Mitochondrial function in patients affected with fibromyalgia syndrome is impaired and correlates with disease severity

Mitochondrial function in patients affected with fibromyalgia syndrome is impaired and correlates with disease severity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mitochondrial function in patients affected with fibromyalgia syndrome is impaired and correlates with disease severity Chiara Macchi, Andrea Giachi, Isabella Fichtner, Silvia Pedretti, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4654793/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Fibromyalgia is a musculoskeletal syndrome characterized by chronic widespread pain that is often associated with systemic manifestations. Since mitochondria are the main source of cellular energy, we hypothesized that fibromyalgia syndrome could be linked to mitochondrial impairment. Aim was to study mitochondrial dysfunction in peripheral blood mononuclear cells isolated from 50 patients with primary fibromyalgia syndrome and 10 apparently healthy controls. Although no differences in mitochondrial basal respiration were observed between patients with primary fibromyalgia syndrome and healthy controls, a lower median bioenergetic health index (BHI; -22.4%, p = 0.03), a proxy of mitochondrial function, was found in patients. According to fibromyalgia severity score (FSS), a composite of widespread pain index and symptom severity scale, a lower median BHI (-18.7%) was found in patients with a FS ≥ 20 compared to those with a FSS < 20. Negative moderate correlations were found only between BHI and FSS (r= -0.36) and widespread pain index (r= -0.38). We demonstrated that patients with fibromyalgia syndrome had an impaired mitochondrial function. Additionally, we found a mild correlation between the widespread pain index and the BHI, possibly indicating that the altered mitochondrial function, in these patients, narrows musculoskeletal rather than central nervous system involvement. Health sciences/Biomarkers Health sciences/Rheumatology Figures Figure 1 Figure 2 Figure 3 1. Background Fibromyalgia, a musculoskeletal syndrome, is characterized by chronic widespread pain that is often accompanied by systemic manifestations, such as persistent fatigue, sleep disturbance and cognitive impairment, hyperalgesia and allodynia ( 1 ). These symptoms significantly impact health-related quality of life. Initially, diagnostic criteria were based on tender points on the body, as defined by the American College of Rheumatology (ACR) ( 2 ). However, the criteria have since shifted to include a more complex set of features including both the widespread nature of pain and associated symptoms ( 3 ). Fibromyalgia syndrome has a prevalence of 2–8% in the general population and it can develop at any age, including childhood, with higher frequency in females ( 4 – 6 ). Despite significantly impacting quality of life, fibromyalgia syndrome remains an under-diagnosed disease ( 7 ). The pathogenic mechanisms and the underlying reason for fibromyalgia-related changes are not fully understood, leading to a lack of specific biomarkers ( 8 ). Research suggests that it may be related to abnormalities in the central nervous system and immune system, as well as to genetic and environmental factors ( 9 ). Initially perceived primarily as a rheumatic disorder, it is now recognized as a disorder involving pain processing and central nervous system sensitization ( 10 , 11 ). One theory proposed, as a potential contributor to fibromyalgia syndrome (FM), involves the dysfunction of mitochondria, which are the energy-producing organelles within our cells and serve as the primary source of cellular energy. When mitochondria malfunction, it can lead to reduced adenosine triphosphate (ATP) production, increased oxidative stress, disrupted calcium homeostasis, and altered mitochondrial membrane potential. These impairments can affect metabolism, cell death and inflammation pathways, innate immune responses, potentially contributing to the pathogenesis of several diseases ( 12 ). In the context of FM pathogenesis, the relevance of mitochondrial dysfunction has been highlighted in a reserpine-induced fibromyalgia rodent model. These animals exhibited reduced functionality of mitochondrial complexes I and II, decreased electron transport, impaired mitochondrial respiration, reduced mitochondrial biogenesis, and altered mitochondrial dynamic. Additionally, they experienced lower ATP synthesis in muscles and spinal cord ( 13 , 14 ). Furthermore, individuals with FM could show decreased ATP production in their muscle cells, contributing to muscle fatigue and overall fatigue experienced by fibromyalgia patients ( 15 ). Notably, the mitochondrial function of peripheral blood mononuclear cells (PBMC) correlates with heart and skeletal muscle mitochondrial function and training status ( 16 , 17 ). Building upon this context, our study was aimed to study the mitochondrial function in PBMC from patients with primary fibromyalgia syndrome and to establish correlations with the clinical phenotype. 2. Methods 2.1 Patients Consecutive patients of all ages and sexes affected with primary fibromyalgia syndrome who were willing to participate in the study were included from June 2023 to January 2024 at the Fibromyalgia syndrome outpatient clinic of the S.C. Medicina – Emostasi e Trombosi at Fondazione IRCCS Ca’ Granda Ospedale Maggiore, Milano, Italy. FM was defined based on the presence of the 2016 criteria ( 3 ). Demographic data, smoke, comorbidities and current therapy were collected. Patients with a diagnosis of inflammatory or systemic autoimmune disease were excluded. 2.2 Study design and ethics committee approval. This is a cross-sectional study. Ethics committee approval was obtained by the Milan Area 2 Ethics Committee on 23rd May 2023 (approval n°310_2023). All patients provided written informed consent. The research was performed in accordance with the Declaration of Helsinki, and it adheres to relevant guidelines/regulations. 2.3 Fibromyalgia severity score. The fibromyalgia severity score (FSS) was used to classify the severity of patients ( 3 ). The FSS is composed of two parts: the widespread pain index (WPI), which ranges between 0–19 and is a summary count of the number of 19 painful regions from the self-reported Regional Pain Scale (RPS) ( 18 ); and the symptom severity scale (SSS), which ranges between 0–12, and is the sum of the severity scores of 3 symptoms (fatigue, waking unrefreshed, and cognitive symptoms) (0–9) plus the sum (0–3) of the score of the following symptoms occurring during the previous 6 months: ( 1 ) headaches (0–1), ( 2 ) abdominal pain or cramps (0–1) and ( 3 ) depression (0–1). The 2016 criteria envisage that the following three conditions are met: a) WPI ≥ 7 and SSS score ≥ 5 OR WPI of 4–6 and SSS score ≥ 9; b) generalized pain, defined as pain in at least 4 of 5 regions, with jaw, chest, and abdominal pain not included; c) symptoms must be present for at least three months. In addition, a diagnosis of fibromyalgia syndrome is valid irrespective of other diagnoses and does not exclude the presence of other clinically important illnesses ( 3 ). 2.4 Isolation of human peripheral blood-derived mononuclear cells (PBMC) PBMCs were isolated by using Ficoll-Hypaque density gradient centrifugation (Merck, Milan, Italy). Briefly, blood was collected and centrifuged (15 min. at 2500 x g) to separate the plasma. The remaining blood was then diluted with RPMI-1640 medium and layered onto the Ficoll-Hypaque media solution. The subsequent density gradient centrifugation (40 min. at 400 x g) allowed to isolate PBMC. 2.5 Mitochondrial functionality assessment Mitochondrial functionality was measured by means of the Seahorse XF Cell Mito Stress Test (Agilent, Santa Clara, USA), which measures key parameters of mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells. OCR was measured at baseline to determine basal respiration and after injection of optimized doses of specific mitochondrial activators and inhibitors to determine non-mitochondrial respiration, maximal respiration and spare respiratory capacity. This assay allows to obtain data relative to the reserve capacity, the ATP-linked mitochondrial respiration, the non-mitochondrial respiration and the proton leak. The spare respiratory capacity was calculated as a percentage, as follows: (maximal respiration/basal respiration)*100. PBMCs were seeded in a pre-coated sterile 24-well plate with a dedicated medium, at a density of 500,000 cells per well. OCR was recorded at the basal level and after the sequential injections of 1µM Oligomycin (ATP synthase inhibitor), 1 µM Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP, uncoupling agent) and a mixture of 0.5 µM Rotenone (complex I inhibitor) and Antimycin A (complex III inhibitor) ( 19 ). OCR data were then normalized per protein concentration in each well. Protein concentration was determined by the Pierce BCA protein assay (Thermo Fisher Scientific, Milan, Italy). 2.6 Mitochondrial bioenergetic health index (BHI) calculation To evaluate the Bioenergetic Health Index (BHI) ( 20 ) of each patient the OCRs of PBMCs, measured through the Seahorse XF Cell Mito Stress Test, were considered. The resulting indices of bioenergetic function were considered and the following formula was used: 2.7 Sample size calculation Based on our preliminary data, we considered a 10% difference between basal and stress as a normal result. Based on preliminary unpublished data from our group, we expected an 80% difference in patients with fibromyalgia. Therefore, to obtain a 5% alpha error and an 80% power, the minimum sample size for each group to detect whether the stated difference was at least 5 subjects per group. 2.8 Statistical analysis Continuous variables were described as median and interquartile range (IQR), correlation between variables was calculated via Spearman’s rho, differences between groups were calculated with Mann-Whitney U. Linear regression was employed to represent the linear model to predict the dependent variable (BHI) as a function of the independent variable (FSS and subscores). Statistical significance was set at a level of p < 0.05. 