Fecal Microbiota Transplantation for Ulcerative Colitis: A Systematic Review and Meta-Analysis

Background Fecal microbiota transplantation (FMT) has been recognized as a novel treatment for ulcerative colitis (UC). However, its efficacy and safety remain unclear. Objective We conducted this systematic review to assess the efficacy and safety of FMT in UC. Data Sources PubMed, EMBASE, Cochrane Central, Web of Science Core Collection, and three other Chinese databases were searched for reports of FMT in UC with clear outcomes. Data Extraction and Synthesis We estimated pooled rates [with 95% confidence interval (CI)] of clinical remission among 15 cohort studies and clinical response among 16 cohort studies. Results Twenty five studies (2 randomized controlled trials, 15 cohort studies, and 8 case studies) with 234 UC patients were included. Overall, 41.58% (84/202) patients achieved clinical remission (CR) and 65.28% (126/193) achieved clinical response. Among the cohort studies, the pooled estimate of patients who achieved CR and clinical response were 40.5% (95% CI 24.7%-58.7%), and 66.1% (95% CI 43.7%-83.0%). Most adverse events were slight and self-resolving. The analyses of gut microbiota in 7 studies showed that FMT could increase microbiota diversity and richness, similarity, and certain change of bacterial composition. Conclusion FMT provides a promising effect for UC with few adverse events. Successful FMT may be associated with an increase in microbiota diversity and richness, similarity, and certain change of bacterial composition.


Introduction
Ulcerative colitis (UC) is a chronic, relapsing and remitting disease characterized by the overaggressive inflammatory response contributing to the destruction of the gastrointestinal tract.Its main symptoms include bloody diarrhea, abdominal pain, urgent and tenesmus, [1][2][3] which produce a miserable influence on the quality of life.
While the exact etiology of UC remains unclear, patients with UC are found to have decreased microbiota diversity and species richness leading to the unbalance between adaptation to environment changes and resistance to natural disturbances.[4] The dysbiosis of UC is also characterized by some alterations of bacterial composition, including decrease in Bacteroidetes, along with Firmicutes (in particular Clostridium IXa and IV groups, Bifidobacteria, Lactobacillus and Faecalibacterium prausnitzii), and an increase in Proteobacteria and Actinobacteria.[5][6][7] The human gut contains, in the assortment of 1000 bacterial species, 100-fold more genes than the human genome.[8] Multifaceted microbial population is considered as an organ with critical function in human health.It has been demonstrated that the changes in gut microbiota reduce the ability of the intestinal environment to fight pathogens and can be relevant with some disease conditions.[9][10][11] With the deep research into the interaction of gut microbiota and host, probiotics, prebiotics and symbiotics began to be used in UC patients to promote intestinal microbiota homeostasis.[12] Some randomized controlled trials (RCTs) and cohort studies of Lactobacillus or VSL#3 have already showed their effect on maintaining UC remission and preventing recurrence.[13][14][15]This promising therapy contributed to more attention to fecal microbiota transplantation (FMT) because they both belong to bacteria-driven therapy.
FMT, which traced back to the 4 th century in China, [16] was first reported formally by Eiseman et al. [17] in 1958 for four patients with pseudomembranous colitis.The fecal retention enemas were successful in all patients, which did not respond to antibiotics.During the past decades, FMT has been applied for refractory Clostridium difficile infection (CDI) on the basic of the idea that the normal microbiota community can be rebuilt by importing the colonic microbiota of the healthy person.Among numerous trials, FMT was reported to be more effective than vancomycin for recurrent CDI patients and its mean successful rate was 87% to 90% for the >500 cases reported.[18][19][20][21] IBD patients are at a higher risk for the development of CDI-associated diseases; therefore, FMT has been regarded as a potential treatment for IBD.Several retrospective trials of FMT in IBD showed attractive results, especially for patients with UC.
The majority of trials about FMT for UC or IBD are one-armed cohort studies or case series.The first two RCTs evaluating the efficacy and safety of FMT were recently published on the Gastroenterology in 2015.[22,23] In the most recent systematic review, Colman et al. [24] found a clinical remission rate of 36.2% in IBD patients for FMT only in cohort studies.Subgroup analysis demonstrated a pooled estimate of clinical remission of 22% for UC.Additionally, several relevant studies were reported after the last systematic review in 2014.As a form of

Data Collection
Data was extracted from eligible articles by two authors independently.The following information was extracted in all included articles: (1) characteristics of participants (including total number, age, sex, diagnosis, duration and severity of UC); (2) type of intervention (including number of exposed and unexposed groups, fecal processing, frequency, route of administration, donor relationship and length of follow-up); (3) clinical outcomes (including clinical remission, clinical response, adverse events, mucosal healing and quality of life assessment); (4) alteration of gastrointestinal microbiota in participants (including similarity, diversity and richness, and composition to the donor).

