The association between gallstone disease and metabolic syndrome related abnormalities: a systematic review and meta-analysis

Bile excretion is one of the important metabolite excretion pathways of the human body. In recent years, it has been reported that metabolic diseases are associated with the occurrence of gallstone disease. The main purpose of this systematic review is to examine the relationship between metabolic syndrome and cholelithiasis, including components of the metabolic syndrome such as abnormal blood glucose regulation, hyperlipidemia, and obesity. PubMed, Cochrane Library, and Embase were searched for all English language articles for the following relevant keywords: Metabolic Syndrome, Reaven Syndrome X, Biliary Calculi, Cholelithiasis Gallstones. Case-control study, cross-sectional study, and cohort study were included. Pooled relative risks (RRs) or odds ratios (ORs) and their corresponding 95% confidence intervals (CIs) were calculated. The pooled mean differences of the outcome measures were compared between patients with and without metabolic syndrome. After screening, a total of 5 cross-sectional studies and 1 cohort study were included in the meta-analysis. The 6 studies evaluated a total of 49,101 people, of whom 9055 had metabolic syndrome and 2308 had gallstone disease. There is a significant correlation between metabolic syndrome and gallstone disease (z = 6.65, p = 0.000), and it’s more significant in female. All studies displayed increasing odds of gallstone disease with increasing number of metabolic syndrome traits, where patients with three or more metabolic syndrome traits tended to have a higher prevalence of nephrolithiasis. Our review shows a definite association of metabolic syndrome with gallstone disease, and the more the components of metabolic syndrome, the higher the prevalence of gallstone disease. Although not as obvious as women, men also support this conclusion.

metabolic syndrome has increased dramatically worldwide [7,10,11]. Studies about the association between gallstone disease and MetS suggested that MetS is a risk factor for gallstone disease [12]. Most of the key components for the diagnosis of MetS are also key risk factors of GSD [8]. As a whole, metabolic syndrome is also associated with cholelithiasis; the purpose of this meta-analysis was to examine the association of MS and gallstones integrated and independent by component.

Search strategy and selection criteria
This systematic review and meta-analysis was performed in accordance with MOOSE guidelines [13]. The major medical databases in the English language, among which were Cochrane, Embase, and PubMed, were searched independently by two authors (ZN and XL) up to September 15, 2019, using the following keywords in combination with both medical subject heading terms and text words: "Metabolic Syndrome X," "Metabolic syndrome," "Insulin Resistance Syndrome X," "Reaven Syndrome X," "Cholecystolithiasis," "Cholelithiasis," "Choledocholithiasis," "Biliary Calculi," and "Gallstones".
Gray trials were excluded in this study. But this may increase the publication bias, because the included studies were not complete and comprehensive and published trials had an overall greater intervention effect than gray trials. On the other hand, unpublished studies may be of lower methodological quality than published studies [14].

Study selection and data extraction
Two reviewers (ZN and XL) independently screened all the titles and abstracts in an effort to minimize selection bias, and a third reviewer was consulted for resolution of disagreement. The following information was extracted from all the literatures: research design type, region, number of patients included, diagnostic methods of cholelithiasis, diagnostic criteria of metabolic syndrome, BMI (Body Mass Index), age, and prevalence of metabolic syndrome and its components such as waist circumference, blood pressure (systolic pressure, diastolic pressure), fasting blood glucose, LDL-C, TG (triglyceride), and other blood biochemical indicators in cholelithiasis group and control group. For continuous data (such as age, blood pressure, fasting blood glucose), mean ± SD and the number of people in the group were extracted respectively. For count data (such as hypertension and diabetes mellitus), the corresponding number of people in the two groups and the total number of people in the group were extracted respectively.
Studies were included if the following criteria were met: (1) retrospective or prospective cohort study, cross-sectional study, case-control study. (2) The experimental group met the diagnostic criteria for MetS. (3) The diagnosis of cholelithiasis was confirmed by imaging evidence or surgical evidence. (4) The control group was included in the study and odds ratios (ORs) in case-control studies or relative risks (RR) in cohort studies were reported with the 95% confidence intervals (CIs) (or, if 95% CIs were not reported, the reported data were sufficient to calculate them). Studies were excluded if (1) literature was not the above research type, or the unpublished. (2) The study did not set up a control, or the number of cases less than 10; patients with long-term use of drugs proved to be conducive to stone formation or stone discharge. (3) The patients in the study had organic lesions, liver dysfunction or viral hepatitis in the hepatobiliary system. (4) Only the latest research involving more cases included in the similar study of the same authors.

