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Therapeutic effect of resveratrol supplementation on oxidative stress: a systematic review and meta-analysis of randomised controlled trials
  1. Mehdi Koushki1,
  2. Mostafa Lakzaei1,
  3. Hadi Khodabandehloo2,
  4. Hossein Hosseini1,
  5. Reza Meshkani1,
  6. Ghodratollah Panahi1
  1. 1 Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran (the Islamic Republic of)
  2. 2 Department of Biochemistry and Nutrition, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran (the Islamic Republic of)
  1. Correspondence to Ghodratollah Panahi, Department of Clinical Biochemistry, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran (the Islamic Republic of); pshahriyar{at}gmail.com

Abstract

Background Resveratrol is a naturally occurring polyphenol compound mainly found in grapes and red wine. The evidence has suggested that resveratrol has an antioxidant effect. However, the results are inconsistent and inconclusive. Thus, we conducted a systematic review and meta-analysis to evaluate the effect of resveratrol supplementation on markers of oxidative stress.

Methods We searched PubMed, ISI Web of Science, EMBASE, Scopus and the Cochrane library up to December 2018 to identify randomised controlled trials (RCTs) assessing resveratrol supplementation effects on oxidative markers. Heterogeneity, publication bias, risk of bias and subgroup analysis were analysed. This meta-analysis was conducted in accordance with the guidelines of the Preferred ReportingItems for Systematic Reviews and Meta-Analysis (PRISMA).

Results Meta-analysis of data from 12 RCTs did not support significant effect of resveratrol supplementation on circulating levels of superoxide dismutase (SOD) (standardized mean difference (SMD) (1.12), (95% CI −0.91 to 3.1), p=0.28), catalase (CAT) (SMD (−0.07), (95% CI −1.4 to 1.3), p=0.92) and glutathione peroxidase (GPx) (SMD (−0.76), (95% CI −2.56 to 1.04), p=0.40). Although, resveratrol supplementation increased significantly circulating total antioxidant capacity (TAC) concentrations (SMD (0.52), (95% CI −0.02 to 1.07), p=0.05). Severe heterogeneity was observed between studies, and no obvious publication bias was observed in included RCTs.

Conclusion Collectively, our findings of available RCTs did no show any benefit of resveratrol supplementation on SOD, CAT and GPx except for TAC. Well-designed RCTs are necessary to confirm these results.

  • resveratrol
  • oxidative markers
  • meta-analysis

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INTRODUCTION

Different studies implicate that oxidative stress plays a pivotal role in the onset and/or progression of many different pathologies including cardiovascular diseases,1 2 cancers,3–5 neurodegenerative,6–8 metabolic,9 10 pregnancy complications11 12 and inflammatory diseases.11–14 Nowadays, oxidative stress has become a major interest of clinical and basic science research. Oxidative stress is a state of imbalance in which production of reactive oxygen species (ROS) overwhelms the intrinsic antioxidant capacity defense.15 ROS are highly reactive and toxic molecules generated from two different sources, respiratory chain during mitochondrial oxidative metabolism and endoplasmic reticulum.16 17 Antioxidants are a group of compounds which inhibit the oxidation of proteins, carbohydrates, nucleic acids and lipids.18 Antioxidants can be divided into enzymatic group including superoxide dismutase (SOD), glutathione reductase (GR), catalase (CAT) and glutathione peroxidase (GPx) and non-enzymatic groups including reduced glutathione (GSH), total thiols, ascorbic acid, carotenoids, α-tocopherol, uric acid and bilirubin.19 20 Oxidative stress occurs when the concentration of ROS is not controlled by internal antioxidants.15 In order to characterise oxidative stress in many pathological conditions markers of oxidative stress, such as total antioxidant status or total antioxidant capacity (TAC) (sensitive and reliable biomarker) malondialdehyde, protein carbonyl and sulfhydryl were evaluated. Given the role of oxidative stress in the development of diseases, the use of effective antioxidants provides an attractive therapeutic strategy to preventing disease progression. In this context, natural products have been shown to reduce oxidative stress and are acknowledged as the antioxidant interventions.

Resveratrol (trans-3,5,4′-trihydroxystilbene), a naturally occurring polyphenolic compound mainly found in grapes and red wine, was shown to prevent and treat chronic conditions such as cardiovascular, neurodegenerative and metabolic diseases.21 A plethora of health beneficial effects of resveratrol is occurring through its antioxidant properties. The antioxidant activity of resveratrol is mediated via multiple mechanisms, including inhibiting lipid peroxidation, increasing TAC and reducing ROS formation.22 Scavenging free radicals and enhancing the levels of endogenous antioxidants by activation of the Nrf-2 pathway are other biological activities described to resveratrol. Resveratrol is known to increase the levels of several antioxidant enzymes, including SOD, GPx, glutathione S-transferase and GR.23 In addition to in vitro and animal studies, randomised clinical trial studies have been shown that resveratrol has a protective effect against several diseases mediated by oxidative stress.24 25 However, the evidence addressing this effects are inconsistent and inconclusive. Thus, we performed a systematic review and meta-analysis of all human clinical trials to determine whether or not consuming resveratrol could modulate oxidative stress. The meta-analysis was conducted in accordance with the guidelines of the 2009 Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement.26

