Dietary Strategies for the Treatment of Fibromyalgia in Overweight and Obese Patients

Fibromyalgia FM is a heterogeneous and disabling syndrome characterized by multisite pain, sleep disturbances, cognitive dysfunction "fibro fog", and fatigue. The prevalence of FM is approximately 1-5% in the adult population, with a global female-to-male ratio of about 3:1.

The etiopathogenesis of the disease remains unclear; however, accumulating evidence suggests that FM is a central disorder that generates pain through altered processing of non-painful or mildly painful stimuli. Pain in FM is classified as nociplastic, defined as "pain arising from altered nociception despite no clear evidence of actual or threatened tissue damage causing activation of peripheral nociceptors." Recently, a theoretical model of FM pathogenesis has been proposed, based on extensive literature suggesting a role for gamma-aminobutyric acid GABA-the main inhibitory neurotransmitter of the central nervous system-in the pathophysiology of chronic pain conditions such as FM. In particular, the proposed model posits that reduced GABAergic neurotransmission at the thalamocortical level-potentially related to neuroendocrine imbalance leading to altered activity of neurosteroids with positive allosteric modulation of GABA-A receptors or NMDA receptors-may contribute to the development of fibromyalgia pain. This hypothesis requires validation through preclinical and interventional studies.

FM is typically managed using a multimodal approach that includes both nonpharmacological and pharmacological strategies. The most common nonpharmacological interventions include physical therapy, relaxation techniques, psychotherapy, acupuncture, and dietary therapy.

Diet is, in fact, the primary factor capable of influencing the microbiota-gut-brain axis, and dysbiosis has been associated with multiple centrally mediated disorders, including altered nociception and FM. Indeed, microbial metabolites-particularly short-chain fatty acids SCFAs-are able to modulate central neurotransmission by altering, for example, the GABA/glutamate ratio. Furthermore, the prevalence of overweight/obesity reaches 70% in patients with FM, a value substantially higher than in the general population. The high prevalence of overweight/obesity, dysbiosis, and gastrointestinal alterations in FM suggests that dietary therapy and the microbiota-gut-brain axis may play an important role in the pathogenesis of FM.

Although data regarding the relationship between various dietary approaches and FM are limited, the Mediterranean diet has been associated with lower pain intensity, according to an observational study conducted in 186 individuals with FM. A pilot study involving 18 patients also reported the effectiveness of the ketogenic diet not only in promoting weight loss but also in improving symptom severity in obese women with FM. Similarly, evidence suggests that the ketogenic diet may enhance GABA levels and, more broadly, increase the GABA/glutamate ratio.

In this context, in addition to dietary therapy, it may be appropriate to consider supplementation with bacterial strains capable of producing neurotransmitters-such as GABA-that may be deficient in patients with FM and could exert beneficial effects on the central nervous system.

The primary objective of this study is to evaluate the impact of a specific dietary approach-ketogenic diet, Mediterranean diet, or Mediterranean diet supplemented with GABA-producing probiotics like Bifidobacterium adolescentis PRL2019-on the severity of FM, specifically focusing on pain, sleep quality, anxiety, and depression, as assessed using validated questionnaires in female subjects diagnosed with fibromyalgia and mild overweight/obesity.

The secondary objectives are to assess the impact of the dietary intervention on anthropometric measures, metabolic parameters, and body composition of the participants; to evaluate the effect of the dietary intervention on gut microbiota composition through metabolomic analysis; and to measure levels of neurosteroids with positive allosteric activity at GABA-A or NMDA receptors in saliva and/or plasma. Additionally, an exploratory pilot substudy aims to measure cerebral GABA and glutamate levels using magnetic resonance spectroscopy in a small subset of patients.

Participants in this study will be recruited from the outpatient clinics of the Rheumatology Unit at IRCCS Policlinico San Martino Hospital in Genoa. Given the high female prevalence of FM, the study sample will include adult female patients diagnosed with fibromyalgia and mild overweight/obesity.

Each patient enrolled in the study will self-administer, at the Rheumatology Unit, a battery of validated questionnaires designed to accurately assess the severity of FM, with particular focus on the following symptoms: pain, sleep quality, depression, anxiety, and bowel function. Patients will be asked to complete the questionnaires at enrollment/baseline, every four weeks during follow-up visits as per the study protocol, and at the end of the intervention (12 week).

The questionnaires include the Widespread Pain Index WPI and the Symptom Severity Scale SSS, both used in the diagnosis of FM, as well as the Fibromyalgia Impact Questionnaire FIQR. The FIQR assesses patients' quality of life, including physical function, overall disease impact, and symptoms. It is the most widely used questionnaire in research and clinical practice for estimating disease severity, and a minimal clinically important difference has been defined as a 14% reduction in the score.

To further investigate pain, an 11-point Numeric Rating Scale will be administered to assess the average global pain intensity over the past 24 hours. The PainDETECT Questionnaire PD-Q will evaluate the type and characteristics of pain, particularly targeting neuropathic/nociplastic components, especially in chronic pain patients.

Anxiety and depression will be assessed using the Hospital Anxiety and Depression Scale HADS, which includes 7 items for anxiety and 7 for depression, yielding two independent scores: HADS-A and HADS-D. Sleep quality will be assessed using the Pittsburgh Sleep Quality Index PSQI, which evaluates sleep latency, duration, efficiency, and disturbances.

To identify functional gastrointestinal disorders, particularly irritable bowel syndrome IBS, the Rome IV criteria and the Bristol Stool Form Scale, which classifies stool form and consistency, will be applied.

