Non-invasive markers of cardiovascular risk in patients with subclinical hypothyroidism: A systematic review and meta-analysis of 27 case control studies

It has been reported that subclinical hypothyroidism (SCH) is closely related to subclinical atherosclerosis. According to the impact of SCH on noninvasive markers of cardiovascular risk, we fulfilled a meta-analysis of included studies to provide an integrated overview. We searched electronic databases and included all relevant studies involving SCH and epicardial adipose tissue (EAT), carotid intima-media thickness (CIMT), pulse wave velocity (PWV), flow-mediated dilation (FMD) and glyceryl trinitrate-induced dilation (GNT- induced dilation). The result was calculated in a meta-analysis to assess the impact of SCH on these markers. A total of 27 studies were entered in the final analysis. Compared with euthyroid subjects, SCH patients exhibited a significantly increased CIMT (SMD: 0.369 mm; 95%CI: 0.038, 0.700; P = 0.029) and EAT (SMD: 1.167 mm; 95%CI: 0.869, 1.466; P = 0.000) and increased PWV (SMD: 3.574 m/s; 95%CI: 0.935, 6.213, P = 0.008). We also found significantly lower FMD (SMD: −1.525%, 95%CI: −2.156, −0.894, P = 0.000) and lower GNT-induced dilation (SMD: −0.384%, 95%CI: −0.625, −0.142, P = 0.002). Sensitivity analysis and subgroup analysis confirmed the above results. Our meta-analysis confirmed a significant association of SCH and cardiovascular risk with arterial wall thickening and stiffening and endothelial dysfunction. These findings will help to establish detailed cardiovascular prevention strategies for SCH patients.

. To further understand the adverse effects induced by SCH, a growing number of studies have focused on CV with disease-related indicators to evaluate the correlation between SCH and atherosclerosis.
CIMT can be measured by noninvasive ultrasound imaging to evaluate subclinical atherosclerosis 11,12 . CIMT has been widely accepted as a formidable predictor of adverse CV disease events (shock and myocardial infarction) 13,14 . Similarly, flow-mediated dilation (FMD), pulse wave velocity (PWV), and glyceryl trinitrate-induced dilation (GNT-induced dilation) as independent predictors of CV events are considered to be surrogate markers of subclinical atherosclerosis [15][16][17][18] . FMD and GNT-induced dilation are widely used to detect vascular endothelial function as a noninvasive and accurate detection technique 19 . In addition, PWV has been used to detect peripheral and central arterial stiffness 20 . The epicardial adipose tissue (EAT) thickness surveyed by echocardiography is also considered an important independent predictor of an increased incidence of CV events for evaluating subclinical coronary atherosclerosis 21 .
For the past few years, many case-control studies have announced that SCH patients exhibit accelerated atherosclerosis, damaged endothelial function, and increased arterial stiffness. However, small sample sizes and potential confounding factors influence the strength of previous evidence. According to the impact of SCH on noninvasive markers of CV risk, we fulfilled a systematic review and meta-analysis of included studies to provide an integrated overview.

Methods
We strictly followed the PRISMA reporting specifications for this meta-analysis.
Search strategy. According to the PRISMA guidelines 22 , we formulated a detailed search strategy, including SCH and noninvasive markers of CV risk (i.e., IMT, EAT, PWV, FMD, and GNT-induced dilation). We systematically searched online databases (PubMed, Embase, Web of Science) through 30 November 2017. All possible combinations of search terms included ('Subclinical hypothyroidism') AND ('atherosclerosis') AND ('arterial stiffness') AND (epicardial adipose tissue OR carotid intima-media thickness OR pulse wave velocity OR flow-mediated dilation OR glyceryltrinitrate-induced dilation). No language or publication date restrictions were imposed in this study.
Furthermore, manual audits were conducted on all retrieved articles. If data were missing from an article, the authors communicated via e-mail to try to obtain the raw data. Two independent authors (YAO K and ZOU X) analysed each paper and independently extracted the data. In the case of differences in opinion, the two authors consulted with a third investigator (ZHAO T). The differences were settled by accordance. The selected results were determined by unanimous agreement of each author and were reported according to the PRISMA guidelines.
Inclusion criteria. Studies adhering to the following inclusion criteria were included: 1) case-control studies; 2) including subjects with normal thyroid function as a control group, 3) CIMT, EAT, PWV, FMD and GNT-induced dilation indicators of patients with SCH compared with euthyroid (EU) subjects reported; and 4) 95% confidence intervals (CIs) were reported. Exclusion criteria. During the literature screening process, studies characterized by the following criteria were excluded: 1) including participants diagnosed with severe SCH or hyperthyroidism; 2) including participants being treated with medicine; 3) including patients with abnormal thyroid function who were diagnosed thyroid disease; 4) studies without a control group, animal studies, and reviews; 5) studies that did not provide the value of at least one variable (mean and standard deviation) among the predictors.
Data extraction and quality assessment. For each included study, we extracted the data corresponding to the demographic variables, the number of cases and controls, and the major clinical variables including CIMT, FMD, GNT-induced dilation, PWV and EAT.
Next, the methodological quality of each study was evaluated based on the characteristics of the study. The Newcastle Ottawa scale (NOS) was used to assess the quality of nonrandomized observational studies 23 . The scoring system consisted of three major areas (selection, comparability, exposure), and the results ranged from 0 to 8 with higher scores representing better methodological quality. The quality evaluation results of the NOS are reported in Table 1.
Statistical analysis and risk of bias assessment. Statistical analysis was performed by STATA 12.0 software. Differences among cases and controls were presented as the standardized mean difference (SMD) with the associated 95%CI for continuous variables 24 .
All reported P values were two-sided with a significance level set at P < 0.05. Heterogeneity between studies was calculated by I 2 statistics; an I 2 value of 0% indicates no heterogeneity, and I 2 values of 25%, 25-50%, and 50% indicate low, moderate, and high heterogeneity, respectively 25 .
Begg's test, Egger's test and funnel plots were used to test publication bias. We visually examined the symmetry of the funnel plots to evaluate possible small sample effects, and we used Begg's test and Egger's test to evaluate publication bias of the included studies. Statistical significance was considered for P < 0.10 26 . When publication bias occurred, the adjusted effect scale was assessed using the Duval and Tweedie's trim and fill method with the random-effect model 27 .
Considering the variability among the studies, we used a random-effect method for all analyses.
Sensitivity analyses. Sensitivity analysis is used to investigate the reliability of a meta-analysis. We evaluated the reliability of the conclusions by examining the impact of individual studies on the total merged effect. For each study, a new meta-analysis was performed upon deleting that study to assess the stability of the results compared with the total effect.

