have reported an increased abundance of Bacteroides abundance in Asian and Chinese postmenopausal women with low BMD (osteopenia (OS) and osteoporosis (OP) at the femoral neck (FN) or lumbar spine (LS) (Wang et al., 2021; Wei et al., 2021b; Akinsuyi and Roesch, 2023). However, contradictory results have been reported in Chinese individuals with decreased BMD (Ozaki et al., 2021) and postmenopausal women with fractures (Yan et al., 2024). Parabacteroides spp. have been found enriched in postmenopausal women with OS and OP across multiple Chinese cohorts (Wang et al., 2017; He et al., 2020; Wei et al., 2021a). In addition, in the FINRISK 2002 cohort, Parabacteroides was identified as fractures risk factor (Grahnemo et al., 2023). Research supports the potential role of Parabacteroides in modulating host metabolism. For instance, Wang et al., demonstrated that P. distasonis influences bile acid metabolism and succinate production, which may contribute to reduced weight gain (Wang et al., 2019). However, further studies are needed to verify these findings and determine whether this association varies by age, gender, or specific microbial strains.

Growing evidence has shown that Barnesiella, Oscillibacter, and Odoribacter might be the key players in the progression of OS and OP in postmenopausal women. Kuo et al., reported that Barnesiella, and Oscillibacter are OP-associated (n = 21) in Taiwanese postmenopausal women (Kuo et al., 2023). A large-scale ethnic GWAS - microbiota study (n=34,024 individuals) reported that the Barnesiella genus was found to be a risk factor for BMD for individuals >60 years old (Wang et al., 2023b). In line, the genus Oscillibacter was increased in postmenopausal women with reduced BMD (Ma et al., 2024; Yan et al., 2024). However, contradictory results have been reported in Chinese postmenopausal women with OP (Dong et al., 2024). The presence of Oscillibacter in the gut is controversial. It may be positively influenced by the intake of soluble corn fiber, which is associated with enhanced calcium absorption (Jakeman et al., 2016). In addition, our results demonstrated a negative association between Odoribacter and BMD in postmenopausal women. This phenomenon was also observed in peri, and early postmenopausal women (Greenbaum et al., 2022). Lai et al., found Odoribacter splanchnicus was significantly higher in Chinese male/female with osteoporosis than in the normal bone density (Lai et al., 2024). A positive correlation between Odoribacter and the number of osteoclasts in femoral tissue in the OVX rat model suggests an essential role in bone resorption. Furthermore, a negative association with Foxp3 expression indicates a potential disruption of immune regulation and a proinflammatory intestinal environment (Zhu et al., 2024). However, contradictory results show that Odoribacter abundance was significantly lower in Chinese postmenopausal women with OP than in the normal BMD group (Liang et al., 2023). Although the data suggest that an increased abundance of Oscillibacter and Odoribacter may be linked to bone resorption and a proinflammatory gut environment, the contradictory results in different populations suggest that the relationship between GM and bone health is complex.

The Sutterella genus was also predominant among aged groups with low BMD. Sutterella, a member of the phylum Proteobacteria, may contribute to non-specific mucosal inflammation due to lipopolysaccharides acting as potent stimulators, potentially predisposing the host to a chronic inflammatory disease and its ability to degrade immunoglobulin A (IgA) (Hiippala et al., 2016). This degradation, mediated by IgA-specific serine endopeptidases, may facilitate bacterial invasion and persistence within host cells (Hiippala et al., 2016). A possible mechanism suggested that Sutterella may produce various metabolites such as short-chain fatty acids (SCFAs)or indirect effects through autoimmune-related bone density alterations (Tyagi et al., 2018; Cao et al., 2021). However, the precise role of this taxon in bone metabolism remains unclear and warrants further investigation.

The association between physiological parameters and gut microbial community is complex and not fully understood. Previous studies have reported that BMI and BMD alter gut microbial community structure. In our research, Bacteroidota members, including Bacteroides, Parabacteroides, Coprobacter, and Odoribacter, were negatively correlated with age and BMD measurements. This supports the hypothesis that these taxa could be responsible for changing the microbial diversity structure and may contribute to bone loss.

Multiple studies highlight short-chain fatty acids (SCFAs) as key regulators of bone composition (Han et al., 2024; Lucas et al., 2018). Our study identified SCFA producers, including Butyricimonas, and Coprobacter in the low-BMD group. In contrast, Qin et al., found Butyricimonas decrease in older adults with OP (n=88). Notably, the decline in butyrate-producing taxa (Lachnospiraceae and Ruminococcaceae) in our low-BMD group aligns with findings in other postmenopausal populations (Li et al., 2019a; Zaplana et al., 2024). Mechanistically, SCFAs (butyrate and propionate) directly suppress osteoclast differentiation by binding to receptors on osteoclast precursors, as demonstrated in vitro (Yan et al., 2018). However, while SCFAs inhibit osteoclast formation, they may lack efficacy against mature osteoclasts (Wu et al., 2023).