3. Results Fifty consecutive adult patients with primary fibromyalgia syndrome (98% females; median age 54.5 years) were included in the study. Ten healthy female subjects with a median age of 54 years were enrolled as the control group. Demographic data, smoking habit, current therapy, and comorbidities of the studied population are reported in Table 1 . None of the patients demonstrated altered erythrocyte sedimentation rate; 18% had Hashimoto’s disease and 22% had a positive antinuclear antibodies (ANA) test although with low titer, no anti-extractable nuclear antigen (ENA) antibody positivity and in the absence of signs and symptoms of connective tissue diseases. Two percent were positive to low titer rheumatoid factor (RF) and none of the patients had anti-citrullinated protein antibodies (ACPAs). To pursue the hypothesis that FM was related with an impairment of mitochondrial functionality, we isolated PBMC from the selected population (Table 1 ) and we evaluated the mitochondrial functionality through the Mito Stress Test (Fig. 1A). To easily represent patient's mitochondrial profile and facilitate statistical analysis, the BHI was calculated. This latter was used as a proxy of the overall mitochondrial functionality of the isolated PBMCs and served as a dynamic index of bioenergetic health. The injection of the uncoupling agent FCCP revealed how primary fibromyalgia syndrome could impair the cellular flexibility, as shown by the diminished spare respiratory capacity (Fig. 1B). This parameter was significantly reduced in patients with primary fibromyalgia syndrome, i.e., 174.7% (129.2%, 218.0%) when compared to controls (228.7% [171.1%, 321.1%]; p = 0.035). The analysis of the key parameters of mitochondrial function, obtained by measuring the cellular OCR, allowed to calculate the BHI, which was also significantly different between controls (median 1.81; 1.35–1.91; p = 0.033) and patients with FM (median 1.41; 0.85–1.65) (Fig. 1C). No significant difference was found in none of the mitochondrial parameters in patients with Hashimoto’s disease. In patients with positive ANA (n = 11), a significantly lower BHI was observed (p = 0.02). As FSS ≥ 20 indicates a more severe clinical phenotype ( 21 ), we stratified fibromyalgia patients in two groups with FSS ≥ 20 and < 20. As shown in Fig. 2, the lower the BHI and spare respiratory capacity were, the more severe the clinical symptoms. In particular, in the group with FSS ≥ 20, the spare respiratory capacity was reduced by 23% and the BHI by 19% compared with patients with FSS < 20. These differences were statistically different for both variables, respectively, p = 0.013 and p = 0.009. Based on these findings, we sought to evaluate the correlation among BHI, spare capacity and the fibromyalgia syndrome, assessed by Spearman correlations. As shown in Fig. 3 (A-C), we found negative moderate correlations between BHI and FSS (r= -0.36) and WPI (r= -0.38). A poor negative correlation was instead found between BHI and SSS (r= -0.1). The correlations between spare respiratory capacity and FSS and WPI were moderate, r= -0.31 and r= -0.37, respectively. A poor negative correlation was instead found between spare capacity and SSS (r= -0.12). A subsequent step involved assessing the linear regression between BHI and the FSS, revealing a beta coefficient of -0.39 (p = 0.005). Similarly, the linear regression between BHI and WPI yielded a beta of -0.43 (p = 0.002). Conversely, the linear regression between BHI and SSS did not show statistical significance (beta − 0.07, ns). 4. Discussion The main findings of the present study were twofold. First, we observed that patients suffering from FM exhibited impaired mitochondrial functionality compared to apparent healthy individuals. Among FM patients, those with a severe clinical presentation (FSS ≥ 20) demonstrated even worse mitochondrial functionality (lower BHI). Second, we identified a negative correlation only between BHI and WPI (a parameter describing musculoskeletal pain), and not between BHI and SSS. Considering that SSS reflects symptoms related to the psychological and functional domains (e.g., fatigue, sleep disturbance, cognitive symptoms, headache, abdominal pain or cramps, and depression), we hypothesize that the reduced mitochondrial functionality in FM patients primarily reflects musculoskeletal impairment rather than central nervous system involvement. The finding of a lower BHI in patients with positive ANA may suggest a role of these antibodies in mitochondrial function impairment, although the small sample size does not allow definitive conclusions. A study specifically designed to investigate the role of autoantibodies in mitochondrial function of patients with fibromyalgia syndrome will provide valuable insights into this field. The choice of evaluating mitochondrial function in PBMCs and using them as a surrogate biomarker to assess fibromyalgia severity can be justified by the following evidence: a) PBMC mitochondrial respiration correlates with walking speed in older human adults ( 22 ) and with heart and skeletal muscle mitochondrial function in nonhuman primates ( 16 ); b) markers of PBMC mitochondrial content (i.e., PGC1α, cytochrome c oxidase subunit IV and mitochondrial NADH dehydrogenase subunit appeared to reflect training status in humans ( 17 ); c) high levels of mitochondrial reactive oxygen species and low levels of citrate synthase, a mitochondrial matrix protein whose activity correlates with mitochondrial mass, have been described in PBMCs of FM patients ( 23 ); d) when the analysis of mitochondrial function was tested in skin biopsies of fibromyalgia syndrome patients, this was similar to the one detected in PBMCs ( 24 ); e) lipid peroxidation levels, one of the consequences of reactive oxygen species overproduction, are better associated to clinical symptoms in FM when measured in PBMCs than in plasma ( 25 ). Regarding the hypothesis that assessing mitochondrial function in PBMCs could serve as a surrogate marker of fibromyalgia syndrome severity, our findings align with the literature for similar clinical conditions. Deficits in mitochondrial function have been linked to chronic pain, and approximately 70% of patients with heritable mitochondrial diseases suffer from chronic pain ( 26 ). Impairment in mitochondrial basal respiration, extracellular acidification rate and ATP-linked OCR have been reported in patients suffering from chronic pain ( 27 ). Mitochondria play a role in pain resolution. For instance, macrophages can control the resolution of inflammatory pain by transferring mitochondria to sensory neurons ( 28 ). Proper regulation of mitochondrial activity appears crucial to prevent the transition from acute to chronic pain. Notably, compared to age- and sex-matched controls, PBMCs from fibromyalgia syndrome patients showed significantly lower mitochondrial coupling efficiency. Large-scale proteomics studies revealed altered mitochondrial metabolism, centered on pyruvate dehydrogenase and coenzyme A metabolism, leading to a decreased capacity to provide adequate intracellular ATP levels ( 18 ). The importance of mitochondria dysfunctionality in fatigue onset has been further highlighted by the ability to use mitochondrial electron transport chain gene expression to distinguish between individuals with systemic sclerosis and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) from non-fatigued patients. In this study, the presence of fatigue was associated with a specific gene expression pattern (i.e., reduced expression of mitochondrially encoded genes ND4 and CyB, and increased expression of nuclear gene Cox7C and ND4 and CyB expression). These expression changes were also correlated with disease severity, providing a distinct mitochondrial signature for systemic sclerosis patients with ME/CFS ( 29 ). Mitochondrial dysfunction has also been implicated in neuropathic pain. In a murine model induced by chronic constriction injury, various mitochondrial genes and mtDNA copy number were decreased, emphasizing the importance of impaired mitochondrial biogenesis and function in neuropathic pain ( 30 ). Furthermore, mitochondrial function may be linked to alterations in neurotransmitter release, including those involved in pain perception. This connection could contribute to the hypersensitivity to pain experienced by patients with fibromyalgia ( 31 , 32 ). Although the relationship between FM and mitochondrial dysfunction remains an active area of research, accumulating evidence suggests that mitochondrial dysfunction may play a role in the development and maintenance of fibromyalgia symptoms. Notably, a study investigating muscle energetics performance in a large cohort of older adults found that lower skeletal muscle energetics were associated with more severe performance fatigability ( 33 ). Interestingly, mitochondria ultrastructural alterations may underlie muscle fatigue. An analysis of murine fibers, particularly the fast-twitch type IIB/IIX fibers prone to fatigue, revealed elongated mitochondrial constrictions in response to fatigue. This effect was accompanied by elevated phosphorylation of Drp1, a key regulator of the fission ( 34 ). Whether the mitochondrial imbalance is the cause, or a consequence of this disease remains an open question ( 35 ). However, we propose mitochondrial dysfunction, and BHI in particular, as a biomarker of fibromyalgia syndrome severity that may support clinicians in the diagnosis of a disease that is still today underestimated and underdiagnosed also due to the absence of validated biomarkers. In addition, mitochondrial dysfunction could serve as a therapeutic target in a disease where pathogenic mechanisms are still unclear, leading to unsatisfactory treatment outcomes. Conclusion In our study, we observed impaired mitochondrial function in patients affected with primary fibromyalgia syndrome. Notably, the degree of impairment was more significant in patients with a more severe clinical phenotype and a more evident correlation with musculoskeletal pain was observed. Our findings support the possibility to use the measurement of this imbalance as a biomarker in the diagnosis and follow-up of patients with fibromyalgia syndrome. Our results underscore the need for further investigations in a larger cohort to identify potential biomarkers and treatment targets for this debilitating condition, which significantly impacts quality of life and currently lacks specific etiopathogenic treatments. Declarations Fundings. This study was partially supported by the Italian Ministry of Health – Bando Ricerca Corrente (2024), by Banca d’Italia (2023) and by Associazione Italiana Sindrome Fibromialgica (AISF). We thank the patients for participating in this study. Conflict of interest. RG is on the advisory boards of Bayer, Roche, Sanofi, SOBI, and Novo Nordisk, and has participated in speaker bureau/educational meetings for Pfizer, SOBI, Takeda, and Novo Nordisk. All the other authors have no competing interests to declare. Contributions CM, MR and RG conceived the study, analyzed the data and wrote the manuscript. CM, AG, IF, SL, SP performed the experiments and the analyses. NM and AC contributed to data interpretation and revised the manuscript. All authors provided critical feedback on drafts and approved the final manuscript. Acknowledgement This study was partially supported by the Italian Ministry of Health – Bando Ricerca Corrente (2024), by Banca d’Italia (2023) and by Associazione Italiana Sindrome Fibromialgica (AISF). We thank the patients for participating in this study. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on request. References Clauw DJ. Fibromyalgia: a clinical review. Jama. 2014;311(15):1547–55. Wolfe F, Smythe HA, Yunus MB, Bennett RM, Bombardier C, Goldenberg DL, et al. The American College of Rheumatology 1990 Criteria for the Classification of Fibromyalgia. Report of the Multicenter Criteria Committee. Arthritis Rheum. 1990;33(2):160–72. Wolfe F, Clauw DJ, Fitzcharles MA, Goldenberg DL, Häuser W, Katz RL, et al. 2016 Revisions to the 2010/2011 fibromyalgia diagnostic criteria. Semin Arthritis Rheum. 2016;46(3):319–29. Vincent A, Lahr BD, Wolfe F, Clauw DJ, Whipple MO, Oh TH, et al. Prevalence of fibromyalgia: a population-based study in Olmsted County, Minnesota, utilizing the Rochester Epidemiology Project. Arthritis Care Res (Hoboken). 2013;65(5):786–92. McBeth J, Jones K. Epidemiology of chronic musculoskeletal pain. Best Pract Res Clin Rheumatol. 2007;21(3):403–25. White KP, Speechley M, Harth M, Ostbye T. The London Fibromyalgia Epidemiology Study: the prevalence of fibromyalgia syndrome in London, Ontario. J Rheumatol. 1999;26(7):1570–6. Choy E, Perrot S, Leon T, Kaplan J, Petersel D, Ginovker A, et al. A patient survey of the impact of fibromyalgia and the journey to diagnosis. BMC Health Serv Res. 2010;10:102. Sarzi-Puttini P, Giorgi V, Marotto D, Atzeni F. Fibromyalgia: an update on clinical characteristics, aetiopathogenesis and treatment. Nat Rev Rheumatol. 2020;16(11):645–60. Ghavidel-Parsa B, Bidari A. The crosstalk of the pathophysiologic models in fibromyalgia. Clin Rheumatol. 2023;42(12):3177–87. Woolf CJ. Central sensitization: implications for the diagnosis and treatment of pain. Pain. 2011;152(3 Suppl):S2-s15. Wolfe F, Rasker JJ. The Evolution of Fibromyalgia, Its Concepts, and Criteria. Cureus. 2021;13(11):e20010. Harrington JS, Ryter SW, Plataki M, Price DR, Choi AMK. Mitochondria in health, disease, and aging. Physiol Rev. 2023;103(4):2349–422. Brum EDS, Fialho MFP, Fischer SPM, Hartmann DD, Gonçalves DF, Scussel R, et al. Relevance of Mitochondrial Dysfunction in the Reserpine-Induced Experimental Fibromyalgia Model. Mol Neurobiol. 2020;57(10):4202–17. Favero G, Bonomini F, Franco C, Rezzani R. Mitochondrial Dysfunction in Skeletal Muscle of a Fibromyalgia Model: The Potential Benefits of Melatonin. Int J Mol Sci. 2019;20(3). Gerdle B, Forsgren MF, Bengtsson A, Leinhard OD, Sören B, Karlsson A, et al. Decreased muscle concentrations of ATP and PCR in the quadriceps muscle of fibromyalgia patients–a 31P-MRS study. Eur J Pain. 2013;17(8):1205–15. Tyrrell DJ, Bharadwaj MS, Jorgensen MJ, Register TC, Molina AJ. Blood cell respirometry is associated with skeletal and cardiac muscle bioenergetics: Implications for a minimally invasive biomarker of mitochondrial health. Redox Biol. 2016;10:65–77. Busquets-Cortés C, Capó X, Martorell M, Tur JA, Sureda A, Pons A. Training and acute exercise modulates mitochondrial dynamics in football players' blood mononuclear cells. Eur J Appl Physiol. 2017;117(10):1977–87. Wolfe F. Pain extent and diagnosis: development and validation of the regional pain scale in 12,799 patients with rheumatic disease. J Rheumatol. 2003;30(2):369–78. Greco MF, Rizzuto AS, Zarà M, Cafora M, Favero C, Solazzo G, et al. PCSK9 Confers Inflammatory Properties to Extracellular Vesicles Released by Vascular Smooth Muscle Cells. Int J Mol Sci. 2022;23(21). Chacko BK, Kramer PA, Ravi S, Benavides GA, Mitchell T, Dranka BP, et al. The Bioenergetic Health Index: a new concept in mitochondrial translational research. Clin Sci (Lond). 2014;127(6):367–73. Wolfe F, Walitt BT, Rasker JJ, Katz RS, Häuser W. The Use of Polysymptomatic Distress Categories in the Evaluation of Fibromyalgia (FM) and FM Severity. J Rheumatol. 2015;42(8):1494–501. Tyrrell DJ, Bharadwaj MS, Van Horn CG, Kritchevsky SB, Nicklas BJ, Molina AJ. Respirometric Profiling of Muscle Mitochondria and Blood Cells Are Associated With Differences in Gait Speed Among Community-Dwelling Older Adults. J Gerontol A Biol Sci Med Sci. 2015;70(11):1394–9. Cordero MD, De Miguel M, Moreno Fernández AM, Carmona López IM, Garrido Maraver J, Cotán D, et al. Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease. Arthritis Res Ther. 2010;12(1):R17. Cordero MD, Moreno-Fernández AM, Carmona-López MI, Sánchez-Alcázar JA, Rodríguez AF, Navas P, et al. Mitochondrial dysfunction in skin biopsies and blood mononuclear cells from two cases of fibromyalgia patients. Clin Biochem. 2010;43(13–14):1174–6. Cordero MD, Alcocer-Gómez E, Cano-García FJ, De Miguel M, Carrión AM, Navas P, et al. Clinical symptoms in fibromyalgia are better associated to lipid peroxidation levels in blood mononuclear cells rather than in plasma. PLoS One. 2011;6(10):e26915. van den Ameele J, Fuge J, Pitceathly RDS, Berry S, McIntyre Z, Hanna MG, et al. Chronic pain is common in mitochondrial disease. Neuromuscul Disord. 2020;30(5):413–9. Chelimsky G, Simpson P, Zhang L, Bierer D, Komas S, Kalyanaraman B, et al. Impaired Mitochondrial Bioenergetics Function in Pediatric Chronic Overlapping Pain Conditions with Functional Gastrointestinal Disorders. Pain Res Manag. 2021;2021:6627864. van der Vlist M, Raoof R, Willemen H, Prado J, Versteeg S, Martin Gil C, et al. Macrophages transfer mitochondria to sensory neurons to resolve inflammatory pain. Neuron. 2022;110(4):613 – 26.e9. van Eeden C, Redmond D, Mohazab N, Larché MJ, Mason AL, Cohen Tervaert JW, et al. Evidence of a Novel Mitochondrial Signature in Systemic Sclerosis Patients with Chronic Fatigue Syndrome. Int J Mol Sci. 2023;24(15). Sun J, Li JY, Zhang LQ, Li DY, Wu JY, Gao SJ, et al. Nrf2 Activation Attenuates Chronic Constriction Injury-Induced Neuropathic Pain via Induction of PGC-1α-Mediated Mitochondrial Biogenesis in the Spinal Cord. Oxid Med Cell Longev. 2021;2021:9577874. Ivannikov MV, Sugimori M, Llinás RR. Synaptic vesicle exocytosis in hippocampal synaptosomes correlates directly with total mitochondrial volume. J Mol Neurosci. 2013;49(1):223–30. Duarte FV, Ciampi D, Duarte CB. Mitochondria as central hubs in synaptic modulation. Cell Mol Life Sci. 2023;80(6):173. Qiao YS, Santanasto AJ, Coen PM, Cawthon PM, Cummings SR, Forman DE, et al. Associations between skeletal muscle energetics and accelerometry-based performance fatigability: Study of Muscle, Mobility and Aging. Aging Cell. 2023:e14015. Lavorato M, Loro E, Debattisti V, Khurana TS, Franzini-Armstrong C. Elongated mitochondrial constrictions and fission in muscle fatigue. J Cell Sci. 2018;131(23). Martínez-Lara A, Moreno-Fernández AM, Jiménez-Guerrero M, Díaz-López C, De-Miguel M, Cotán D, et al. Mitochondrial Imbalance as a New Approach to the Study of Fibromyalgia. Open Access Rheumatol. 2020;12:175–85. Tables Table 1 Demographic data, smoking habits, current treatments and comorbidities of the studied population Cohort (n = 50) Control (n = 10) AGE (years) Median (IQR) 54.5 (51.0–61.0) 54 (36.0–56.0) SEX M, number (%) 1 (2.0) 0 (0.0) F, number (%) 49 (98.0) 10 (100) SMOKER Yes, number (%) 3 (6.0) 1 (10.0) No, number (%) 46 (92.0) 9 (90.0) FIBROMYALGIA SCORE Total fibromyalgia score, median (IQR) 21 ( 18 – 24 ) Widespread Pain Index score, median (IQR) 13 ( 10 – 16 ) Symptom Severity Score, median (IQR) 9 ( 7 – 10 ) CLINICAL CHARACTERISTICS History of Hashimoto’s thyroiditis, number (%) 9 ( 18 ) ANA positivity, number (%) 11 ( 22 ) ACPA positivity, number (%) 0 (0) RF positivity, number (%) 1 ( 2 ) ESR, mm/h, median (IQR) 13 ( 5 – 21 ) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 17 Oct, 2024 Reviews received at journal 07 Oct, 2024 Reviewers agreed at journal 14 Sep, 2024 Reviews received at journal 06 Sep, 2024 Reviewers agreed at journal 29 Aug, 2024 Reviewers invited by journal 07 Jul, 2024 Editor assigned by journal 07 Jul, 2024 Editor invited by journal 02 Jul, 2024 Submission checks completed at journal 01 Jul, 2024 First submitted to journal 28 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4654793","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":328390561,"identity":"d040c6f0-da12-49eb-8b02-dc0be9906aac","order_by":0,"name":"Chiara Macchi","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Chiara","middleName":"","lastName":"Macchi","suffix":""},{"id":328390562,"identity":"ae1feefa-69fd-4622-bbba-ac4b5339c1cc","order_by":1,"name":"Andrea Giachi","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Giachi","suffix":""},{"id":328390563,"identity":"f44ba0b2-8674-43b1-ae19-9729400e9a81","order_by":2,"name":"Isabella Fichtner","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Isabella","middleName":"","lastName":"Fichtner","suffix":""},{"id":328390565,"identity":"1f65b52a-e02a-43f2-970d-eb18b66cd8ef","order_by":3,"name":"Silvia Pedretti","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Silvia","middleName":"","lastName":"Pedretti","suffix":""},{"id":328390566,"identity":"bf3c8495-0729-46d3-9371-2df19f19149b","order_by":4,"name":"Piercarlo Sarzi Puttini","email":"","orcid":"","institution":"IRCCS Galeazzi-S.Ambrogio Hospital","correspondingAuthor":false,"prefix":"","firstName":"Piercarlo","middleName":"Sarzi","lastName":"Puttini","suffix":""},{"id":328390569,"identity":"6ab653d8-ac70-4900-9f38-f7e44df7a303","order_by":5,"name":"Nico Mitro","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Nico","middleName":"","lastName":"Mitro","suffix":""},{"id":328390572,"identity":"3b8784af-b66b-4151-8559-eea7286cd384","order_by":6,"name":"Alberto Corsini","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Alberto","middleName":"","lastName":"Corsini","suffix":""},{"id":328390573,"identity":"0dde2641-fcc0-468f-8c93-e77ea37ed265","order_by":7,"name":"Massimiliano Ruscica","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYLCCBAYJGTY4jxmIPzAw8IAYB/Bo4UHRwjgDpgWnHpACZMAM52LRott+/PGHhzkWPHzsZw8+YNxz2J6/nfnhZ9u2OzK67QyMhz9gajE7k2MmkbgN6DCevGQDhmeHE2ccZjOWzm17xmN2GLvDzA7ksDGAtTAA9TIcOJxgAHQVUMth3FrOP3/8AayF/w1Yiz1QC/NvS3xabiQYQBwmAbGFcQMzD5s0I14tb6B+kXhjbJBwIB3kFzPLnnMgLYwNB85gc1j6448/t9XJyffnGD74cMDanr//8OMbP8oO25udP3z4QwWmFlSQgMplbCCkYRSMglEwCkYBdgAA++Zk/NSy0vQAAAAASUVORK5CYII=","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":true,"prefix":"","firstName":"Massimiliano","middleName":"","lastName":"Ruscica","suffix":""},{"id":328390577,"identity":"0a1e4503-3939-4a84-95bc-1412f984150c","order_by":8,"name":"Roberta Gualtierotti","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Gualtierotti","suffix":""}],"badges":[],"createdAt":"2024-06-28 12:31:50","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4654793/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4654793/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-81298-x","type":"published","date":"2024-12-04T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61183933,"identity":"cb60f747-9d34-4ed3-a179-f8ceea98fea6","added_by":"auto","created_at":"2024-07-26 17:09:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80662,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of mitochondrial functionality in peripheral blood mononuclear cells (PBMC) of controls and patients with fibromyalgia syndrome (FMS). Panel A) shows the oxygen consumption rate (OCR) measured through the Seahorse XF Cell Mito Stress Test. Panel B) shows the spare respiratory capacity (as expressed in %) and panel C) the distribution of the BHI. Light grey line and bars represent the control group (n=10), and dark grey line and bars represent patients with fibromyalgia syndrome (FMS) (n=50). Yellow dots represent patients with FSS score \u0026lt; 20, while red dots represent patients with FSS score ≥ 20. Results are expressed as median (Q1, Q3). Differences between groups were assessed by Mann-Whitney U test, *p\u0026lt;0.05. AA, Antimycin A; BHI, Bioenergetic Health Index; FCCP Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; FMS, Fibromyalgia patients; FSS, Fibromyalgia Severity Score; OCR, Oxygen Consumption Rate; Rot, rotenone.