The Norwegian Microbiota Study in Anorexia Nervosa (NORMA): integrating a clinical trial with preclinical experiments - a study protocol

Technical Summary: The Norwegian Microbiota Study in Anorexia Nervosa (NORMA)

Problem Statement
Anorexia nervosa (AN) is a severe eating disorder with high mortality rates and limited treatment efficacy, often complicated by co-occurring gastrointestinal (GI) symptoms and psychiatric comorbidities. Emerging evidence suggests a link between AN and gut microbiota dysbiosis, potentially mediated through the gut-brain axis (GBA). However, existing literature is characterized by small sample sizes, cross-sectional designs, inconsistent findings regarding microbial diversity (alpha and beta), and a lack of comprehensive dietary data. Crucially, it remains unclear whether microbiota alterations are a consequence of undernutrition or a causal driver of AN pathogenesis. Furthermore, the persistence of dysbiosis following weight restoration and the potential for prebiotic interventions to normalize the microbiome in AN remain understudied.

Methodology
The NORMA study is a comprehensive, multi-disciplinary protocol integrating three work packages (WPs) to investigate the role of gut microbiota in AN. The study is led by the Norwegian University of Life Sciences (NMBU) in collaboration with seven specialized inpatient eating disorder units across Norway.

• Work Package 1 (WP1): Clinical Observational Trial

  • Participants: 90 female patients with AN (age 16–50, BMI <18.5) and 90 healthy controls (HCs, age 16–50, BMI 18.5–27).
  • Design: Longitudinal observational design. AN patients are assessed at three time points: baseline (within the first week of admission), 6 weeks, and 12 weeks. HCs are assessed once.
  • Data Collection:
    • Biological Samples: Fasting blood samples (serum, EDTA, heparin) and fecal samples (DNA-preserving buffer and anaerobic collection for fermentation/FMT).
    • Clinical Metrics: Anthropometrics, medical records, and registry data (antibiotic use, medication).
    • Questionnaires: A battery of validated self-reports covering eating disorder symptoms (EDE-Q6.0), psychosocial impairment (CIA3.0), GI symptoms (GSRS-IBS), quality of life (SF-36), anxiety (GAD-7), depression (PHQ-9), obsessive-compulsive symptoms (OCI-R), and physical activity.
    • Dietary Intake: Detailed 3-day digital food diaries (or structured interviews) analyzed via the NutriFoodCalc system to quantify energy, macronutrients, micronutrients, and food groups.
  • Exclusion Criteria: History of inflammatory bowel disease, celiac disease, GI surgery, recent antibiotic use (past 2 months), or systematic probiotic intake.

• Work Package 2 (WP2): In Vitro Fermentation Experiments

  • Objective: To test the fermentability of prebiotics by AN-derived microbiota.
  • Method: Anaerobic fecal samples from a subset of AN patients and HCs are cultured in bioreactors.
  • Intervention: The prebiotic acetylated galactoglucomannan is tested against a control to assess its ability to stimulate the growth of beneficial bacteria (e.g., Roseburia, Faecalibacterium) that are often reduced in AN.

• Work Package 3 (WP3): Animal Experiments

  • Objective: To investigate causal links between AN microbiota and AN-relevant phenotypes.
  • Method: Fecal Microbiota Transplantation (FMT) from AN and HC donors into newly weaned female C57BL/6JRj mice.
  • Groups: AN-FMT, HC-FMT, and Sham (PBS/glycerol) groups.
  • Protocol: Mice receive FMT via oral gavage over a 5-week period.
  • Assessments: Weekly monitoring of body weight and food intake; behavioral testing for anxiety (Open Field, Elevated Plus Maze) and compulsive-like behaviors (Marble Burying); collection of tissue for gene expression and gut barrier function analysis.
  • Future Scope: If initial FMT results are positive, follow-up experiments will test if prebiotic interventions can reverse AN-associated phenotypes in the mice.

Key Contributions and Study Design Features

• Scale and Longitudinal Depth: With 180 participants and three assessment time points over 12 weeks, NORMA aims to be one of the largest longitudinal studies of gut microbiota in AN, addressing the limitation of small sample sizes in previous research.
• Comprehensive Dietary Data: Unlike many prior studies, NORMA integrates rigorous, detailed dietary registration to disentangle the effects of diet from the disease state itself on microbiota composition.
• Transdisciplinary Integration: The study uniquely combines clinical observation with mechanistic in vitro and in vivo models to move beyond correlation toward causation.
• Standardized Treatment Context: By recruiting primarily inpatients undergoing standardized re-nutrition and psychotherapy, the study minimizes treatment heterogeneity, a common confounder in outpatient studies.
• Ethical Considerations: The use of preclinical models (WP2 and WP3) allows for the testing of prebiotic interventions and mechanistic hypotheses without disrupting the delicate clinical treatment of vulnerable AN patients.

Results
As a study protocol, this paper does not report experimental results. It outlines the hypotheses and statistical plans for future analysis:

• WP1 Hypotheses: To identify baseline differences in microbiota composition between AN and HCs; to track changes in microbiota, GI symptoms, and mental health scores during treatment; and to correlate baseline microbiota with treatment outcomes (BMI gain, EDE-Q scores).
• WP2 Hypothesis: To demonstrate that specific prebiotics can increase the abundance of beneficial, SCFA-producing bacteria in AN-derived cultures.
• WP3 Hypotheses: To determine if AN-FMT mice exhibit distinct phenotypes (reduced weight gain, lower food intake, increased anxiety/compulsivity) compared to HC-FMT mice, and whether these phenotypes can be modulated by prebiotics.

Significance and Claims
The paper positions NORMA as a pioneering effort to address critical gaps in the understanding of the gut-brain axis in AN. By integrating clinical, in vitro, and animal data, the study aims to:

1. Clarify whether gut microbiota dysbiosis is a cause, consequence, or maintaining factor of AN.
2. Determine the extent to which standard treatment normalizes the microbiome.
3. Identify specific microbial targets and prebiotic candidates that could lead to personalized, non-energy-yielding pharmaceutical or nutritional interventions.
4. Ultimately, improve treatment outcomes and quality of life for individuals with AN by developing microbiota-informed therapeutic strategies.

The authors acknowledge limitations, including the focus on female patients (limiting generalizability to males), the 12-week duration (potentially insufficient for long-term microbiota normalization), and the inherent constraints of translating animal models to human physiology. However, they argue that the study's rigorous design, large sample size, and multi-modal approach provide a robust foundation for future translational research.