Fibromyalgia (FM) is a complex disorder whose main symptoms are chronic widespread pain and fatigue. Frequently, these symptoms are accompanied by a significant variety of related physical and psychological disturbances (Longley, 2006; Clauw, 2014). Despite the lack of knowledge about the etiopathogenesis and the complex diagnosis of the disorder, the prevalence ranges between 0.2 and 6.6% of the world population (Marques et al., 2017), with women accounting for approximately 89% of patients. To date, the non-specificity of the symptoms, the huge increase in worldwide prevalence, and the economic impact that it entails have turned FM into a pressing health and social issue. This evidence, together with the absence of molecular biomarkers that could facilitate diagnosis, underlines the extreme necessity of basic research on this chronic disorder.

Several physical and psychological disturbances such as migraine, muscle cramps, intestinal discomfort, sleep alterations, lack of short- and long-term memory, inability to concentrate, depression, stress, and anxiety, are recurrently reported by FM patients (Valera-Calero et al., 2022). To unveil the source of these symptoms, the latest efforts by the researchers have focused on studying problems at the level of central nervous system (CNS) sensitivity (Giorgi et al., 2023), inflammatory and oxidative disorders (Coskun Benlidayi, 2019), and even imbalances related to the intestinal microbiota (Liu et al., 2023).

Increasing evidence indicates that inflammatory processes occurring in peripheral tissues and the CNS, primarily in the spinal cord and the brain, are responsible for the pathophysiology of FM (Bains et al., 2023; O'Mahony et al., 2021). The release of chemokines and cytokines leads to the activation of both the innate and adaptive immune systems, as well as neuroinflammation (Littlejohn and Guymer, 2018). These mechanisms are reflected in many of the peripheral clinical features reported by FM patients, such as swelling, dysesthesia, cognitive changes, and fatigue. This lack of energy that accompanies FM patients has been related to mitochondrial dysfunction and oxidative stress. In this sense, it has been shown that there is a correlation between oxidative processes and pain sensitization in FM patients. In this way, a deficiency in the function of enzymes such as superoxide dismutase 1 (SOD1), catalase (CAT), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has been identified in FM patients (Assavarittirong et al., 2022), which correlates with the severity of pain and fatigue (Rus et al., 2021; Altindag and Celik, 2006). Likewise, certain studies have identified a lower total antioxidant capacity, accompanied by a greater presence of reactive oxygen species (ROS) in women with FM compared to the control group (Rus et al., 2021). As a whole, all these processes cause immunity deregulation and mitochondrial imbalances that, ultimately, can trigger peripheral neuroinflammatory processes (Coskun Benlidayi, 2019).

The gut-brain axis stands out as one of the recent alterations linked to FM. The main hypothesis indicates a connection between intestinal microbiota imbalance and the increase of intestinal permeability in patients, with the consequent alteration in digestion, immune response, and CNS signaling (Erdrich et al., 2020; Goebel et al., 2008). When the intestinal epithelial barrier is compromised, it can lead to increased intestinal permeability, allowing gram-negative bacteria to move through the mucosal lining and reach the blood flow. Their ability to produce neurotransmitters and their interaction with the primary function of the vagus nerve allows these bacteria to modulate the activity of the hypothalamic-pituitary-adrenal (HPA) axis (Tomasello et al., 2018). This situation allows direct interactions between intestinal microbiota components and humoral and cellular mediators from the immune system and the CNS, triggering an immune response characterized by increased production of inflammatory mediators (Moloney et al., 2016). Thus, several studies have observed changes in the composition of the gut microbiota in various disorders, including gastrointestinal, rheumatic, and metabolic conditions (Wang et al., 2022; Afzaal et al., 2022). In the FM context, a recent study has found a reduced bacterial diversity, as well as altered levels of glutamate and serine, suggesting changes in the metabolism of crucial neurotransmitters in the CNS signaling (Clos-Garcia et al., 2019). This innovative approach based on intestinal microbiota opens a new field for searching for biomarkers. Those biomarkers could also be modulated through diet therapy, which has been gaining relevance in FM management.

