Gut microbiota–derived metabolite trimethylamine-N-oxide and multiple health outcomes: an umbrella review and updated meta-analysis

ABSTRACT Background Trimethylamine-N-oxide (TMAO) is a gut microbiota–derived metabolite produced from dietary nutrients. Many studies have discovered that circulating TMAO concentrations are linked to a wide range of health outcomes. Objectives This study aimed to summarize health outcomes related to circulating TMAO concentrations. Methods We searched the Embase, Medline, Web of Science, and Scopus databases from inception to 15 February, 2022 to identify and update meta-analyses examining the associations between TMAO and multiple health outcomes. For each health outcome, we estimated the summary effect size, 95% prediction CI, between-study heterogeneity, evidence of small-study effects, and evidence of excess-significance bias. These metrics were used to evaluate the evidence credibility of the identified associations. Results This umbrella review identified 24 meta-analyses that investigated the association between circulating TMAO concentrations and health outcomes including all-cause mortality, cardiovascular diseases (CVDs), diabetes mellitus (DM), cancer, and renal function. We updated these meta-analyses by including a total of 82 individual studies on 18 unique health outcomes. Among them, 14 associations were nominally significant. After evidence credibility assessment, we found 6 (33%) associations (i.e., all-cause mortality, CVD mortality, major adverse cardiovascular events, hypertension, DM, and glomerular filtration rate) to present highly suggestive evidence. Conclusions TMAO might be a novel biomarker related to human health conditions including all-cause mortality, hypertension, CVD, DM, cancer, and kidney function. Further studies are needed to investigate whether circulating TMAO concentrations could be an intervention target for chronic disease. This review was registered at www.crd.york.ac.uk/prospero/ as CRD42021284730.

Most evidence on the health effects of plasma TMAO concentrations has been generated by observational studies with conflicting results. In addition, some studies were conducted among patients with specific diseases, which calls into question whether such associations can be generalized to a healthy population. Hence, it is necessary to synthesize the current evidence to provide a comprehensive overview of the claimed associations of TMAO concentrations with health outcomes.
Umbrella review is designed to provide a comprehensive overview of evidence from systematic review with or without meta-analysis (20). Several meta-analyses on the relations between increased TMAO concentrations and risks of obesity (21), stroke (22), diabetes (23), hypertension (24), and all-cause mortality (25) have been conducted. A comprehensive credibility assessment of these associations will help elucidate the role of TMAO in human health. Using a standardized approach, we performed an umbrella review to evaluate the validity and credibility of the evidence from updated meta-analyses of observational studies. In detail, we summarized the range of related health outcomes; presented the magnitude, direction, and significance of the reported associations; assessed the potential biases; and identified the most convincing evidence in relation to the health impact of TMAO concentrations.

Study design
In this umbrella review, all meta-analyses on the associations between plasma TMAO concentrations and health outcomes were identified. Original studies that evaluated the associations between TMAO and health outcomes were also identified to update the identified meta-analyses. The protocol of the present study was registered in PROSPERO (CRD42021284730).

Literature search
Two investigators (DL and YL) independently searched the Embase, Medline, Web of Science, and Scopus databases from inception to 15 February, 2022 using a search strategy to identify meta-analyses of observational studies. The literature search algorithm was as follows: "((((meta-analysis) OR (meta)) OR (systematic overview)) OR (systematic review)) AND ((((trimethylamine oxide) OR (trimethylamine N-oxide)) OR (trimethylammonium oxide)) OR (TMAO))." We also searched for individual observational studies to update the identified meta-analyses and reported the results in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist (26). All identified publications went through a 3-step parallel review of title, abstract, and full text based on predefined inclusion and exclusion criteria, and any discrepancies were resolved by consensus.

Eligibility criteria
Meta-analyses performing quantitative analysis of plasma TMAO concentrations and health outcomes were included in the umbrella review. All relevant population-based observational studies including prospective cohort, nested case-control, case cohort, case-control, or analytical cross-sectional studies were combined in the updated meta-analysis, and we conducted subgroup analysis by study design. Guidelines, narrative reviews, literature reviews, and genetic studies were excluded. We further excluded studies in which TMAO was not the primary exposure. Meta-analyses or original studies that had inadequate data (e.g., lack of information on RRs, ORs, HRs, or 95% CIs) were also excluded.