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4654793/v1/9521cacf32b97db13df126a6.png"},{"id":61183931,"identity":"21964531-c02b-4648-9f8e-d4ef6521b6df","added_by":"auto","created_at":"2024-07-26 17:09:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":93600,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of mitochondrial functionality in peripheral blood mononuclear cells (PBMC) of patients with fibromyalgia syndrome and a FSS\u0026lt;20 (n=18) and in the group with FSS ≥20 (n=32). Panel A) shows the oxygen consumption rate (OCR) measured through the Seahorse XF Cell Mito Stress Test. Panel B) indicates the spare respiratory capacity (as expressed in %) and Panel C) the distribution of BHI. Orange bars represent the group with FSS\u0026lt;20 and red bars represent the FSS≥20 group. Results are expressed as median (Q1, Q3). Differences between groups were by Mann-Whitney U test, *p\u0026lt;0.05; **p\u0026lt;0.01. AA Antimycin A; BHI, Bioenergetic Health Index; FCCP Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone; FSS, Fibromyalgia Severity Score; OCR, Oxygen Consumption Rate; Rot, rotenone.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4654793/v1/1eaacf00ffee1ba1be91115c.png"},{"id":61184881,"identity":"707225d6-ca5f-41ab-85ad-426f66d6e2f6","added_by":"auto","created_at":"2024-07-26 17:17:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43488,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation analysis between the bioenergetic health index (BHI) vs total fibromyalgia severity score (panel A); and vs widespread pain index (panel B). BHI, bioenergetic health index; WPI, widespread pain index.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4654793/v1/e2ca5cdd44f0f55038c582d6.png"},{"id":70964722,"identity":"43b9cc00-78db-4a27-bb5f-4edc5a305e81","added_by":"auto","created_at":"2024-12-09 16:14:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":649231,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4654793/v1/88cb6937-0423-4e36-9cf6-e598c3857c78.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial function in patients affected with fibromyalgia syndrome is impaired and correlates with disease severity","fulltext":[{"header":"1. Background","content":"\u003cp\u003eFibromyalgia, a musculoskeletal syndrome, is characterized by chronic widespread pain that is often accompanied by systemic manifestations, such as persistent fatigue, sleep disturbance and cognitive impairment, hyperalgesia and allodynia (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). These symptoms significantly impact health-related quality of life. Initially, diagnostic criteria were based on tender points on the body, as defined by the American College of Rheumatology (ACR) (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). However, the criteria have since shifted to include a more complex set of features including both the widespread nature of pain and associated symptoms (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Fibromyalgia syndrome has a prevalence of 2\u0026ndash;8% in the general population and it can develop at any age, including childhood, with higher frequency in females (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite significantly impacting quality of life, fibromyalgia syndrome remains an under-diagnosed disease (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The pathogenic mechanisms and the underlying reason for fibromyalgia-related changes are not fully understood, leading to a lack of specific biomarkers (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Research suggests that it may be related to abnormalities in the central nervous system and immune system, as well as to genetic and environmental factors (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Initially perceived primarily as a rheumatic disorder, it is now recognized as a disorder involving pain processing and central nervous system sensitization (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOne theory proposed, as a potential contributor to fibromyalgia syndrome (FM), involves the dysfunction of mitochondria, which are the energy-producing organelles within our cells and serve as the primary source of cellular energy. When mitochondria malfunction, it can lead to reduced adenosine triphosphate (ATP) production, increased oxidative stress, disrupted calcium homeostasis, and altered mitochondrial membrane potential. These impairments can affect metabolism, cell death and inflammation pathways, innate immune responses, potentially contributing to the pathogenesis of several diseases (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the context of FM pathogenesis, the relevance of mitochondrial dysfunction has been highlighted in a reserpine-induced fibromyalgia rodent model. These animals exhibited reduced functionality of mitochondrial complexes I and II, decreased electron transport, impaired mitochondrial respiration, reduced mitochondrial biogenesis, and altered mitochondrial dynamic. Additionally, they experienced lower ATP synthesis in muscles and spinal cord (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Furthermore, individuals with FM could show decreased ATP production in their muscle cells, contributing to muscle fatigue and overall fatigue experienced by fibromyalgia patients (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Notably, the mitochondrial function of peripheral blood mononuclear cells (PBMC) correlates with heart and skeletal muscle mitochondrial function and training status (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBuilding upon this context, our study was aimed to study the mitochondrial function in PBMC from patients with primary fibromyalgia syndrome and to establish correlations with the clinical phenotype.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Patients\u003c/h2\u003e \u003cp\u003e Consecutive patients of all ages and sexes affected with primary fibromyalgia syndrome who were willing to participate in the study were included from June 2023 to January 2024 at the Fibromyalgia syndrome outpatient clinic of the S.C. Medicina \u0026ndash; Emostasi e Trombosi at Fondazione IRCCS Ca\u0026rsquo; Granda Ospedale Maggiore, Milano, Italy. FM was defined based on the presence of the 2016 criteria (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Demographic data, smoke, comorbidities and current therapy were collected. Patients with a diagnosis of inflammatory or systemic autoimmune disease were excluded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Study design and ethics committee approval.\u003c/h2\u003e \u003cp\u003eThis is a cross-sectional study. Ethics committee approval was obtained by the Milan Area 2 Ethics Committee on 23rd May 2023 (approval n\u0026deg;310_2023). All patients provided written informed consent. The research was performed in accordance with the Declaration of Helsinki, and it adheres to relevant guidelines/regulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fibromyalgia severity score.\u003c/h2\u003e \u003cp\u003eThe fibromyalgia severity score (FSS) was used to classify the severity of patients (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The FSS is composed of two parts: the widespread pain index (WPI), which ranges between 0\u0026ndash;19 and is a summary count of the number of 19 painful regions from the self-reported Regional Pain Scale (RPS) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e); and the symptom severity scale (SSS), which ranges between 0\u0026ndash;12, and is the sum of the severity scores of 3 symptoms (fatigue, waking unrefreshed, and cognitive symptoms) (0\u0026ndash;9) plus the sum (0\u0026ndash;3) of the score of the following symptoms occurring during the previous 6 months: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) headaches (0\u0026ndash;1), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) abdominal pain or cramps (0\u0026ndash;1) and (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) depression (0\u0026ndash;1).\u003c/p\u003e \u003cp\u003eThe 2016 criteria envisage that the following three conditions are met: a) WPI\u0026thinsp;\u0026ge;\u0026thinsp;7 and SSS score\u0026thinsp;\u0026ge;\u0026thinsp;5 OR WPI of 4\u0026ndash;6 and SSS score\u0026thinsp;\u0026ge;\u0026thinsp;9; b) generalized pain, defined as pain in at least 4 of 5 regions, with jaw, chest, and abdominal pain not included; c) symptoms must be present for at least three months. In addition, a diagnosis of fibromyalgia syndrome is valid irrespective of other diagnoses and does not exclude the presence of other clinically important illnesses (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Isolation of human peripheral blood-derived mononuclear cells (PBMC)\u003c/h2\u003e \u003cp\u003ePBMCs were isolated by using Ficoll-Hypaque density gradient centrifugation (Merck, Milan, Italy). Briefly, blood was collected and centrifuged (15 min. at 2500 x g) to separate the plasma. The remaining blood was then diluted with RPMI-1640 medium and layered onto the Ficoll-Hypaque media solution. The subsequent density gradient centrifugation (40 min. at 400 x g) allowed to isolate PBMC.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Mitochondrial functionality assessment\u003c/h2\u003e \u003cp\u003eMitochondrial functionality was measured by means of the Seahorse XF Cell Mito Stress Test (Agilent, Santa Clara, USA), which measures key parameters of mitochondrial function by directly measuring the oxygen consumption rate (OCR) of cells. OCR was measured at baseline to determine basal respiration and after injection of optimized doses of specific mitochondrial activators and inhibitors to determine non-mitochondrial respiration, maximal respiration and spare respiratory capacity. This assay allows to obtain data relative to the reserve capacity, the ATP-linked mitochondrial respiration, the non-mitochondrial respiration and the proton leak. The spare respiratory capacity was calculated as a percentage, as follows: (maximal respiration/basal respiration)*100. PBMCs were seeded in a pre-coated sterile 24-well plate with a dedicated medium, at a density of 500,000 cells per well. OCR was recorded at the basal level and after the sequential injections of 1\u0026micro;M Oligomycin (ATP synthase inhibitor), 1 \u0026micro;M Carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP, uncoupling agent) and a mixture of 0.5 \u0026micro;M Rotenone (complex I inhibitor) and Antimycin A (complex III inhibitor) (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). OCR data were then normalized per protein concentration in each well. Protein concentration was determined by the Pierce BCA protein assay (Thermo Fisher Scientific, Milan, Italy).