This study included MS patients who completed 1–4 courses of WMT treatment or ordinary treatment in the First Affiliated Hospital of Guangdong Pharmaceutical University from January 2017 to December 2023 during the first WMT. Inclusion criteria were patients aged 18–80 with informed consent and voluntary acceptance of WMT. Exclusion criteria were pregnant women and patients who changed their hypoglycemic, antihypertensive, or lipid-lowering medications during the observation period. The study was approved by the Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University, adhering to the Helsinki Declaration (No.2021-13). Among the enrolled participants, 210 met the requirements of this study and provided written informed consent to participate.

The diagnostic criteria for MS in this study refer to the Chinese Guidelines for the Prevention and Treatment of Type 2 Diabetes (2020 Edition) (17) and the diagnostic criteria for metabolic syndrome of the Diabetes Society of the Chinese Medical Association (18). Metabolic syndrome was diagnosed with three or more of the following: (1) BMI ≥ 25 kg/m². (2) Hyperglycemia: fasting blood glucose ≥6.1 mmol/L or blood glucose ≥7.8 mmol/L 2 h after glucose load and (or) those who have been diagnosed with diabetes and received treatment. (3) Hypertension: Blood pressure ≥ 130/85 mmHg (1 mmHg = 0.133 kPa) and (or) diagnosed with hypertension and receiving treatment. (4) Fasting triglycerides (TG) ≥ 1.70 mmol/L. (5) Fasting HDL-c < 1.04 mmol/L. The 210 patients included in this study were divided into WMT group and DT group based on whether they received WMT treatment. There were a total of 68 patients in the WMT group, and 142 patients in the DT group who were hospitalized at the same time but did not receive WMT treatment or take medication such as microbial preparations and probiotics.

In accordance with the Chinese Guidelines for the Prevention of Cardiovascular Diseases (2017 Edition) (19), ASCVD risk stratification was carried out according to the baseline and blood lipid status, which were classified extremely high risk group, high risk group, medium risk group, and low risk group. Subsequently, patients were divided into the single-course WMT group, double-course WMT group, and multi-course WMT group (three or more courses) for WMT. After receiving 1–4 rounds of WMT or drug treatment and completing follow-up, statistical analysis and evaluation of height, weight, blood glucose, blood lipids, and blood pressure results were conducted for all patients.

The WMT program adhered to the Nanjing Consensus on the Methodology of Washed Microbial Transplantation (20). All healthy donors aged between 18 and 25 must undergo rigorous counseling, psychological and physical examinations, biochemical tests, and screening for infectious diseases. The specific fecal preparation procedures can refer to the Nanjing Consensus (20) and our previous research. Relevant indicators were collected before completing each course of treatment, including baseline values, values after the first course (single-course), values after the second course (double-course), and values after the third or more courses (multi-course).

The study collected data from MS patients before (baseline) and after treatment, and compared the partial clinical efficacy between the WMT group and the DT group using the difference before and after treatment as the improvement value. Mainly including BMI indicators: weight (kg), height (m), weight/height² = BMI (kg/m²). Blood glucose indicators: fasting blood glucose (FBG, mmol/L), fasting insulin (FI, mU/mL), and the insulin resistance value (HOMA-IR, insulin resistance value = fasting blood glucose ^* fasting insulin/22.5). Blood lipid indexes: total cholesterol (TC, mmol/L), triglyceride (TG, mmol/L), low density lipoprotein cholesterol (LDL-c, mmol/L), high density lipoprotein cholesterol (HDL-c, mmol/L). Blood pressure indicators: systolic blood pressure (SBP, mmHg) at admission, and diastolic blood pressure (DBP, mmHg) at admission. And various influencing factors: age, gender, smoking and alcohol history, medication and infection status, chronic disease, pathways of WMT, selection of donor type and number of WMT courses. Smoking index = number of cigarettes smoked/day × Years of smoking (smoking index < 200 represents mild smoking, while smoking index 200 ≥ moderate to severe smoking). The middle digestive tract is defined as microbiota transplantation through a jejunal tube, while the lower digestive tract is microbiota transplantation through a colon tube. Adverse events (AEs): diarrhea, fever, fatigue, nausea, abdominal pain, etc.