Advances in identifying inflammatory markers involved in FM have been largely made thanks to the development of omics techniques. Proteomics studies have been conducted in patients, primarily in saliva, serum, and cerebrospinal fluid. Therefore, (Ramírez-Tejero et al. 2018) identified 33 plasma proteins with different expression in FM patients compared with volunteers, with most proteins linked to inflammatory processes. Another study in patients revealed differences in plasma proteins involved in inflammatory, metabolic, and immunological processes and demonstrated a correlation between these proteins, patients' pain perception, and psychological distress (Wåhlén et al., 2020). Additionally, 22 differentially expressed proteins were identified in FM patients compared with pain-free volunteers, mainly associated with blood coagulation processes, immune response, and interactions with extracellular matrix receptors (Han et al., 2020). Among other findings, these results confirmed the promising utility of proteomics in the study of FM and demonstrate the roles of coagulation, inflammation, and the immune response in the disorder's pathogenesis.

In spite of the above-mentioned research, nowadays, the diagnosis and treatment of FM remain controversial. Traditionally, the diagnostic criteria of the American College of Rheumatology (ACR) have been followed (Wolfe et al., 1990, 2011, 2016), and most healthcare professionals treat their patients with numerous drugs focused on symptoms such as pain, depression, and fatigue. However, clinical recommendations from the European League Against Rheumatism (EULAR) and other organizations point to a multidisciplinary point of view of FM and its treatment (Giorgi et al., 2023; Macfarlane et al., 2017). Likewise, the latest trend is toward the use of highly individualized therapies combined with non-pharmacological approaches indeed (Kundakci et al., 2022).

In this context, this study is focused on biomarker discovery for FM in plasma and feces from 199 FM patients and 43 women as a control group, aiming to combine the analysis of both samples in a diagnostic panel for clinical practice.

FM is a chronic pain-related disorder that is still underdiagnosed due to the lack of molecular markers. The present study is focused on the search for these markers in the plasma proteome and intestinal microbiota. The results showed a significant differential pattern of 30 proteins and 19 bacterial taxa between FM patients and controls, and a significantly higher α diversity in controls compared to patients, suggesting a worse intestinal environment and, consequently, a weaker intestinal and general health in FM.

Main symptoms of FM (pain and cognitive impairment) suggest an evident and deleterious nervous system disruption. In fact, several authors have agreed on classifying this disorder as one of the most common conditions among the so-called central sensitization syndromes (Boomershine, 2015), which are characterized by a CNS imbalance that affects the homeostasis of the whole nervous system. This altered neurological performance has been demonstrated in a substantial number of studies utilizing various imaging, electrodermal, and molecular techniques. For instance, different groups have found that nerve fibers participating in nociception in FM patients are less abundant, thinner, and even abnormally hyperexcited when compared with controls (Serra et al., 2014; Vecchio et al., 2020). Similarly, autonomic nervous system-dependent processes such as sweating have been reported as compromised by pathological traits in nerve fibers identified through electrodermal measurements (Reyes del Paso and de la Coba, 2020; Falco et al., 2024), adding further evidence to support the hypothesis of a complete nervous system failure in these patients.

At the molecular level, the long list of neurological imbalances is usually accompanied by chaotic neuroimmunological signaling. An extensive and varied record of altered molecular signaling in the immune system has been reported (Ovejero et al., 2020; Verma et al., 2022; Maes et al., 2024; Goebel et al., 2021), resulting in marked neuroinflammation, a common finding in FM patients (Littlejohn and Guymer, 2018; Mueller et al., 2023). In line with this hypothesis, nervous system impairment was also inferred by proteomic analysis in our cohort, with CHLE (FM/C ratio: 0.85), DOPO (FM/C ratio: 0.85) and G3P (FM/C ratio: 0.82) as affected proteins among others (Table 3); the first and the second, participate actively in the regulation of cholinergic and dopaminergic synapses (Darvesh et al., 2003; Silman, 2021; Rush and Geffen, 1980), and the third one, is annotated in the GO term neuron apoptosis process (GO:0051402). In fact, the role of G3P protein (GAPDH) seems to be essential in the gut-brain-mitochondrial axis, as discussed in the upcoming paragraphs. Inflammation traits were as well identified in our study, since some proteins among the 30 differentially expressed in the FM patients were narrowly related with pro-inflammatory pathways (Figure 1b), such as acute phase signaling, with AMBP (FM/C ratio: 1.10), APOH (FM/C ratio: 1.32), CO4B (FM/C ratio: 0.86), and KLKB1 (FM/C ratio: 0.88) as altered proteins; and IL-12 signaling and production in macrophages, represented by APOA4 (FM/C ratio: 0.83), IGKC, (FM/C ratio: 0.78), APOA (FM/C ratio: 0.83), and S100A8 (FM/C ratio: 1.43) as Table 3 showed. The last one, a component of calprotectin, has recently been proposed as a biomarker for rheumatoid arthritis and FM (Inciarte-Mundo et al., 2022; Vega-Ramírez et al., 2024), given its implication in the activation of innate immunity and inflammation.

