The study aims to evaluate the role of the gut microbiome and phageome in explaining interindividual variability in the metabolic response to polyphenol-rich nutraceuticals among menopausal women. Insights from this research will support the development of personalized nutrition strategies to improve quality of life and reduce cardiovascular disease (CVD) risk during menopause.

This trial aims to investigate the role of the gut microbiome and phageome in mediating interindividual variability in the metabolic response to polyphenol-rich plant extracts among postmenopausal women. Polyphenols are widely recognized for their potential benefits in cardiovascular health, cognitive function, and overall metabolic regulation. However, the magnitude and direction of their effects vary significantly between individuals. Mounting evidence suggests that the gut microbiota plays a critical role in the biotransformation of polyphenols into bioactive metabolites, giving rise to the so-called gut microbiota metabotypes (i.e., metabolic fingerprints of the gut microbiota that produce specific metabolites depending on the individual), thereby modulating the physiological effects of ingested polyphenols.

A cohort of postmenopausal women will be studied at four timepoints in a randomized, double-blind, placebo-controlled, crossover trial. Participants will undergo a controlled dietary intervention with standardized polyphenol-rich plant extracts (pomegranate, soy, and resveratrol). The primary outcome will be a ≥15% change in serum oxidized LDL (oxLDL). At each timepoint, gut microbiome and phageome profiles will be characterized using shotgun metagenomic sequencing. TMAO, bile acids, and short-chain fatty acids will be determined by UPLC-QTOF-MS and GC-MS. Additional biomarkers of cardiovascular and metabolic health will be determined, including serum total cholesterol, LDLc, HDLc, and LPS-binding protein (LBP); fecal microbial enzymatic activities (β-glucuronidase and sulfatase); and serum neurotransmitters (GABA, dopamine, serotonin, melatonin, epinephrine, norepinephrine).

The distribution of polyphenol metabotypes in this cohort will be assessed through circulating and urinary phenolic-derived metabolites. This will allow exploration of differential individual responses to polyphenol intake and their derived health effects. Untargeted urinary metabolomics will further explore potential changes in metabolites relevant to cardiovascular prevention. Quality of life will be evaluated using the validated Cervantes Scales, which include domains related to menopause-associated symptoms, cognitive issues, and overall health.

The study is designed to integrate multi-omic data, combining microbial and phageomic composition with metabolomic and biochemical readouts, to identify patterns and predictors of individual responses to polyphenol intake. Statistical and bioinformatic analyses will focus on associations between microbiome/phageome signatures and metabolic outcomes, aiming to elucidate the mechanisms by which gut microbes and their viruses influence polyphenol biotransformation and subsequent systemic effects. This study therefore seeks to advance precision nutrition strategies for postmenopausal women. Insights gained may inform personalized dietary recommendations aimed at enhancing cardiovascular health, metabolic regulation, and overall quality of life during menopause. Moreover, the inclusion of the phageome represents a highly innovative and largely unexplored aspect of the study, offering new avenues for microbiota-targeted interventions.

Overall, this research will contribute to understanding the complex interactions between diet, the gut ecosystem, and human metabolism, ultimately supporting the development of evidence-based, individualized nutritional strategies to reduce cardiovascular disease risk and improve health outcomes in the postmenopausal population.

Inclusion Criteria:

• Women aged 45 to 57 years.
• Diagnosed menopause (defined as 12 consecutive months without menstruation).
• Presenting at least one climacteric symptom (hot flashes and/or sweating episodes and/or low mood and/or irritability and/or decreased libido and/or insomnia and/or joint/muscle pain).
• Body Mass Index (BMI) ≥ 18 kg/m².
• Adequate cultural level and ability to understand the clinical study.
• Willing to voluntarily participate in the study and provide written informed consent.

Exclusion Criteria:

• Presence of severe, chronic-degenerative, or psychiatric conditions, or any contraindication to the use of nutritional supplements.
• History of major gastrointestinal surgery.
• Swallowing difficulties (e.g., inability to ingest capsules).
• BMI < 18 kg/m² or > 30 kg/m².
• Currently following a weight-loss regimen.
• Known or suspected allergy or intolerance to red clover extract, resveratrol (grape, wine), soy, or pomegranate.
• Use of chronic preventive medication for cholesterol, glucose, blood pressure, etc. (e.g., statins, metformin, beta-blockers).
• Use of antibiotics within one month prior to study initiation.
• Undergoing hormone replacement therapy.
• Alcohol consumption exceeding 1 beer or 1 glass of wine per day.
• Regular use of dietary supplements (e.g., probiotics, isoflavones, resveratrol, others).
• Following a vegetarian diet.
• Current smoker or having smoked at any time during the past year.
• Individuals unwilling to comply with study guidelines.

Only individuals assigned female at birth who self-identify as women are eligible to participate, provided they meet the biological criteria for postmenopause as defined in the study protocol. While gender identity is fully respected, eligibility is determined by biological sex due to the physiological nature of the condition under investigation.

De-identified individual participant data underlying the results (including raw metabolomics datasets from UPLC-QTOF-MS and GC-MS, and raw microbiome/phageome sequencing data) will be available upon reasonable request. Data will be shared after publication of the primary outcomes and will require a data use agreement to ensure appropriate use.

Time Frame: Beginning 12 months after publication of the results; available for 5 years.

Access Criteria: Qualified researchers may request access by contacting the Principal Investigator. Data will be shared in a controlled manner to ensure confidentiality of participants.