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Mitochondrial bioenergetic health index (BHI) calculation\u003c/h2\u003e \u003cp\u003eTo evaluate the Bioenergetic Health Index (BHI) (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e) of each patient the OCRs of PBMCs, measured through the Seahorse XF Cell Mito Stress Test, were considered. The resulting indices of bioenergetic function were considered and the following formula was used:\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cimg 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hL24uq+LPFgmTbEDUmAm045Oojbcimt1hqXOg0ZpwbaPv/fdd98QREb80PFoyAQm3xofHZ9N1eAIJh0OrcVIvoblYxTsOcKuolHrEPudOAh4mo8RN1+iuNI2CdhNNtkk7vGc+BOe4t99nxE23x2twmh/PMRZODqeiToZQpvGQjgYQOjkdbQEoLhNlfo+4RLAb3rTm2IwQavQietMu8JCvup8F6YzR7/sK+JAQMsjGibBJc916oSfcpgMHXRNj7/v6c2aHYZBAr+rvKRhGRypcwZi/H4wW5YAnQrezRKintKaCU4+PnWJsIJ4EkCElbz34QvmB3TPeHk1EcPqMLp1SrjKkNWCr1zZGyjJg4pyFhb/cr8+i+ff/M3fxGBNuPW5mq4u3fcuc5SMTJJpgQ+TT6h0RPebcVo6+raMWsMHVTqnwdXRuP7669sDDjgg1rYddthhcY1/kt+RX2sy+DD5zsx27fuqKnw1wjv44INj1qOZgPyNpaOI3/kbvZuvbWF9NaXTCp8Xf9V4vroiuGKGrHwqmsF98pH/ib+rO8u0+jBLpz24Mp8inMPXWjq4MV9aEerhxzr55JPv48csHWqkq/ri+DL5z5Rj9V8OY5gPE3Ud5kc+8pHBlQV4r3SYSXvWWWfF3/K9+psngp+NH7IIm/AvSpvZyZPxsY99LPyx0lOEQPjzyoAq8m7u3Lnt05/+9IjDueeeO3hi4XyYC8NUfZhFS4/Z3V34LYugC3+1tgI+THlcBpPtb3/72whPHnzgAx8InyaqT175T4Y5CfLozDPPHFxZQK7DTJLFDG2A2Y1Zzlqv/mjeSLf65ox8u+ZXvjtmVmEwpYG2RUNimmNK7cPsNxUTGy2A9kMTMeNQ/Ph2qgmLeYr267q1fLTFLkbzw3yLXWhytCuj/WqSrLjGzyXcav6idZVOKn5nDjYj1Qi/P6Iv7TpM0+6p0NxocbRTWrh7isCJ56VLOcA7+e/8X8OVt0yV/FvdeArjmmuuCW0K42kW1cc1TGPznhe+8IXxrFm88pKvtW/q6yNfzOqVLuZCf/N9m90rXeNBI+cHl26zRM0qNcPTDG0znM0WZt1wH7PsVDV4eTKexjcVxsvLRYUWbLcfmrX2Yta1Nb1Q5/m1i+Ac6sNVn2sb4jtWx4tQjPZZ4YpglVFftc9ljRSYybSh49DA+Jh0JH2YwUyYYQZ7+9vfHmYwDc6+npYL1M2nTbgxQcGSCAJMA7esQYNkToNJDibwuNekC9PghcWUyvTkGp8SdII6DnEbFi8wvxIkJjkQPoSGjrx2Ar6ZfnX2/KnMr4S1+Fnu8a53vSviO1744Ht69atfHX9b1iIceaDz3nPPPcOX6B5+WHGVH4Se8Ov7dEzDOmjPmhQjH5gICQcCjxlbWsRLuASPSTMmHxEQwrXcQodd466DLdppdJjMciaMiKfOljlRfCAeNW+7ENLS4T4bO1hG0l0KY4KTZQ0XX3xxDAJMYJH/4yFe0lS0m5hkwuQpLUzLBhfveMc7xt18Qf1QJ9QXA6JhqJNFww7fYx0MeCcB4TNMiPq91qmpCtkutc10hZCwXRN+N2xxcd13lxqG+2sZusaFUMPlN9XmlKk9c9VzAzgDUYNQcwbksfosvywvka91yZDyMtdA/RKOumjAox5zk2gf4rDMUTI0SaaFSy+9NExYpul3TYldmLdKJx4mQIvsLQuwJKN06mEeQ9EiYknHCiusEFPW/cZsZfH6bbfdFvfg5ptvDvOfKfXMV8JiIhOu5+uSFBsX+G2//fZri+Ya14bBpFw6ljA92RCgdDiDXxYgjUxx4mPRv/gx9xYh0RbhM7hrfIQ5b968MPmati8c6WQutCQBlm7ssMMO8bv8tIGBd8q3oqHex0xXTbKWZAhDWD4WtDMvl0HD4M754TIzMqMWjS7CYqqzxRtznrRVSscaeWvJAbOd5UDCtaygLvQvwjBM8N7dhYnQBgnewyzI5FmXtqB0+GEWFm/5MGyzgi7C22mnnWLpS/dey0OYHJVD0VQHV++Lre+Uz1FHHTW4cn9KRx+bDIirNEHdkueW68i3Pup3GTxE+Qzb+GBUmL+lqwjzwZX5Jtk5c+bcL2/kmbZSBpeDK/ORPza1WH/99ceW2JTBUrQDptqKumeDCOXIvSAN2oNyVn/VRd/KtGiekec1PBRBGu8og6u41302gWDm1kZHWUbzQCNPK0mmDTNITd83bd0klfFMNLY/s2zBaN5kgtKAw1RpBFsnflgyYBmKiSHMaX6n8dEEutBmvZNJSbhMp0yQwvIN2gezkZmpTH/CG4YRuaUktCEnPdDMhuF37zT6NmK3REb8jOL78RsGDUH6pdGEFFuumXxDM6vQnNxjiQftSH7SLk0sEbeaT7Qs5kmmTWZUJlThFyEVGwj042OCjeU6zLdM3cJ1v/cJt3u/vBUHzzCRmxyiDHxDPjCH01SY27vUpSPeY+IJzVw5V8TTMgzapQ0rmFfHg2lXfjOPS1NdjgP5QUOS/9LfhwmXBsSkaILOeDA1ywP1Q72RdnWBhsYU35/1TYOTdnHr59tUUH9o+9pM1YCVB+uAmdLCLsIprstT6ZFO5tWKuFpC5Vt50u61K/kvz+vENQhT+UuPe+Wl98lf5aw+dtuQslbvKvJEnitX9ZVVRhpo8u5n4l2WSIGZJMsQVWAyyXZnBc9kdOx8j/xqzMNmA3c75SSZKaQPM0mWIfiNqi/xgQLfss3cac60nxSWyUwlBWaSLEMwMzPZ1dm8DwSY/pj0rPFjxkuSmUqaZJNkGcKuPHyJ/Elmtz4Q4A+1nIXvc1nzeSXLFktcYDIXdV85kfml3muyyLB1SX5zT//38a7PJEwoMNHB5IN6mkOSJEkyc1miAtOC4rolFcy+MuvKDD4z9Ori6Yr1dhZwW2s1bEabWWlmfjk2yKw4SI53eJeF3uPNbFyaiKPF+tYN2v6trjdMkiRJZi5LzIdpEoIdU+wBagGss9Ys7rZHob0KbQhsGn8X08stviY0+xA6FkDbF9FGxxWapQW0FlhbjDsTofVWzdqU7yRJkmTms0QFpnVxsLOE3V3sGGK3EELjqKOOip01uhB+GKYEu1Z/r99w3bsIpe71JEmSJFkUlpjAJMDqwvXNN988zKhmxTFJ2lrJaQlMsN3p8FULG2/B+7Dfu9rbTPVfjoLFwxYNmwwx2Vl5NgiwWNv0fIuGfZi9l8mtqZIkSZYSS0xgogqwvum17lKhkx+mFQ6bGERIjidIH+jwyx588MHNXnvtFcc62W/UYnQzILtY8G0/Tb/bI9T99oI84IADmjlz5sQekEmSJMniYalInO7kHpqUbbxcszXTsG3KhglMLIrAZLrl6yRc+EkJmu6n+lqrGXlJwSzt9HgbH9tizabStvKyg4udUOoG4hB/u6KIowXf7nf+nk20nWrRn0SVJEmSLDxLTGASUFXw8V/SmJxIsffee8f/jtSpJzdUqkA0QcgBwLQuH0LCt5MPMJ5AnQjxsWejkzGcvOC7+3HNso/+sU3Tib1EHTfExMq/67QLk6HOOuus2ObMtU9/+tNxrz1FTYqyO4rTOOSREwVoltbf+Wb6TpIkSRYTRXAsEe6+++62aHJm78QpAHbOnzVrVvzvM3v27PudAuDAXr85LcIzdtX3WXHFFeP/Iijj9yJQBk/MP2nAwcBOqyja2uDqcJxecc0114z7cfhq0YAHdy9ePvWpT0XcnYJRKQIy0uRg5XvvvXdwdT5O2JAmp1Y4ANZJGk4QKJpn+8c//nFwV9sW4RlhDDvYNUmSJFl4ltg6TKZXS0BoQzZYZkLkr7T7PxPiySefHGcbnn766bEmE9YpfuITnwgty+L+6t+keZoc5N4zzjgjPvvss0/85jpfHi3RdWs4ZyI0RWcJOqdx3rx5kTZnPpo1zCzbj7eTCSzBMZHHvpv8wU52YLJlvmV+5dMUnnMVaaMLm3YarXPuhvmTkyRJljTElHX7zul00s1Sm79CYC4J7rnnnjgTzysvuOCCwdX53Hnnne2ee+4Z5+GddNJJg6ttnPfn/vHOlzvmmGPi949+9KODK6NrmLSyCy+8sC2CJbSx/sd1Z+E5M2466GuYNPA5c+bENdpkH+cYOnvQOYXf+973Iv41z8pApC2Dg/aNb3xjWwRnnIXXPSdyqgiPlloqZX7yk5/8zJjPhz70oftZ35YkS1TDNLmmnipPO+pCI+LH5Kt04jyqhlkEbGxu0IX2Y+MDJ8QXgRnPYlQN030777zz2OnyfWSLMwYtdemeS7i46GuY3veWt7wljjn68pe/HKe+dzHxZ9ddd401q0XYRpx333330Dhvv/32OIfPhKnZs2fHBKGqpS8MdlByzt4SqhpJkiSTos/baKONoj8e1mcvEQjMJUFXw+yfqP6HP/whTv722zve8Y7B1QUaZl8jhVGGe/2+sBqm082dGu5k+/7HdZqtuE0Hw3yYH/zgB0Ozc2I5jbPL+eefHyfd77fffvE/jdIJ9qeddlqchu+kdN9JkiTJ9LBEDcG0OjjKx3Z3Ptdcc03M9jRrdu2112622WabuAf1/hLP+O5TfWz93113bbznYITiVHj28C233PJ+H9eNZqZraUaNW00Dttpqqzil33IRPkR+S7NhL7300uboo4+O09L5MfG4xz0u9tf9h3/4h9CybTvIDywvL7nkkvutdU2SJEkWjSUmMAkoHX7RoGJZiI3Rt9tuuzDNzp07N9Rs6x5N/KlUoTJM8AmvCpuu0HHvKAJzadKNVzfu66yzTphTmYItsWGCtSHBLrvsEpvJH3bYYSHMsdlmmzXPfvazY8Bh0pT1l5aVuIep+aSTToodf5IkSZLFw4OPsonrEmC55ZYL/9tqq63WrLnmmuFjs8OPk0r48vjvaFhdzKB1GO6OO+4Y310IzHvuuad56EMfGsLXqScVs0UJHQL5sY997ODqzEHc+XT5H2nU8qBCCNIyDS5o2DYgMIh485vfHL7ZurGDmbJmCe+///5x2gl/Jr/n+uuv31x11VWxy8+mm246Lf7XJEmSP0Vm9AHSBAqh6PR4QqYP7czGAsymNNeK6/amfeQjHzn0uZmAbBdHcTddehgm8mCVVVaJ7wphayODiy++OIRj3VqwQrieeOKJzWc+85nQTpMkSZJFZyktZhkNgoRWOp7Qq1prV1jCdRraTBWWEDcCfTxhCYKyLyzhWf5LfkqmWObam2++OXYKsmsS/6eDqZl4kyRJksXDjNYwk/GhWR555JHNeeedF4dkGzjQtu+8885m3XXXjfNAmXv7g4kkSZJk4UiB+QDGbGOHZzsGzFrMWbNmxexZm9gTojNZw06SJHmgkQJzGYDPlr+XeZc5Oll4NIelNdBQjjbpcHqPiXCWNc00WDAsX3rCE55wn0lok1G7mYXJWy4Hy6ZMYjNzPJl+lJflbJ/85Cdj1n2dnT8dqPe2TOVSspHLyiuvPPhl5pG96zIAIWm2cArLhcdEqkMPPTQElQ56aSAOBKY1ydbSzkTuuOOOWBPMd14npU3GRRddFJPULHsy832q2HnKftIGEsmSwQx91it10eTC6USdIDC9a9Q6tbTIHjZJCjoIx7np3LtnoBIQlvB8/etfj+VK0wnty6daC2Yi4scvLo6jaos33nhjLHOy3aLnpkp930zNk8UFwWSbTxP4lja1LmoXo1oRFgXvMGActU4tLVJgJknBelfrWu1p7GScys9+9rPm8MMPj1NkciOI+dTOdFSsD/7c5z4X+0SvuOKKg6tTZ6Z3pouKHb5sWEKj/lNjqnVqaZECM5kQvoy77rorNC0Y6d9yyy2hNfBnjYfnPGN7v5///OdDTS3CMlmpmul+97vfNb/4xS9iO8CpmO5qONa1QjjeaVKUdbwVYbruHcPCt571ec97Xox2xd893XhLD+1z2LaD8kjY0vurX/1qbFvHYXTvrfnap47qhVXDnAiHjgtTuYhj//38RN7l3TCjWv54ZqKBgLS6T5l4Zpi2QfO+9dZbQ0OAe8W55jEhaRDypCc9Kf7vUsv8hhtuiG//Lwq1vgqrbxGQB+I5Ub2Vb/26JE/Hi5t8r2UjrwywTMLr5r84eadw5Le/XavIN/WsaljCUVbu65ej+iwNDmOgiQ6Lk3wXB+lFfXc/XouKtIuHsvb3MGp+yhf3TiUO4i+N8remZWmzxHb6SR6Y6DiMfGkIZuA6e/P4448P05GlLXZUWn311Qd3z0eH7TQWOxC5zwYKfHIayhprrBH+VthL2HaIOjgdgZNabOl39tlnRye91lprNSuttFLcOxEaoskCOhkd0QknnBBhffGLXwxhYE2qjuq4446LjzTo1O02Vde5itsxxxwTp+M4GYcZkClWfMRTh8C36RQcHZIdleDv888/P9Iqn5wk861vfSvul1ZrbSsE+4UXXhjaKn8ePyA/EVNj3XyCWY4f017GOgr7BJ9yyilj98mTrq9a5+p0GxtVmBjj/d6hA7bLk+VG0FFJO+1Fntpz2KlAzj6Vvic+8YlRvl2Ur3DdJy90jNJjna91wLvttluE/93vfrd5+9vfHuH+4Ac/iF27TBZ51KMeFbtWXXHFFbFlo7Kxf7P4ywsmcOFLn/CFy2xrQpG4VI3jxz/+cdQheT7ZGa/eoYxs3qEMvb8urXIykTNnCX07ag1DvTjiiCOi/Nz37ne/O9KvTsonO4dJe0U9E3fl528nKH3/+9+PuEq/OqktSCcLhnTIL8Jc/bBxiTJRJ9QjApCQ4Br49re/3Tz3uc8dawN18pOwlJv6/aMf/ah5zGMeE3Gq+fWVr3wl2qjr8t7f9pyW3977tKc9bawNjod8NOnHyVIvetGLYivOijrvujrv9CntSfuWFmmq+a3N8U2Kr3Yl7Uzz4mC3t7pPt4GC9ePavG1TTfohIJ0LrLzcLx9mhEm+ZEySjEvptNtddtmlXWGFFdrSCbSlw2tf/vKXt9tss01bGmi7zjrrtKWTG9w9/2zTuXPnxkkqpZLHCTW77rprW4RqWzSNtjTeOLkGToRZbbXV2tKA23XXXbfdaKON2t13371db731DL/b17zmNW3pQOLeiSgNuy2NrC0CIp7dcMMNI85PecpT2tIxtGVM2O6www5tEZDt7Nmz2yJAI/xDDjmkLcI6wiiNMtInjtJTOoW2dHBt6SjilJjSgbc77bRThFuETTxTGnrcUzqmtnQUcT6pjzgUoRAny5SON+4tHUBbOvK2dKztrFmz2pe97GWRVvkn3kUoRByKgIx8FYb823bbbdsiJCL/vcepNRUn6TjhRtpLB9TutddeccKPvJQ+55rW9F1zzTVt0Z7b0rFGuEVgtK94xSvaTTfdNO5VnqWjjntx9dVXR9qL0Gi32mqrSLt3iL9r2223XVs0h7i3dJpR3htssEFbtMgoR7+ffPLJ8Xs9mUd+izOKxhblUARau8cee7RFyMYzpVOMuJTONu5D6Wjj+XpSz2R85zvfaYvQjU8ROnFNXLfccsvIq2GnH1Uuv/zyeE7+yy/xKwODdvPNN4+ThIRRBOfg7jbiLO1+V65bb711lIH8c3rQ4YcfHvklrc67lefqoWfmzZsXZe6d6rr3KvvnP//50cYOOOCAsXwoA5F2iy22iPpZBlNx//bbb98uv/zy0SadsFR5z3veE3EtWn1bBlhtEXhRb9Uf9chJR5NRBjRtEYiR78KrqMfOLF511VWjvbzpTW9qDzrooEjTk5/85PucEKXdqQ/yRNm/9rWvjfiUQVdbBGmEBW1cmrXVmt4yGIj8cP5vEc5L9QzMLikwkwkpI+R27733joajEynaTAiTokW2+++/fzTwY489Nu7VAMpIMgTMzjvv3F577bVR0d3vsG5CpWgyYweCOyj76U9/eoStM7nhhhuiA7nyyiujoRBStcObCPdrmDomcRKO9+qoiwYZcdSZe684ul8HJi46K3hv0Syj0+sOAIpmF4JC56STd5/OBEWTiIGANEgfAeo3wk/n5r1FE4h7y+g5Oi4djQ5LJ2Hg4Ii5ol20119/ffx/9NFHR34Q9joVArdo+W3RdKITVBb1/fJPGgg/8fR8DVN6dUBF04t7r7vuuhB8wiaQLrvsshBeRWtsX/ziF0cHXkb5ca806gjdq8M3aBIHaSQMXfdM0VrjfmVeNK+4/oY3vKEtGn/UD3UHRXMMQVi0zDGBSYAZeBDkdQB16623xoBDOAYXlakKTPlTtNZ4pg66ivYXcTBgmwhlagDnWUKL4BM/5XfggQfGdYKulgFhqlzUVemUbvnid/WPgJJX6pRr2gKB7R2eqcJb3ZF2YZ177rmRT8oBBK98VZ8cmq881G91g1Crg4w6OCLQtIVHP/rRMaAyiPVeh+LXAdBvfvObuHc8xHWYwFS3tGODrjpwEBflRcBpJ7fccktclw7Crr7LfQShNkmA18FkFZjqoLhq8waRBpPq9UwiBWYyITo9GoCR7Zlnnjm4Oh+N3Qh33333jf/da3StoXY1oYpOX+M+7rjj4n8dvsa3ySabhJDr4kxQnc3HP/7x+J+g04HoSOqnQgB6p1E+oVYRHw1b3PvxoX3pDM4555z4X6c4TGBeccUVMSp+yUteEh1VRQd3wgknhCZJ4PX5whe+EHkjP3DRRRfFQMJZrTqvPjV9NEzxctZrHYHDyFvc5JXn3ese7z/11FMHdy3gs5/9bPy2zz77xP8EMo2RhinfuyhX9xLWuPHGG9uNN944NIauNgVC82EPe1hoOVXDJDB1gsIn9PoME5jjQWOX/tNOO20s/VMVmCC0aObiRfjTgAiKYfHrQmC61wCsXyel3YBHuDXtNEaCjEDuQsgpa3V4WFtwv+eUd0U7IjDPO++8wZX5XHXVVdFO1M+bbrppcHU+v//972Nwpv5X4XLiiSdGfmlDhFSF4DIQJawNoCZiPIHpGqF79tlnD64sgAapftHwx0Mc1BNt1WANVWDSPLUbv9HuRxksL2ly0k8yKaWexCzSMoIcXJlPEUTxXYRNfPObWXzMR1I6nbjWpXT24bvic0TpMMJ3KNzuYmX38C2WDjP+RunUY7alzw477BAfPiPUcPhs+FQr/G38Pz7i1KX6FksHHt/jIVzpFxd/V/hd+Na8swjlwdUF2G2pdCyRVs/x5/AH299XXvaRBngX+BTrNfAVes57ISw+Lb6rYXsG1/eb7FGEa4QlDU796Z/gI3/8VsuR79REEf4/5dCldNzhS+rmBfzvnf1ThSbD8XT8xHycc+bMCR9djWvNi4VBufBj+ra+T76ZrjHK6UXe6zl528VJS9LPx6iuV8pgI3zOXfiMlT3f5rC2IK+8h1+4lmnN01oOFROO+Fb5f8WhS9HKov55RlhdxFXcKkUYh19V3tZ3TgXxrfVZeywCcuxTNPnw3ZfBXPjVK/7mi+SLf/3rXx9lXAakY37siripp3yWfLp8mTaqmGmkwExGQiPrVnDU/2vHrhFqIJz5GkCfOtFAw6h4VjjDOmDUsN1DSAi/jEjjI06VGs6wjkBD73dCtTOu4U8V7zahiPAYNoGCsBK290qLiU2oEx0mo5+Ofl77Xx54tzj0cV2nJK+7eevvGlaln9eeEW8Cup8/4lXzrov7XO+WyUR4p4lPRXOPySMmwlx77bUxAxTi3n/3VDEZzcBDnAny/uS0iRhW38VJvvqtnwfdPIY8NBgz0