In FM patients, the presence of neuroinflammation has been proposed as a multifactorial process driven by the co-occurrence of low vitamin D levels, mitochondrial dysfunction, and intestinal microbiota dysbiosis (Vasquez, 2016). In this sense, our proteomic results identified four proteins participating in the 24-dehydrocholesterol reductase (DHCR24) signaling pathway, according to IPA: AMBP (FM/C ratio: 1.10), APOA4 (FM/C ratio: 1.25), APOH (FM/C ratio: 1.32), and CO4B (FM/C ratio: 0.86). DHCR24 is the final enzyme in the biosynthesis pathway of cholesterol, a ubiquitous molecule that serves as a precursor to several by-products participating in a wide variety of physiological and pathological pathways, such as the metabolism of lipids and vitamin D, the synthesis of steroid hormones, and liver X-receptor (LXR)/retinoid X-receptor (RXR) activation, among others (Warren et al., 2021). LXR/RXR activation appears to be supported by stronger evidence in previous studies from our group and other authors. In the present study, the LXR/RXR activation pathway is the most differential canonical pathway between FM and C groups according to the p-value (Figure 1a), and is represented by the differential expression of AMBP (FM/C ratio: 1.10), APOA4 (FM/C ratio: 1.25), APOH (FM/C ratio: 1.32), CO4B (FM/C ratio: 0.86), APOA (FM/C ratio: 0.83), and S100A8 (FM/C ratio: 1.43), with a clear tendency toward upregulation. LXR/RXR receptors are steroid- and lipidic metabolites-dependent transcription factors involved in biological processes as diverse as lipid metabolism, immune system maintenance, neurobiological homeostasis or reproductive health homeostasis. Not in vain, this route has been significantly enriched and activated in numerous cohorts of FM patients in previous research (Ramírez-Tejero et al., 2018; Han et al., 2020; Frigo et al., 2021; Mohapatra et al., 2024), leading to altered lipid metabolism. Since steroid synthesis takes place inside the mitochondria, starting from cholesterol, the complete disruption found in lipid and steroid metabolism of FM patients that, at the end, alters the performance of the neuroendocrine system might be mainly triggered by this organelle.

Not surprisingly, mitochondria may be the common link among the various symptoms experienced by FM patients. By far, the nervous and musculoskeletal systems have the highest energy demand in the body, and they have been found to be dramatically affected by mitochondrial dysfunction (Cantó-Santos et al., 2020). Similarly, mitochondria have been repeatedly mentioned as a dysfunctional organelle in FM, not only by the scientific community, but also in previous studies from our research group (Cordero et al., 2013; Martínez-Lara et al., 2020; Castro-Marrero et al., 2013; Sánchez-Domínguez et al., 2015; Gerdle et al., 2020; Alcocer-Gómez et al., 2015; Cordero et al., 2010, 2016), which makes perfect sense, considering that pain, fatigue, joint stiffness, muscle cramps, and neurocognitive disorders stand out as main features of FM. Specifically, our previous findings suggested a profound imbalance of the mitochondrial membrane-associated porin voltage-dependent anion channel 1 (VDAC1) in FM patients (Martínez-Lara et al., 2020; Ramírez-Tejero et al., 2023), which directly cooperates with G3P in a well-defined classical mitochondrial-related apoptosis pathway (Tarze et al., 2007). Additionally, VDAC1 was identified in the plasma proteome analysis of our cohort, although it did not show statistically significant differences between groups (data not shown). This result may be due to technical limitations of the nLC-MS/MS approach, as even though a depletion step was introduced, the most abundant proteins could mask subtle changes in the less present ones. However, G3P was differentially expressed in FM patients, as shown in Table 3. This specific enzymatic activity appeared as the top functionally decreased activity in the intestinal microbiome of FM patients (Figure 3b) and is involved in the main pathways altered in FM patients, as indicated by our data. For this reason, the G3P enzyme and its by-products emerge as a promising diagnostic and therapeutic target for FM patients, and its enzymatic increase inferred from the metagenomic functional analysis could be a counteracting response to the lower plasma levels in FM patients.

