Impact of a single fecal microbiome transplantation in adult women with anorexia nervosa: an open-label feasibility pilot trial

Domain: biology
Published: 2026-01-14
Editorial outcome: major_revision

Abstract
Anorexia nervosa (AN) is a severe mental disorder characterized by restrictive eating and disturbance in the way one’s body weight or shape is experienced, often accompanied by depression and anxiety. Current evidence-based treatments for AN have limited efficacy, with less than half of the patients achieving full recovery in long-term follow-up studies. Recent findings have identified gut microbiota (GM) dysbiosis as a potential contributor to AN pathology through the gut-brain axis. This open-label, non-randomized, feasibility trial (Clinicaltrials.gov Identifier: NCT05834010) evaluated the feasibility of utilizing fecal microbiota transplantation (FMT) to modify the GM and GM-associated signaling in females with AN and to examine biological effects following a single FMT procedure. Adult female participants diagnosed with AN were recruited. FMT was administered either orally via capsules or as rectal enema. Stool and blood samples were collected pre- and one week post-FMT to assess GM composition, hormonal changes, and biomarkers. Primary endpoints: Feasibility of FMT in individuals with AN and preferred route of FMT. Secondary endpoints: A single FMT treatment can alter GM composition in individuals with AN short term and relevant gut brain signaling in serum. 18/22 participants (81%) completed FMT and sampling and 19/22 participants chose oral capsules, with no serious adverse effects reported. GM analysis showed significant shifts toward donor composition 1-week post-FMT, with improved stool consistency. No significant changes were observed in psychopathology measures or appetite-related biomarkers. Oral FMT is a feasible intervention for adult women with AN, leading to changes in GM profile. Future studies should focus on placebo-controlled trials to assess the efficacy of repeated oral treatments and explore long-term effects on GM, appetite, body weight, sex hormones, disorder-specific symptoms, and overall well-being.

Introduction
Anorexia nervosa (AN) is a complex disorder characterized by disturbances in the way one’s body weight or shape is experienced, difficulty in gaining weight, restrictive food intake, and depression and anxiety. The evidence base for treatment is still very limited and AN carries one of the highest fatality rates amongst psychiatric disorders1. Fewer than half of treated adults with AN achieve recovery2. In fact, there is little evidence that the prognosis for AN in adults has improved over the last decades3. So far, attempts at pharmacologic treatment have focused on a narrow range of compounds and only a few controlled studies have been completed4. Furthermore, large placebo-controlled trials have proven to be very difficult to conduct in individuals with AN because of its egosyntonic nature, low treatment acceptability, and low participant retention5.

In some cases AN is preceded by depression, trauma, gastrointestinal symptoms or infection. However, its etiology is multifactorial with both genetic and environmental factors playing a role6. The role of genetic factors is well-established7,8. Further, AN is associated with multiple endocrine alterations9, and it has been proposed that AN should be reframed as a metabo-psychiatric disorder10.

The human digestive tract harbors complex microbial communities, with the gut microbiota (GM) playing key roles in host metabolism. The GM aids nutrient and energy acquisition, synthesizes amino acids, vitamins, and metabolites, and drives enteroendocrine hormone signaling. These processes and GM-host interactions support homeostasis, affecting immunity, pathogen defense, gastrointestinal function, metabolism, appetite, emotions via the gut-brain axis, and functions beyond the gastrointestinal tract11,12.

Growing evidence shows that in the active phase of illness, individuals with AN have a dysbiotic GM, with lower microbial diversity and reduced stool levels of serotonin, GABA, dopamine, butyrate, and acetate compared to healthy controls (HCs)13–15. A multi-omics study uncovered profound and complex disruptions of the GM and altered serum metabolites in individuals with active AN15. While constipation and dyspepsia stem from neurologic and hormonal adaptations to starvation16,17, returning to a normal diet with more complex foods, like fiber, does not fully reverse dysbiosis in AN18.

Two recent reviews on GM in individuals with AN highlight overlapping findings. A 2021 review (n=9) identified reduced butyrate-producing taxa, particularly Roseburia, and increased levels of Parabacteroides, Alistipes, and mucin-degrading bacteria like Akkermansia in AN patients19. A 2024 review (n=14) similarly found lower abundances of Roseburia inulinivorans and Faecalibacterium prausnitzii, key butyrate producers linked to healthy GM, and higher levels of Methanobrevibacter smithii in AN20. F. prausnitzii, an anti-inflammatory bacterium, and a potent butyrate producer associated with mental health, is notably reduced in AN21. Increased Methanobrevibacter smithii may enhance energy extraction and contribute to symptoms like constipation and gas22.

Fecal microbiota transplantation (FMT) involves the transfer of fecal matter (the GM including all metabolites, hormones and more) from chosen donors to recipients with GM dysbiosis. Two single human case studies have been published where FMT was used as experimental treatment of AN23,24. Both studies suggest that FMT can overcome persistent dysbiosis, metabolic, and clinical symptoms if implemented as an adjunctive to treatment as usual. FMT may “reset” gut-brain signaling, boost appetite, and relieve gastrointestinal symptoms like constipation caused by the lack of essential GM-derived molecules and enzymes needed for complex food digestion.

During the last decade FMT has become well established as a highly effective routine treatment for recurrent Clostridioides difficile gut infections (rCDI) in many countries25. FMT trials for recurrent C. difficile infection (rCDI) have demonstrated over 90% cure rate, regardless of the preparation method or administration route26. However, our recent findings indicate that capsules were more effective than both FMT enemas and rectal bacteriotherapy27. Despite the non-specific nature of an FMT, it has shown remarkably few mild side effects and a very low rate of serious adverse events28.

This study aimed to assess the feasibility of using FMT to restore the GM in adult females with AN, exploring a single FMT administered orally or rectally based on participant preference, without pretreatment GM clearing. We used metagenomic sequencing to analyze GM changes before and one week after FMT, assessing shifts toward the donor’s GM, including diversity, species transfer, and functional gene pathways. We examined whether a single FMT could influence serum levels of sex-, appetite-, and metabolism-related hormones. We examined gut microbial β-glucuronidase (gmGUS), which can reactivate conjugated hormones excreted in the gut like estrogen, influencing metabolism. Its reduction in AN may impair estrogen reabsorption and contribute to symptoms10,29.

We also hypothesized that individuals with AN have low fecal gmGUS levels, which might be restored by FMT. Furthermore, we assessed participants self-reported stool consistency and transit time using the Bristol Stool Form Scale (BSFS)30 and eating disorder symptomatology. Participant evaluations and observations were collected to provide qualitative insights and guide future study designs. Here, we show that a single oral or rectal FMT in adult women with AN is feasible, well-tolerated, and leads to shifts in GM composition toward donor profiles, along with improved stool consistency, while producing no significant changes in appetite- sex-related hormones or gmGUS.