The fecal samples from 5 MS patients who excluded independent influencing factors and 5 donors were collected before and after WMT for sequencing. After collection, all samples were stored at −80°C until DNA was extracted. DNA quality and concentration were checked by NanoDrop™ 2000 (Thermo Fisher Scientific, Wilmington, DE, USA) (21). Primers 338F (50-ACTCCTACGGGAGGCAGCAG-30) and 806R (50-GGACTACHVGGGTWTCTAAT-30) were used to amplify bacterial 16S rRNA gene fragments (V3-V4) from extracted DNA. The PCR products were subjected to agarose gel electrophoresis to determine the size of the amplicon. The constructed library was quantified by Qubit and Q-PCR, and the NovaSeq6000 (Illumina, San Diego, CA, USA) sequencing platform was used for onboard sequencing until the library was qualified (22).

From all the sample data split from plane data and amputation of barcode and primer sequences after the use of FLASH software (version 1.2.11, http://ccb.jhu.edu/software/FLASH/) to splice the sample reads, raw tags were obtained (23). Then, fastp software version 0.23.1 (Shenzhen Hypros, Shenzen, China) was used to obtain high-quality clean tags (24). Finally, clean tags were compared with the database to detect and remove chimeras, so as to obtain the effective tags (25). The DADA2 Variants in QIIME2 were used to obtain the final ASV variants and the feature list of the variant. The resulting ASVs were then compared with the database by the classify-sklearn module in QIIME2 software version 2.0 (QIIME 2 development team, https://docs.qiime2.org) to obtain species information for each ASV (26). The representative sequences of ASVs using the classification sklearn (Naive Bayes) algorithm were analyzed, obtaining the relative abundance of ASVs at the genus level.

Statistical analysis was performed using SPSS 22.0 (IBM Corp, Armonk, NY, USA) and Prism 8 (GraphPad, San Diego, CA, USA). The Categorical variables were analyzed by Chi-squared test or Fisher exact test. For comparison of continuous variables between two independent groups, unpaired Student's t-test (Normal distribution) and Mann Whitney test (non-Normal distribution) could be used. The paired data were compared by paired Student's t-test (Normal distribution) and Wilcoxon signed rank test (non-Normal distribution). Logistic regression analysis is used to summarize the main factors that affect therapeutic efficacy. Two-tailed p-values < 0.05 were considered statistically significant.

A total of 210 patients met the inclusion criteria for WMT treatment or ordinary treatment at the First Affiliated Hospital of Guangdong Pharmaceutical University from January 2017 to December 2023, including 126 males (60.00%) and 84 females (40.00%), with an average age of 60.12 ± 11.66 years. Due to different patient compliance, each WMT treatment might not be completed as scheduled. The average number of microbiota transplants is 2.68 ± 1.22, which is close to 3 times. This study calculated the time interval of selected patients, expressed in days as the median (25%−75%). The blood test results of the patient before the first treatment were the baseline values, with a treatment interval of 36 days (32–46 days) in single-course WMT, 95 days (76.75–104.25 days) in double-course WMT, 192 days (152.75–212.50 days) in multi-course WMT, and 42 days (36.42–50.48 days) in the drug treatment (DT) group.

The comparison of demography and clinical characteristics between the WMT and the DT group is shown in Table 1. Due to different compliance, not all patients had complete data, so the number of patients in each group was different for each indicator. There was no significant difference in age, gender, medical history, and laboratory indicators between the WMT group and DT group in MS patients, which reduces the interfering factors for our study of efficacy differences between treatment groups. Compared to MS patients, the various indicators of the donor, including BMI, blood glucose, blood lipid, and blood pressure, were healthier.

Figure 1 and Supplementary Table S1 show the impact of WMT on BMI, blood glucose, blood lipids and blood pressure in MS patients in the WMT group. The results showed that single-course treatment had a significant reducing effect on BMI (from 26.61 ± 4.67 to 26.05 ± 4.61, p = 0.007), FBG (from 6.36 ± 2.37 to 5.83 ± 1.76, p = 0.038), TG (from 3.20 ± 4.04 to 2.34 ± 1.87, p = 0.013), HDL-c (from 0.99 ± 0.25 to 1.10 ± 0.45, p = 0.029), SBP (from 133.12 ± 12.01 to 125.78 ± 12.56, p < 0.001) and DBP (from 82.78 ± 9.69 to 77.81 ± 9.40, p = 0.002). At the same time, the double-course treatment also showed a significant reducing effect on BMI (from 26.44 ± 4.30 to 25.74 ± 4.28, p = 0.033) and SBP (from 132.49 ± 11.82 to 125.64 ± 11.64, p = 0.006), DBP (from 82.56 ± 9.65 to 79.18 ± 8.23, p = 0.037), while the multi-course treatment only had a significant reducing effect on SBP (from 129.82 ± 12.25 to 118.68 ± 10.51, p = 0.010) and DBP (from 82.05 ± 9.96 to 75.18 ± 10.05, p = 0.024).