Sticking to the mitochondrial environment, several other molecular functions take place inside this organelle, apart from energy metabolism. Specifically, mitochondria have been linked to oxidative stress and subsequent inflammatory processes in a wide range of pathological conditions (Chen et al., 2023). Among the differentially expressed proteins in FM patients participating in this study, some proteins related to these processes were found. For instance, GPX3 (FM/C ratio: 0.85), LX12B (FM/C ratio: 1.33), and APOA4 (FM/C ratio: 1.25) differences shown in Table 3, suggested a deficiency in oxidative stress defense as well as an increased lipid peroxidation, results already reported in FM and even linked to pain intensity and inflammation in those patients (Assavarittirong et al., 2022; Meeus et al., 2013; Akkuş et al., 2009; Ozgocmen et al., 2006). In line with these previous findings, some of the top-rated canonical pathways according to the p-value were complement cascade and complement system, implying proteins such as CO4B (FM/C ratio: 0.86), CO8B (FM/C ratio: 0.89), CO8G (FM/C ratio: 0.81), CFAH (FM/C ratio: 1.15), and IGKC (FM/C ratio: 0.78). This molecular pathway is narrowly related to chronic inflammation (Rawish et al., 2021).

All the previously identified imbalances found in our study could have a common link in the intestinal microbiome, defined as the unique signature of microbes, their genes, and metabolites, as well as their interaction with the host. Intestinal dysbiosis has been identified as a main source of CNS-related syndromes and symptoms, often via the vagus nerve and immune system (Rutsch et al., 2020). This connection has already been evaluated in FM, finding a low vagal nerve tone that might be related to pain and inflammation, as reported also for other conditions (Martins et al., 2021). One of the latest hypotheses links FM with this exact connection, dysbiosis being the main triggering cause of CNS symptoms. In FM patients, those deviations have lately been blamed for associated complications, such as irritable bowel syndrome (Garofalo et al., 2023) or psychological distress (Nhu et al., 2024). Besides that, a fast-growing hypothesis for some FM-overlapping sex-related conditions, such as endometriosis, breast and endometrial cancer, points to the role of β-glucuronidase activity from intestinal microbiota metagenome (estrobolome), specifically from the phyla Bacteroidetes (Bacteroidota) and Firmicutes (Bacillota) (Pollet et al., 2017), as the primary source of estrogens disruptions (Kwa et al., 2016; Baker et al., 2017; Ervin et al., 2019; Komorowski and Pezo, 2020; Salliss et al., 2022; Siddiqui et al., 2022; Sobstyl et al., 2022; Pai et al., 2023; Hu et al., 2023). This hypothesis reinforces the crucial role of the intestinal microbiota in a considerable list of human diseases and syndromes. In terms of hormone signaling, and following the estrobolome hypothesis, cortisol, estradiol, and progesterone levels have been investigated in FM patients and in vitro models; however, the findings on the impact of these hormones on pain and neuropsychological complications were preliminary and contradictory (Okifuji and Turk, 2006; Rus et al., 2016; Schertzinger et al., 2018; Hernandez-Leon et al., 2018; Lin et al., 2021).

