We conducted a comprehensive literature search in scientific databases such as PubMed/Medline, Scopus, Web of Science, EMBASE, the Cochrane databases, and Google Scholar to find relevant prospective studies on the effects of CLA supplementation on inflammatory cytokines, adiponectin, and leptin published up to March 2022. There were no restrictions on the length of time or language of publications. The Participant, Intervention, Comparison/Control, Outcome (PICOS) search framework was used (32), which stands for population (both healthy and unhealthy adults), intervention (CLA supplementation), comparison (placebo/control group), outcome (changes in CRP, IL-6, TNF, adiponectin, and leptin), and research design (parallel and crossover randomized clinical trials).

The combination of MESH and non-MESH terms were used for the search, as follows: (“conjugated linoleic acid” OR “conjugated fatty acid” OR “boric acid” OR “rumenic acid” OR “CLA”) AND (intervention OR “intervention study” OR “intervention studies” OR “controlled trial” OR randomized OR random OR randomly OR placebo OR “randomized controlled trial” OR “randomized clinical trial” OR RCT OR blinded OR “double blind” OR “double blinded” OR trial OR “clinical trial” OR trials OR “Pragmatic Clinical Trial” OR “Cross-Over Studies” OR “Cross-Over” OR “cross-over study” OR parallel OR “parallel study” OR “parallel trial”).

By reading the titles, abstracts, and full text of the papers as needed, two researchers independently (FS and OA) chose the appropriate articles. The effects of CLA supplementation on inflammatory cytokines and adipokines variables in adults with different health statuses in all human randomized clinical trials)RCTs(.

We incorporated studies that satisfied the following criteria: (1) RCTs (parallel or crossover); (2) used oral intake of CLA; (3) examined the effects of CLA supplementation on CRP, IL-6, TNF-α, adiponectin, and leptin; (4) had an intervention duration of at least 2 weeks (RCTs with two or more eligible arms were considered as separate studies); (5) were performed on adults (≥18 years old); (6) provided means and standard deviations (SDs) for all outcome variables, or any other effect sizes from which the calculation of mean and SD was possible. The searches were limited to human studies with no language restrictions. Animal studies, reviews, in vitro research, research on kids and teenagers, grey literature, conference abstracts, opinion pieces, books, and RCTs without a placebo or control group were excluded. Studies that used CLA in combination with vitamins or minerals were also excluded.

In the present study, we searched for studies that assessed the effects of CLA supplementation on all inflammatory cytokines. After screening and finding eligible studies, we found that most studies evaluated CRP, IL-6, and TNF. In addition, a limited number of studies have evaluated other inflammatory factors such as IL-1 and IL-8. Therefore, we included studies that evaluated the effects of CLA on CRP, IL-6, and TNF.

After quickly skimming the titles, abstracts, and full text to choose the most pertinent research following a separate review of each qualifying RCT, the following data were gleaned by two independent researchers (OA and FS). Name of the first author, country of origin, year of publication, type of clinical trial, participant characteristics (mean age, body mass index (BMI), sex), randomization, blinding, sample size, number of participants in the intervention and control groups, type and dosage of supplemented CLA, duration of the study, and related data were extracted for additional measurements. The CLA dosages were converted to milligrams per day, whether they were given in grams per day or another unit.

To rate the quality of the studies, the Cochrane Collaboration technique was utilized (33). Two researchers (SR and GS) independently assessed the methods, and any disagreements between their assessments were resolved through discussion. Each study was evaluated for any bias, including those caused by randomized sequence generation, allocation concealment, participant and staff blindness, outcome assessor blindness, insufficient outcome data, selective reporting, and other biases. Thus, three categories were established: high risk of bias (general risk of bias > 2 high risks), low risk of bias (general risk of bias < 2 high risks), and undetermined risk of bias (general risk of bias = 2 high risk).