Materials and methods

Data source and search strategy

We searched the published articles assessing the effect of resveratrol supplementation on circulating oxidative markers (enzymatic and non-enzymatic) up to December 2018 using databases PubMed, EMBASE, Web of Science, Scopus and the Cochrane library. We followed the standard PRISMA criteria when conducting this meta-analysis. We performed the search using Medical Subject Heading terms: (“Oxidative markers” OR “Oxidative stress”) OR (“Enzymatic oxidative/anti-oxidative markers” OR “Non-enzymatic oxidative/anti-oxidative markers”) OR (“Superoxide dismutase” OR “Erythrocuprein” OR “Hemocuprein”) OR (“Catalase” OR “Mn catalase”) OR (“Glutathione peroxidase” OR “Selenoglutathione peroxidase”) AND (“Resveratrol” OR “trihydroxystilbene”) AND (“Clinical trial” OR “Randomized controlled clinical trial”). Furthermore, grey literature, conference abstracts and reference lists were manually searched to identify potentially relevant studies. Searches were limited to human and English language.

Inclusion/ exclusion criteria

Two reviewers independently screened titles, abstract and full-text publications by inclusion criteria form. The studies were included in the meta-analysis, if they met the following criteria: (1) measurement of the circulating levels of enzymatic and non-enzymatic markers in (plasma/serum), (2) English language, (3) oxidative/antioxidative markers as a primary outcome, (4) the studies with an appropriate controlled design and (5) presented sufficient information (SDs, SE or 95% CIs) on oxidative/antioxidative levels at the baseline and at the end of the study in both resveratrol and control groups. In addition, we excluded studies due to (1) insufficient data such as review article, letters, brief report and comments, (2) duplicate articles, (3) lack of access to studies data even by contacting to the first author or corresponding author, (4) non-clinical studies and (5) studies without control or placebo groups.

Data extraction and quality assessment

The following data were extracted by two reviewers independently using a predefined data extraction sheet: the first authors last name, publication year, country, study design, sample size (intervention/placebo), total sample size, type of sample, age (years), population disease, dose of resveratrol (mg/day), treatment duration of resveratrol (days), primary outcome such as SOD (intervention/placebo), TAC (intervention/placebo), CAT (intervention/placebo) and GPx (intervention/placebo) and Jadad score. The quality of the studies was assessed by two authors using the Jadad scale.27 In cases of disagreement, a third author will examine such articles. Results will be discussed until reaching consensus among all three authors. The items included were (1) randomisation, (2) double blinding, (3) reporting of the number of dropouts and reasons for withdrawal, (4) allocation concealment and (5) generation of random numbers. Randomised controlled trials (RCTs) with a jadad score ≥3 were considered high quality.

Statistical analyses

We analysed results qualitatively and quantitatively. Our primary outcome measure was mean change in circulating oxidative markers. We analysed our findings using meta-analysis random-effect models to calculate the pooled standardised mean difference (SMD) with 95% CI, due to both of resveratrol consumption and the levels of oxidative markers, variables are continuous. Because of severe heterogeneity of reported between-studies, we analysed our findings using random effect models (using the inverse variance-weighted method). SDs of the mean difference were calculated using the following formula: SD=square root ((SDpretreatment)2+(SDpost-treatment)2−(2R×SDpretreatment×SDpost-treatment)), assuming a correlation coefficient (R)=0.5. In case of reporting SEM, SD was estimated using the following formula: SD=SEM×sqrt (n), where n is the number of subjects. We also used the following formula for calculation of pooled mean and pooled SD at the baseline and at the end of the study in both the intervention and placebo groups. Pooled mean=N1*mean (baseline)+N2*mean (the end study)/N1+N2, Pooled SD = (N1−1) S1 (baseline)+(N2−1) S2 (the end study)/N1+N2. If circulating oxidative markers (SOD, TAC, CAT and GPx) were reported in the median and IQR, mean and SD values were estimated using a method previously defined.28 Statistical heterogeneity was measured using the Q and the Higgins I2 (p<0.1, I2>50 %) statistics. We performed subgroup analysis according to the ‘type of sample’, ‘dose of resveratrol’, ‘treatment duration of resveratrol’ and ‘Jadad score’ for identifying the potential source of heterogeneity among included studies in the meta-analysis. Publication bias was assessed with the Begg and the Egger regression test as statistically and also funnel plot as graphically. Stata V.12.0 software was used for this meta-analysis and p-value <0.05 was considered to be statistically significant.

Results

Literature search

Our literature search identified 143 published articles in five databases. After screening titles and abstracts, we excluded 17 duplicates and 46 articles that were apparently not relevant to the topic. Eighty full-text articles were further reviewed. We excluded 35 articles due to the following reasons: 15 studies had no insufficient data, six studies were not English language, 16 articles were brief report, reviews or letters, 20 articles not measuring the effect of resveratrol supplementation on circulating levels of oxidative markers (enzymatic and non-enzymatic) and additional 11 articles were excluded because of unavailability of full-text. Finally, 12 articles were found to meet the inclusion criteria in the systematic review and meta-analysis. The study selection result is shown in figure 1.