SCFAs may influence bone homeostasis providing a direct link between the gut microbiota and bone via immunomodulatory response (Tyagi et al., 2018; Li et al., 2019b). Propionate and butyrate act as histone deacetylase (HDAC) inhibitors, modulating NF-κB activity to exert anti-inflammatory effects. Butyrate enhances IL-10 production while suppressing proinflammatory cytokines (IL-12, TNF-α, IL-1β, and NO) and dampening inflammatory responses in intestinal macrophages (Liu et al., 2023). SCFAs promote the differentiation of naive T cells into Th1 and Th17 effector cells, likely through their HDAC-inhibiting effects. In this regard, certain Bacteroides and Bifidobacterium strains could contribute to IFNα production (Schirmer et al., 2016; López et al., 2010) by producing SCFAs and exopolysaccharides (Ai et al., 2021). For instance, Bifidobacterium bifidum strains enhance IL-17 secretion while suppressing IFNγ and TNFα, suggesting a possible Th17 profile. Conversely, Bifidobacterium pseudocatenulatum and Bifidobacterium adolescentis exhibit an opposite correlation with IFNα and TNFg (Schirmer et al., 2016), and Bifidobacterium longum exhibits protective effects by suppressing osteoclastogenesis and increasing bone mass density in experimental models (Sapra et al., 2022). Unexpectedly, higher Bifidobacterium abundance was associated with low BMD in our data, underscoring the need to elucidate strain-level mechanisms and contextual interactions within the gut-bone axis. Additionally, previous studies have reported that Odoribacter and Barnesiella, both Gram-negative bacteria, exhibit a negative correlation with TNFα production in response to LPS, stimulated ex vivo (Schirmer et al., 2016). This finding contrasts with our initial expectations and highlights the need for further environment analysis of bacterial interactions to clarify the bone-specific roles of immunomodulatory taxa and optimize SCFA-targeted interventions for skeletal health.

The Firmicutes/Bacteroidota (F/B) ratio has been associated with maintaining homeostasis, and changes in this ratio can lead to various pathologies. For example, increases in the abundance of specific Firmicutes or Bacteroidetes species lead to obesity (Clarke et al., 2014). In low BMD the results are contradictory. A meta-analysis (175 healthy controls vs. 177 OP patients) from five studies found no significant differences in the F/B ratio between groups (Akinsuyi and Roesch, 2023). In contrast, our study, observed a significantly decreased F/B ratio in the low BMD group. Similar data have been reported in Chinese postmenopausal women (>64 years) (Wang et al., 2017; Li et al., 2019a). These discrepancies may reflect variations in gut microbiota composition across different populations, size sample (Wang et al., 2017, 2022, 2023a; Das et al., 2019; He et al., 2020; Palacios-González et al., 2020; Xu et al., 2020; Qin et al., 2021; Rettedal et al., 2021), and methodological such as DNA extraction protocols, 16S rRNA gene targeted for amplification, and quality filtering parameters (e.g. Phred score). Maintaining a balanced intestinal ecosystem is crucial for normal body function, and many therapeutic strategies aim to achieve a suitable Firmicutes to Bacteroidetes (F/B) ratio.

The functional profiling of microbial communities reveals significant insights into metabolic pathways associated with bone health. In this study, the metabolic pathway analysis shows a significant depletion of valine, leucine, and methionine pathways in women with low BMD. Evidence suggests that the gut microbiota composition explains 19% of the variance of circulating, branched-chain amino acid (valine, leucine and isoleucine) (BCAA) concentrations (Dekkers et al., 2022). An enriched of Bacteroidaceae could explain the differences in serum BCAA concentrations between the groups, potentially contributing to bone loss. Similarly, a large population-based study (n=1776) reported negative correlations between leucine and valine concentrations and BMD in a Chinese population with OS and OP. The authors proposed that enrichment of ABPVCR consortium (Actinobacillus –Bacteroides – Phascolarctobacterium – Veillonellaceae – Collinsella - Ruminococcaceae) is associated with BCAAs degradation (Ling et al., 2021). Interestingly, Wang et al., identify fecal metabolites D-alanyl-D-alanine and serum serine-valine as inversely correlated with BMD (Wang et al., 2023a), confirming our results and reinforcing the link between microbial metabolism and bone health.

The potential benefits of BCAAs in bone maintenance have been extensively documented, highlighting their role in preserving bone integrity and supporting muscle-bone crosstalk (Zhao et al., 2018; Su et al., 2019; Grahnemo et al., 2023). Leucine, in particular, enhances mTOR-mediated protein synthesis, promoting osteoblast activity and bone formation while reducing bone resorption markers (Jennings et al., 2016). Meanwhile, valine has also been strongly correlated with bone health since it shares metabolic pathways with leucine, and has been proposed to exert a protective effect against fractures (Grahnemo et al., 2023). Additionally, dietary methionine supplementation improves hepatic steatosis, insulin resistance, inflammation, fibrosis, and bone health. Conversely, methionine deficiency has been linked to impairing osteoblast function, reduced bone formation and increased osteoclast activity (Ouattara et al., 2016). Although, these findings highlight emphasize the intricate relationship between microbial metabolism-AA production in skeletal integrity, experimental approaches (such as GM transplantation and metagenomic sequencing) are needed to identify key bacterial strains involved in amino acid metabolism and their role in low BMD.