Data extraction and quality assessment
From each eligible meta-analysis, we extracted information on the lead author's name, study design, publication year, study sample, number of studies included, the reported summary risk estimates [RR, OR, HR, or weighted mean difference (WMD)] with 95% CIs, the number of participants and cases, and the investigated outcomes. For meta-analyses on >1 health outcome, each outcome was recorded separately. Furthermore, we searched for recently published original articles on TMAO and combined them with studies identified from the previous meta-analyses to update the meta-analyses. When updating the meta-analyses, we added the newly identified studies and re-estimated the summary effect estimates using random-effects models. To account for potential confounding and reverse causality, we performed subgroup analyses by confining the meta-analyses to include only cohort studies with adjustment for renal function and diet (if possible). Data extraction at this stage covered information on study design, number of cases, total number of participants, RR estimates, and 95% CIs. Two investigators (DL and YL) extracted data independently using a predesigned data extraction form.
The quality of individual studies was assessed by the Newcastle-Ottawa Scale (NOS) for observational studies (27).

Statistical analysis
For each unique meta-analysis of observational studies, several metrics were estimated, including the summary effect and corresponding 95% CI using the random-effects model; the heterogeneity among studies (Q statistic and I 2 metric); and the 95% prediction interval (95% PI) to predict the range of effect size that would be expected in a new original study after accounting for both the heterogeneity among individual studies and the uncertainty of the summary effect estimated in the random-effects model (28) (the calculation of the 95% PI is based on the predicted distribution derived from a function of the degree of heterogeneity, number of studies included, and within-study SEs) (29,30). Egger's regression test was used to evaluate the small-study effects (31). The excess significance test was conducted to investigate whether the observed number of studies with significant results differed from the expected number of significant studies using the χ 2 test (32)(33)(34). The expected number of significant studies for each meta-analysis was calculated by summing the statistical power estimates for each component study. We estimated the power of each study for an effect equal to the effect of the largest study (the study with the smallest variance), as previously described (35). All statistical analyses were performed using the "metafor" and "forestplot" R packages, R software version 4.0.2 (The R Foundation, Boston, MA).

FIGURE 2
High compared with low TMAO concentrations and associations with multiple health outcomes. Estimates are RRs and meta-analyses are based on random-effect models. An I 2 value ≥50% is considered to indicate substantial heterogeneity. All results are presented as HR with 95% CIs, using the Mantel-Haenszel method with a random-effects model. CRC, colorectal cancer; CVD, cardiovascular disease; DM, diabetes mellitus; GDM, gestational diabetes mellitus; MACE, major adverse cardiovascular events. revealed that participants with high TMAO concentrations were more likely to die from CVDs than those with low TMAO concentrations (HR: 2.02; 95% CI: 1.74, 2.34; P = 6.01 × 10 −21 ) ( Figure 2, Supplemental Figure 9). The association remained significant when the meta-analysis was restricted to cohort studies (HR: 2.00; 95% CI: 1.72, 2.33; P = 3.06 × 10 −19 ) ( Figure 2).

Renal function
The umbrella review identified 1 meta-analysis reporting that circulating TMAO was associated with a decrease of GFR (WMD:

Discussion
Our updated meta-analyses included a total of 82 individual studies and examined the associations of TMAO with 18 unique health outcomes. Among them, 14 outcomes (all-cause mortality, CVD, MACE, stroke, hypertension, CVD mortality, SBP, BMI, CRP, TC, DM, GDM, GFR, CRC) were found to be significantly associated with TMAO concentrations. When we restricted metaanalyses to only include cohort studies, 11 outcomes (all-cause mortality, MACE, hypertension, CVD mortality, SBP, CRP, HDL cholesterol, LDL cholesterol, TC, DM, GFR) were still significantly associated with TMAO concentrations. The doseresponse analyses revealed that circulating TMAO concentrations were positively associated with the risk of hypertension and MACE. After assessment of the evidence credibility, we found highly suggestive associations of TMAO concentrations with 6 health outcomes, including all-cause mortality, CVD mortality, MACE, hypertension, DM, and GFR.
Former published meta-analyses (25,(38)(39)(40)45) demonstrated that high TMAO concentrations were related to an increased risk of all-cause mortality and the updated meta-analysis showed consistent results. When conducting subgroup analysis by disease status, TMAO showed a significant association with all-cause mortality only in patients with CVD. In addition, our study revealed a positive association between TMAO concentrations and CVD risk. Given that the majority of evidence was from case-control studies, we cannot rule out reverse causality. It has been reported that TMAO may affect platelet reactivity, lipid metabolism, and endothelial dysfunction, which could result in the acceleration of atherosclerotic plaque formation (123). Because atherosclerosis is one of the major causes of CVD, high concentrations of TMAO could be related to high incidence of CVD, due to TMAO's contribution in the development of atherosclerosis. However, no causal association between TMAO and CVD was identified in a recent bidirectional Mendelian randomization study (124). Taken together, current evidence suggests that TMAO might be a novel biomarker indicating the risk of CVD.
Our umbrella review reported a highly suggestive association between TMAO concentrations and hypertension, and both the former published study (42) and the updated meta-analysis revealed that this association displayed a dose-response relation. Previous studies have found that hypertensive patients had more gut microbial enzymes involved in TMA production than those without hypertension (125). Animal studies have also found that elevated plasma concentrations of TMAO can prolong the duration of elevated blood pressure (126)(127)(128). TMAO could also promote Ang II-induced vasoconstriction via the PERK/ROS/CaMKII/PLCβ3 (protein kinase r-like endoplasmic reticulum kinase (PERK), reactive oxygen species (ROS), calmodulin-dependent protein kinase (CaMK), phospholipase c β3 (PLCβ3) axis, thereby facilitating Ang II-induced hypertension (126).
Both the former published study (23) and the updated meta-analysis revealed a positive association between TMAO concentrations and risk of DM. Previous studies reported supportive evidence on associations between TMAO and diabetesrelated traits, including insulin resistance, impaired glucose metabolism, and metabolic syndrome (17,129,130). Animal studies also found that TMAO may exacerbate impaired glucose GFR, glomerular filtration rate; MACE, major adverse cardiovascular events; NA, not available; NP, not pertinent (because the number of expected significant studies was larger than the number of observed significant studies); NS, not significant; PI, prediction interval; SBP, systolic blood pressure; TC, total cholesterol; WMD, weighted mean difference. 2 Egger's regression test was used to evaluate the small-study effects. 3 Interstudy heterogeneity was tested using the Cochran Q statistic (t 2 ) at a significance level of P < 0.10 and quantified by the I 2 statistic. An I 2 value ≥50% is considered to indicate substantial heterogeneity. All results are presented as RR/OR/HR/WMD with 95% CIs, using the Mantel-Haenszel method with a random-effects model. 4 Excess significance test was conducted to investigate whether the observed number of studies with significant results differed from the expected number of significant studies using the tolerance and hyperglycemia by blocking the hepatic insulin signaling pathway and causing inflammation in adipose tissue (131), whereas a decrease of plasma TMAO could reduce plasma glucose and insulin resistance in mice by inhibiting the main TMAO-generating enzyme FMO3 (flavin-containing monooxygenase-3) (132). Furthermore, we found evidence from 2 studies (133,134) reporting a positive association between TMAO concentrations and GDM, but the involvement of TMAO in any causal or compensatory pathway has not been proven. Therefore, further studies should be conducted to understand the mechanism of TMAO influencing GDM. The former published study (47) and updated meta-analysis showed that an increase of TMAO concentrations was associated with lower GFR. Previous studies showed that chronic dietary exposures that increased TMAO concentrations appeared to directly contribute to progressive renal fibrosis and dysfunction (10,135), which is one of the main end-stage renal diseases and a common outcome of almost all progressive chronic kidney diseases (CKDs) (136). Animal studies demonstrated that inhibition of TMAO production attenuated CKD development and cardiac hypertrophy in mice, suggesting that TMAO concentrations may play an important role in CKD development and TMAO reduction may be a novel strategy in treating CKD and its CVD complications (137). However, in this umbrella review, we only assessed the observational association of TMAO with GFR as an intermediate surrogate trait of CKD. Future studies focusing on CKD as an endpoint need to be performed to examine the association with TMAO concentrations.
It is widely known that TMAO is produced from the fermentation of dietary nutrients (choline, betaine, and carnitine) by the gut microbiota. Considering high concentrations of TMAO being associated with gut microbiota balance and several diseases, nonpharmacologic strategies, including foods and dietary supplements rich in bioactive compounds or nutrients, have the potential to modulate the gut microbiota to reduce TMAO concentrations, and therefore decrease the risk of several diseases. There is evidence showing that TMAO concentrations can be reduced by some bioactive compounds, such as resveratrol, allicin, capsanthin, and dietary components present in the apple, oolong tea, natural wheat bran, and low-fat diet, whereas strategies such as the paleolithic diet, high-fat diet, and highprotein diet promote increased TMAO concentrations (138). Because TMAO is a metabolite produced by the gut microbiota, targeting the gut microbiota and the metabolic pathway of TMAO might provide new strategies for the prevention of these related diseases (139). Further studies should be conducted to evaluate these dietary components' effectiveness, dose, and intervention time on TMAO concentrations and whether their health effects could be mediated through regulating TMAO concentrations.