Methodology quality appraisal
Quality assessment of each RCT and cohort study was carried out by two authors independently and disagreements were resolved by discussion.The methodological quality of two RCTs was assessed using the Cochrane risk of bias tool.[27] For the one-arm cohort studies, quality was assessed by the adjusted Newcastle-Ottawa Scale on the following criteria: representativeness of the UC cohort, ascertainment of FMT, demonstration that outcome of interest was not present at start of study, assessment of outcome, the length (at least 3months) and adequacy of follow-up.[28,29] It should be noted that we considered "demonstration that outcome of interest was not present at start of study" as "evidence of no prior FMT exposure".

Statistical methods
The meta-analysis of the two included RCTs cannot be performed due to their different control groups.Therefore, for RCTs as well as case studies, descriptive summaries of efficacy and safety of FMT were reported.To provide more information for further studies and make comparison with results of RCTs, meta-analysis of cohorts based on random effects model was conducted to evaluate clinical remission rate and clinical response rate with their corresponding 95% confidence interval (CI).The random effects model was conducted using Der Simonian and Laird method.[30] The presence of heterogeneity was assessed using Q statistic (Chi-square test) and the I 2 statistic was used to assess the degree of heterogeneity between the trials.Funnel plot was employed to measure the potential publication bias.Sensitivity analyses were undertaken in clinical remission and response.Subgroup analysis was stratified by donor type, the route of delivery, and number of treatment.All meta-analyses were performed using Meta-Analyst software (version Beta 3.13; Tufts Medical Center, Boston, MA) to pool effect sizes of the above two main outcomes.[31]

Methodological quality of included studies
The methodological quality of two RCTs was showed in S2 Table .The RCT conducted by Rossen et al. was rated as low risk of bias on five out of seven items, and the study of Moayyedi et al. on three items.The total scores of each cohort study ranged from 3 to 5 points with a mean score as 4.3.(S2 Table ), indicating the quality of single-arm studies was medium to high.All the cohort studies showed ascertainment of exposure and assessment of outcome. 1 study did not have adequacy of follow-up and 5 studies did not report enough length of follow-up as long as three months.Seven of 15 studies were considered to have representativeness of the exposed cohort.Only 3 studies demonstrated that outcome of interest was not present at start of study.

Patient demographics
Among the 25 studies, of the 234 patients treated with FMT, 3 patients were excluded due to enema intolerance (n = 2 patients) or micro-perforation (n = 1 patient).[36,40,44] The age range of the remaining 231 patients varied widely from 18 months to 66 years.As for the severity, 98(42.4%)patients were described as "mild or mild-moderate disease", 47(20.3%)as "moderate-severe or severe disease".Other descriptions of the disease severity included 149 (64.5%) "active disease", 19(8.2%)"therapy dependent" and 26(11.3%)"therapy refractory".And the duration of UC extended from 1 year to 40 years.Clinical follow-up time of participants ranged from 3 weeks to 13 years.

Patient preparation
Patient preparations were reported in 14 of 25 studies (n = 100 patients) and were unclear in the other 11 studies (n = 131 patients).6 studies (n = 35 patients) received antibiotic pretreatment before FMT, such as vancomycin, rifampicin and omeprazole, while 2 studies including 15 patients declared that any antibiotic pretreatment was not allowed.[36,40] As for bowel lavage, polyethylene glycol was used in 52 patients of 5 studies.[23,35,40,44,49] Other than the above two kinds, proton pump inhibition therapy was used to inhibit the secretion of gastric acid in 4 studies (n = 25 patients) as another kind of patient preparations before FMT procedure via gastroscopy or nasojejunal tube.[34,39,45,46]