On the other hand, estrogen deficiency is known to influence bone remodeling and alter lipid profiles; however, the application of plasma lipidomics in studying menopausal osteoporosis remains underexplored. In this study, we identified dysregulation in key lipid metabolism pathways, including those involved in alpha-linolenic acid, glycosaminoglycan, sphingolipid, and glycerolipid metabolism. It has been reported that plasma lipids and polar metabolites differ between women with normal and low BMD and are involved in several metabolic pathways, including sphingolipid and phospholipid metabolism, as well as fatty acid β-oxidation (Cabrera et al., 2018). Notably, two studies have linked specific sphingolipid species to low BMD in postmenopausal women, suggesting that sphingolipids may modulate bone metabolism via bone marrow-derived macrophages and could be attributed to estrogen deficiency (Cabrera et al., 2018; Lee et al., 2012). In contrast, other studies report inconsistent associations between triacylglycerol levels and hip BMD in this population (Cui et al., 2005; Brownbill and Ilich, 2006; Makovey et al., 2009), highlighting the need for further investigation.

Differences in the gut microbiome across populations are significant and driven by factors such as geography, ethnicity, diet, and lifestyle. Among these, diet plays a pivotal role in shaping the composition, function, and diversity of gut microbial communities. For instance, western diets rich in protein and fat are strongly associated with Bacteroides-dominant microbiomes, as observed in the US and Europe (Clemente-Suárez et al., 2023).

Regional variations in gut microbiota also have been documented (Gaulke and Sharpton, 2018). East Asian populations, particularly in China, Japan, and Taiwan, exhibit higher Bacteroides abundance, “BB-type”, compared to Southeast, Southern, and Central Asian populations (Nakayama et al., 2015; Gaulke and Sharpton, 2018; Gorvitovskaia et al., 2016). The “BB-type” gut microbiota is more prevalent in these regions, whereas the “P-type” microbiota, dominated by Prevotella, is more common in Southeast Asia (Gorvitovskaia et al., 2016).

Notably, older adults in both Asian and Mexican urban settings demonstrate higher protein and fat intake (Cho and Choi, 2021; Nabuco et al., 2018; López-Montoya et al., 2023) which could influence microbial composition and metabolic outcomes. However, while Asian gut microbiota research has identified specific bacterial taxa associated with BMD, the Mexican microbiome remains understudied in this context. These findings highlight the challenges in developing microbiome-based personalized medicine and underscore the necessity for region-specific research to account for such variations.

This study has several strengths. First, it comprehensively characterizes GM composition in a large cohort of Mexican postmenopausal women (n=535), offering valuable insights into this understudied demographic. Second, it identifies specific GM taxa associated with BMD status and explores their metabolic pathways, particularly those involved in AA metabolism, which may influence BMD. However, the study also has present limitations. First, the regulation of circulating AA concentrations is a complex physiological process influenced by multiple factors, including dietary intake, protein turnover, the novo synthesis and metabolic clearance. Thus, the specific contribution of gut microbiota to AA metabolism remains unclear and requires further validation.

Second, GM composition is influenced by diet, age, geography, and host health status. For instance, diet can account for up to 20% of the variation in GM composition, which also fluctuates seasonally (Farhat et al., 2023). Notably, phytoestrogen intake has been shown to enhance the diversity and abundance of beneficial gut bacteria, thereby modulating their effects on sex hormones (Gyriki et al., 2025). Although this variable was not included in the questionnaire, its potential impact warrants consideration in future research. Third, GM undergoes age-related changes, including reduced diversity of commensal bacteria and increased in pathogenic species, which may contribute to disease susceptibility. Another key factor in gut dysbiosis is antibiotic use, which has been shown to disrupt sex hormone metabolism, bile acid metabolism, and the synthesis of fats and vitamins (e.g., vitamin K)— (LeBlanc et al., 2013; Collins et al., 2023) all of which may influence bone health regulation.

Four, several studies have determined the impact of single-nucleotide variants (SNVs) related to amino acid metabolism (Imaizumi et al., 2019; Lares-Villaseñor et al., 2024) on serum concentrations. However, the bidirectional interactions between host genetics, GM, and serum amino acids remain underexplored. Future studies should investigate whether these genetic variants exhibit a causal relationship with bone loss over time and elucidate the underlying mechanisms, including potential mediation by microbial metabolites or host-microbe metabolic crosstalk.

Lastly, while probiotics and prebiotics have demonstrated osteoprotective effects in other studies (Sapra et al., 2021; Farhat et al., 2023), their role in this specific population remains unexplored. Although this study partially accounts for some of these factors, the heterogeneity in bacterial associations with bone loss across the literature underscores the need for further research to fully elucidate these complex interactions.

The results showed that postmenopausal women with low bone mineral density (BMD) experience significant changes in their gut microbiota and serum metabolites. These changes are closely correlated with BMD measurements, similar to the Caucasian and Asian populations. This correlation offers potential insights into the mechanisms behind the low BMD and may serve as an early diagnostic indicator. This study could pave the way for new interventions to improve bone health in Mexican postmenopausal women.