At the T0 visit, all clinical data necessary for study evaluation will be collected, including sociodemographic information, adherence to the Mediterranean diet (assessed via the validated PREDIMED questionnaire), basal metabolic rate calculated using the Mifflin formula, dietary, clinical, familial, and medical history, smoking habits, blood pressure measurement, and the most recent laboratory test results.

Participants will then undergo blood sampling for routine laboratory evaluations as per clinical practice, with a portion of the sample reserved for metabolomic analysis. Metabolites will be extracted from blood samples using methanol and MTBE for untargeted metabolomic profiling, and the MSK-QC-KIT will be added prior to injection into the UHPLC system. Chromatographic separation will be performed under C18 and HILIC conditions. Mass spectrometry data will be acquired using a hybrid quadrupole-Orbitrap Q Exactive Plus in full-scan mode, both in positive and negative ionization. Data will be processed for deconvolution, peak picking, alignment, and compound identification. A procedural blank sample will be used for background subtraction and noise removal during preprocessing. Compound annotation will be performed using MS-FINDER ver. 3.26, followed by bioinformatic analyses for metabolite and pathway enrichment.

Routine laboratory tests will be conducted at the laboratories of Policlinico San Martino, while untargeted metabolomic analysis of the gut microbiota will be performed at the DINOGMI university laboratories IRCCS Giannina Gaslini.

Finally, participants will also be asked to provide saliva samples, in which levels of neuroactive steroids such as cortisol, progesterone, and DHEA will be measured via ELISA analysis. This analysis will be conducted at the DIFAR university laboratories UNIGE.

Within the following seven days, a visit will take place at the same outpatient clinic designated by the Rheumatology Unit, during which the results obtained at T0 will be reviewed with the participant. If no issues are identified related to potential screening failures, participants will proceed with randomization and receive their dietary plan from the dedicated staff at the same clinic, in collaboration with the Dietetics and Clinical Nutrition Unit of the same hospital.

During the T1 visit, participants will be randomly assigned to one of three dietary groups: hypocaloric ketogenic diet, Mediterranean diet, or Mediterranean diet supplemented with probiotics. They will then be provided with the corresponding dietary plan, accompanied by detailed instructions both in writing and through an individual consultation. No adverse effects are expected from the proposed dietary plans, except for the ketogenic diet, where rare side effects are transient and may include nausea, vomiting, temporary lethargy, anorexia, hypoglycemia, dehydration, acidosis, constipation, abdominal bloating, headache, and fatigue.

Anthropometric measurements will be performed, including weight, height, calculation of Body Mass Index BMI, waist circumference, and waist-to-hip ratio. Body composition analysis may be performed using bioelectrical impedance analysis BIA. Anthropometric measurements and biological samples will be collected again during follow-up visits every four weeks and at the end of the 12-week study period.

For the pilot substudy, GABA and glutamate levels will be measured in specific brain regions using magnetic resonance spectroscopy MRS. To this end, a subset of 18 patients-6 per dietary arm-will be selected for this analysis and will sign a specific informed consent form. The procedure will be performed twice: first at T1 depending on scanner availability, prior to the start of the diet, and a second time after 4 weeks.

The spectroscopy results, focusing on GABA and glutamate levels in the patients, will be compared with spectra obtained from six control subjects. Control subjects will be screened using remote and recent medical history to exclude the potential presence of any organ-specific pathologies at the time of assessment by the neuroradiologist. As per routine clinical practice, a pre-examination questionnaire will be administered to evaluate any contraindications, and it will be signed by both the supervising neuroradiologist and the participant. No contrast agent will be administered.

Recruitment of control subjects is necessary because measuring the GABA-glutamate peak in CNS regions is challenging and complex. Establishing baseline GABA and glutamate levels in healthy controls is essential for comparison with FM patients, who may exhibit altered levels. If any pathological findings are suspected during the MRS exam in either controls or FM patients, the neuroradiologist will inform the subjects and recommend a complete morphological MR study, with contrast if necessary.

Magnetic resonance imaging MRI uses radiofrequency waves rather than ionizing radiation, unlike computed tomography, and with appropriate sequences, no specific risks are currently known for participants. Data processing will be performed offline using Gannet software version 3, developed for single-voxel 1H-MRS data analysis to measure GABA and glutamate. Metabolite concentrations will be expressed in institutional units, relative to water, with a correction factor derived from the cerebrospinal fluid fraction in the voxel. The software enables co-registration of the structural image with the voxel and segmentation into the three main tissue types to compute this correction factor. Chang

• Assess adherence to the Mediterranean diet and the impact of the dietary approach on anthropometric measurements. [Срок оценки: Baseline, Week 4, Week 8, and at the end of treatment (up to 12 weeks).]
• Evaluate the impact of dietary intervention on the composition of the gut microbiota through metabolomic analysis. [Срок оценки: Baseline, Week 4, Week 8, and at the end of treatment (up to 12 weeks).]
• Assessment of neurosteroids with positive allosteric action on GABA-A receptors or NMDA receptors in saliva and/or plasma. [Срок оценки: Baseline, Week 4, Week 8, and at the end of treatment (up to 12 weeks).]
• Exploratory pilot sub-study to measure brain levels of GABA and glutamate using magnetic resonance imaging (MRI) in a small sample of patients (18 patients + 6 controls). [Срок оценки: Baseline, Week 4.]
• Assess the impact of the dietary approach on metabolic parameters. [Срок оценки: Baseline, Week 4, Week 8, and at the end of treatment (up to 12 weeks).]
• Assess the impact of the dietary approach on the body composition of the participants. [Срок оценки: Baseline, and at the end of treatment (up to 12 weeks).]