Quality assessment
The Newcastle-Ottawa scale was used to assess the quality of the included studies [15]. Briefly, the instrument contains 8 items categorized into 3 dimensions: the selection of the study groups; the comparability of the groups; and the ascertainment of either the exposure or outcome of interest for case-control or cohort studies respectively. An 8-point scoring system is used for a semiquantitative assessment of study quality; score of 5-8 were classified as qualified studies [16]. Crosssectional studies use the quality evaluation criteria recommended by AHRQ (Agency for Healthcare Research and Quality) which includes eleven items. Article quality was assessed as follows: low quality = 0-3; moderate quality = 4-7; high quality = 8-11 [17].

Outcome measures and data analysis
The primary outcome was the interrelation between the period/point prevalence of cholelithiasis with metabolic syndrome patients and that in non-metabolic syndrome patients, and the prevalence of cholelithiasis in different sexes, the relationship between the components of metabolic syndrome and the prevalence of cholelithiasis, and the relationship of number of components of metabolic syndrome and cholelithiasis. The effect measures of interest were odds ratios for casecontrol studies and cross-sectional studies, relative risks for cohort studies, and the corresponding 95% confidence intervals are given.
The heterogeneity of the study was evaluated by I 2 statistics. The I 2 > 50% was taken to indicate high levels of heterogeneity and the random effect analysis model is used. Otherwise, the fixed effect model will be used. Subgroup analysis was performed to explore potential heterogeneity. A p value < 0.05 was considered statistically significant, and 95% confidence intervals (CIs) are given. Publication bias was evaluated by using the Egger test [18]. A p value < 0.05 indicates statistically publication bias. The meta-analyses were performed using Stata software (version 14; Stata Corp, College Station, TX). The Z test was used to examine differences in experiment group and control group. A Z value more than 1.96 and p < 0.05 was considered statistically significant, and the 95% confidence intervals (CIs) are given. Cochran-Armitage test for trend was used in the SAS system (version 9.2); p < 0.05 was considered statistically significant.

Results
A flow diagram of study selection is shown in Fig. 1. After the initial literature searching and eliminating duplicate articles, 94 were excluded from 432 identified articles (Fig. 1). Three hundred nineteen unrelated articles and reviews were excluded after examination. Read the full text carefully to rule out inconsistencies in the type or purpose of the study. Finally, 6 articles were included in the study which including 5 crosssectional studies and 1 cohort study [6,12,[19][20][21][22]. We found no related or relevant meta-analyses in the Cochrane Library. A summary of the 6 included studies is given in Table 1. The 6 studies included in the meta-analysis contained a total of 48,858 people, wherein 5451 had MetS and 43407 did not have MetS.