Stata 11.0 was used to conduct the statistical analysis (Stata Corp, College Station, TX, USA). p-values <0.05 were deemed statistically significant for all two-tailed tests. The pooled weighted mean difference (WMD) was calculated using a random-effects model to take into account any existing heterogeneity (due to the different intervention doses, duration, participant health, sample sizes, and length of intervention) developed by Der Simonian and Laird (34). We computed the mean differences in CRP, IL-6, TNF-α, adiponectin, and leptin between the CLA supplementation and control groups from the preintervention to the postintervention. Formula: SD = square root [(SD at baseline)² + (SD at end of study)² (2rSD at baseline, SD at end of study)] was used to get the SD of the mean difference (35). For RTCs that only provided standard error of the mean (SEM) information, SD was determined using the following formula: Standard Error of Mean (SEM): SD =, where n is the number of subjects in each group (36). After visual inspection of forest plots or Cochrane’s Q test, heterogeneity was evaluated using the I square (I ²) statistic (p < 0.05 and I ² > 40%), with I ² > 40% considered clinically significant (37). The baseline CRP, IL-6, TNF-α, adiponectin, and leptin levels were analyzed, while the intervention duration (< 12 weeks, ≥ 12 weeks) and dosage of CLA (≥ 3 and < 3 g/day) were based on the median values of the included studies and health status (healthy: people without any disease; unhealthy: people suffering from a disease such as atherosclerosis, rheumatoid arthritis, diabetes, etc.). Other subgroup analyses were performed according to gender (man, woman), baseline BMI (normal (18.5–24.9 kg/m²), overweight (25–29.9 kg/m²), and obese (>30 kg/m²)) and isomer’s type (CLA-c9t11, CLA-t10c12, both). Studies examining the effect of CLA supplementation on inflammatory cytokines and adipokines used the Begg’s test and the funnel plot test to evaluate publication bias (38). We used the leave-one-out approach to do a sensitivity analysis to identify how many inferences were dependent on a single sample to examine the influence of each study on the pooled effect size (39). The possible linear association between CLA (mg/day) dose and duration on outcome variables was evaluated using meta-regression, whereas a nonlinear dose–response analysis was used to assess the effect of CLA supplementation dose and duration (40, 41).

The flow chart presented in Figure 1 describes the selection process and the references retrieved from the database.

A total of 8,351 studies were identified in the first step of the literature search of electronic databases, PubMed (n = 3,582), ISI Web of Science (n = 1,991), Scopus (n = 2,310), and the Cochrane library (n = 468). After excluding duplicated (n = 2,419) and irrelevant studies based on titles and abstracts (n = 2,804), 104 potentially relevant articles were considered for full-text review. Out of 104 studies, 62 did not have the desired data. Finally, 42 studies (11, 12, 16, 18, 19, 27, 28, 42–76) were included in the present meta-analysis, and their characteristics are illustrated in Table 1 . The risk of bias assessment is summarized in Table 2 .