Figure 2 and Supplementary Table S2 show the comparison of improvement values for various indicators under different conditions during the single-course of treatment in the WMT group. Regarding blood glucose indicators and BMI in Figure 2A, the WMT plus drug (WMT-D) group showed superior effects to the WMT without drug (WMT-ND) group in reducing FBG, while the WMT-ND group was superior in reducing BMI, HbA1c, FI, and HOMA-IR. For blood lipid indicators in Figure 2B, the WMT-D group had a better improvement effect than the WMT-ND group on TC, TG, LDL-c, and HDL-c. However, there was not much difference between the two groups in terms of blood pressure reduction in Figure 2C. In our study, even though some indicators of the WMT-D group were superior to those of the WMT-ND group, there was no statistically significant difference between the two groups as a whole, indicating that WMT can improve indicators such as blood glucose, blood lipids and blood pressure in MS patients.

As shown in Figure 3 and Supplementary Table S3, compared to the DT group, the WMT group had greater advantages in reducing blood glucose, blood pressure, lipid, and weight, such as FBG improvement value (0.53 ± 1.99 vs. −0.41 ± 2.57, p = 0.012), TG improvement value (0.96 ± 2.97 vs. 0.02 ± 1.67, p = 0.006), SBP improvement value (7.34 ± 15.86 vs. 1.97 ± 18.00, p = 0.037), DBP improvement value (4.97 ± 12.52 vs. 0.99 ± 13.74, p = 0.045), and BMI improvement value (0.56 ± 1.64 vs. 0.12 ± 1.33, p = 0.047).

As shown in Table 2, WMT had the effect of treating MS patients as non-MS patients (p = 0.011), with 29 (42.65%) MS patients improving to non-MS patients in the WMT group. Therefore, WMT treatment was superior to drug therapy in treating MS patients effectively.

In addition, according to ASCVD risk stratification (19), MS patients were divided into extremely high-risk group, high-risk group, medium-risk group and low-risk group. Acute coronary syndrome, stable coronary heart diseases, ischemic cardiomyopathy, peripheral atherosclerosis and ischemic stroke were included in the extremely high-risk group. This group of patients was not reassigned after WMT treatment and is not listed in Table 2.

In the high-risk group, 14 (45.16%) patients experienced a grade decline, which reduced high-risk ASCVD stratification from 45.59% to 25.00% in the WMT group (p = 0.011). In the medium-risk group, 14 (48.28%) patients experienced a grade decline, which reduced medium-risk ASCVD stratification from 42.65% to 22.06% (p = 0.038). In the low-risk group, this change was not statistically significant, indicating that WMT treatment did not increase the risk of ASCVD. In conclusion, WMT has a significant ASCVD downgrade effect on high-risk and medium-risk MS patients.

Among the completed WMT treatments, the total adverse reaction rate was 2.96%. Only one patient experienced fever with a maximum body temperature of 37.6°C, along with diarrhea after treatment completion. All symptoms were alleviated after < 3 days of symptomatic treatment.

This study included 135 MS patients in the WMT group, including 68 patients who completed a single course of WMT, 45 patients who completed a dual course of WMT, and 22 patients who completed multiple courses of WMT. Among the 135 MS patients, 55 patients recovered to non-MS patients after treatment, with a success rate of 40.74%.

It showed the influencing factors that may affect the efficacy of WMT in treating MS patients in Tables 3, 4, including antibiotics and immunosuppressants use, infection with pathogenic bacteria or viruses during observation, hypertension, diabetes, hyperlipemia, obesity, other chronic diseases, the pathways of WMT, the selection of donor type and number of WMT courses. As shown in Table 4, in both the MS group and non-MS group formed after WMT treatment, the MS group had a significantly higher proportion of age < 60 years old, smoking index ≥200, using of antibiotics or immunosuppressants during observation, infection with pathogenic bacteria or viruses during observation, having hypertension, diabetes, obesity, single donor selection and single-course WMT treatment compared to the non-MS group (p < 0.050), suggesting the main factors affecting the efficacy of WMT in treating MS patients. However, there was not currently statistical significance in gender, alcohol consumption, having hyperlipidemia, suffering from other chronic diseases and pathways of WMT.