Fecal microbiota transplantation characteristics
Donor identification was reported in 20 studies (n = 162 patients), including related donor (n = 55 patients), unrelated donor (n = 75 patients) and unclear relationship (n = 32 patients).Related donors are traditionally dominated by patients including family member, partner, spouse and close friend.And health screening for donors was carried out in 18 studies (n = 155 patients).The information of stool processing was available in 17 studies, including the amount of fecal infused into normal saline (9.6-100g/100mL) and the volume of fecal suspension in per treatment (30 to 500mL).
Routes of administration included enema in 5 studies, colonoscopy in 4 studies, gastroscopy in 3 studies, nasogastric tube in 2 study, nasoduodenal tube in 1 study, percutaneous endoscopic cecostomy in 1 study and combination of two routes in 5 studies.The number of total FMT treatments in each patient was categorized into three levels: only 1 treatment (n = 7 studies), more than 1 treatment (n = 15 studies) and unclear times (n = 3 studies).In the RCT of Moayyedi et al., [22] FMT was performed via enema weekly for 6 weeks with significant outcome for participants.In the RCT of Rossen et al., [23] FMT was performed via nasoduodenal tube and there was no significant statistically difference at 0 and 3 weeks.
In the RCT performed by Moayyedi et al., [22] FMT (9/38, 24%) achieved significantly better clinical remission than those receiving water (2/37, 5%) at week 7 (P = 0.03).In RCT conducted by Rossen et al., [23] 30.4% patients (7/23) receiving FMT from healthy donors (FMT-D group) and 20.0% patients (5/25) receiving FMT from their own fecal microbiota (FMT-A group) achieved clinical remission and endoscopic response (P = .51).47.8% patients (11/23) in the FMT-D group and 52.0%patients (13/25) in the FMT-A group had a clinical response.Besides, there was no statistical significance between the two groups on clinical response, clinical remission or endoscopic outcomes.Sensitivity analyses and assessment of bias.In the meta-analysis of clinical remission and clinical response, the result of leave-one-out analysis did not materially change the overall effect, indicating that clinical remission rate and clinical response rate were not sensitive to any individual study included in the analysis.This result showed the robustness of our evidence.The funnel plots showed evidence of asymmetry revealing possible existence of publication bias.(S3 File) Subgroup analysis.Subgroup analyses were conducted for donor type, the route of delivery, and number of treatment.The details of all subgroup analyses were shown in S3 File.Differences in these subgroups were not associated with heterogeneity in the estimated clinical remission rate and clinical response rate.Mucosal healing.Mucosal healing was assessed in terms of endoscopy and histology.The descriptions of mucosal healing were "mucosal healing", "endoscopic remission" (Mayo endoscopic subgroup sore = 0) and "normal", while the description of histology just included not active inflammation.Overall, the mucosal healing was reported in 25 of 81 (30.87%) patients in endoscopy and 30 of 48 (62.50%) patients in histology.In one RCT, 7 patients in remission had no active inflammation and the remaining 2 patients in remission had mild patchy inflammation.[22] Quality of life assessment.Of the 25 studies included, 4 studies including 2 cohort studies and 2 RCTs reported quality of life assessment.[22,23,37,44] In the cohort study conducted by Wei et al., [44] the mean Inflammatory Bowel Disease Questionnaire score (IBDQ) score increased significantly to 177.30±20.88 at 4 weeks after FMT.With regard to Landy et al, [37] there was no improvement in Cleveland Global Quality of Life Score (CGQoL).In the RCT conducted by Moayyedi et al., [22] the IBDQ and EuroQol score (EQ-5D) were not statistically significant between FMT group and placebo group at 6 weeks.In the RCT performed by Rossen et al., [23] the total IBDQ score improved shortly in responders after treatments with statistical significance but no change was found in nonresponders.

