The design of this study has been described in detail previously[12]. In brief, patients completed three questionnaires to assess their symptoms and quality of life at the baseline and 2 wk, 1 mo and 3 mo after FMT. They also provided faecal samples at the baseline and 1 mo after FMT. Polyethylene glycol and loperamide were allowed as rescue medication during the study. The patients were randomized 1:1:1 to placebo (own faeces), 30-g (superdonor faeces) or 60-g (superdonor faeces) FMT[12]. The 30- and 60-g superdonor-faeces groups were pooled together and called the active treated group in order to increase the sample size and reduce the probability of type-II statistical errors.

This study included 164 patients who had participated in our previous study[12]. The characteristics of these patients are given in Table 1. The patients enrolled in this study have been described in detail previously[12]. In brief, patients attending the outpatient clinic at Stord Hospital who fulfilled the Rome IV criteria for a diagnosis of IBS were recruited. All of the recruited patients had previously not responded to consuming the National Institute for Health and Care Excellence (NICE)-modified diet for at least 3 mo[12]. They also received a course of IBS treatment that slightly improved their symptoms.

The inclusion criteria were being aged between 18 and 75 years and having moderate-to-severe IBS symptoms, as indicated by a score of 175 on the IBS severity scoring system (IBS-SSS). The exclusion criteria were being pregnant or planning pregnancy, lactating, the presence of systemic disease, having immune deficiency or being treated by immune-modulating medication, or having a psychiatric illness, excessive alcohol consumption or drug abuse. Patients who took probiotics, antibiotics or IBS medications within 8 wk prior to study inclusion were also excluded[12].

The single superdonor used in this study has been described in detail previously[12]. Briefly, he was screened according to the European guidelines for FMT donors[26]. He was a healthy 36-year-old male, non-smoker, not taking any medication regularly and had a normal body mass index. He had been born via a vaginal delivery, breastfed and had taken only a few courses of antibiotics during his life. He exercised regularly and took sport-specific dietary supplements, which made his diet richer than average in protein, fibre, minerals and vitamins. He was normobiotic, but his faecal bacteria profile deviated from the healthy subjects abundance in 14 of the 39 bacteria markers[12].

Faecal samples were frozen immediately and kept at -20 °C until they were delivered frozen to the laboratory, where they were kept at -80 °C. The process of FMT has been described in detail previously[12]. In brief, the patients randomized to the placebo FMT group received 30 g of their own faeces (autologous), while those in the 30-g and 60-g FMT groups received 30 g and 60 g of the superdonor’s faeces (allogenic), respectively. The transplant was administered to the distal duodenum via a gastroscope[12].

Symptoms were assessed using the IBS-SSS and the Fatigue Assessment Scale (FAS)[27-31]. Quality of life was measured using the IBS quality of life scale (IBS-QoL)[32-34]. Response was defined as a decrease of ≥ 50 points in the IBS-SSS total score after FMT.

The faecal bacteria profile and dysbiosis were determined by the GA-map Dysbiosis Test (Genetic Analysis, Oslo, Norway) using the 16S rRNA gene polymerase chain reaction DNA amplification covering V3-V9; probe labelling by single nucleotide extension and signal detection by BioCode 1000A 128-Plex Analyzer (Applied BioCode, Santa Fe Springs, CA, United States)[6]. The bacterial markers used detected bacteria within 5 phyla (Firmicutes, Proteobacteria, Bacteroidetes, Tenericutes and Verrucomicrobia) that cover 10 bacterial classes, 36 genera and 32 species[6]. This test assesses > 300 bacteria at different taxonomic levels[9]. The dysbiosis index (DI) was measured on a 5-point scale from 1 to 5, where DI values 1-2 indicates normobiosis, 3-5 indicates dysbiosis[6].

The method used to determine faecal SCFA levels has been described in detail previously[25]. Briefly, the faecal samples were homogenized with a solution containing 3 mmol/L 2-ethylbutyric acid and 0.5 mmol/L H₂SO₄. The homogenate was vacuum distilled, and the SCFA levels were determined by gas chromatography (Agilent 7890 A, Agilent, CA, United States) using a capillary column (serial no. USE400345H, Agilent J&W GC columns, Agilent) and flame ionization[35,36] levels of total SCFAs, acetic, propionic, iso-butyric, n-butyric, iso-valeric, n-valeric acid, isocapronic and n-capronic acids, were determined and were expressed in units of mmol/kg wet weight.

The sample size required in each arm of the previously published trial was calculated by assuming that a placebo effect was 40% and an effect response was 80%. The total sample size was estimated to be 60 patients, with 20 in each arm (α = 0.05, 1-β = 0.80)[12]. In the present study a new calculation for the sample size was done based on the response rates obtained from our previous RCT[12]. Thus, assuming that the females’ response is 90% and males’ response is 60%, The total sample size was estimated to be 22 with 11 females and 11 males (α = 0.05, 1-β = 0.80). The 30- and 60-g superdonor-faeces groups were pooled together and called the active treated group in order to increase the sample size and reduce the probability of type-II statistical errors. Differences in response and dysbiosis between females and males in the placebo and the active treated group were analyzed using the χ²test. Differences between females and males in the total scores on the IBS-SSS, FAS and IBS-QoL, and in faecal bacteria and SCFA levels were analyzed using the Mann-Whitney test. These analyses were performed using GraphPad Prism (version 8, La Jolla, CA, United States).