Subgroup analysis.
Taking into account the potential impact of SCH confounding factors on the outcomes of the study, we conducted subgroup analyses for smoking, obesity, NOS score, and national factors.

Meta-regression analyses.
Meta-regression is often used to explore the sources and sizes of heterogeneity among studies and to further explain the effects of heterogeneity in the meta-analysis. We hypothesized that the included studies could exhibit differences in the demographic variability (sample size) and the combined traditional CV risk factors (smoking habit, diabetes mellitus, obesity, hyperlipidaemia and hypertension). To assess the possible effects of these variables in explaining different results observed across studies, a regression model with changes in CIMT, FMD, PWV and EAT values as dependent variables (y) and the abovementioned co-variates as independent variables (x) was constructed.
Availability of data and materials. All the data we get was from public sources.

Results
After excluding the repeated citations, we retrieved 423 articles. Among these studies, 217 were excluded because they deviated from the theme according to the title and/or abstract, 15 because they were animal/non-case-control studies or because they lacked the data of interest. Thus, 27 studies (on 1065 SCH cases and 866 subjects) were included in the final analysis 28-54 (Fig. 1). Thirteen studies compared the CIMT between 494 SCH patients and 390 EU subjects. PWV was evaluated in 5 studies
We performed sensitivity analysis by deleting single studies one-by-one and performing an additional meta-analysis for each study removed. For CIMT, PWV, FMD, EAT and GNT-induced dilation, the influence of the omission of each individual study on pooled SMD was assessed. However, no single study had an impact on the overall effect according to the sensitivity analysis, and thus, the meta-analysis was statistically stable. To eliminate the effects of confounding factors on the outcomes of the study, we conducted a subgroup analysis for high-heterogeneity markers. Subgroup analysis using the high-quality studies (n = 10) indicated that CIMT was significantly increased in patients with SCH (SMD: 0.35 mm; 95%CI: 0.10, 0.59; P = 0.005), and the heterogeneity of the result was markedly reduced (I 2 = 59%; P = 0.009). For PWV, subgroup analysis revealed that smoking habit (n = 3) was significantly increased in patients with SCH (SMD: 6.138 m/s; 95%CI: 4.493, 7.783; P = 0.005), with significant heterogeneity among studies (I 2 = 87.9%; P = 0.00). For FMD, we assessed the variability of heterogeneity and effects by region classification, and we found that the heterogeneity of China studies the pooled effects were statistically significant, and the heterogeneity of the results was significantly decreased. For EAT, we assessed the variability of heterogeneity and effects of non-obesity (n = 2), and we found that SCH was increased (SMD: 1.538 mm; 95%CI: 1.202, 1.874; P = 0.000) without heterogeneity among studies (I 2 = 0%; P = 0.978). Regression models for studies comparing SCH patients and EU subjects revealed that small-size studies significantly affected CIMT, indicating that small-sample-size studies are the source of heterogeneity (p = 0.075, tau 2 = 0.2467, Adj R-squared = 23.39%, I-squared_res = 78.41%, Fig. 5A). Similarly, a small sample size was also associated with FMD heterogeneity (p = 0.041, tau 2 = 0.5246, Adj R-squared = 44.21%, I-squared_res = 82.69%, Fig. 5C).According to the regression models for studies of SCH patients and EU subjects, smoking habit (p < 0.01, tau 2 = 0.9931, Adj R-squared = 92.41%, I-squared_res = 82.44%, Fig. 5B) as a confounding factor significantly affected PWV. No other demographic or clinical factors influenced the assessment results.