Safety and adverse events
Adverse events were monitored and reported in 19 of 25 studies.Among 19 studies, no severe adverse event was noted in 3 studies after FMT without more details.[33,38,53] On the whole, FMT was of safety and tolerance.Fever occurred in 18 patients from 8 studies after FMT.Fever severity was mild to moderate or self-resolving in most patients, while 2 subjects from 2 studies required antibiotics or antivirus to deal with the fever caused by infection.[42,52] Patients in 2 studies suffered from nasal stuffiness and sore throat probably due to nasogastric tube or nasojejunal tube placement.[34,43] Additionally, C difficile diarrhea was reported in 2 patients from the cohort study conducted by Suskind et al. [43] The majority of adverse events were self-resolving including bloating, abdominal pain, cramping, blood in stool, diarrhea and fatigue.
In the RCT conducted by Moayyedi et al., [22] 3 patients treated with FMT developed adverse events including colonic patchy inflammation (n = 2 patients), rectal abscess (n = 2 patients) and worsening abdominal discomfort (n = 1 patient).In the RCT performed by Rossen et al. with 78.3% adverse events related to FMT, most events were self-resolving within 2 days including fever.[23] Additionally, 2 patients in the intervention group developed serious adverse events including severe small bowel Crohn's disease and cervix carcinoma, but these were not related to the FMT.[23]

Microbiota analysis
Effects on gastrointestinal microbiota in UC after FMT were reported in 7 studies including 2 RCTs and 5 cohort studies.[22,23,34,35,40,41,45] Similarity to donors was reported in 16 of total 21 patients in 4 cohort studies in the condition of positive response to FMT, except one trial conducted by Kump et al. showed no clinical improvement in 3 of 7 patients.[34,35,40,45] Moayyedi et al. demonstrated that patients in the intervention group received higher similarity to their related donors than the control group.[22] Data from Rossen et al. indicated that responders in FMT-D group had a significant higher similarity to their donors than nonresponders.[23] Among the 16 patients, 10 patients were reported to experience a long-term resemblance more than 90 days; 5 patients experienced a rapid slight or moderate increase with subsequent decline, even back to the baseline at 2-3 months.
5 studies reported the change of microbiota diversity and richness, using the Shannon diversity index or the number of different operational taxonomic units (OTUs).[23,35,40,41,45] 4 trials, which had specific data for every subject respectively, demonstrated the increase diversity and richness in 17 of total 36 patients after FMT from healthy donors.[23,35,41,45] Studies from Damman et al. showed that there was no significant difference in diversity among subjects after transplantation compared to the baseline.[40] In addition, Kump et al. found that the change was temporal, meaning that the diversity as well as richness peaked at day 7 and thereafter back to baseline.[35] The alteration of microbiota composition after positive FMT treatment has been the central point in recent trials and 6 studies we included focused on it.[23,34,35,40,41,45] The magnitude and persistence of the alterations in baterial composition were highly variable between patients.On the phylum level, the increase of Bacteroidetes was observed in 3 trials, [35,40,45] while the decrease of Bacteroidetes in the responders in FMT-D group in 1 RCT.[23] Evidence also showed that the increase of Actinobacteria, Firmicutes and Clostridium clusters IV, XIVa, XVIII but the decrease of Proteobacteria in some subjects.[23,35,40,45] On the family level, the increase of Lachnospiraceae was observed in 2 trials and the increase of Bacteroidaceae was reported in Kump et al. [34,35,41] The reduction of Enterobacteriaceae was demonstrated in 2 trials.[34,35] On the genus level, Kellermayer et al. showed the increase of Coprococcus.[41] Additionally, Angelberger et al. and Kellermayer et al. found that, at the family level, the abundance of Enterobacteriaceae and Lachnospiraceae were associated with clinical outcomes or UC disease activity.[34,41] In the study of Kump et al. the alterations of bacterial composition had no association with clinical performance, [36] while Cui et al showed that the degree of microbiota reconstruction was associated with the clinical response in the patients with steroid-dependent UC. [45]

Discussion
This systematic review involved 231 patients from 2 RCTs, 15 cohorts and 8 case studies.For clinical remission and response in 2 RCTs, Moayyedi and his colleagues reported a statistically significant effect of 24% and 39% respectively, comparing with the study of Rossen et al., which reported effects of 30.4% and 47.8% respectively with no statistically significant.In comparison with the results of RCTs, meta-analysis based on 15 cohorts with poor quality showed higher clinical remission and response rate of 40.5% and 66.1% respectively.The results from RCTs and cohort studies showed similarly high clinical response and clinical remission in FMT for the treatment of UC, showing that FMT is a promising therapy to some extent.According to microbiota analysis of 2 RCTs and 5 cohorts, FMT generally resulted in a manipulation of microbiota towards donor microbiota, especially in the patients with favorable outcomes.However, these results needed further certification with more well-designed RCTs.
As for heterogeneity in meta-analysis, the I 2 statistic revealed that heterogeneity was moderate and sensitivity analysis showed the robustness of results.But the subgroup analyses did not find the specific causes of heterogeneity.In short, the results of meta-analysis were trusted and believed to some degree.And the important clinical heterogeneity, including age of patients, the choice of donor, and the route and number of treatments administered, were well stressed and discussed as follows.