Our results revealed a differential pattern in intestinal microbiota composition in FM patients, as shown in the volcano plot resulting from the metagenomic analysis (Figure 2c), with a total of 19 bacterial taxa that were differentially expressed. Specifically, at the phylum level, the distribution of zOTUS is as follows: 12 Bacillota, six Actinobacteria, and one Bacteroidota. Streptococcus salivarius appeared as the most differential bacterium between the FM and C group, with the lowest p-value and the highest FC. Interestingly, this bacterium has demonstrated regulatory properties for immunological and inflammatory responses in vitro (Kaci et al., 2014), suggesting that this marked difference may represent an outstanding research hypothesis for further studies. Several other species, such as Mogibacterium neglectum, Streptococcus parasanguinis, Schaalia odontolytica, Pseudoruminococcus massiliensis, Ruminococcus gnavus, and Bifidobacterium pseudocatenulatum showed the same trend as S. salivarius. On the other hand, Bacteroides cellulositycus and Intestinimonas massiliensis were found to be less abundant in FM patients, according to our results. To our knowledge, this is the first paper to report differential abundance in FM patients for bacteria from these genera, except for Bifidobacterium and Streptococcus. The first one was previously identified as a reduced genus in FM patients (Clos-Garcia et al., 2019), and the second one showed differences between men and women with FM in a pilot, descriptive study of our group (Ramírez-Tejero et al., 2023). In this regard, different research groups have demonstrated differentially abundant bacterial groups in the gut of FM patients compared with controls. (Minerbi et al. 2019) reported a distinct microbiota signature in FM patients, composed of a high prevalence of bacteria from the Firmicutes phylum. Besides, species such as Prevotella copri, Faecalibacterium Praustnitzii, and Bacteroides uniformis, classically associated with a healthy gut, showed less abundance in those patients. In a parallel study, (Clos-Garcia et al. 2019) found a similar pattern, where beneficial bacteria, such as Bifidobacterium spp. and Bacteroides spp., were clearly less abundant in patients compared to controls, as mentioned before. Moreover, this group demonstrated some correlation between these differences and the concentration of several metabolites with a crucial role in CNS signaling. Our results showed a striking coincidence, since Bacteroides cellulosyticus appeared with the same exact tendency as that found in both studies for their genus relatives. Several species from this genus have exerted a remarkable ability to participate in γ-aminobutyric acid (GABA) synthesis in the human gut (Otaru et al., 2021). GABA is the primary calming CNS neurotransmitter, and previous authors have demonstrated that it is present in the brain at a lower concentration in FM patients compared to controls, while its precursor and main excitatory neurotransmitter, L-glutamate, exhibits the opposite behavior (Minerbi et al., 2019; Otaru et al., 2021). In this sense, the generalized Bacteroides spp. deficiency that seems to be present in FM patients might be a promising therapeutic target for CNS imbalances in them, although further research is needed.

Functional prediction with PICRUSt of bacterial differences in our FM cohort provided promising results. Several pathways with a marked link to mitochondria were enriched in those patients. As shown in Figure 3a, glycolysis/gluconeogenesis and pyruvate metabolism were, by far, the most affected routes. Additionally, some vital routes for mitochondrial health were also compromised. For instance, the PPAR signaling pathway and folate biosynthesis (Xiu and Field, 2020; Mello et al., 2016). In line with this hypothesis, typically mitochondrial enzymatic activities also showed a differential behavior in ontology prediction based on intestinal dysbiosis found in these patients. That was the case of hydroxymethylglutaryl-CoA reductase (K00054) and glyceraldehyde-3-phosphate dehydrogenase (NADP+) (K00131). Mitochondrial dysfunction could be a triggering factor, according to further PICRUSt predictions, since antioxidant activities, such as glutathione reductase (NADPH) (K00383), also appear to be compromised. Taking into account that plasma results were suggesting a mitochondrial blockade, microbiota might be reacting as a counterbalance to this deficiency.