Results
Feasibility
Over a nine-month recruitment period in 2023, 69 individuals expressed interest in participating. Nineteen were excluded during pre-interview screening due to age (n=4), sex (n=3), or BMI outside our criteria (n=12). Of the 50 who completed screening, 28 were further excluded based on our inclusion criteria, most commonly due to BMI (n=20), laxative abuse (n=7), or recent probiotic use (n=2); several individuals met more than one exclusion criterion. Ultimately, 22 females with AN were enrolled and provided informed consent, with 18 (81%) completing all study procedures. These numbers suggest overall feasibility, though the strict inclusion criteria limited final enrollment. The dropouts either failed to deliver the second fecal sample or complete the 1 month follow-up questionnaire (Supplementary Fig. 1).

No serious FMT-related adverse effects were reported. However, one participant with a history of depression was hospitalized for severe depression post-FMT, underscoring the need for careful monitoring in vulnerable populations. For the administration route 19 chose oral FMT with capsules and only 3 chose the rectal route. None had problems swallowing the frozen capsules within the required time. Overall, most participants were young students recruited from Odense or Ballerup hospitals or through social media with a mean age of 24.5 (±5.7 years) and mean BMI of 16.1 (±1.9).

Psychopathology measures of quality of life (EDQLS), depression (MDI) eating disorder symptoms (EDE-Q) and participation data
There were no significant differences in psychopathology measures of eating disorder symptoms (EDE-Q), depression (MDI), or disease-specific quality of life (EDQLS), as tested by paired t-tests (Supplementary data 1). The small sample size limits detection of associations with donor, closer-to-donor-status, or clinical variables. All 19 participants completed the FMT feasibility questionnaire one week post-FMT. Among capsule recipients (16/19), none reported disgust, found swallowing acceptable, and preferred capsules for future use. They considered a 6-week regimen reasonable and 30–50 capsules per FMT acceptable. Most would adhere to treatment if no alternatives existed. However, the three enema recipients (3/19) remained uncomfortable with swallowing capsules.

Gut Transit Time
Based on self-reported stool type using the BSFS at the time of fecal sampling—one day prior to FMT and one week following it—seven participants reported no change in stool type, while 11 participants reported looser stools. (Wilcoxon test P < 0.01; Fig. 1).

Fecal moisture content
To validate the BSFS scores and analyse differences stratified on BSFS response, we measured the fecal moisture content in the pre-post-FMT samples. A Spearman’s rank correlation test showed a strong positive association between fecal moisture content and self-reported BSFS scores both before (rho=0.74, p=0.0017) and after FMT (rho=0.88, p<0.0001). Overall, no significant difference in fecal moisture content was seen comparing pre-FMT (68.45±12.02%) and post-FMT (69.32±12.29%) (paired t-test: p=0.55) regardless of BSFS scores. Pre-FMT, the fecal moisture content ranged from 53.67% to 96.22% and post-FMT from 47.03% to 89.64%. Fecal moisture content did not change significantly after FMT in participants, regardless of stool consistency (when based on BSFS response) or microbiota closer-to-donor status (when based on Bray-Curti’s dissimilarity metrics) (See Supplementary Figs. and Methods).

Microbiome analysis
The GM diversity of the FMT donations (n=13) from two distinct donors (Don1: n=3; Don2: n=10) and of participants (n=18) pre- and post-FMT is illustrated in Fig. 2A, using both the Chao1 and Fig. 2B Shannon indices. Individual participants’ pre-(w0) and post-(w1) FMT are shown connected with lines. The Chao1 (total richness) index of both donors was significantly different from that of their eventual recipients. In particular Don2 recipients also showed a significant difference in diversity between pre-FMT (w0) and post-FMT (w1), while the Shannon diversity (evenness) index no statistically significant changes were observed between pre- and post-FMT. Using a Distance-based Redundancy Analysis (dbRDA) model FMT-recipients experience a clear shift toward donor GM composition post-FMT (Fig. 2C, p=0.001).

To assess whether the FMT treatment effectively shifted the recipients’ GM composition toward that of the donor, we calculated the distance to the donor both pre- and post-FMT. Prior to FMT, the average distance between recipients and donors was 0.65 (interquartile range [0.65; 0.69]), indicating a notable distinction between their microbiota compositions (Fig. 2D). Following FMT, this distance significantly decreased for all but two individuals, reaching an average of 0.59 [0.53; 0.64] (Wilcoxon test, P < 0.0001), demonstrating a substantial shift in recipients’ microbiota composition toward that of the donor. To further assess donor specificity, we also calculated distances to the ‘other’ donor and found no significant change from baseline to w1 (p=0.46). Additionally, recipients’ w1 profiles were significantly closer to their assigned donor than to the ‘other’ donor (p < 0.001), supporting a donor-specific shift (see Supplementary Figs. 9 and 10).

To assess GM compositional changes from pre- to post-FMT differential abundance analysis was conducted separately for each donor due to their distinct microbial profiles. Figure 2E shows a volcano plot, where positive effect sizes indicate taxa (family level) enriched by FMT, and negative values indicate suppression. Figure 2F recasts these results, displaying effect sizes relative to significance. Together, these plots suggest FMT generally enriches a range of taxa without depleting or suppressing specific ones. Most enriched taxa are abundant in donor samples, except Ruminococcaceae at low abundance and Christensenellaceae, that were significantly increased in Don2 recipients, despite being absent in Don2 samples.

FMT-closer-to-donor status was defined by evaluating the changes in IQR (Interquartile Range) Bray-Curtis dissimilarity between donor and recipient pre- and post-FMT. Closer-to-donors were participants with a reduction of at least 1.5 standard deviations in Bray-Curtis dissimilarity metrics, classifying recipients as closer-to-donor (n=9) and not-closer-to-donor (n=9). All three participants receiving FMT via enema were not-closer-to-donor. Among Don1 recipients (n=4), 2 were closer-to-donor and 2 were not-closer-to-donor.

We also explored potential microbial predictors of not-closer-to-donor status before FMT. To achieve this, we applied a sparse partial least squares discriminant analysis (sPLS-DA) model to baselines stool samples to identify bacterial families associated with FMT response. The regression coefficients from this model (Fig. 3) highlight bacterial families associated with either an increased or decreased likelihood of FMT success. Positive coefficients identify families promoting closer-to-donor status, including some Lachnospiraceae, Bacteroidaceae, and UBA1381. Conversely, negative coefficients highlight families such as Coriobacteriaceae, Oscillospiraceae other Lachnospiraceae and Rikenellaceae, which are associated with reduced response likelihood.

Furthermore, we performed a species-level analysis to pinpoint key differences between the two FMT donors and their respective recipients, both pre- and post-FMT, offering exploratory insights into the microbial factors involved in the FMT treatment. A qualitative comparison between healthy donors (Don1 (n=3) and Don2 (n=10)) revealed notable differences in 34 taxa, including significant higher abundances of Catenibacterium misuokai and Methanobrevibacter smithii in Don2, and Ruminococcus bicirculans and Bifidobacterium longum in Don1 (Supplementary Fig. 2).

We further analyzed the specific species level differences between donors and eventual recipients before FMT. Notably, Don1 had a higher abundance of Prevotella copri, Ruminococcus callidus, and Coprococcus eutactus, while the Don1 recipients showed higher abundances of Fecalibacillus intestinalis and a Ruminococcus species sp003526955 (Supplementary Fig. 3) pre-FMT. For Don2, Catenibacterium misuokai was markedly more abundant compared to eventual recipients. The recipients had higher abundances of the same Ruminococcus sp003526955 and Bifidobacterium longum compared to Don2 (Supplementary Fig. 4).