Based on the logistic regression analysis in Table 5, not only the age < 60 years old (OR = 3.525, p = 0.023), smoking index ≥200 (OR = 11.705, p = 0.034), infection of pathogenic bacteria or viruses during observation (OR = 8.783, p = 0.037), single donor selection (OR = 3.003, p = 0.026) and single-course WMT (OR = 2.721, p = 0.041), but also suffering from hypertension (OR = 3.375, p = 0.026), diabetes (OR = 3.928, p = 0.008), and obesity (OR = 2.945, p = 0.038) were independent influencing factors. As shown in Figure 4A, the area under the AUC curve of some independent influencing factors was >0.700, including smoking index (X4), area under AUC curve = 0.706 (0.621–0.792), infection with pathogenic bacteria or viruses during observation (X7), area under AUC curve = 0.719 (0.634–0.803), having obesity (X11), area under AUC curve = 0.712 (0.621–0.803), selection of donor type (X14), and area under AUC curve = 0.712 (0.624–0.796), indicating that the model had a certain degree of predictability. However, some independent influencing factors had an area under the AUC curve < 0.700 in Figure 4B.

We analyzed the gut microbiota composition of MS patients who excluded independent influencing factors before and after WMT and donors. At the phylum level, the gut microbiota mainly included Firmicutes, Fusobacteriota, Bacteroidota, Actinobacteriota, and Proteobacteria. At the phylum level, the relative abundance of Firmicutes, Bacteroidota, and Actinobacteriota increased after WMT. The relative abundance of Fusobacteriota and Proteobacteria decreased (Figure 5A). At the family level, the relative abundance of Bacteroideaceae, Lachnospiraceae, Bifidobacteriaceae, and Ruminococcaceae increased, with a great increase of Bifidobacteriaceae in MS patients after WMT and donors, while the relative abundance of Enterococcaceae and Fusobacteriaceae decreased, with a great decrease of Enterococcaceae in MS patients after WMT and donors (Figure 5B). At the genus level, the relative abundance of Bifidobacterium, Bacteroides, Faecalibacterium, and Prevotella increased after WMT. The relative abundance of Enterococcus, Fusobacterium, Escherichia–Shigella, and Klebsiella reduced (Figure 5C).

The phylogenetic relationships of the top 100 gut microbiota at the genus level were analyzed, with the top seven being Bacteroides, Fusobacterium, Bifidobacterium, Escherichia–Shigella, Faecalibacterium, Enterococcus, and Prevotella (Figure 5D). Among them, not only could WMT increase the relative abundance of beneficial bacteria such as Bifidobacterium and Bacteroidetes, playing the basic functions of beneficial bacteria, but also reduce the relative abundance of pathogenic bacteria such as Fusobacterium and Escherichia-Shigella. WMT increased gut microbiota α diversity, such as Chao1 index (Figure 5E), revealing that WMT could significantly improve the diversity of gut microbiota in MS patients.

LEfSe analysis was performed on MS patients before and after WMT and donors to identify the biomarkers with statistical differences between groups. It was found that Enterococcus was the distinct species before WMT, while UCG-002 was the distinct species after WMT in MS patients, and the distinct species was Lactobacillaceae in donors (Figure 5F). The species with significant differences between the MS patients before and after WMT were identified by Metastat. We found that it could still significantly increase the relative abundance of Paraprevotella, Fournierella, and Senegalimassilia compared to baseline WMT, with a great reduction of the relative abundance of Hungatella and Ruminococcus at the genus level (Figure 5G).

In conclusion, compared to baseline, the relative abundance of Firmicutes, Bacteroidota, and Actinobacteriota increased after WMT, while the relative abundance of Fusobacteriota and Proteobacteria decreased at the phylum level, maintaining the balance of gut microbiota (Figure 5H). In addition, the proportion of Firmicutes/Bacteroidota decreased after WMT and in the donor group. The relative abundance of gut microbiota in MS patients after WMT gradually tended toward healthy donors, suggesting that maintaining the homeostasis of gut microbiota might be a new approach for treating MS patients.