All of these studies were RCTs published between 2000 and 2020. Study design characteristics are presented in Table 1 . The WMD and 95% CI of CRP (mg/dl), IL-6 (pg/ml), TNF-α (ng/ml), adiponectin (µg/ml), and leptin (ng/ml) and their changes are presented in Figures 2A–E , respectively. The two studies were conducted in the USA (42, 67), three in Sweden (43, 45, 49), three in the Netherlands (44, 52, 65), four in Canada (12, 14, 46, 68), two in Norway (27, 47), four in the UK (28, 48, 51, 56), two in France (50, 53), two in Turkey (11, 72), one in Japan (55), one in Ireland (57), one in Korean (58), one in Finland (59), one in Denmark (61), one in China (62), three in Germany (62, 69, 70), one in Italy (64), one in Spain (16), one in Poland (75), and the others in Iran (18, 19, 60, 66, 71, 73, 74, 76). Thirteen studies included only men and seven women, and 22 included both sexes. There were 37 parallel (11, 12, 16, 18, 19, 27, 42–45, 47–63, 65–67, 69, 71–76) and five crossover (28, 46, 64, 68, 70) studies. A total of 1,192 subjects (intervention: 597/control: 595) in terms of CRP, 817 subjects (intervention: 418/control: 399) for IL-6, 779 subjects (intervention: 402/control: 377) for TNF-α, 845 subjects (intervention: 424/control: 421) for adiponectin, and 1,274 subjects (intervention: 649/control: 625) for leptin were enrolled. The duration of the intervention varied from 4 to 104 weeks. The CLA supplements were used in doses of 1.6 to 10.8 mg/day. The mean age and baseline BMI ranged from 20 to 61.55 years and 22.2 to 45.71 kg/m², respectively. Participant’s health conditions were as follows: obese men with metabolic syndrome (43), overweight subjects with LDL phenotype B (52), both older obese and young lean, older lean, young obese (28), birch pollen allergy (59), obesity-related hypertension (62), postmenopausal women with type 2 diabetes mellitus (63), overweight mild asthmatics (12), postmenopausal women (66), overweight and hyperlipidemic (68), overweight type 2 diabetes (71), healthy overweight people (16, 47, 56), healthy subjects (11, 42, 44, 48, 49, 51, 53, 54, 57, 58, 61, 64, 67, 70, 72), atherosclerotic patients (73), rheumatoid arthritis (19, 60), athletic (74), overweight/obese (27, 45, 46, 50, 55, 65, 69, 75), nonalcoholic fatty liver disease (76), and chronic obstructive pulmonary disease (COPD) (18). Iwata et al. (55), Albers et al. (44), and Steck et al. used two different CLA doses in their studies. Out of the 42 studies, CRP, IL-6, TNF-α, adiponectin, and leptin were reported in 20, 15, 16, 12, and 20, respectively. For supplementation, 47 study arms used mixed isomers (9cis,11trans-CLA and 10trans,12cis-CLA), four study arms used 9cis,11trans-CLA isomer, and five study arms used 10trans,12cis-CLA isomer. In these studies, detection methods of CRP were Behring latex-enhanced high-sensitivity assays, Eckman Synchron CX7 System, enzyme-linked immunosorbent assay, enhanced turbidimetric immunoassay, highly sensitive immunoassay with a monoclonal antibody coated with polystyrene particles, immunoturbidimetric assay, and rabbit antihuman.

Although the visual inspection of funnel plots showed slight asymmetries for all of the outcome variables, no significant publication bias was detected according to Begg’s test for most of the variables, such as CRP (p = 0.189), IL-6 (p = 0.441), adiponectin (p = 0.198), and leptin (p = 0.050). Publication bias was considerable just for TNF-α (p = 0.042) ( Figures 3A–E ).

The present study conducted a nonlinear dose–response regression to analyze the dose–response relationship between CLA supplementation and inflammatory cytokines and adipokines. We did not find a significant nonlinear relationship between dose (g/day) and changes in CRP [(coefficients = −0.059; p _nonlinearity = 0.353)] ( Figure 4A ), IL-6 [(coefficients = −10.437; p _nonlinearity = 0.244)] ( Figure 4B ), TNF-α [(coefficients = −791.208; p _nonlinearity = 0.081)] ( Figure 4C ), adiponectin [(coefficients = 1.337; p _nonlinearity = 0.522)] ( Figure 4D ), and leptin [(coefficients = −77.607; p _nonlinearity = 0.841)] ( Figure 4E ). Moreover, there was a nonlinear association between the duration of intervention and CRP [(coefficients = −0.798; p _nonlinearity = 0.164)] ( Figure 5A ), IL-6 [(coefficients = 2.635; p _nonlinearity = 0.247)] ( Figure 5B ), TNF-α 9[(coefficients = −8.156; p _nonlinearity = 0.622)] ( Figure 5C ), adiponectin [(coefficients = −0.343; p _nonlinearity = 0.752)] ( Figure 5D ), and leptin [(coefficients = 0.056; p _nonlinearity = 0.748)] ( Figure 5E ).