For Don1-recipients (n=4), a comparison between baseline stool samples (w0) and those collected one week after FMT (w1) revealed a significant increase in the abundance of three taxa: Ruminococcus callidus, Prevotella copri, and Coprococcus eutactus following FMT. (Supplementary Fig. 5). For Don2 recipients (n=14) the most prominent significant change from pre- to post-FMT, was a higher abundance in Catenibacterium mitsuokai (out of 10 significantly increased taxa) post-FMT. Two taxa CAG-41 sp900066215 (Firmicutes) and Phocaeicola dorei (Bacteroides) were at a lower abundance (Supplementary Fig. 6).

The final analysis of changes in individual taxa from pre-FMT (w0) to post-FMT (w1), we focused on identifying bacteria that had 1: higher abundance post-FMT, 2: were absent in the recipient before FMT, and 3: were present in the corresponding donor. This was to investigate possible direct transfer of species from donor to recipient. Following FMT, we observed a moderate transfer of 11 species with significant changes in abundance that were originally present in the donors but absent in the recipients pre-FMT. These species include Catenibacterium misuokai, Alloprevotella sp900541575, Anaerotardibacter sp900553605, CAG-177 sp003538135 (Ruminococcus), Cryptobacteroides sp000434935, Duodenibacillus massiliensis, Faecousia sp900546415, Phocaeicola sp000434735, UBA1829 sp900548385, UMGS1502 sp900552365 (Eggerthellaceae), and [UMGS1590]_unknown (Peptococcaceae). See (Supplementary Fig. 7) for further details.

Blood samples and gut microbial β-glucuronidase
We assessed the effects of FMT on sex hormones (estradiol, FSH, and LH), appetite-regulating hormones (GLP-1, insulin, glucagon, C-peptide, PYY, GiP, and leptin) using blood samples, and gmGUS. Figure 4 depicts the pre-(w0) and post-FMT (w1) changes for each biomarker, stratified by microbiota-derived closer-to-donor status. While biomarker responses were highly individual, no clear patterns linked to closer-to-donor status emerged. Notable changes included a two-fold reduction in estradiol levels across participants, though this did not reach statistical significance (OR=0.51, CI: 0.26–1.01, p=0.054). Insulin levels showed a modest 10% increase overall (OR=1.1, CI: 1.00–1.21, p=0.047), while PYY levels in closer-to-donors exhibited a small but significant increase (OR=1.22, CI: 1.02–1.46, p=0.037). Exploratory analyses showed that most metabolites and hormones were unrelated to self-reported BSFS scores, except blood estradiol, which modestly correlated with BSFS at week 1 (Supplementary Table 1). No significant changes pre- to post-FMT in GmGUS were observed (Fig. 4).

Metagenome gene pathway KEGG modules and single gene analysis
The KEGG modules analysis was conducted to investigate the changes to metabolic pathways and functional gene pathways present within the GM pre- to post-FMT. Overall, we see no changes to module relative abundances pre- to post-FMT. For more details see Supplementary Fig. 3. We further performed a single-gene analysis to assess functional gene changes in the GM pre- and post-FMT. While the total annotated gene count remained stable, constrained correspondence analysis revealed a significant reduction in distance to Don2 post-FMT for Don2 recipients (p=0.01), indicating greater similarity to the donor’s gene composition (Fig. 5). Don1 post-FMT distance to Don1 recipients was not significantly different from pre-FMT (P > 0.05).

Methods
This study was conducted in accordance with all relevant ethical regulations and was approved by the Danish National Committee on Health Research Ethics (VEK protocol H-22030034), registered at Clinicaltrials.gov (NCT05834010) and written informed consent was obtained from all participants. Further ethical oversight provided through Copenhagen University Hospital Hvidovre (CUHH). This study was an open-label, non-randomized feasibility study performed in 2023 at CUHH, Denmark. For the clinical trial protocol see supplementary materials ”Protocol”. Individuals with AN were recruited55,56. Participants completed validated questionnaires before and four weeks after FMT, as specified below. After inclusion, participants were given the choice of receiving a single FMT either via enema or via oral capsules. Blood and stool samples were collected before and again 7 days after the single FMT dose. Likewise, a custom participation questionnaire (Supplementary File) about the FMT procedure and stool type according to the Bristol Stool chart was collected30. Blood samples were analyzed for appetite- and sex-related hormones, while stool samples were examined for GM composition, β-glucuronidase, bacterial species and genes transfer from donor to recipient.

Statistics & reproducibility
As a feasibility study, this work lays the foundation for future power calculations. We predetermined the sample size based on the power of planned GM analysis, no data were excluded, and the study was neither randomized nor blinded.

Recruitment
Participants were recruited actively from Odense and Ballerup hospitals, Denmark and passively through public web postings and trial promotion. Interested individuals received recruitment documents detailing inclusion/exclusion criteria and instruction to confirm their participation. After a prescreening of age, sex, and body mass index (BMI) they were instructed to fill out the initial Eating Disorder Examination Questionnaire (EDE-Q)57. Based on these scores suitable candidates were invited to a meeting for the informed consent process. During the meeting, participants received informed consent documents, Danish pamphlets on rights and ethics, and details about study procedures, time commitments, potential risks, and data collection. All participants were in psychotherapy, many with prior hospitalizations, and their AN diagnoses were confirmed by their treatment centers. Travel expenses were reimbursed, but no monetary compensation was provided.

Inclusion criteria
Adult (age >18 years) biological females referred to the specialized centers at Ballerup or Odense, Denmark, or by advertising who met Diagnostic and Statistical Manual of Mental Disorders version 5 diagnostic criteria for AN58.

Exclusion criteria
Age <18 and >45 years, known ongoing use of laxatives, antibiotic treatment within the last three months, probiotics within the last month, inflammatory bowel disease, colorectal cancer, other chronic bowel disorders, or any other somatic chronic diseases requiring medical treatment.

FMT production and administration
Fecal donors were screened and tested according to the international guidelines for organization of FMT and donor recruitment including questions on normal BMI (18.5–25.0 kg/m2), normal bowel habits (1–2 daily of normal consistency) and with no use of antimicrobial therapy in the past 6 months59,60. We did not specifically ask questions about histories of EDs or other psychopathology. Donors were further selected to be female and in the reproductive age. Manufacturing of capsules was done according to patent61. One portion of FMT capsules consist of 50 grams of un-lyophilized single donor material stored at −80°C freezer and transferred to −18°C freezer on the morning of treatment. Frozen FMT capsules (20-25), from a single donor portion, were swallowed under supervision using either fruit juice or flat, diet soda within 10-15 min after 6 h of fasting. Participants opting for rectal FMT received approximately 150ml (50g) of donor FMT, instilled 30–40 cm rectally following standard procedures62. Both FMTs as enema or capsules were administered once as a single treatment.