OkKrUq3LfSf7aPOS4Py6IclLuqU7267Qv//bvksTN7KD5OhhKN+EI7apI93bbjhhiEU6wCBYCxadnPAAQfEBKWrr746ZstqN/3yFaY8ISSFZVKRyYMzjRSYyWJDw6WRGH13R5kVDUAjqaP8fqczjHqPhqfRnXvuufHtc9BBB8Vvo4SzqIh3t4Fr8LRZnWJX26jQ0HRO0kp4Vm2c5rGo8fW8MGkLhOawvL7jjjuig/N+WshU0HH5SFs/rv186DKVdJkBajaqPY/N+DSD0kxsM2wNtvoDnKki7Wa2/vCHPwzNjOXDLNXJLAqVYen0LIFB+BKEXaogqvh9hRVWiHIY9s4qDJRPHUTV/Ou/l/VFGQ4Ly73qmjBoxdOJgZm4eKeD7mnu3Y8tE81WNzBRfmZOm5nLeqBslbGyZmnqC3P5J1yHRtBCzTxmQeoPWpY2KTCTxYbGZNmDqe6m4HdR8Qk5nYgObKrogGbNmhWdQv1Us+p0oiPy0VF2Gy+BwhSqE6Md9TG1n1aw3nrrRUegExFfSw5Mn+9C4PU7kInQsQrLwePMp1deeeXglwV4v/uYVb2/36FPhCUdTH/Cvf322wdX579X/GkUw7TaqWDZBA3lVa96VRyQ7p3KVDzlszgvCpbEvPe97439eHXS1tdaujEsr/oYDCkjJvcu6jQzuLKczPRMEFrWQ2umWXWRbktI1CsnC9W0ahu0zb5QtH7VAfmEPw2tizXRliExqw8zzS9Oaj1W9pdffnm0dwNkH3/7VI36uuuui6UmXCEHHnhgxF/5SqP0Dytf5S4sG4Wot5YbEbQziRSYyaToKOunS71WO2OVnYlNg7L2S0NmfiFUVP4zzjgjfDf8jxgvXEz0Wx/3iMOwe10fJizqvd1nhoWhI5IufhVaivTwR9KCttlmmzA/8efolI30/W5goHPWae69994Rjvu23nrrGHFbA0kQufecc84J7VkHpBMZFq9KTYvf5PG2224bHZj1n9b60XSFqZOR/965xx57DJ5ekKd9+u9kxtx8883DPGodYk23NPItgYbbfa5+hjHsd/lKOPB7iTdNmfXAekH0w+9+TwbtjZnXQGRO0Vasw+VjM8jhS1NOEyFvlc8+++zTfOlLX4q06/yVqXirv1Vrl4Zh8fK7NaOsANa/Wn8oHAMv+ajMDBT45CsGDeqAwYQ48l0anBC83mkQwN/LjygswptGxncpnXyWmCifhtXx8RiW7zvttFMM1KyttG5WPJSd+Dpq0H6/BL4BnQ/BKT3uMQihNV522WVRFl1q3ZZedVq9g32L3T9qnKebFJjJpGjERo0qdB8dW1fz2m677aIR81845YD5xSifqc23TocAgkYg3Pp3xd/eSShN5t/p0vebCoeWpwH3G5y06Bi7aarXuvca3dcJHRbqE/jMT7D4et68efFeo+iNN944PjZwpz3YMHzttdeOe00gkQcWzHuGv8dnzz33jIk5Osua7n68Kq4xddX4WRxv4wdptKWad8tvWpuOnYalc4NnxNP3sLC9UzgVgl5ZMo05qULYRv7+pjXTEio1bOHWuHVxTTnXsoaOV16aEEJwCJc5T92Qd9IpL1DzpFvPxkMcDBjEe/bs2WODs1122aXZfvvtQ3DZTGNYHlS8R3n5KEvxFF8a5yGHHBIDlYpw+nWmsuOOO8b9djHyfmUjrIMPPjisLDbK6E6kE78qjDbbbLM4mYUwJHi4H4Rn0CkcA5qtttoqBmcGAyY4VWqcuvndZbz61aWWWT/f1XmTp7gY5I16scUWW0R8tXdm8Cr0DDgIRgNCdckGCEzx2oQwaxyUr7/dW/NR2tQ3Grp8MtFqJpA7/SQTojKrxHxNKnF3Fmqt6HbG0YlCZ6fx+F8DcI8Rsk6coGCyrfhNo9SJmOlXR501XCYejaw/O7dPDYcAIkSq4HRNx6tzIvS6vjzhM6vp/Jgf6zurJlh9VMJgNmMOJrzdq2NgavKbsHVe9X5apU762GOPjXzpohMhGKSrvn/33XeP0bT3dtMtXsxYFb8pB3mlw5RG76c9+V+n6lmC18kg3r9pZ5ZhDVtaxNf9XQx85LUOEcpZuOLoWQMHnf/cuXMjrdIvv5WZeCh36X3GM54R//cxYcV7CQS/E4zywnPM0eJt0GGCCGEv7vKrouMWv8nMjsqbYJMO4dGWIX3qnrh7l/o2LJ60arvSKGe7FUkf7cjWfoQSTa6WNZSJ+qm8xLuLOEuztiCdkCYChOakLLrIY21FPVW+8pMVQ94pD0KHEBdv16TB/rwGYjV8KC+/q/PdPKx1QD4Iu18HutR7pdV7q/YKAk9Z1HiKj/6BAH3d6143lucGyN4vj4RlYCTd4i1/CdtumxSvml7hqmM0dPHUrutAe2mS52EmSZIMsLWfARHhZztCAkNnP0y4JvMxkKyD1GWdrAVJkiQDCEcf5unqDkhhOTF/KsISWROSJEkGMKMybzILMksmSZc0ySZJkgwwi9XpLwSmSTZ8n0lSSYGZJEmSJCOQJtkkSZIkGYEUmEmSJEkyAikwkyRJkmQEUmAmSZIkyQikwEySJEmSEUiBmSRJkiQjkAIzSZIkSUYgBWaSJEmSjEAKzCRJkiQZgRSYSZIkSTICKTCTJEmSZARSYCZJkiTJCKTATJIkSZIRSIGZJEmSJCOQAjNJkiRJRiAFZpIkSZKMQArMJEmSJBmBFJhJkiRJMgIpMJMkSZJkBFJgJkmSJMkIpMBMkiRJkhFIgZkkSZIkk9I0/w9mK0EY9nWwkwAAAABJRU5ErkJggg==\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Sample size calculation\u003c/h2\u003e \u003cp\u003eBased on our preliminary data, we considered a 10% difference between basal and stress as a normal result. Based on preliminary unpublished data from our group, we expected an 80% difference in patients with fibromyalgia. Therefore, to obtain a 5% alpha error and an 80% power, the minimum sample size for each group to detect whether the stated difference was at least 5 subjects per group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eContinuous variables were described as median and interquartile range (IQR), correlation between variables was calculated via Spearman\u0026rsquo;s rho, differences between groups were calculated with Mann-Whitney U. Linear regression was employed to represent the linear model to predict the dependent variable (BHI) as a function of the independent variable (FSS and subscores). Statistical significance was set at a level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eFifty consecutive adult patients with primary fibromyalgia syndrome (98% females; median age 54.5 years) were included in the study. Ten healthy female subjects with a median age of 54 years were enrolled as the control group. Demographic data, smoking habit, current therapy, and comorbidities of the studied population are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. None of the patients demonstrated altered erythrocyte sedimentation rate; 18% had Hashimoto\u0026rsquo;s disease and 22% had a positive antinuclear antibodies (ANA) test although with low titer, no anti-extractable nuclear antigen (ENA) antibody positivity and in the absence of signs and symptoms of connective tissue diseases. Two percent were positive to low titer rheumatoid factor (RF) and none of the patients had anti-citrullinated protein antibodies (ACPAs).\u003c/p\u003e \u003cp\u003eTo pursue the hypothesis that FM was related with an impairment of mitochondrial functionality, we isolated PBMC from the selected population (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and we evaluated the mitochondrial functionality through the Mito Stress Test (Fig.\u0026nbsp;1A). To easily represent patient's mitochondrial profile and facilitate statistical analysis, the BHI was calculated. This latter was used as a proxy of the overall mitochondrial functionality of the isolated PBMCs and served as a dynamic index of bioenergetic health. The injection of the uncoupling agent FCCP revealed how primary fibromyalgia syndrome could impair the cellular flexibility, as shown by the diminished spare respiratory capacity (Fig.\u0026nbsp;1B). This parameter was significantly reduced in patients with primary fibromyalgia syndrome, i.e., 174.7% (129.2%, 218.0%) when compared to controls (228.7% [171.1%, 321.1%]; p\u0026thinsp;=\u0026thinsp;0.035). The analysis of the key parameters of mitochondrial function, obtained by measuring the cellular OCR, allowed to calculate the BHI, which was also significantly different between controls (median 1.81; 1.35\u0026ndash;1.91; p\u0026thinsp;=\u0026thinsp;0.033) and patients with FM (median 1.41; 0.85\u0026ndash;1.65) (Fig.\u0026nbsp;1C). No significant difference was found in none of the mitochondrial parameters in patients with Hashimoto\u0026rsquo;s disease. In patients with positive ANA (n\u0026thinsp;=\u0026thinsp;11), a significantly lower BHI was observed (p\u0026thinsp;=\u0026thinsp;0.02).\u003c/p\u003e \u003cp\u003eAs FSS\u0026thinsp;\u0026ge;\u0026thinsp;20 indicates a more severe clinical phenotype (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), we stratified fibromyalgia patients in two groups with FSS\u0026thinsp;\u0026ge;\u0026thinsp;20 and \u0026lt;\u0026thinsp;20. As shown in Fig.\u0026nbsp;2, the lower the BHI and spare respiratory capacity were, the more severe the clinical symptoms. In particular, in the group with FSS\u0026thinsp;\u0026ge;\u0026thinsp;20, the spare respiratory capacity was reduced by 23% and the BHI by 19% compared with patients with FSS\u0026thinsp;\u0026lt;\u0026thinsp;20. These differences were statistically different for both variables, respectively, p\u0026thinsp;=\u0026thinsp;0.013 and p\u0026thinsp;=\u0026thinsp;0.009.\u003c/p\u003e \u003cp\u003eBased on these findings, we sought to evaluate the correlation among BHI, spare capacity and the fibromyalgia syndrome, assessed by Spearman correlations. As shown in Fig.\u0026nbsp;3 (A-C), we found negative moderate correlations between BHI and FSS (r= -0.36) and WPI (r= -0.38). A poor negative correlation was instead found between BHI and SSS (r= -0.1). The correlations between spare respiratory capacity and FSS and WPI were moderate, r= -0.31 and r= -0.37, respectively. A poor negative correlation was instead found between spare capacity and SSS (r= -0.12).\u003c/p\u003e \u003cp\u003eA subsequent step involved assessing the linear regression between BHI and the FSS, revealing a beta coefficient of -0.39 (p\u0026thinsp;=\u0026thinsp;0.005). Similarly, the linear regression between BHI and WPI yielded a beta of -0.43 (p\u0026thinsp;=\u0026thinsp;0.002). Conversely, the linear regression between BHI and SSS did not show statistical significance (beta \u0026minus;\u0026thinsp;0.07, ns).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe main findings of the present study were twofold. First, we observed that patients suffering from FM exhibited impaired mitochondrial functionality compared to apparent healthy individuals. Among FM patients, those with a severe clinical presentation (FSS ≥ 20) demonstrated even worse mitochondrial functionality (lower BHI). Second, we identified a negative correlation only between BHI and WPI (a parameter describing musculoskeletal pain), and not between BHI and SSS. Considering that SSS reflects symptoms related to the psychological and functional domains (e.g., fatigue, sleep disturbance, cognitive symptoms, headache, abdominal pain or cramps, and depression), we hypothesize that the reduced mitochondrial functionality in FM patients primarily reflects musculoskeletal impairment rather than central nervous system involvement. The finding of a lower BHI in patients with positive ANA may suggest a role of these antibodies in mitochondrial function impairment, although the small sample size does not allow definitive conclusions. A study specifically designed to investigate the role of autoantibodies in mitochondrial function of patients with fibromyalgia syndrome will provide valuable insights into this field.