The Regional Committee for Medical and Health Research Ethics West, Bergen, Norway approved the study (approval No. 2017/1197/REK vest). All subjects provided both oral and written consents to participate. The study was registered at www.clinicaltrials.gov (NCT03822299) and www.cristin.no (ID657402).

In the placebo group, the response did not differ between females and males at 2 wk, 1 mo and 3 mo after FMT (P = 0.4, 0.9 and 0.8, respectively). The responses in the active treated group did not differ between females and males after 2 wk, 1 mo and 3 mo (P = 0.6, 0.8 and 0.3, respectively) (Figure 1). The response rate was significantly higher in females with IBS-D than that of males at 1 mo, and 3 mo after FMT (Table 2 and Figure 2). There was no significant difference of response rates between female and male patients with either moderate or severe IBS symptoms (Table 3 and Figure 3).

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Figure 1 Response to faecal microbiota transplantation in females and males at different intervals after faecal microbiota transplantation in the two groups. A: Placebo group; B: Active treated group.

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Figure 2 Response rates to faecal microbiota transplantation of female and male irritable bowel syndrome patients. A: Irritable bowel syndrome with diarrhoea-predominant; B: Irritable bowel syndrome with constipation-predominant; C: Irritable bowel syndrome with mixed diarrhoea and constipation. ^cP < 0.001. FMT: Faecal microbiota transplantation.

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Figure 3 Response rates to faecal microbiota transplantation and the total irritable bowel syndrome-severity scoring system scores of irritable bowel syndrome patients with moderate irritable bowel syndrome symptoms (irritable bowel syndrome-severity scoring system total score between 175 and 300) and with severe irritable bowel syndrome symptoms (irritable bowel syndrome-severity scoring system total score of ≥ 300). A: Faecal microbiota transplantation; B: Irritable bowel syndrome severity scoring system total score. MS: Moderate irritable bowel syndrome symptoms; SS: Severe irritable bowel syndrome symptoms; FMT: Faecal microbiota transplantation.

The IBS-SSS total score did not differ significantly between female and male IBS patients in either the placebo or the active treated group (Table 4 and Figure 4). However, IBS-SSS total score was significantly lower in female patients with IBS-D than that of male patients both 1 mo and 3 mo after FMT (Table 5 and Figure 5). The IBS-SSS total score did not differ significantly between females and males in patients with moderate or severe IBS symptoms (Table 6 and Figure 3).

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Figure 4 The total irritable bowel syndrome-severity scoring system scores in females and males. A: Placebo group; B: Active treated group. FMT: Faecal microbiota transplantation.

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Figure 5 The total irritable bowel syndrome-severity scoring system scores in females and males. A: Irritable bowel syndrome with diarrhoea-predominant; B: Irritable bowel syndrome with constipation-predominant; C: Irritable bowel syndrome with mixed diarrhoea and constipation. ^aP < 0.05. IBD-SSS: Irritable bowel syndrome-severity scoring system; FMT: Faecal microbiota transplantation.

The FAS total score also did not differ significantly between female and male IBS patients in the active treated group (Table 7 and Figure 6), but it was lower in males than females in the placebo group at 3 mo after FMT. This could have been due to a type-I statistical error. There was no significant difference between female and male IBS patients belonging to different IBS-subtypes IBS symptoms (Table 8 and Figure 7). However, the FAS total score was lower in males IBS patients with IBS-D than that of females 2 wk after FMT.

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Figure 6 The total Fatigue Assessment Scale scores in female and male irritable bowel syndrome patients. A: Placebo group; B: Active treated group. ^aP < 0.05. FAS: Fatigue Assessment Scale; FMT: Faecal microbiota transplantation.

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Figure 7 Total Fatigue Assessment Scale scores in female and male patients. A: Irritable bowel syndrome with diarrhoea-predominant; B: Irritable bowel syndrome with constipation-predominant; C: Irritable bowel syndrome with mixed diarrhoea and constipation. FAS: Fatigue Assessment Scale; FMT: Faecal microbiota transplantation.

The IBS-QoL total score did differ between females and males in both the placebo and active treated groups (Table 9 and Figure 8), being higher in males than in females at the baseline. IBS-QoL total scores did not differ significantly between female and male patients belonging to different IBS-subtypes (Table 10 and Figure 9).

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Figure 8 Total irritable bowel syndrome quality of life scale scores in female and male patients. A: Placebo group; B: Active treated group. ^aP < 0.05. IBS-QoL: Irritable bowel syndrome quality of life scale; FMT: Faecal microbiota transplantation.