Sample collection
Fecal samples were collected at home by the participants. Participants were instructed to collect about 4 × 1-5 g in 4 specialized collection tubes with an in-tube-spoon attached and immediately store their samples in their own freezer at −18°C. Samples were transported in a cold, insulated box with freezing elements, delivered within 24 hours, and stored at −60°C until DNA extraction. Standard blood samples were taken twice, one week apart at the hospital using 10 mL EDTA, 10 mL, Heparin-tubes, 10 mL PAX.

Hormone and appetite biomarker measurements
From the blood plasma, we analysed the following biomarkers: active GLP-1, insulin, Glucagon, C-peptide, PYY, GiP and leptin levels using the U-PLEX® Multiplex sandwich immunoassay (MSD®, Rockville, MD, USA), following the manufacturer’s protocol. We measured Estradiol, P-Follitropin (FSH) and Lutropin (LH) pre- and post-FTM treatment using the CUHH standard pipeline electrochemilumininscence (ECL) immunoassay (Cobas 8000, Roche Denmark).

Gut microbial β-glucuronidase
To assess gmGUS levels, we modified a previously published method54. For full methodology, see Supplementary Methods.

Participation questionnaire and fecal sample consistency according to Bristol Stool Form Scale (BSFS)
Participants completed a questionnaire (Supplementary File) on “gut feeling”, FMT evaluation, future participation limits, and stool consistency using the BSFS30, a validated but indirect proxy for gut transit time (TT)30.

Fecal moisture content measurements. To further validate the BSFS we measured fecal moisture content. Samples (pre-post-FMT) from 15 of 18 participants were thawed (3 had insufficient material), homogenized, and briefly vortexed. Moisture content was determined by drying samples at 50°C for 48 hours and reweighing using a Mettler Toledo AT200 scale (±0.1g). Data normality was assessed with the Shapiro-Wilk test.

Psychopathology
We utilized several validated psychometric instruments to assess various aspects of psychopathology: (A) Eating Disorder psychopathology: The assessment of eating disorder symptoms was conducted using the Eating Disorder Inventory (EDI-3), which has been validated for Danish patients63. (B) Eating Disorder symptoms: We administered the 22-item Eating Disorder Examination Questionnaire (EDE-Q), a self-report version of the semi-structured Eating Disorder Examination (EDE) interview, to assess eating disorder symptoms. (C) Quality of Life: The Eating Disorder Quality of Life Scale (EDQLS), validated for Danish anorexia nervosa populations, was used to measure the impact of eating disorders on patients’ quality of life64,65. (D) Depressive Symptoms: To assess the severity of depressive symptomatology, we employed the Major Depression Inventory (MDI), a widely used self-report instrument66,67.

Microbiome analysis. DNA extraction and library preparation: Fecal genomic DNA extraction was conducted using (250mg of stool) QIAamp PowerFecal Pro DNA kit according to manufacturer. Purity was tested on Nanodrop, ≥10ng/μL, OD260/280=1.8-2.0. DNA sequencing was done by Novogene: The DNA libraries were prepared for the Whole Genome Shotgun Sequencing (WGS) NovaSeq X Plus Series (PE150) (15 GB reading depth per sample). The metagenomics DNA was randomly sheared into short fragments and end repaired, A-tailed and further ligated with Illumina adapter. This was then PCR amplified, size selected, and purified. For further info on metagenomics assembly, binning, taxonomy functional annotations and bioinformatics see Supplementary methods2.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The metagenomic sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive under accession number PRJNA1184021. De-identified biological data and study materials are available from the corresponding author upon reasonable request for up to five years after publication. The study protocol and analytic code are provided in the Supplementary Files. Individual participant data cannot be shared due to the Danish Data Protection Act, which supplements the EU General Data Protection Regulation (GDPR), but aggregated data are available as described or upon request. All study documents and materials are stored in accordance with the Act on Processing of Personal Data and relevant provisions of the Danish Health Act governing biobanks. Participants were informed of these protections during the consent process. After analyses, biological samples will be stored in a biobank for 15 years.