\u003c/p\u003e \u003cp\u003eThe choice of evaluating mitochondrial function in PBMCs and using them as a surrogate biomarker to assess fibromyalgia severity can be justified by the following evidence: a) PBMC mitochondrial respiration correlates with walking speed in older human adults (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e) and with heart and skeletal muscle mitochondrial function in nonhuman primates (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e); b) markers of PBMC mitochondrial content (i.e., PGC1α, cytochrome c oxidase subunit IV and mitochondrial NADH dehydrogenase subunit appeared to reflect training status in humans (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e); c) high levels of mitochondrial reactive oxygen species and low levels of citrate synthase, a mitochondrial matrix protein whose activity correlates with mitochondrial mass, have been described in PBMCs of FM patients (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e); d) when the analysis of mitochondrial function was tested in skin biopsies of fibromyalgia syndrome patients, this was similar to the one detected in PBMCs (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e); e) lipid peroxidation levels, one of the consequences of reactive oxygen species overproduction, are better associated to clinical symptoms in FM when measured in PBMCs than in plasma (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRegarding the hypothesis that assessing mitochondrial function in PBMCs could serve as a surrogate marker of fibromyalgia syndrome severity, our findings align with the literature for similar clinical conditions. Deficits in mitochondrial function have been linked to chronic pain, and approximately 70% of patients with heritable mitochondrial diseases suffer from chronic pain (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Impairment in mitochondrial basal respiration, extracellular acidification rate and ATP-linked OCR have been reported in patients suffering from chronic pain (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Mitochondria play a role in pain resolution. For instance, macrophages can control the resolution of inflammatory pain by transferring mitochondria to sensory neurons (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Proper regulation of mitochondrial activity appears crucial to prevent the transition from acute to chronic pain. Notably, compared to age- and sex-matched controls, PBMCs from fibromyalgia syndrome patients showed significantly lower mitochondrial coupling efficiency. Large-scale proteomics studies revealed altered mitochondrial metabolism, centered on pyruvate dehydrogenase and coenzyme A metabolism, leading to a decreased capacity to provide adequate intracellular ATP levels (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe importance of mitochondria dysfunctionality in fatigue onset has been further highlighted by the ability to use mitochondrial electron transport chain gene expression to distinguish between individuals with systemic sclerosis and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) from non-fatigued patients. In this study, the presence of fatigue was associated with a specific gene expression pattern (i.e., reduced expression of mitochondrially encoded genes ND4 and CyB, and increased expression of nuclear gene Cox7C and ND4 and CyB expression). These expression changes were also correlated with disease severity, providing a distinct mitochondrial signature for systemic sclerosis patients with ME/CFS (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMitochondrial dysfunction has also been implicated in neuropathic pain. In a murine model induced by chronic constriction injury, various mitochondrial genes and mtDNA copy number were decreased, emphasizing the importance of impaired mitochondrial biogenesis and function in neuropathic pain (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, mitochondrial function may be linked to alterations in neurotransmitter release, including those involved in pain perception. This connection could contribute to the hypersensitivity to pain experienced by patients with fibromyalgia (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough the relationship between FM and mitochondrial dysfunction remains an active area of research, accumulating evidence suggests that mitochondrial dysfunction may play a role in the development and maintenance of fibromyalgia symptoms. Notably, a study investigating muscle energetics performance in a large cohort of older adults found that lower skeletal muscle energetics were associated with more severe performance fatigability (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Interestingly, mitochondria ultrastructural alterations may underlie muscle fatigue. An analysis of murine fibers, particularly the fast-twitch type IIB/IIX fibers prone to fatigue, revealed elongated mitochondrial constrictions in response to fatigue. This effect was accompanied by elevated phosphorylation of Drp1, a key regulator of the fission (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhether the mitochondrial imbalance is the cause, or a consequence of this disease remains an open question (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). However, we propose mitochondrial dysfunction, and BHI in particular, as a biomarker of fibromyalgia syndrome severity that may support clinicians in the diagnosis of a disease that is still today underestimated and underdiagnosed also due to the absence of validated biomarkers. In addition, mitochondrial dysfunction could serve as a therapeutic target in a disease where pathogenic mechanisms are still unclear, leading to unsatisfactory treatment outcomes.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eIn our study, we observed impaired mitochondrial function in patients affected with primary fibromyalgia syndrome. Notably, the degree of impairment was more significant in patients with a more severe clinical phenotype and a more evident correlation with musculoskeletal pain was observed. Our findings support the possibility to use the measurement of this imbalance as a biomarker in the diagnosis and follow-up of patients with fibromyalgia syndrome. Our results underscore the need for further investigations in a larger cohort to identify potential biomarkers and treatment targets for this debilitating condition, which significantly impacts quality of life and currently lacks specific etiopathogenic treatments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFundings.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was partially supported by the Italian Ministry of Health \u0026ndash; Bando Ricerca Corrente (2024), by Banca d\u0026rsquo;Italia (2023) and by Associazione Italiana Sindrome Fibromialgica (AISF). We thank the patients for participating in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRG is on the advisory boards of Bayer, Roche, Sanofi, SOBI, and Novo Nordisk, and has participated in speaker bureau/educational meetings for Pfizer, SOBI, Takeda, and Novo Nordisk. All the other authors have no competing interests to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCM, MR and RG conceived the study, analyzed the data and wrote the manuscript. CM, AG, IF, SL, SP performed the experiments and the analyses. NM and AC contributed to data interpretation and revised the manuscript.\u0026nbsp;All authors provided critical feedback on drafts and approved the final manuscript.\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eThis study was partially supported by the Italian Ministry of Health \u0026ndash; Bando Ricerca Corrente (2024), by Banca d\u0026rsquo;Italia (2023) and by Associazione Italiana Sindrome Fibromialgica (AISF). We thank the patients for participating in this study.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eClauw DJ. Fibromyalgia: a clinical review. Jama. 2014;311(15):1547\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolfe F, Smythe HA, Yunus MB, Bennett RM, Bombardier C, Goldenberg DL, et al. The American College of Rheumatology 1990 Criteria for the Classification of Fibromyalgia. Report of the Multicenter Criteria Committee. Arthritis Rheum. 1990;33(2):160\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolfe F, Clauw DJ, Fitzcharles MA, Goldenberg DL, H\u0026auml;user W, Katz RL, et al. 2016 Revisions to the 2010/2011 fibromyalgia diagnostic criteria. Semin Arthritis Rheum. 2016;46(3):319\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVincent A, Lahr BD, Wolfe F, Clauw DJ, Whipple MO, Oh TH, et al. Prevalence of fibromyalgia: a population-based study in Olmsted County, Minnesota, utilizing the Rochester Epidemiology Project. Arthritis Care Res (Hoboken). 2013;65(5):786\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcBeth J, Jones K. Epidemiology of chronic musculoskeletal pain. Best Pract Res Clin Rheumatol. 2007;21(3):403\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhite KP, Speechley M, Harth M, Ostbye T. The London Fibromyalgia Epidemiology Study: the prevalence of fibromyalgia syndrome in London, Ontario. J Rheumatol. 1999;26(7):1570\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoy E, Perrot S, Leon T, Kaplan J, Petersel D, Ginovker A, et al. A patient survey of the impact of fibromyalgia and the journey to diagnosis. BMC Health Serv Res. 2010;10:102.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarzi-Puttini P, Giorgi V, Marotto D, Atzeni F. Fibromyalgia: an update on clinical characteristics, aetiopathogenesis and treatment. Nat Rev Rheumatol. 2020;16(11):645\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhavidel-Parsa B, Bidari A. The crosstalk of the pathophysiologic models in fibromyalgia. Clin Rheumatol. 2023;42(12):3177\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoolf CJ. Central sensitization: implications for the diagnosis and treatment of pain. Pain. 2011;152(3 Suppl):S2-s15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolfe F, Rasker JJ. The Evolution of Fibromyalgia, Its Concepts, and Criteria. Cureus. 2021;13(11):e20010.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrington JS, Ryter SW, Plataki M, Price DR, Choi AMK. Mitochondria in health, disease, and aging. Physiol Rev. 2023;103(4):2349\u0026ndash;422.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrum EDS, Fialho MFP, Fischer SPM, Hartmann DD, Gon\u0026ccedil;alves DF, Scussel R, et al. Relevance of Mitochondrial Dysfunction in the Reserpine-Induced Experimental Fibromyalgia Model. Mol Neurobiol. 2020;57(10):4202\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFavero G, Bonomini F, Franco C, Rezzani R. Mitochondrial Dysfunction in Skeletal Muscle of a Fibromyalgia Model: The Potential Benefits of Melatonin. Int J Mol Sci. 2019;20(3).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGerdle B, Forsgren MF, Bengtsson A, Leinhard OD, S\u0026ouml;ren B, Karlsson A, et al. Decreased muscle concentrations of ATP and PCR in the quadriceps muscle of fibromyalgia patients\u0026ndash;a 31P-MRS study. Eur J Pain. 2013;17(8):1205\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTyrrell DJ, Bharadwaj MS, Jorgensen MJ, Register TC, Molina AJ. Blood cell respirometry is associated with skeletal and cardiac muscle bioenergetics: Implications for a minimally invasive biomarker of mitochondrial health. Redox Biol. 2016;10:65\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBusquets-Cort\u0026eacute;s C, Cap\u0026oacute; X, Martorell M, Tur JA, Sureda A, Pons A. Training and acute exercise modulates mitochondrial dynamics in football players' blood mononuclear cells. Eur J Appl Physiol. 2017;117(10):1977\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolfe F. Pain extent and diagnosis: development and validation of the regional pain scale in 12,799 patients with rheumatic disease. J Rheumatol. 2003;30(2):369\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreco MF, Rizzuto AS, Zar\u0026agrave; M, Cafora M, Favero C, Solazzo G, et al. PCSK9 Confers Inflammatory Properties to Extracellular Vesicles Released by Vascular Smooth Muscle Cells. Int J Mol Sci. 2022;23(21).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChacko BK, Kramer PA, Ravi S, Benavides GA, Mitchell T, Dranka BP, et al. The Bioenergetic Health Index: a new concept in mitochondrial translational research. Clin Sci (Lond). 2014;127(6):367\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolfe F, Walitt BT, Rasker JJ, Katz RS, H\u0026auml;user W. The Use of Polysymptomatic Distress Categories in the Evaluation of Fibromyalgia (FM) and FM Severity. J Rheumatol. 