Children and adolescents
Children and adolescents have vulnerable nature and their gut microbiota might not be as stable as adults.These characteristics might be beneficial to implantation and manipulation of gut microbiota in younger people.But children and adolescents may have more risks than adults due to the unknown causal associations between gut microbiota and a number of diseases.The unknown long-term effects on children and adolescents also need to be taken into account.[55]

Donor selection and screening
Donor selection is one of the crucial unresolved questions in FMT.Related donors share common genetic and/or environmental factors with recipients, so they could have greatest common microbial species with the recipients.This similarity minimized the risk of transmission of infectious diseases and led to more tolerance of FMT.However, there is a possibility that the similar genetics and environments lead to altered microbiota of the related donor, though the donor does not suffer from UC.On this condition, the altered microbiota of related donors may result in re-development of UC in patients; therefore the unrelated donors may be preferable in UC.In addition, unrelated donors reduce screening cost.Therefore unrelated donors rather than related donors facilitate and standardize the process of FMT if industrialization come true one day.[55] Due to the differences between CDI and UC, the screening methods for selection of donors in CDI are not adequate in UC and more aspects should be taken into consideration in UC.Trials with high quality investigating the effect of donor types on outcomes are surely needed.

The route of delivery
Delivery method could be a key factor affecting the efficacy of FMT.A number of delivery routes have been used in FMT for UC: gastroscopy, nasogastric / nasojejunal tube, colonoscopy and retention enemas.Previous studies and reviewers have suggested a slight superiority of the colonoscopy in CDI patients but without sufficient evidence.[19] Although some hypotheses said the gastric acid of upper delivery may denature microbiota, such as Bacteroidetes and Firmicutes, [56] the exact association between them has not been verified 7 cohort studies and 1 RCT involved reported the upper gastrointestinal delivery, including gastroscopy in 3 studies, nasogastric tube in 2 studies, the combination of nasojejunal tube and enema in 1 study, the combination of gastroscopy and colonoscopy in 1 study and nasojejunal tube in the RCT.Among them, 4 of 7 cohort studies reported the use of proton pump inhibitor to inhibit the secretion of gastric acid before and during the FMT procedure, and the use of nasojejunal tube or gastroscopy rather than nasogastric tube can deliver microbiota directly into the mid-guts.Therefore, we consider the influence of gastric acid to be limited.The concerns for the use of nasogastric / nasojejunal tube were: small volumes, vomiting, aspiration, injury of upper gastrointestinal tract and the necessity to be verified by x-ray before transplant.Colonoscopy could visualize the relevant pathology and deliver larger volume suspensions directly into the site of inflammation in the colon, while endoscopy procedure increases the risk of perforation.In addition, the retention of the infused material via colonoscopy was better than via enema.Enema is accessible, safe and inexpensive, but intolerance was reported in some patients.[21] Additional well-designed trails are needed to identify the best route of delivery.

Number of treatment
Number of treatment to obtain beneficial outcome in UC also constitutes additional concerns.However, most of cured patients with CDI only received single administration of FMT.[21] UC is a chronic, relapsing and remitting disease; therefore unlike CDI, gut microbiome in UC was stable and resilient to change on condition that the manipulation of gut microbiome was in short-term.This phenomenon suggested that several treatments or more drastic microflora manipulation is needed to permanently reconstitute and remain a balance bacterial community in UC patients.[4] More high quality trials are required to determine the number of treatments needed for different UC patients.