Code availability
All custom R scripts used for data processing, statistical analyses, and figure generation (Supplementary data 2) are publicly available at https://github.com/farhadm1990/fmt_trial. A DOI-minted archive of the analysis workflow is available on Zenodo (https://doi.org/10.5281/zenodo.17949926).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Panah et al describe the results of a feasibility study of fecal microbial transplant in 18 Adults with Anorexia Nervosa. This was an open label, non-randomized trial and demonstrates the feasibility of a larger FMT trial. This was a single treatment study with two stools samples. While 80% of enrolled subjects completed the trial, the 4 dropouts failed to provide the second stool sample. In a larger placebo control trial, would the design be the same, a single treatment with two stool collections? (although the stool collection is less important than clinical measures as an outcome). Outside of C difficile treatment, most FMT studies have more frequent treatments, and more treatment usually correlates with better outcomes. If a larger trial design would involve more treatments, does this feasibility study address dropout and adverse events that would be observed in such a trial?\nFrom the description of the metagenomic analysis, the microbiome data was limited to the analysis of metagenome assembled genomes (MAGs). The following comments are based on the methods used as understood from the methods as described. An important missing detail is the thresholds of completeness and contamination that were used to define MAGS as this does vary between studies. It seems that the taxonomic analysis was limited to the species and coverage of the MAGs and this is significant limitation. Many genomes are present in metagenomic data that are not present at sufficient depth to assemble MAGs and these would be considered absent from those samples in the methods as described. Using a short read based taxonomy (such as MetaPhlAn4) are more sensitive and would be an improvement by the MAG based approach. Regardless species level analysis (particularly with shallow profiling such as MAGs as described) are insufficient to predict engraftment and the authors should avoid conclusions about this. The authors should also not conclude presence and absence of specific taxa based on MAG analysis.\nProkka is no longer a recommended annotation tool for microbial genomes or MAGs. Even the developer of the software recommends not using it. Bakta or RAST would be two common alternatives that will generally give better gene prediction and annotation.\nThe functional analysis, it did appear that the data was restricted to MAGs. If this was done on all the assembled data it should be clearly stated and if there was a contig size threshold applied (to minimize artifacts due to partial genes). Using clustering approaches to define functional groups (such a linclst) does reduce the ability to distinguish engraftment. The clustering thresholds are critical to understand the analysis but this was not mentioned. As these are set by the user, it is not sufficient to reference previous the software or paper, and these values should be included in the methods. If as described, all of the analysis was based on MAG data, it means most of the sequenced data is unused.\nSince there was no obvious clinical response (unlikely from a single FMT and short term follow-up) the definition of responders and “likelihood of FMT success” are based primarily in the Bray -Curtis distances. The fact that some patient microbiomes become more donor-like is not evidence of engraftment (and none of the subjects get that close to the donor). A good control for Fig 2D would be to plot the distance between the two donors and the distance between the two subjects (w0 and w1) to the donor they did not receive. If the change in the microbiome reflects improve transit time (and consequently changes in the microbiome towards a more “healthy one” this distance might be donor “independent”.\nThe results in Fig 1 are quite striking (although self reported). This might be a proxy for clinical response and it would be interesting to stratify the subjects for comparing microbiome data and other measured variables (fig 4). Ofcourse we do not know if this may represent normal weekly fluctuations in this patient population or a placebo response to choosing to be in the trial.\nFor a small study with no control group and the chosen analysis approached restricted to MAGs, the focus on the microbiome is not justified and the conclusions are overstated.\nMinor comments\nThere were do details of donor selection and screening for pathogens which is standard in the field. This is not mention in the paper or in the Study Protocol provided in the supplementary material.\nFigure 2A-C, this is a very small data set for violin plots and not very useful here (these are useful for seeing distributions in large datasets). With the subject samples (w0, w1) the individual data points are larger obscured and there is no real structure.\nThe change in the Bristol Stool scores reporter in Fig 1 was one of the only ‘clinical’ findings. Considering that the Bristol Stool Index was a self reported, a more quantitative measure stool consistency would have been useful. The authors could consider measuring water content of the samples (not an onerous task for a small study).\nIn the limitations, the authors could comment on the possibility of a placebo effect. This is common in disorders of the gut-brain axis and other eating disorders, although not to my knowledge for AN. Because this is open label, and the patients are self selected, this could be a confounding factor.\nThe quality of supplementary figures are low and the font very small, the figures could be made larger and more legible.", "reviewer_2": "The paper “Investigating the Impact of a Single Fecal Microbiome Transplantation to 18 Adults with Anorexia Nervosa - A Feasibility Study” reports a series of single dose FMT in individuals with anorexia nervosa exploring both the efficacy of the transfer and some data on psychological outcomes (although they correctly state that they were not powered to see differences).\nThis paper adds to a growing body of literature related to the gut microbiota (GM) in anorexia nervosa and here a case series of FMT delivered via frozen capsule or enema (patient’s choice). Overall, the authors demonstrate feasibility and acceptability with patients generally choosing frozen capsule administration and an 81% completion rate. I find myself wishing that they had just taken a few extra steps to make this a more definitive feasibility study. As conducted several important questions remain unanswered and unanswerable with the data they have collected.\nAlthough the paper is billed as a feasibility study, the authors give one sentence of feasibility-type information on line 241-243 (“Most participants preferred oral FMT, completed all study requirements, and were satisfied with administration method; those using capsules found them easy to swallow and were open to future doses”), however, no additional feasibility data were provided.\nInclusion and exclusion criteria are not entirely clear from the paper. The protocol is somewhat helpful, but this information needs to be clearer in the manuscript. They state:\n\"69 volunteers expressed interest in participating. Of these, 19 were excluded based on a pre-interview assessing age, sex, and current BMI. Fifty completed the EDI-3 and our participation questionnaire and further 28, were not included as they did not meet our inclusion criteria (BMI, age, probiotics, antibiotics, laxative abuse, intercurrent disease).\"\nI would like to see how many individuals were excluded for each reason.\nPresenting the study as a feasibility study can explain why they opted for a single FMT administration. However, it seems like a disappointing missed opportunity not to have collected subsequent stool samples to see how long the observed changes lasted. In fact there is no commentary on the importance of determining how long any changes toward donor composition last. In the absence of any subsequent measurements, the actual value of the single fecal transfer is unclear.\nI am also unclear why they collected the second fecal sample a week later and then completed questionnaires at one-month follow-up.\nThey used the Bristol Stool Scale to self-report stool consistency and transit time. Although use of the Bristol Stool Scale is adequate for estimating stool consistency, it is an imperfect measure of gut transit time. Are the authors aware of any literature validating it against more conventional gastric transit time measures? If not, this should be listed as a weakness (in addition to the general weakness of relying on self-report).\nNo information on selection of donors was provided in the manuscript. In the protocol there is additional information, but it is not sufficient. They state:\n“Donor identification and FMT material: Through our established procedures and guidelines for FMT in C. diff treatments at Hvidovre Hospital, we will, as before, recruit and screen donors via either via the voluntary blood donor system/ blood and tissue transplant service or among already established donors at Hvidovre Hospital.”