Case Studies
Iron supplements and Gut Microbiome in Reproductive-age Women: Randomized Placebo-Controlled Trial
Clinical Overview
This randomized, double-blind, placebo-controlled trial in South Australia evaluated whether 21 days of oral iron supplementation alters the gut microbiome in healthy, nonpregnant females aged 18–45 years. Participants received 65.7 mg/d elemental iron as ferrous fumarate or placebo; fecal microbiota were profiled by 16S rRNA sequencing at baseline and day 21. Among 79 women with adequate sequencing data, iron did not significantly change microbiota β-diversity (mean weighted UniFrac difference vs placebo: 0.003; 95% CI –0.007 to 0.014; P = 0.523) or Escherichia-Shigella relative abundance (adjusted OR 0.524; 95% CI 0.186–1.475; P = 0.22).
Study Setting
The study was conducted virtually in South Australia, a high-income setting, recruiting primarily from a university environment. Participants were nonpregnant females aged 18–45 years, largely healthy and with BMI mostly in the normal range; recent iron deficiency anemia, iron supplementation, or antibiotic use were exclusion criteria. Iron exposure consisted of one oral capsule daily for 21 days providing 65.7 mg elemental iron as ferrous fumarate versus an identical-appearing placebo capsule with 0 mg iron; no additional metal exposures or clinical comorbidities were targeted.
Study Design and Methods
| Aspect | Details |
|---|---|
| Design | Single-center, prospective, 2-arm, parallel, double-blind randomized controlled trial; virtual conduct with no in-person visits; trial registration NCT05033483. |
| Participants & setting | 82 nonpregnant females (18–45 y) randomized (iron n = 40; placebo n = 42); 80 completed; 79 provided paired fecal samples with adequate sequencing depth; predominantly Australian-born university students. |
| Metal exposure / measurement | Intervention: 65.7 mg/d elemental iron as ferrous fumarate in gelatin capsules vs placebo (0 mg iron) for 21 days. Iron status or iron concentrations in blood or stool were not measured; exposure defined by randomized allocation. |
| Microbial characterization | Fecal samples collected at baseline (day 0) and day 21 into DNA-preserving kits. DNA extracted (DNeasy Powersoil), 16S rRNA V4 region sequenced on Illumina MiSeq. Taxonomy and β-diversity derived using QIIME2 with weighted UniFrac dissimilarity. |
| Endpoints, follow-up, analysis | Primary endpoint: β-diversity change (paired-sample weighted UniFrac dissimilarity) between baseline and day 21, comparing iron vs placebo via linear regression adjusted for BMI and covariates. Exploratory endpoints: relative abundance of genera present in ≥50% of samples and Escherichia-Shigella, analyzed with ordinal logistic regression and false discovery rate correction. Washout (day 42) stool was collected but not sequenced because no group differences were observed at day 21. |
Major Findings
The trial links a defined oral iron formulation and dose to gut microbial community structure in healthy reproductive-age women and shows that, in this context, the fecal microbiome is largely resilient to short-term iron loading.
Across 79 participants with paired fecal samples, iron supplementation for 21 days did not significantly alter overall microbial community composition compared with placebo, as assessed by weighted UniFrac β-diversity. Baseline microbiota were typical of a healthy adult cohort, dominated by Bacteroides, Blautia, Bifidobacterium, and Faecalibacterium (>95% prevalence). Exploratory analyses suggested nominal increases in several Clostridiales taxa and a decrease in Bacteroides in the iron arm, but all lost significance after false discovery rate correction. Escherichia-Shigella relative abundance, a key taxon implicated in iron-driven dysbiosis in low-income pediatric cohorts, did not differ between groups. No downstream clinical outcomes were reported; side effects were minor and adherence high.
| Findings | Details |
|---|---|
| Global community structure | No significant difference in weighted UniFrac dissimilarity between iron and placebo at day 21 (mean difference 0.003; 95% CI –0.007 to 0.014; P = 0.523), indicating similar overall microbiota trajectories. |
| Baseline microbiome | Core genera Bacteroides, Blautia, Bifidobacterium, and Faecalibacterium were present in >95% of baseline samples, consistent with a healthy adult gut community. |
| Taxa with nominal changes | Four taxa within the order Clostridiales (including Clostridium [sensu stricto], Clostridiales AD3011 group, and Romboutsia) were enriched, and Bacteroides decreased in the iron group before correction; all q values >0.05. |
| Escherichia-Shigella | No significant difference in Escherichia-Shigella relative abundance between groups (adjusted OR 0.524; 95% CI 0.186–1.475; P = 0.22). |
| Intervention feasibility | High adherence (98% took >80% of capsules; 78% took all) and only minor side effects, supporting feasibility of 65.7 mg/d ferrous fumarate for similar microbiome-focused trials. |
Mechanistic Interpretation & Microbial Metallomics
Above mentioned findings suggest that, in iron-replete, low-pathogen, high-income adult settings, short-term ferrous fumarate supplementation does not meaningfully perturb colonic microbial ecology.