Microbiota change
FMT generally produced a major alteration in the patients' microbiota towards donor microbiota, especially in the patients with favorable outcomes.This suggested that donor selection required more attention due to their potential influence on outcomes.The similarity maintained over 2-3 months over half of the patients.However, in some subjects, the increased similarity was transient and not stable for a long time.In addition, the patients who shared the increased similarity had different clinical outcomes, suggesting that the only presence of healthy microbiota signature was not sufficient to lead to positive effects of FMT.Thus, the exact alterations of composition, diversity and richness may produce more useful diagnostic indications of clinical outcomes.
Successful FMT was associated with an increase in diversity and richness generally, and reversals of some of the reported dysbiotic changes in UC.However, the most recent RCT showed an opposite result that the abundance of Bacteroidetes in FMT-D responders decreased, which used to be found increasing after FMT, suggesting that UC patients could get clinical remission although the gut microbiota changed in an opposite direction.As Cui et al reported, one patient who did not benefit from the first FMT due to perianal abscess experienced a surgery presented at 7 days after the first FMT, and antibiotics were given before and after surgery.[45] This patient was the only one who did not experienced the increased similarity to donor and increased diversity in their fecal microbiota analyses, indicating that the diversity and composition of fecal microbiota were affected by antibiotic greatly.Therefore, the assumption could be made that some changes of microbiota in UC are induced by inflammation or previous treatment and not the causes of UC universally.Another possibility is that other significant factors of dysbiosis outweighed the composition of bacterial, but no studies have discovered them yet.
Although dysbiosis and its corresponding changes after FMT were similar, the FMT for UC was not as successful as CDI in which FMT led to cure rates of more than 90%.[20] Two different classifications of the gut microbiota, luminal microbiota and mucosal microbiota, [57] may account for this phenomenon.The infection of Clostridium difficile may result in the change of luminal microbiota which could be restored easier by FMT.In contrast, the disruption of mucosa microbiota rather than luminal microbiota is observed in UC patients.Another reason could be that the causative role of dysbiosis in UC and CDI maybe totally different.

Safety
FMT is generally of safety and tolerance with few serious adverse events.As many patients need to receive more than one FMT therapy, more procedural complications will probably be reported due to the invasion of procedure.Despite rigorous donor selection and screening for infectious agents, known and unknown risks still remain a major problem for widely application of FMT in UC.One of unknown risks is the long-term influence of microbiome on host after FMT.Moreover, short follow-up time increases the risk of underreporting latent adverse events.Therefore, besides the efficacy of FMT, the safety of FMT also needs to be determined especially in long-term follow-up.

Limitations
The evidence for FMT in this systematic review was mostly depended on two RCTs and 15 cohort studies.The numbers of participants in each study were too small to avoid publication bias and patient selection bias.As an important source of grey literature, conference abstracts are susceptible to report more negative results honestly.So 9 studies we included were available in abstract/letter form.As for the synthesis of cohorts, low or moderate heterogeneity, the results of sensitivity analysis and publish bias all reflected the stability of synthesis.So the synthesis of cohorts was reliable and had reference value for further studies to some extent.There was significant heterogeneity in several aspects.As a novel therapy, FMT has no standard practical guideline for UC, thus we were supposed to take the variations of intervention into account, including the variations in the patient and donor preparation, dosage and frequency, and route of administration.Due to the lack of standardized definitions of clinical outcomes, several indices of disease activity were used including Mayo score, Pediatric Ulcerative Colitis Activity Index (PUCAI), etc., which definitely led to heterogeneity.Mucosal healing offers more objective and accurate evidence to judge the disease activity and therapeutic effects, but only small patients were reported the outcome of mucosal healing.

Conclusion
Overall, FMT is a promising therapy for UC patients.It could be safe and effective for partial patients.However, the present evidence of FMT for UC is limited and many serious concerns and questions are required to be resolved before it is widely applied.Therefore, more welldesigned RCTs and long-term follow-up are necessary to confirm the effects of FMT.Doctors should think over its advantages and disadvantages carefully before recommending and applying it to UC patients.

Table 1 .
Characteristic of cohort studies.
t Characteristics of patients Study (Year, Reference) Country Type Number (M/F) Age Severity (duration)

Table 2 .
Characteristic of cohort studies.(Continued) Clinical remission and clinical response.The results of the synthesis of outcomes from cohorts were shown in Fig 2 and in Fig 3. Data on clinical remission and clinical response were extracted from 13 studies assessing 141 patients and 11 studies assessing 132 patients respectively.The pooled estimate of clinical remission rate was 40.5% (95% CI: 24.7%-58.7%)with low heterogeneity (Cochrane's Q, P = 0.005; I 2 = 36.5%),whereas the pooled estimate of clinical

Table 4 .
Characteristics of randomized controlled trails.