Discussion
This meta-analysis encompassing 27 studies and including 1065 SCH patients and 866 EU subjects provides compelling evidence that SCH was associated with increased arterial stiffness. We also found a marked increase in EAT and impaired endothelial function (as expressed by a reduced FMD and GNT-induced dilation) in SCH patients compared with EU controls. The sensitivity analysis results strongly confirmed our findings. In addition, the regression models provide evidence that smoking habits and small sample sizes have significant effects on the results of assessments. A former published meta-analysis reported an increase in CIMT in patients with SCH 55 . Our meta-analysis confirms this result and further reveals significant adverse effects of PWV, EAT, FMD and GNT-induced dilation as other CV risk factors.
There is frequent concern regarding the long-term adverse effects of various factors on increasing the risk of CV disease in SCH patients. SCH is associated with increased levels of total cholesterol and low-density lipoprotein cholesterol. In a population-based study of the Health Aging and Body Composition, the authors prospectively investigated the adverse effects of TSH levels on CV outcomes through a 4-year follow-up. The study found that when the patient's TSH levels exceeded 7 mIU/L and 10 mIU/L, the risk ratios for developing heart failure were 2.58 and 3.26, respectively 56 . Another study conducted by Rodondi et al. revealed that more than 55,000 individuals exhibited a positive association of elevated TSH levels with CV events rates and mortality 8 .
Many CV risk factors are deemed to play causal roles in the atherosclerotic process 57 . SCH contributes to vascular endothelial dysfunction by promoting lipid metabolism disorder. A study carried out in DaDong district of Shenyang city (China) revealed that the elevated serum TSH levels are positively correlated with serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol and negatively correlated with high-density lipoprotein cholesterol 58 . Although the majority of SCH patients are hyperlipidaemic, the correlation between subclinical atherosclerosis and SCH appear to be more complicated, and dyslipidaemia may not fully explain the accelerating atherosclerosis in this clinical setting 10,59 . There is increasing evidence that long-term exposure to high levels of TSH has a deleterious effect on the CV system beyond the mechanism of dyslipidaemia 9,33,49,60-62 . In addition, chronic inflammation may initiate and promote atherosclerosis and its complications through adverse effects on the vascular endothelium, which may be one of the causes of endothelial dysfunction. The clinical importance of SCH in CV disease and mortality remains controversial, and many studies offer conflicting results. This can be explained by the choice of heterogeneous patient groups, arbitrary definitions of TSH reference limits when defining SCH, lack of stratification based on elevated TSH levels, and different study designs.
To fully understand the correlation between SCH and subclinical atherosclerosis, our meta-analysis involves major CV risk markers. In addition, to prevent SCH-related dyslipidaemia from affecting the results of our assessment, we compared hyperlipidaemic and non-dyslipidaemic SCH patients, and the results were consistent. In addition, a meta-regression analysis was conducted to assess whether clinical data and demographic variables affect the outcome. As expected, the regression models revealed that the combination of traditional CV risk factors (e.g., smoking habits, hyperlipidaemia) and small-sample-size studies affect the outcomes. In addition, FMD and GNT-induced dilation were significantly impaired and PWV was significantly increased in SCH patients compared with EU subjects. However, given the small number of studies included in the PWV results, these results should be interpreted with caution. Studies have shown that when CIMT increases by 0.163 mm, the risk of myocardial infarction increases by 43% 63,64 ; furthermore, the risk of CV events increases by 14% when PWV increases by 1 m/s 16 . These data illustrate the clinical significance of our assessment of these indicators and the need for regular examination of the relevant indicators of subclinical atherosclerosis in patients with SCH.
Some potential limitations of this study need to be considered. First, among the studies included in this meta-analysis, each study had their own inclusion and exclusion criteria. Some of these patients were characterized by CV risk factors (smoking, obesity, diabetes mellitus, hyperlipidaemia and hypertension). Although the results of the regression analysis could be improved by assessing the impact of most clinical and demographic variables on the outcomes of the observation, caution is needed in interpreting the overall outcome. Finally, the evaluation of heterogeneity among studies is very important. Although all sources of possible heterogeneity could not be conclusively determined, the stability of the outcomes was confirmed after adjusting for potential publication bias.
In conclusion, SCH has a significant association with arterial wall thickening and stiffening and endothelial dysfunction and increased risk of CV events. Therefore, Whether SCH patients can benefit from early assessments of measures of CV risk markers that may require large-scale, long-term clinical studies to further confirmation.