Primary outcome
The main findings of the study were the prevalence of cholelithiasis in MetS and non-MetS populations (Fig. 2). In the six studies included, significant heterogeneity was observed. Heterogeneity chi-squared = 18.69 (d.f. = 5), p = 0.002, Isquared (variation in RR attributable to heterogeneity) = 73.2%, and thus a random-effects model of analysis was performed. The result indicates that patients with MetS had a significantly higher GSD prevalence than patients without MS (z = 6.65, p = 0.000). Egger's test suggested no statistically significant publication bias for the study, p = 0.959 ( Supplementary Fig. 1). Funnel plots can't be used for the reviews as insufficient numbers of included studies (10 or more studies) [23].
A subgroup analysis conducted to investigate people from different regions that led to strong heterogeneity. Four and two studies were included in the analysis of the associations between Asian and European and American countries in the prevalence of GSD (Fig. 3). The pooled estimates indicated that neither Asian populations (p = 0.007, I-squared = 75.4%) nor European and American (p = 0.026, I-squared = 79.9%) populations contribute to the heterogeneity of prevalence of cholelithiasis in MetS and non-MetS populations.
An analysis was also conducted to investigate the effect of gender on the prevalence of GSD between MetS populations and non-MetS. Three studies reported the gender data were included (Fig. 4a.). Significant heterogeneity was observed in male patient (p = 0.030, I 2 = 71.5%) and overall patient (p = 0.000, I 2 = 78.5%), and thus a random-effects model of analysis was performed. Male patients (z = 10.22, p = 0.000) and female patients (z = 8.45, p = 0.000) and overall patients (z = 12.45, p = 0.000) with MS have more rate for gallstone disease than without MS.
Four studies reported age data (Fig. 4b). No significant heterogeneity was observed (heterogeneity chi-squared = 5.82, p = 0.121, I-squared = 48.5%), and thus a fix-effects model of analysis was performed. We found old patients (z = 21.16, p = 0.000) have higher prevalence for gallstone disease than the youngers.
Four studies reported BMI data (Fig. 5a). Significant heterogeneity was observed (heterogeneity chi-squared = 12.19, p = 0.007, I-squared = 75.4%) and thus a random-effects model of analysis was performed. Patients with high BMI are more likely to suffer from GSD (z = 5.74, p = 0.000).
Four studies reported WC (waist circumference) data (Fig. 5b). Significant heterogeneity was observed (heterogeneity chi-squared = 11.23, p = 0.011, I-squared = 73.3%) and thus a random-effects model of analysis was performed. Patients with high WC are more likely to get GSD (z = 6.57, p = 0.000). Four studies reported TC (total cholesterol) data (Fig. 5c). Significant heterogeneity was observed (heterogeneity chisquared = 8.44, p = 0.015, I-squared = 76.3%) and thus a random-effects model of analysis was performed. There was no significant difference in two groups of people (z = 0.66, p = 0.508) indicating that there was no significant increase in the prevalence of cholelithiasis among people with higher TC levels.
With regard to the number of components and the prevalence of GSD, we merged data from three articles; participants were divided into 4 levels according to the counts of their metabolic disorders. The Cochran-Armitage test for trend was used in the SAS system (version 9.2). Among the subjects with different numbers of metabolic abnormalities, there was an increased prevalence of gallstones as the number of metabolic abnormalities increased (test for trend, p < 0.001, Table 2). Even if the diagnostic criteria for MS are not met, the independent component remains a risk factor for GSD, and as the number increases, the risk of GSD increases.