The multi-omics approach provided valuable insights into the relationships between proteins, bacteria, and health questionnaire results. In this sense, as shown in Figure 4, Fisher's z-transformed ρ highlighted evident differences in protein-to-bacteria and protein/bacteria-to-health questionnaire scores between controls and patients. Thus, strong positive differences in correlations were found between DSG1 and IGKC proteins and several bacterial genera, including Clostridium, Eubacterium, Faecalibacterium, Coprococcus, Collinsella, and Mogibacterium, all of which belong to the phylum Firmicutes. Desmogleins (DSG1 and DSG2) are essential proteins in cell-to-cell adhesion, serving as the primary components of desmosomes in tissues subjected to strong mechanical stress, such as the intestinal barrier layer (Gross et al., 2018; Sherrill et al., 2014). IGKC, as the constant region of the light chains from all immunoglobulin types, participates in a wide range of adaptive immune responses. Combined, these findings suggest that, in FM patients, the highest abundance of these genera may trigger an intestinal barrier breakdown, followed by an immunological response, whereas controls do not experience such a disturbance. Since FM has been classically linked to several gastrointestinal challenges (Slim et al., 2015), the intestinal microbiome composition of these patients should be a clinical indicator in their initial evaluations, as well as a therapeutic target to address. Furthermore, DOPO protein exerted a marked positive correlation with the Bifidobacteriaceae family, specifically with Bifidobacterium pseoudocatenulatum and Bifidobacterium alomucense. According to our results, the increase in these bacteria is linked to a higher abundance of Dopamine beta-hydroxylase, which is responsible for converting dopamine (a pleasure neurotransmitter) to noradrenaline (a concentration neurotransmitter). The link between these bacteria and dopaminergic signaling has been widely depicted in the scientific literature (Hamamah et al., 2022). For instance, in murine models, the supplementation with Bifidobacterium infantis elevated the levels of noradrenaline after maternal separation (Desbonnet et al., 2010). Whether these differential effects of Bifidobacterium spp. on dopamine/noradrenaline signaling could be acting as a gut-brain axis regulator checkpoint in FM symptoms needs to be addressed in further studies. An additional relevant finding concerns Bacteroides cellulosyliticus and the marked and opposite relationship established with the proteins PRG4 and APOH, both of which have higher levels in FM patients (Table 3). According to the heatmap of correlation differences, the presence of B. cellulosyliticus is somewhat related to PRG4. This proteoglycan plays a significant role in joint lubrication, but is also implicated in the management of neuroinflammation by interfering with TLR2/4 signaling (Bennett et al., 2021). In parallel, this bacterium showed a positive difference in correlations with APOH, which was previously described as a biomarker for antiphospholipid syndrome (Sibilia, 2003), a condition that worsens FM symptoms when it appears as a comorbidity (Costa et al., 2011). Given that, as previously mentioned, Bacteroides spp. was found to be a reduced genus in FM patients in both previous studies and our results, its differential correlation with these two proteins could be proposed as a molecular mechanism affected in patients.

Finally, regarding health questionnaires, the highest differences between FM and C correlation coefficients are shown by CFAH and Faecalibacterium hattori. The former showed positive differences with physical function, while the latter showed negative differences with energy/fatigue, physical health, general health, and pain, as shown in Table 2. Hence, CFAH and Faecalibacterium hattori could be potential therapeutic targets to address, thereby enhancing patients' quality of life.

This is the first multi-omics approach for FM patients, highlighting the feasible role of the gut-brain-mitochondrial axis in FM etiopathogenesis in such a large cohort. This study identifies several promising markers for classifying patients with FM. Plasma and fecal multi-omics analysis and its integration into an algorithm allows discrimination between cases and controls with high AUC values, pointing to an intricate and multifactorial connection between gut microbiota and mitochondria-derived oxidative stress and inflammation, with glyceraldehyde-3-phosphate dehydrogenase and Streptococcus salivarius as leading actors. All biomarkers identified in this study could play an important role in the disease, suggesting their potential as prognostic, diagnostic, and therapeutic targets for personalized, preventive, or palliative/curative treatment of FM in the future. However, further blinded studies linking these potential biomarkers to FM could define a specific signature of women suffering from FM, which would be very useful for developing a robust diagnostic tool and focusing on probiotic-based treatments or other effective procedures for the patient.