Study strengths and limitations
Although previous meta-analyses of TMAO and the risk of disease outcomes have been conducted, our study is the first to summarize and present the evidence for the associations between TMAO concentrations and a wide spectrum of health outcomes systematically and thoroughly by incorporating information from meta-analyses of observational studies. In addition, our dose-response analyses revealed that there were no critical concentrations of TMAO in terms of varying degrees of risk in patients with all-cause mortality, diabetes, hypertension, and MACE disease. Subgroup analyses further evaluated the associations by only including prospective studies or studies adjusted for certain confounding factors. Although previous studies reported multiple health outcomes associated with TMAO concentrations, our study evaluated the reliability of these associations based on established credibility criteria.
Our study also has limitations. First, because all the included studies were observational, causal associations between circulating TMAO and related outcomes cannot be inferred. Second, sexand ethnicity-specific findings could not be obtained owing to limited data. Diet-specific findings could not be obtained owing to limited data, and therefore we were not able to perform subgroup analyses to further explore the associations by minimizing the potential confounding of dietary patterns. Third, there was high heterogeneity in the current meta-analyses, possible reasons being the inclusion of different populations and different study designs. Further, our evidence grading was not sensitive to the use of 95% PIs or excess significance bias because the evidence grading remained the same when we removed them consecutively. In addition, when updating the meta-analyses, we added the newly identified studies, re-estimated summary effect estimates using random-effects models, and applied a set of wellestablished criteria to properly classify the evidence according to the reported P values, heterogeneity, and excess significance bias, with consideration of the inflated risk of false positives inherited by the updated meta-analyses (140). Finally, the underlying mechanisms between TMAO and the development of various diseases have not been explored in depth.

Conclusions
In conclusion, our umbrella review and updated meta-analyses identified multiple health outcomes associated with TMAO concentrations. Evidence assessment demonstrated that TMAO concentrations are associated with several health conditions, including all-cause mortality, CVD, hypertension, diabetes, and CKD. Our dose-response meta-analyses indicated that there were no critical concentrations of TMAO in terms of its health impact. Further studies are needed to investigate whether circulating TMAO concentrations could be an intervention target for chronic disease.
The authors' responsibilities were as follows-XL and YZ: conceived the study; ET: contributed to the study design; DL, YL, and SY: performed the systematic review and data extraction and wrote the manuscript; DL and YL: performed the statistical analysis; other authors (XC, YH, JC, QW, DH, AF, YB, PS, DB, KT, SCL, HY, and HZ): provided significant advice and consultation; and all authors: critically reviewed the manuscript, contributed important intellectual content, and read and approved the final manuscript. The authors report no conflicts of interest.

Data Availability
All data relevant to the study are included in the article or as supplementary information.