| Concept | Implication |
|---|---|
| Iron exposure without biomarker deficiency data | In a population not selected for iron deficiency, unabsorbed supplemental iron over 21 days may remain insufficient to remodel an already stable adult gut community. |
| High-income, low baseline pathogen burden | Low baseline Escherichia-Shigella abundance (0.14% vs 3.3% reported for Kenyan infants in prior work cited by the authors) may limit iron-driven expansion of iron-hungry pathogens, blunting dysbiosis. |
| Clostridiales trends with iron | Nominal increases in several commensal Clostridiales genera suggest that any iron effect, if real, might subtly redistribute commensal anaerobes rather than enrich enteric pathogens in this setting. |
| Resilient adult microbiome | The absence of β-diversity change over 21 days emphasizes the stability of adult gut communities to a single-nutrient perturbation at therapeutic iron doses. |
| Methodological choice of weighted UniFrac | Phylogeny-aware β-diversity may reduce spurious signals from minor taxonomic shifts, strengthening the conclusion that large-scale community reorganization did not occur. |
| Lack of functional readouts | Without metagenomic or metabolomic data, potentially subtle iron-driven shifts in microbial metabolism (e.g., siderophore pathways, SCFA production) remain unresolved. |
Limitations
The cohort was small and demographically narrow, with mostly young, healthy university students, limiting generalizability to older, comorbid, or non-university populations. Iron status and anemia were not measured, preventing stratified analyses by iron deficiency or iron load. Microbiome profiling relied on 16S rRNA amplicon sequencing, restricting taxonomic resolution to approximately genus level and precluding direct functional inference. Only a 21-day exposure window was studied; longer-term effects or later washout dynamics in the day-42 samples were not characterized.
Future perspectives
Next studies should extend this design to longer supplementation periods and explicitly enroll women with iron deficiency and/or anemia, with baseline and follow-up iron indices to link iron status, luminal iron exposure, and microbiome responses. Including pregnant women and participants from low- and middle-income settings would align directly with current blanket iron-supplementation policies. Adding shotgun metagenomics, metatranscriptomics, or metabolomics alongside 16S rRNA profiling would clarify whether iron alters microbial functional capacity even when community structure remains stable. Finally, simultaneous measurement of stool iron could directly connect metal availability, microbial taxa, and potential pathogen overgrowth.
Key takeaways for Researchers and Clinicians
In this Australian randomized controlled trial of nonpregnant females aged 18–45 years, daily 65.7 mg elemental iron as ferrous fumarate for 21 days did not significantly change overall fecal microbiota composition or Escherichia-Shigella abundance compared with placebo. Iron was delivered orally without measured systemic or fecal iron biomarkers, and the gut community remained dominated by typical health-associated genera with no significant β-diversity shift or taxa surviving multiple-comparison correction.
Methodologically, the study demonstrates that carefully controlled 16S rRNA sequencing with phylogeny-aware metrics (weighted UniFrac) can robustly document microbiome stability under therapeutic iron dosing in a high-income, low-pathogen context. Clinically, the findings support the microbiome safety of short-term oral ferrous fumarate in healthy reproductive-age women, while underscoring that risk–benefit considerations may differ in populations with higher enteric pathogen carriage. Translationally, this work positions microbial metallomics to move beyond presence/absence of dysbiosis toward context-specific evaluation of when luminal iron actually becomes a microbial selective pressure.
Citation
Elms L, Hand B, Skubisz M, Best KP, Grzeskowiak LE, Rogers GB, Green TJ, Taylor SL. The Effect of Iron Supplements on the Gut Microbiome of Females of Reproductive Age: A Randomized Controlled Trial. The Journal of Nutrition. 2024;154:1582–1587. doi:10.1016/j.tjnut.2024.03.014