Meta-regression analyses were performed to assess whether inflammatory cytokines and adipokines were affected by CLA supplementation doses and intervention duration. We found no significant linear association between intervention dose (g/day) and changes in CRP [(coefficients = 0.279; p _linearity = 0.304)] ( Figure 6A ), IL-6 [(coefficients = 0.162; p _linearity = 0.384)] ( Figure 6B ), TNF-α [(coefficients = 0.051; p _linearity = 0.538)] ( Figure 6C ), adiponectin [(coefficients = −0.152; p _linearity = 0.616)] ( Figure 6D ), leptin [(coefficients = 0.138; p _linearity = 0.693)] ( Figure 6E ), nor duration of supplementation (week) and changes in CRP [(coefficients = 1.001; p _linearity = 0.365)] ( Figure 7A ), IL-6 [(coefficients = 0.327; p _linearity = 0.528)] ( Figure 7B ), TNF-α [(coefficients = 0.038; p _linearity = 0.737)] ( Figure 7C ), adiponectin [(coefficients = −0.094; p _linearity = 0.944)] ( Figure 7D ), and leptin [(coefficients = −0.006; p _linearity = 0.994)] ( Figure 7E ).

The sensitivity analysis demonstrated that no study had a significant impact on the overall findings. Although the overall result of studies reporting data on TNF-α was sensitive to the study by Song et al. (48) (WMD = −0.644; 95% CI, −1.353, 0.065), and IL-6 was sensitive to the study by Sofi et al. (64) (WMD = −0.284; 95% CI, −0.601, 0.033), by omitting these studies, the significance of the correlation between CLA and TNF-α and IL-6 was lost.

In the case of CRP, some studies had an impact on the overall effect size, including that of Whigham et al. (54) (WMD = 0.253; 95% CI, −0.013, 0.520), Smedman et al. (49) (WMD = 0.247; 95% CI, −0.010, 0.506), and Pfeuffer et al. (69) (WMD = 0.257; 95% CI, −0.007, 0.523); the arm “a” and “b” of the study of Ramakers et al. (50) (WMD = 0.258; 95% CI, −0.013, 0.529; WMD = 0.245; 95% CI, −0.028, 0.518), Taylor et al. (51) (WMD = 0.251; 95% CI, −0.009, 0.512), Gaullier et al. (27) (WMD = 0.190; 95% CI, −0.053, 0.434), and Mullen et al. (57) (WMD = 0.226; 95% CI, −0.036, 0.490); the arm “a” and “b” of the study of Steck et al. (56) (WMD = 0.245; 95% CI, −0.010, 0.501; WMD = 0.237; 95% CI = −0.011, 0.485), Raff et al. (61) (WMD = 0.219; 95% CI, −0.046, 0.484), and Sluijs et al. (65) (WMD = 0.262; 95% CI, −0.003, 0.529); and the arm “b” of the study of Joseph et al. (68) (WMD = 0.237; 95% CI, 0.026, 0.501) and Lopez-Plaza et al. (16) (WMD = 0.249; 95% CI, −0.016, 0.515). It was shown that the exclusion of every individual study by the sensitivity analysis could not change the direction of the correlation but eliminated the significant effect of CLA on CRP.

Table 4 displays the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) profile of CLA supplementation on inflammatory cytokine and adipokine variables together with the certainty in outcomes. For CRP, because of serious limitations in inconsistency, the quality of evidence was moderate. In the case of IL-6, because of very serious limitations in inconsistency and for TNF-α because of very serious limitations in inconsistency ad serious limitations for publication bias, the quality of evidence was low. For both outcomes, including leptin and adiponectin, because of very serious limitations in inconsistency and serious limitations in imprecision, the quality of evidence was very low.

Using the GRADE methodology, which was previously outlined, the total degree of evidence certainty across the studies was evaluated and summarized (77).