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Figure 9 Total irritable bowel syndrome quality of life scale scores in females and males. A: Irritable bowel syndrome with diarrhoea-predominant; B: Irritable bowel syndrome with constipation-predominant; C: Irritable bowel syndrome with mixed diarrhoea and constipation. IBS-QoL: Irritable bowel syndrome quality of life scale; FMT: Faecal microbiota transplantation.

The faecal bacteria levels in the placebo group did not differ between female and male IBS patients at the baseline and 1 mo after FMT (Table 11 and Figure 10). Similarly, there were no differences in the faecal bacteria levels between female and male IBS patients in the active treated group (Table 12 and Figure 11).

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Figure 10  Bacteria levels in the faeces of female and male irritable bowel syndrome patients in the placebo group at the baseline and 1 mo after faecal microbiota transplantation. A: Baseline; B: 1 mo after faecal microbiota transplantation. The bacterial levels are relative values to a normobiotic microbiota profile of 165 healthy subjects. FMT: Faecal microbiota transplantation.

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Figure 11  Faecal bacteria levels in the female and male irritable bowel syndrome patients in the active treated group at the baseline and 1 mo after faecal microbiota transplantation. A: Baseline; B: 1 mo after faecal microbiota transplantation. The bacterial levels are expressed as in Figure 10. FMT: Faecal microbiota transplantation.

In the placebo group, 26 females (55%) and 4 males (50%) had dysbiosis (P = 0.8) at the baseline, while 25 females (53%) and 4 males (50%) had dysbiosis (P = 0.9) at 1 mo after FMT. In the active treated group, 52 females (61%) and 13 males (54%) had dysbiosis (P = 0.3) at the baseline, while 41 females (48%) and 9 males (38%) had dysbiosis (P = 0.2) at 1 mo after FMT.

The faecal levels of total SCFAs and acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocapronic and capronic acids did not differ between female and males IBS patients in both the placebo and active treated groups at the baseline and 1 mo after FMT (Table 13 and Figure 12).

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Figure 12  The faecal short chain fatty acids concentration of female and male patients in both the placebo and active treated groups at the baseline and 1 mo after faecal microbiota transplantation. The values were expressed as mmol/kg wet weight (mean ± SD).

Irritable bowel syndrome (IBS) is a common chronic disorder, where intestinal microbiota plays a pivotal role in its pathophysiology. Faecal microbiota transplantation for IBS appears to be a promising treatment of IBS.

In Western countries, there is a female predominance in IBS with female:male ratio of 2:1. In a recent randomized double-blind placebo-controlled trial on faecal microbiota transplantation (FMT) in IBS females responded better to FMT than did males.

We aimed to investigate whether there is a sex difference in the response to FMT in terms of symptoms, dysbiosis, and bacteria and short-chain fatty acids (SCFAs) profiles in the same cohort of patients that we had investigated in our previous randomized controlled trial.

This study included 164 patients who fulfilled the Rome IV criteria for the diagnosis of IBS. These patient’s cohort included IBS diarrhoea-predominant (IBS-D), IBS-constipation predominant (IBS-C) and mixed diarrhoea and constipation (IBS-M) subtypes. They were randomized to placebo (own faeces), 30 g or 60 g donor’s faeces at a ratio of 1:1:1. The faecal transplant was administered via gastroscope to the duodenum. Patients completed IBS severity scoring system (IBS-SSS), the Fatigue Assessment Scale (FAS) and the IBS quality of life scale (IBS-QoL) questionnaires at the baseline and 2 wk, 1 mo and 3 mo after FMT. They also provided faecal samples at the baseline and 1 mo after FMT. Response was defined as a decrease of ≥ 50 points in the IBS-SSS total score after FMT. The faecal bacteria profile and dysbiosis were determined by the GA-map Dysbiosis Test (Genetic Analysis, Oslo, Norway) using the 16S rRNA gene. The levels of faecal SCFAs were determined by gas chromatography.

There was no sex difference in the response to FMT either in the placebo group or active treated group. There was no difference between females and males in either the placebo group or actively treated groups in the total score on the IBS-SSS, FAS or IBS-QoL, in dysbiosis, or in the faecal bacteria or SCFA level. However, the response rate was significantly higher in females with IBS-D than that of males at 1 mo, and 3 mo after FMT. Moreover, IBS-SSS total score was significantly lower in female patients with IBS-D than that of male patients both 1 mo and 3 mo after FMT.

There is no sex difference in the response to FMT in IBS patients with moderate-to-severe IBS symptoms belonging to the three of IBS subtypes of IBS-C and IBS-M in patients who did not responded to National Institute for Health and Care Excellence-modified diet. However, female patients with IBS-D had a significant higher response rate to FMT and lower IBS-SSS score after FMT than males.

The present observation that female patients with IBS-D respond better to FMT than males raise several questions as to the cause of this difference. Further studies are needed to explore the difference in diet and life style between females and males as possible causes for this difference.