Discussion
Gallstones can be divided into the following types: cholesterol stones, pigment stones, and mixed stones. Cholesterol stones must be at least 80% cholesterol by weight. Other common constituents are primarily of bilirubin (insoluble bilirubin pigment polymer) and calcium (calcium phosphate) salts that are found in bile. Between 35% and 90% of stones are cholesterol stones [24]. During the formation of cholesterol stones,     [25][26][27]. Long bile retention time in gallbladder may lead to high concentration in gallbladder bile and promote cholesterol precipitation in epithelial cells [28]. Occasionally chronic bacterial infections, although asymptomatic, can also be a cause of GSD [29,30]. Metabolic syndrome was first proposed by Reaven in 1993, which was then called X syndrome [31]. The main clinical manifestations were insulin resistance (IR), hyperinsulinemia, impaired glucose tolerance (IGT), hypertension, and abnormal atherogenic lipid metabolism [32]. Different definitions of MetS have been proposed, reviewed, recommended, and even questioned over the decades. The World Health Organization (WHO) first proposed a working definition centering on IR or hyperglycemia [33]. The National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) of the USA proposed diagnostic criteria for MS in 2001 [34].
In the past 20 years, the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) of the USA and IDF have proposed new definitions successively, but they have these core components including central observatory, dyslipidemia, hypertension, and insulin resistance. In fact, it also proved to be reliable and universally accepted diagnostic tool [35][36][37]. As a result of different diagnostic criteria, the results of our statistical analysis may have deviation, which may lead to a decrease in the reliability of the conclusions to a certain extent, but this is inevitable.  This study aimed to evaluate the association of MS with characteristics of gallstone. There are great differences in body shape or WC among different ethnic groups, men, and women where studied as one group, a fact that it could have introduced a bias in the final analysis, even if appropriate adjustments of data were performed in the final statistics. We included 5 cross-sectional studies in the literature, which means that these studied under normal living conditions in only one point in time; thus, the relationships found in the analysis have limitations. And the exclusion of gray literature from the meta-analysis will exaggerate the positive results of the meta-analysis and lead to decision errors.
In spite of the limitations described above, conclusions can also be drawn from these studies that the prevalence of gallstones in MetS patients is increased compared with patients without MetS. A subgroup analysis by sex analysis showed that both men and women had an increased prevalence. Analysis of various factors including MS components, the prevalence of cholelithiasis increased in older patients. Obesity-related factors like high blood lipids TC, WC, BMI, fasting blood glucose were positively correlated with the prevalence of cholelithiasis. Diabetes and hypertension are also positively correlated with the prevalence of cholelithiasis. In male subjects, metabolic syndrome was associated with presence of gallstones, and the size of gallstones [38].
In recent years, insulin resistance is another hot topic among metabolic abnormalities associated with GSD. Not only in obese people but insulin resistance can also occur in people with normal weight. A study in a Hispanic population found that GS was associated with insulin resistance, fatty liver, and metabolic syndrome. Insulin resistance is a risk factor for GS [39]. Insulin resistance may play an important role in the pathogenesis of GS favoring the production of cholesterol supersaturated bile and altering gallbladder function [39]. Nakeeb et al. [40] demonstrated that insulin resistance alone may be responsible for gallbladder dysmotility, which may result in acalculous cholecystitis or gallstone formation.
Even evidence supports the contention that insulin resistance not only directly induces gallbladder inflammation increase mucus production and alter gallbladder function but could also favor the secretion by the liver of cholesterol supersaturated bile [41]. The latter seems more accepted pathogenic link. GS increases cholesterol saturation in gallbladder bile, a phenomenon related to increases of body cholesterol synthesis and hypersecretion of biliary cholesterol as observed in obesity [30]. Adiponectin acted as a critical element in the development of insulin resistance; the lower the rate of obesity, weight, and BMI, the higher the concentration of adiponectin and that this information would be helpful for the treatment of diabetes [42].
The definition presented by the IDF that emphasizes abdominal obesity as a sine qua non diagnostic factor [43]. In fact, it has been confirmed that some indicators such as larger waist circumference, higher BMI, and hyperlipidemia are obese manifestations. Recent data from animal and human studies have shown that the gallbladder of obese people is enlarged and their response to neurotransmitters is usually reduced. A high-fat diet can lead to abnormal emptying of the gallbladder. In addition, obese and high carbohydrate diet had increased gallbladder tissue levels of tumor necrosis factor-alpha, interleukin-6, and interleukin-1 beta. These changes lead to decreased smooth muscle function and diminished gallbladder absorption [44].
The function of bile components secretory also increased in GS patients. In a study from Chile, increased bile synthesis was found in GS patients [45]. Hepatic bile acid and body cholesterol were significantly increased by 50% in patients with cholelithiasis, hepatic hypersecretion of cholesterol, and supersaturated bile which give life to precipitating cholesterol crystals that accumulate and grow in a sluggish gallbladder.
Although there is no parallel relationship with blood lipids level, increased lipid components excretion through bile directly promote the formation of cholesterol stones. Total cholesterol, direct bilirubin, and especially lean body weight might provide simple stratification tools for obese women, outlining a high-risk profile for developing gallstones [46].
In the next step of the study, the number of included literature should be increased as much as possible, especially the gray literature, to reduce publication bias, and subgroup analysis of each component, thereby reducing heterogeneity, and, more importantly, investigating the priority or order of Mets and GSD in cross-sectional studies.

Conclusions
Despite there may be some heterogeneity due to different diagnostic criteria of metabolic syndrome or some other errors, the result trend shows that metabolic syndrome is closely related to cholelithiasis. Even some researchers propose that GD is another member of the metabolic syndrome [43]. These results also suggest that the metabolic syndrome can even be regarded as another indication for prophylactic surgery in patients with GSD.
Authors' contributions PJ and HH conceived and designed the study. ZN and XL performed the experiments. PJ and SH wrote the paper. PJ, ZN, XL, YL, and HH reviewed and edited the manuscript. All authors read and approved the manuscript.
Funding This study was funded by the Zhejiang Medical and Health Science and Technology (grant number 2021KY927) and Zhejiang Traditional Chinese Medicine Administration (CN) (grant number 2021ZB208).