\nWithout knowing their standard screening procedures, we do not know if the donors had histories of eating disorders or other psychopathology that could affect GM. Donor selection should be mentioned in the article.\nIn addition it is unclear why Donor 1 was n=3 and Donor 2 was n=10).\nFigure 2A: the donor2 spread is quite massive. They describe a significant difference in chao1 between w0 and w1, but the figure is not convincing.\nPlus why only plot Donor 2 in Figure 5?\nThe authors report “notable trends” in their data, which if corrected for multiple comparisons would not at all be significant. Could the authors please comment on the potential clinical significance of these trends. If they are not clinically significant, then they should not be reported as trends.\n\"Notable trends included a two-fold reduction in estradiol levels across participants, though this did not reach statistical significance (OR = 0.51, CI: 0.26–1.01, p = 0.054). Insulin levels showed a modest 10% increase overall (OR = 1.1, CI: 1.00–1.21, p = 0.047), while PYY levels in responders exhibited a significant increase (OR = 1.22, CI: 1.02–1.46, p = 0.037).\"\nThe authors highlight gmGUS as a key hypothesis in the introduction but then brush over it in the results (219-227). If something is explicitly mentioned as a hypothesis, the results should also be explicitly described in text, not just relying on a figure).\nInteresting that the authors mention:\n“Future studies should measure stool energy content, such as through bomb calorimetry, to determine if decreased transit time leads to energy loss, especially in these sensitive patients.”\nAccordingly, I recommend that they cite a recent paper on this topic PMID: 39569450.\nMinor points:\nAbstract: It is odd that they chose body image distortion as a defining feature of AN as this is not required to be present for a diagnosis. Later, in the text, they also refer to “disturbed body image,” which is an odd phrase.\nSpelling error: Bristol Stoll (Stool) and line 37 of introduction starts with a misplaced “Of”."}, "author_response": "Reviewer #1 Author Response\nClinical FMT, metagenomics, microbiome - We have planned the next trial as a\n(Remarks to the Author): The manuscript randomized, double-blind, two-period,\nby Panah et al describe the results of a placebo-controlled crossover trial (with 3\nfeasibility study of fecal microbial months follow-up) to assess the efficacy of\ntransplant in 18 Adults with Anorexia (5+5weeks) repeated oral FMT (5x 25\nNervosa. This was an open label, non- capsules)/placebo capsules (5x25).\nrandomized trial and demonstrates the (participant: n=60) Four sampling times.\nfeasibility of a larger FMT trial. However, funding of this trial is still\nThis was a single treatment study with two pending funding.\nstools samples. While 80% of enrolled\nsubjects completed the trial, the 4 dropouts\nfailed to provide the second stool sample.\nIn a larger placebo control trial, would the\ndesign be the same, a single treatment with\ntwo stool collections? (although the stool\ncollection is less important than clinical\nmeasures as an outcome).\nOutside of C difficile treatment, most FMT We asked participants if they would be\nstudies have more frequent treatments, willing to take capsules over multiple\nand more treatment usually correlates with weeks, and most said yes. We’ve clarified\nbetter outcomes. If a larger trial design this in the manuscript. We also added a\nwould involve more treatments, does this note to the Discussion (LINE 338)\nfeasibility study address dropout and acknowledging that repeated treatments\nadverse events that would be observed in may carry unknown risks in AN, which\nsuch a trial? would need evaluation in future trials.\nFrom the description of the metagenomic The missing information is now included in\nanalysis, the microbiome data was limited material and method supplementary file\nto the analysis of metagenome assembled (SL186-189).\ngenomes (MAGs). An important missing\ndetail is the thresholds of completeness\nand contamination that were used to define\nMAGS as this does vary between studies.\nIt seems that the taxonomic analysis was Only MAGs with no read coverage was\nlimited to the species and coverage of the filtered out.\nMAGs and this is significant limitation.\nMany genomes are present in metagenomic\ndata that are not present at sufficient depth\nto assemble MAGs and these would be\nconsidered absent from those samples in\nthe methods as described.\nUsing a short read based taxonomy (such We are aware that Megaphlan4 is a\nas MetaPhlAn4) is more sensitive and powerful tool. However, instead we have\nwould be an improvement over the MAG- used marker-based classification by\nbased approach. genome taxonomy database tool kits\n(GTDB-tk) and CheckM tools that proved to\nbe robust for taxonomy classification.\nRegardless, species level analysis We agree that MAGs are insufficient to fully\n(particularly with shallow profiling such as predict engraftment, therefore we have no\nMAGs as described) is insufficient to conclusions on this.\npredict engraftment and the authors\nshould avoid conclusions about this.\nThe authors should also not conclude On presence/ absence we have added a line\npresence and absence of specific taxa based in the discussion of manuscript as not to\non MAG analysis. overstate. (Line323)\nProkka is no longer a recommended At the time of analysis Prokka was still\nannotation tool for microbial genomes or maintained and only later Torsten\nMAGs. Even the developer of the software Seemann made the announcement, that\nrecommends not using it. Bakta or RAST Prokka would no longer be maintained.\nwould be two common alternatives that However, it is still using updated databases\nwill generally give better gene prediction Isfinder, NCBI reference genes, and\nand annotation. UniProtKB (SwissProt). We will definitely\nconsider using Bakta in any future analysis.\nThe functional analysis appeared to be Functional annotation was done on MAGs\nrestricted to MAGs. If this was done on all binned with genomes > 5000bp, >50%\nthe assembled data it should be clearly completeness, and <10% contamination as\nstated, and if there was a contig size well as 97.5% average neuclotide identity.\nthreshold applied (to minimize artifacts The text in Supplementary files has been\ndue to partial genes). updated (SL 191).\nUsing clustering approaches to define The text on clustering thresholds is now\nfunctional groups (such as linclst) reduces updated in supplementary material and\nthe ability to distinguish engraftment. The supplementary methods (SL191)\nclustering thresholds are critical to\nunderstand the analysis but were not\nmentioned. These values should be\nincluded in the methods.\nIf all of the analysis was based on MAG\ndata, it means most of the sequenced data\nis unused.\nSince there was no obvious clinical We use the definition “responder” here\nresponse (unlikely from a single FMT and only for a definition to use for further\nshort-term follow-up) the definition of exploring the data on “Bray-Curtis\nresponders and “likelihood of FMT success” distance- responders”\nare based primarily on the Bray-Curtis\ndistances.\nThe fact that some patient microbiomes We agree that changes are not evidence of\nbecome more donor-like is not evidence of engraftment, therefore we have no\nengraftment (and none of the subjects get conclusions on this.\nthat close to the donor).\nA good control for Fig 2D would be to plot This is an interesting suggestion. We have\nthe distance between the two donors and therefor defined a distance to “the other\nthe distance between the two subjects (w0 donor” and repeating the analysis in Fig2D\nand w1) to the donor they did not receive. with this metric reveal no change in this\ndistance from w0 to w1 (p = 0.46).\nIf the change in the microbiome reflects Further, comparing the w1 distances to the\nimproved transit time (and consequently “own” donor versus the “other/wrong”\nchanges in the microbiome towards a more donor reveal significantly longer distance\n“healthy” one) this distance might be donor to the “wrong” donor (p < 0.001)]\n“independent”.\nWe have added a few lines (L176) to the\nmanuscript and 2 supplementary figures\n(SL 134) to make this clearer.\nThe results in Fig 1 are quite striking We have conducted an exploratory analysis\n(although self-reported). This might be a to reveal whether the range in Bristol Stool\nproxy for clinical response and it would be Form Scores (BSFS) causes differences in\ninteresting to stratify the subjects for the resulting [metabolite/hormone] levels.\ncomparing microbiome data and other In almost all cases these measures act\nmeasured variables (Fig 4). Of course we independently, except Estradiol, where the\ndo not know if this may represent normal resulting w1 levels are somewhat\nweekly fluctuations in this patient correlated to BSFS also at w1.\npopulation or a placebo response to\nchoosing to be in the trial. Added lines (L247) to manuscript to make\nthis clearer as well as a Table reporting\nthese comparisons is added to\nSupplementary files.