2015;42(8):1494\u0026ndash;501.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTyrrell DJ, Bharadwaj MS, Van Horn CG, Kritchevsky SB, Nicklas BJ, Molina AJ. Respirometric Profiling of Muscle Mitochondria and Blood Cells Are Associated With Differences in Gait Speed Among Community-Dwelling Older Adults. J Gerontol A Biol Sci Med Sci. 2015;70(11):1394\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCordero MD, De Miguel M, Moreno Fern\u0026aacute;ndez AM, Carmona L\u0026oacute;pez IM, Garrido Maraver J, Cot\u0026aacute;n D, et al. Mitochondrial dysfunction and mitophagy activation in blood mononuclear cells of fibromyalgia patients: implications in the pathogenesis of the disease. Arthritis Res Ther. 2010;12(1):R17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCordero MD, Moreno-Fern\u0026aacute;ndez AM, Carmona-L\u0026oacute;pez MI, S\u0026aacute;nchez-Alc\u0026aacute;zar JA, Rodr\u0026iacute;guez AF, Navas P, et al. Mitochondrial dysfunction in skin biopsies and blood mononuclear cells from two cases of fibromyalgia patients. Clin Biochem. 2010;43(13\u0026ndash;14):1174\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCordero MD, Alcocer-G\u0026oacute;mez E, Cano-Garc\u0026iacute;a FJ, De Miguel M, Carri\u0026oacute;n AM, Navas P, et al. Clinical symptoms in fibromyalgia are better associated to lipid peroxidation levels in blood mononuclear cells rather than in plasma. PLoS One. 2011;6(10):e26915.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan den Ameele J, Fuge J, Pitceathly RDS, Berry S, McIntyre Z, Hanna MG, et al. Chronic pain is common in mitochondrial disease. Neuromuscul Disord. 2020;30(5):413\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChelimsky G, Simpson P, Zhang L, Bierer D, Komas S, Kalyanaraman B, et al. Impaired Mitochondrial Bioenergetics Function in Pediatric Chronic Overlapping Pain Conditions with Functional Gastrointestinal Disorders. Pain Res Manag. 2021;2021:6627864.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan der Vlist M, Raoof R, Willemen H, Prado J, Versteeg S, Martin Gil C, et al. Macrophages transfer mitochondria to sensory neurons to resolve inflammatory pain. Neuron. 2022;110(4):613\u0026thinsp;\u0026ndash;\u0026thinsp;26.e9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003evan Eeden C, Redmond D, Mohazab N, Larch\u0026eacute; MJ, Mason AL, Cohen Tervaert JW, et al. Evidence of a Novel Mitochondrial Signature in Systemic Sclerosis Patients with Chronic Fatigue Syndrome. Int J Mol Sci. 2023;24(15).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun J, Li JY, Zhang LQ, Li DY, Wu JY, Gao SJ, et al. Nrf2 Activation Attenuates Chronic Constriction Injury-Induced Neuropathic Pain via Induction of PGC-1α-Mediated Mitochondrial Biogenesis in the Spinal Cord. Oxid Med Cell Longev. 2021;2021:9577874.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIvannikov MV, Sugimori M, Llin\u0026aacute;s RR. Synaptic vesicle exocytosis in hippocampal synaptosomes correlates directly with total mitochondrial volume. J Mol Neurosci. 2013;49(1):223\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuarte FV, Ciampi D, Duarte CB. Mitochondria as central hubs in synaptic modulation. Cell Mol Life Sci. 2023;80(6):173.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQiao YS, Santanasto AJ, Coen PM, Cawthon PM, Cummings SR, Forman DE, et al. Associations between skeletal muscle energetics and accelerometry-based performance fatigability: Study of Muscle, Mobility and Aging. Aging Cell. 2023:e14015.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLavorato M, Loro E, Debattisti V, Khurana TS, Franzini-Armstrong C. Elongated mitochondrial constrictions and fission in muscle fatigue. J Cell Sci. 2018;131(23).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMart\u0026iacute;nez-Lara A, Moreno-Fern\u0026aacute;ndez AM, Jim\u0026eacute;nez-Guerrero M, D\u0026iacute;az-L\u0026oacute;pez C, De-Miguel M, Cot\u0026aacute;n D, et al. Mitochondrial Imbalance as a New Approach to the Study of Fibromyalgia. Open Access Rheumatol. 2020;12:175\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eDemographic data, smoking habits, current treatments and comorbidities of the studied population\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eCohort\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e(n\u0026thinsp;=\u0026thinsp;50)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003eControl\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003e(n\u0026thinsp;=\u0026thinsp;10)\u003c/div\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eAGE (years)\u003c/div\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eMedian (IQR)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e54.5 (51.0\u0026ndash;61.0)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e54 (36.0\u0026ndash;56.0)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eSEX\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003eM, number (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1 (2.0)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0 (0.0)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eF, number (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e49 (98.0)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e10 (100)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eSMOKER\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eYes, number (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e3 (6.0)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e1 (10.0)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eNo, number (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e46 (92.0)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e9 (90.0)\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eFIBROMYALGIA SCORE\u003c/span\u003e\u003c/div\u003e \u003cdiv class=\"SimplePara\"\u003eTotal fibromyalgia score, median (IQR)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e21 (\u003cspan additionalcitationids=\"CR19 CR20 CR21 CR22 CR23\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eWidespread Pain Index score, median (IQR)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e13 (\u003cspan additionalcitationids=\"CR11 CR12 CR13 CR14 CR15\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eSymptom Severity Score, median (IQR)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e9 (\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eCLINICAL CHARACTERISTICS\u003c/span\u003e\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eHistory of Hashimoto\u0026rsquo;s thyroiditis, number (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e9 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eANA positivity, number (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e11 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eACPA positivity, number (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e0 (0)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eRF positivity, number (%)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e1 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cdiv class=\"SimplePara\"\u003eESR, mm/h, median (IQR)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003e13 (\u003cspan additionalcitationids=\"CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e)\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003cbr/\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4654793/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4654793/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFibromyalgia is a musculoskeletal syndrome characterized by chronic widespread pain that is often associated with systemic manifestations. Since mitochondria are the main source of cellular energy, we hypothesized that fibromyalgia syndrome could be linked to mitochondrial impairment. Aim was to study mitochondrial dysfunction in peripheral blood mononuclear cells isolated from 50 patients with primary fibromyalgia syndrome and 10 apparently healthy controls. Although no differences in mitochondrial basal respiration were observed between patients with primary fibromyalgia syndrome and healthy controls, a lower median bioenergetic health index (BHI; -22.4%, p\u0026thinsp;=\u0026thinsp;0.03), a proxy of mitochondrial function, was found in patients. According to fibromyalgia severity score (FSS), a composite of widespread pain index and symptom severity scale, a lower median BHI (-18.7%) was found in patients with a FS\u0026thinsp;\u0026ge;\u0026thinsp;20 compared to those with a FSS\u0026thinsp;\u0026lt;\u0026thinsp;20. Negative moderate correlations were found only between BHI and FSS (r= -0.36) and widespread pain index (r= -0.38). We demonstrated that patients with fibromyalgia syndrome had an impaired mitochondrial function. Additionally, we found a mild correlation between the widespread pain index and the BHI, possibly indicating that the altered mitochondrial function, in these patients, narrows musculoskeletal rather than central nervous system involvement.\u003c/p\u003e","manuscriptTitle":"Mitochondrial function in patients affected with fibromyalgia syndrome is impaired and correlates with disease severity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-26 17:09:41","doi":"10.21203/rs.3.rs-4654793/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-18T02:48:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-07T16:55:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290759906990274351642636643648960100095","date":"2024-09-14T10:54:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-06T18:24:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326079766807792794510384135235116517820","date":"2024-08-29T14:36:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-07T06:59:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-07T06:55:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-02T15:37:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-02T03:45:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-06-28T12:30:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6830ff38-9770-4d62-8332-85b1f9982a69","owner":[],"postedDate":"July 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":34757253,"name":"Health sciences/Biomarkers"},{"id":34757254,"name":"Health sciences/Rheumatology"}],"tags":[],"updatedAt":"2024-12-09T16:01:52+00:00","versionOfRecord":{"articleIdentity":"rs-4654793","link":"https://doi.org/10.1038/s41598-024-81298-x","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-12-04 15:57:27","publishedOnDateReadable":"December 4th, 2024"},"versionCreatedAt":"2024-07-26 17:09:41","video":"","vorDoi":"10.1038/s41598-024-81298-x","vorDoiUrl":"https://doi.org/10.1038/s41598-024-81298-x","workflowStages":[]},"version":"v1","identity":"rs-4654793","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4654793","identity":"rs-4654793","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}