We identified 1594 potentially relevant trials from our initial electronic search, and excluded 1528 trials after a preliminary review. The remaining 66 studies were retrieved for detailed assessment, and 16 randomized controlled trials met the inclusion criteria ( and Protocol S1 
), which consisted of data of 44841 individual patients. summarized the baseline characteristics of the participants and the design of the studies included. The trials included in this study compared folic acid supplementation (with or without B vitamins) with placebo. The follow-up for patients ranged from 8.3 to 87.6 months, with a mean of 43.2 months. The population of the trials ranged from 114 to 12064 individuals, with a mean of 2803. We restricted the inclusion criteria to randomized placebo-controlled trials with at least 100 patients and a minimum of 6 months follow-up to ensure that high-quality literature was included in our research, and to ensure a reliable conclusion. One trial had a Jadad score of 5, 6 trials had a score of 4, 7 trials had a score of 3, and the remaining 2 trials had a score of 2.
Flow diagram of the literature search and trial selection process.
Design and patient characteristics for trials included in the systematic review and meta-analysis.
Data for the effect of folic acid on major cardiovascular events were available from 12 trials, including 38015 individuals with 8238 cardiovascular events. shows the effect of folic acid (with or without B vitamins) on major cardiovascular events as compared to placebo. The pooled RR showed a 2% reduction in cardiovascular event rates, and with no evidence showed that folic acid therapy protected against cardiovascular event risk (RR, 0.98; 95%CI, 0.93–1.04). Although there was some evidence of heterogeneity across the studies included, a sensitivity analysis indicated that the results were not affected by sequential exclusion of any particular trial from all pooled analysis.
Effects of folic acid supplementation on the risk of major cardiovascular events.
Data for the effect of folic acid on stroke were available from 12 trials, including 42960 participants with 2001 events of stroke. Overall, folic acid therapy reduced the risk of stroke by 11%, but was not associated with a statistically significant decrease in the risk of stroke (fatal or nonfatal) events (RR, 0.89; 95% CI, 0.78–1.01, with unimportant heterogeneity, ).
Effects of folic acid supplementation on the risk of stroke.
Data for the effect of folic acid on myocardial infarction were available from 11 trials, including 39923 patients and 2917 events of myocardial infarction. No effect of folic acid therapy on the risk of myocardial infarction events was observed (RR, 1.00; 95% CI, 0.93–1.07, without evidence of heterogeneity of effect, ).
Effects of folic acid supplementation on the risk of myocardial infarction.
Fourteen trials including 44340 patients and 6314 total events of mortality were recorded, with 10 trials providing separate data for vascular death and 8 studies providing separate data for non-vascular death. There was no evidence to show that folic acid therapy could reduce the risk of mortality, whether total mortality, vascular death, or non-vascular death (). According to a sensitivity analysis, we excluded the (SEARCH) Collaborative Group study 
. This trial specifically included individuals with pre-existing myocardial infarction, which may have contributed to a high mortality rate. After this, we could conclude that folic acid therapy was associated with a reduction in the risk of vascular death, which was decreased by 11% (RR, 0.89; 95% CI, 0.81–0.98, ).
Summary of the relative risks of all outcomes assessed.
Ten of the trials included 38068 patients with 2939 revascularization events. There was no evidence to show that folic acid therapy protected against revascularization, although heterogeneity was observed in the magnitude of the effect across the trials included (RR, 1.05; 95% CI, 0.95–1.16, ). However, after sequential exclusion of each trial from all pooled analysis, the results were not affected by exclusion of any specific trial.
The risk of coronary syndrome was reported in 5 trials, including 19050 individuals and 3148 events of coronary syndrome. No evidence indicated that folic acid therapy protected against coronary syndrome risk (RR, 1.06; 95%CI, 0.97–1.15, without evidence of heterogeneity of effect, ).
Six trials reported data for the incidence of cancer, including 26544 patients and 2472 events of cancer. Reduction in the risk of cancer with folic acid therapy was not statistically significant (RR, 1.08; 95%CI, 0.98–1.21, with unimportant heterogeneity, ).
Of the 16 trials included in our meta-analysis, only 2 provided data about dialysis and amputation, and included 2294 participants, 725 dialysis events, and 116 amputation events. The pooled analysis showed no significant differences between folic acid therapy and placebo therapy for dialysis or amputation ().
Subgroup analyses were carried out for major cardiovascular events, stroke, and myocardial infarction. We noted that folic acid therapy was associated with a reduction in the risk of major cardiovascular events, when trials with less than 36 months follow-up period were included. Furthermore, compared with placebo, folic acid therapy showed a clear effect on stroke events when the mean age of the patients was less than 60 years. However, no other significant differences were identified between the effect of folic acid therapy and placebo, based on additional subset factors ().
Subgroup analysis for the effect of folic acid supplementation on major cardiovascular events, stroke, and myocardial infarction.