\nFor a small study with no control group We appreciate this point and have tried to\nand the chosen analysis approach make clear that microbiome results are\nrestricted to MAGs, the focus on the exploratory. We tempered language\nmicrobiome is not justified and the throughout, expanded the Limitations to\nconclusions are overstated. note the open-label design, small sample,\nand constraints of MAG-based profiling,\nand removed any over-interpretation (e.g.,\nengraftment or presence/absence claims).\nThe primary objective was feasibility;\nmicrobiome findings are presented only as\nhypothesis-generating for a future\nrandomized, adequately powered trial.\nMinor comments: There were no details of The information is in (L472), additional\ndonor selection and screening for information was added (L474- L477).\npathogens, which is standard in the field.\nThis is not mentioned in the paper or in the\nStudy Protocol provided in the\nsupplementary material.\nFigure 2A-C: This is a very small data set We agree and have removed the violin\nfor violin plots and not very useful here. aesthetic and made the individual points\nWith the subject samples (w0, w1) the larger. Figure 2A-F has been updated.\nindividual data points are largely obscured\nand there is no real structure.\nThe change in the Bristol Stool scores Thank you for this good suggestion. We\nreported in Fig 1 was one of the only have now measured moisture content (MC)\n‘clinical’ findings. Considering that the and have added the information to the\nBristol Stool Index was self-reported, a manuscript M&M results and discussion\nmore quantitative measure of stool (L148, L279 and L501). Added\nconsistency would have been useful. The Supplementary table 2. BSFS scores were\nauthors could consider measuring water significantly correlated to MC both before\ncontent of the samples. (rho = 0.74, p = 0.0017) and after FMT (rho\n= 0.88, p < 0.0001). However, when\ncomparing pre-postFMT no significant\ndifferences in MC were found.\nIn the limitations, the authors could Limitation added: L 267-275\ncomment on the possibility of a placebo\neffect. This is common in disorders of the\ngut-brain axis and other eating disorders,\nalthough not to my knowledge for AN.\nThe quality of supplementary figures is low Noted with thanks. We have now replaced\nand the font very small; the figures could them with the highest quality and made the\nbe made larger and more legible.\nReviewer #2 -\nThe paper “Investigating the Impact of a We fully agree that a more definitive\nSingle Fecal Microbiome Transplantation feasibility study — with extended follow-\nto 18 Adults with Anorexia Nervosa - A up and additional fecal sampling, such as a\nFeasibility Study” reports a series of single second sample several months after\ndose FMT in individuals with anorexia treatment — would have been ideal.\nnervosa exploring both the efficacy of the However, before initiating this study, we\ntransfer and some data on psychological were genuinely uncertain whether it would\noutcomes (although they correctly state be feasible at all to conduct such a trial in\nthat they were not powered to see this particularly vulnerable and hard-to-\ndifferences).\nreach patient population. As such, we\nThis paper adds to a growing body of deliberately chose a minimalistic design,\nliterature related to the gut microbiota focusing on obtaining essential feasibility\n(GM) in anorexia nervosa and here a case data while keeping participant burden and\nseries of FMT delivered via frozen capsule study complexity to an absolute minimum\nor enema (patient’s choice). Overall, the to ensure successful completion.\nauthors demonstrate feasibility and\nacceptability with patients generally The experience gained from this initial\nchoosing frozen capsule administration feasibility study has provided us with\nand an 81% completion rate. I find myself critical insights and reassured us that a\nwishing that they had just taken a few extra more ambitious trial is indeed possible.\nsteps to make this a more definitive Based on what we have learned, we have\nfeasibility study. As conducted several now planned a future (pending funding)\nimportant questions remain unanswered randomized, double-blind, placebo-\nand unanswerable with the data they have controlled, two-period crossover trial\ncollected. (n=60), with repeated oral FMT capsules\n(5+5 weeks) and extended follow-up over 3\nmonths, including four fecal sampling time\npoints. We are confident that this design\nwill allow us to address the remaining\nquestions raised by the reviewer and to\nmore robustly assess the efficacy and\nlonger-term impact of the intervention.\nAlthough billed as a feasibility study, the We have now added additional feasibility\nauthors give only one sentence of data and added to the discussion (L 115)\nfeasibility-type information on line 241-\n243. No additional feasibility data were\nprovided.\nInclusion and exclusion criteria are not Below is a list of specific exclusion criteria.\nentirely clear from the paper. The protocol This is now added to the text and updated\nis somewhat helpful, but this information at (L115-121). Some individuals had\nneeds to be clearer in the manuscript. They multiple exclusion criteria. Therefore, the\nstate:\n\"69 volunteers expressed interest in sum is higher than the number of excluded\nparticipating. Of these, 19 were excluded individuals\nbased on a pre-interview assessing age, sex, BMI >18.5: 20\nand current BMI. Fifty completed the EDI-3 Daily use of laxatives: 7\nand our participation questionnaire and Lactic acid bacteria supplementation: 2\nfurther 28, were not included as they did Celiac disease: 1\nnot meet our inclusion criteria (BMI, age, Hypothyroidism: 1\nprobiotics, antibiotics, laxative abuse,\nintercurrent disease).\"\nI would like to see how many individuals\nwere excluded for each reason.\nPresenting the study as a feasibility study We have clarified this further (L321)\ncan explain why they opted for a single\nFMT administration. However, it seems like\na disappointing missed opportunity not to\nhave collected subsequent stool samples to\nsee how long the observed changes lasted.\nIn fact there is no commentary on the\nimportance of determining how long any\nchanges toward donor composition last. In\nthe absence of any subsequent\nmeasurements, the actual value of the\nsingle fecal transfer is unclear.\nI am unclear why they collected the second The four-week interval was chosen to\nfecal sample a week later and then reduce practice effects, especially in the\ncompleted questionnaires at one-month absence of a control group. A shorter\nfollow-up. interval (e.g., one week to match the GM\nsampling) risked recall bias and may have\nonly captured transient GM effects. While\narbitrary, one month was considered a\nmore reasonable window for detecting\npotentially meaningful (although not\nexpected) psychological changes from 1\nsingle FMT treatment. Added to (L296)\nThey used the Bristol Stool Scale to self- We have added a reference in line (L 499)\nreport stool consistency and transit time. and performed validating fecal moisture\nAlthough use of the Bristol Stool Scale is content measurements (L501-505).\nadequate for estimating stool consistency,\nit is an imperfect measure of gut transit\ntime. Are the authors aware of any\nliterature validating it against more\nconventional gastric transit time\nmeasures? If not, this should be listed as a\nweakness (in addition to the general\nweakness of relying on self-report).\nNo information on selection of donors was We have now added further information to\nprovided in the manuscript. In the protocol the manuscript (L 471-475)\nthere is additional information, but it is not\nsufficient. They state:\n“Donor identification and FMT material:\nThrough our established procedures and\nguidelines for FMT in C. diff treatments at\nHvidovre Hospital, we will, as before, recruit\nand screen donors via either via the\nvoluntary blood donor system/ blood and\ntissue transplant service or among already\nestablished donors at Hvidovre Hospital.”\nWithout knowing their standard screening\nprocedures, we do not know if the donors\nhad histories of eating disorders or other\npsychopathology that could affect GM.\nDonor selection should be mentioned in the\narticle.\nIt is unclear why Donor 1 was n=3 and We used available safe donor portions. It\nDonor 2 was n=10. ended up undesirably uneven due to\nrestriction on recruiting and drop-outs. We\naddress this in the Discussion (L326).\nFuture studies should strive to balance this\nbetter and use several different donors\nboth for feasibility and purposeful\nheterogeneity. (L328-331)\nFigure 2A: The Donor 2 spread is massive. The NS not significant is added to the\nThey describe a significant difference in figure.\nChao1 between w0 and w1, but the figure\nis not convincing.\nWhy only plot Donor 2 in Figure 5? Donor 1 was not significant. Added to the\ntext.\nThe authors report “notable trends” in Now stated in text (L 259) We acknowledge\ntheir data, which if corrected for multiple that the “trends” in in Estradiol, Insulin and\ncomparisons would not at all be significant. PYY are not statistically robust and their\nCould the authors please comment on the clinical relevance in AN remains unclear\npotential clinical significance of these and should be interpreted cautiously,\ntrends. If they are not clinically significant, especially given the small sample size and\nthen they should not be reported as trends. lack of dietary or weight data. They may\nhowever still be useful as a hypothesis-\n\"Notable trends included a two-fold generating observation for future studies\nreduction in estradiol levels across and we have thus decided to maintain the\nparticipants, though this did not reach findings in the manuscript, but of course\nstatistical significance (OR = 0.51, CI: 0.26– leave them out, should the editor request\n1.01, p = 0.054). Insulin levels showed a so. We have added more text on the\nmodest 10% increase overall (OR = 1.1, CI: limitations in the discussion (L410)\n1.00–1.21, p = 0.047), while PYY levels in\nresponders exhibited a significant increase\n(OR = 1.22, CI: 1.02–1.46, p = 0.037).\"\nThe authors highlight gmGUS as a key Now emphasized in (L248) and discussed\nhypothesis in the introduction but then in (L413-417)\nbrush over it in the results (219-227). If\nsomething is explicitly mentioned as a\nhypothesis, the results should also be\nexplicitly described in text, not just relying\non a figure).\nInteresting that the authors mention: This was an oversight and very valuable\nrecommendation and its now cited (L289)\n“Future studies should measure stool energy\ncontent, such as through bomb calorimetry,\nto determine if decreased transit time leads\nto energy loss, especially in these sensitive\npatients.”\nAccordingly, I recommend that they cite a\nrecent paper on this topic PMID:\n39569450.\nMinor points: Abstract: It is odd that they Thank you for this helpful comment. While\nchose body image distortion as a defining DSM-5 includes “disturbance in the way\nfeature of AN as this is not required to be one’s body weight or shape is experienced”\npresent for a diagnosis. Later, in the text, as a diagnostic criterion, we agree that its\nthey also refer to “disturbed body image,” expression can vary. We used established\nwhich is an odd phrase. psychometric tools that include this\nfeature, but appreciate the point and have\nuniformed the language in the text.\nSpelling error: Bristol Stoll (Stool) and line Stool Corrected, but could not find the\n37 of introduction starts with a misplaced misplaced “of”?\n“Of”."}, {"round": 2, "reviewer_comments": {"reviewer_3": "I have been asked to assess whether the authors have adequately dealt with reviewer #1's comments.\nI commend the authors for largely addressing the technical issues raised, however there are several points that have not ben adequately addressed.\n1. The authors still overstate the reported changes in stool consistency. On P4 the authors state \"The change reflects a significant decrease in estimated gut transit time...\", which is simply incorrect when fecal moisture content ( a more objective measure) did not change.\n2. As already stated the term responders should not be used based upon Bray Curtis and not clinical response. It is misleading and incorrect to use responders in this way. I suggest you consider instead :greater similarity to donor microbiomes\".\n3. The authors state that \"these numbers (of participants) suggest good interest, when they do not. Recruiting 22 subjects in 9 months suggests that any larger RCT will likely need to includer multiple centers to achieve adequate recruitment. Recruitment of anorexia nervosa trial patients for clinical trials has previously been found to be challenging.\n4. Whilst the supplementary figures labels have been made more readable, all of the figure labels in the manuscript are almost unreadable. Of note Figure 4 X axis labels run over each other and are completely unreadable. The X and Y axes of Fig 4 should be symmetrical. As presented there is an illusion created of a far steeper gradient of the regression lines than should exist.", "reviewer_4": "- Although the authors added in a few feasibility metrics, they still excuse the small sample size as a \"feasibility\" matter without reporting much on data. More specifically, they report the following in the discussion without presenting any data elsewhere: \"Most participants preferred oral FMT, completed all study requirements, and were satisfied with the administration method; those using capsules found them easy to swallow and were open to future doses\". If the authors are going to claim this is a feasibility study, it must be treated as such in structure. The readers cannot be expected to take their word for it.\n- There is no evidence of engraftment.\n- The authors use of the term \"responder\" is specific to being \"a reduction of at least 1.5 standard deviations in Bray-Curtis dissimilarity metrics\". I worry that the use of responders later in the manuscript, for example in the blood samples results section, can misdirect a reader to forget the absolute definition of the term.\n- The most striking findings are in Figure 1, but this is all based on self-report. this limitation must be highlighted further. It is additionally complicated by the fact that there were no changes in fecal moisture content (in which the authors also omit freeze/thaw/storage protocols that may impact these results)"}, "author_response": "Reviewer #3 Author response\n1. The authors still overstate the We agree. This line \"The change reflects a significant\nreported changes in stool consistency. decrease in estimated gut transit time.\"(L144) has been\nOn P4 the authors state \"The change deleted\nreflects a significant decrease in\nestimated gut transit time...\", which is\nsimply incorrect when fecal moisture\ncontent ( a more objective measure) did\nnot change.\n2. As already stated the term We agree and have reworded to “closer-to-donor”\nresponders should not be used based instead in the entire manuscript and figures\nupon Bray Curtis and not clinical\nresponse. It is misleading and incorrect\nto use responders in this way. I suggest\nyou consider instead :greater similarity\nto donor microbiomes\".\n3. The authors state that \"these We agree and have removed “good interest (L121) and\nnumbers (of participants) suggest good added a discussion point as a challenge instead (L271)\ninterest, when they do not. Recruiting\n22 subjects in 9 months suggests that\nany larger RCT will likely need to\nincluder multiple centers to achieve\nadequate recruitment. Recruitment of\nanorexia nervosa trial patients for\nclinical trials has previously been found\nto be challenging.\n4. Whilst the supplementary figures All figures in the manuscript have been thoroughly\nlabels have been made more readable, checked for the flagged issues and improved and\nall of the figure labels in the manuscript finalized.\nare almost unreadable. Of note Figure 4\nX axis labels run over each other and\nare completely unreadable. The X and\nY axes of Fig 4 should be symmetrical.\nAs presented there is an illusion\ncreated of a far steeper gradient of the\nregression lines than should exist.\nReviewer #4 Author response\nAlthough the authors added in a few We have added an anonymized “Supplementary data2”\nfeasibility metrics, they still excuse the which includes data on: Clinical characteristics including\nsmall sample size as a \"feasibility\" preferred FMT route =N, Eating Disorder Inventory-3\nmatter without reporting much on data. subscales (total scores), MDI and EDQLS\nMore specifically, they report the questionnaires. A table with Gut feeling and Bristols\nfollowing in the discussion without score results.\npresenting any data elsewhere: \"Most\nparticipants preferred oral FMT, This is now mentioned in (L135)\ncompleted all study requirements, and\nwere satisfied with the administration\nmethod; those using capsules found\nthem easy to swallow and were open to\nfuture doses\". If the authors are going\nto claim this is a feasibility study, it must\nbe treated as such in structure. The\nreaders cannot be expected to take\ntheir word for it.\nThere is no evidence of engraftment. No new data.\nThe authors use of the term We agree and have reworded to “closer-to-donor”\n\"responder\" is specific to being \"a instead in the entire manuscript and figures\nreduction of at least 1.5 standard\ndeviations in Bray-Curtis dissimilarity\nmetrics\". I worry that the use of\nresponders later in the manuscript, for\nexample in the blood samples results\nsection, can misdirect a reader to forget\nthe absolute definition of the term.\nThe most striking findings are in Figure We have further added and emphasized this in the\n1, but this is all based on self-report. Discussion (L 228)” It is a limitation that BSFS is on self\nthis limitation must be highlighted reported data and MC was done on once-frozen\nfurther. It is additionally complicated by samples”.\nthe fact that there were no changes in\nfecal moisture content (in which the\nauthors also omit freeze/thaw/storage\nprotocols that may impact these results)\n(figures) All figures in the manuscript have been thoroughly\nchecked for the flagged issues and improved and\nfinalized.\nSupplementary figures and Methods In Supplementary Figures and Methods (Table 2 (L145)),\nwe have changed the “non-responder/responder\nDouble check all figures and tables to nomenclature to the current “closer-to-donor” in the\nremove “non-responder/responder entire table.\nnomenclature."}]

Decision letter