Figure 1

Processes selection of included studies in this meta-analysis.

Studies’ characteristics

This systematic review and meta-analysis included 12 RCTs published up to December 2018 with a total of 290 cases in the intervention group and 285 individuals in the placebo group. The characteristics of selected studies were summarised in table 1. Among 12 studies, four studies used plasma and eight studies used serum samples. Of these studies, one was conducted in North America, three in South America, four in Asia, four in Europe and one in Australia. In 12 RCTs included in the meta-analysis, three trials used type 2 diabetes (T2D) patients, three trials reported healthy subjects with or without obesity and smoking. One used non-alcoholic fatty liver disease (NAFLD) individuals, one trial performed on chronic kidney disease (CKD) participants, one trial on obesity-related nephropathy and three trials reported data on asymptomatic hypercholesterolemic, military firefighters and ulcerative colitis. All of the selected studies were measured oxidative markers (enzymatic and non-enzymatic) as primary outcome. With regard to studies quality, the Jadad score ranged from 2 to 5. A dose of resveratrol used in included studies ranged between 40 mg/daily and higher proportion 3000 mg/daily. In all trails, the oxidative/antioxidative markers in intervention group were compared with the placebo group. The mean patient age of intervention and placebo groups ranged between 22 and 65 years; and duration of patient follow-up for resveratrol supplementation ranged from 28 to 180 days. A total of 300 individuals participated in 12 RCTs. Participants were mostly male (53.7%) and female (46.2%). Articles were published between 2012 and 2018 years.

Table 1

Baseline characteristic of included studies in this systematic review and meta-analysis

Meta-analysis results

The effect of resveratrol supplementation on oxidative markers

Twelve studies with 290 cases and 285 placebo assessing the effect of resveratrol consumption on the levels of oxidative markers were included for overall effect size analysis in our meta-analysis. The combined effect size (SMD) using random-effects model was presented in figure 2. Results of this study showed that resveratrol consumption significantly increase TAC (SMD (0.52), (95% CI −0.02 to 1.07), p<0.05), although it had no significant effects on SOD (SMD (1.12), (95% CI −0.91 to 3.1), p=0.28), CAT (SMD (−0.07), (95% CI −1.4 to 1.3), p=0.92) and GPx (SMD (−0.76), (95% CI −2.56 to 1.04), p=0.40). There was statistically significant heterogeneity among the trials for SOD (I2=97.7%, p<0.001), TAC (I2=84.2%, p<0.001), GPx (I2=94.3%, p<0.001) and CAT (I2=94.5%, p<0.001). Publication bias was assessed by funnel plot as graphically and the Begg and the Egger tests as statistically. Although symmetrical distribution (figure 3) and the Egger, s-weighted regression intercept (p=0.19 for SOD, p=0.83 for TAC, p=0.85 for CAT) showed no publication bias in the case of SOD, TAC and CAT (figure 4), GPx with the Egger, s-weighted regression intercept (p=0.002) showed statistically publication bias.

Figure 2

Forest plots indicating the pooled estimate SMD of resveratrol supplementation on oxidative markers of (A) SOD, (B) TAC, (C) GPx and (D) CAT. CAT, catalyse; GPx, glutathione peroxidase; SMD, standardised mean difference; SOD, superoxide dismutase; TAC, total antioxidant capacity.

Figure 3

Visual inspection of the funnel plots evaluating publication bias in the RCTs included for the analysis of resveratrol consumption on (A) SOD, (B) TAC, (C) GPx and (D) CAT. CAT, catalyse; GPx, glutathione peroxidase; RCTs, randomised controlled trials; SMD, standardised mean difference; SOD, superoxide dismutase; TAC, total antioxidant capacity.

Figure 4

Funnel plots detailing publication bias as statistically with the Egger-weighted regression method in the RCTs selected for the analysis of resveratrol consumption on oxidative markers of (A) SOD, (B) TAC, (C) GPx, (D) CAT. CAT, catalyse; GPx, glutathione peroxidase; RCTs, randomised controlled trials; SOD, superoxide dismutase; TAC, total antioxidant capacity.

Subgroup analysis

To explore the reasons for the heterogeneity, we performed subgroup analysis based on type of sample (plasma/serum), resveratrol dosage (mg/day), duration of treatment (days) and Jadad score (≤3,>3). The results of subgroup analysis were shown in table 2. In the subgroup analysis stratified by type of sample, despite a higher statistical significant heterogeneity in oxidative markers of TAC (SMD (1.08) 95% CI (0.39 to 1.77), p=0.002), and GPx (SMD (−1.6) 95% CI (−5.01 to 1.6), p<0.001) in plasma samples as well as oxidative marker of CAT (SMD (−0.3) 95% CI (−2.1 to 1.5) p<0.001) in serum sample, there was no significant heterogeneity in oxidative markers of SOD and CAT in plasma samples as well as oxidative marker of GPx in serum sample (online supplementary figure 1). When subgroup analysis conducted based on resveratrol dosage, we find statistically significant heterogeneity in doses≥800 mg/day for SOD (SMD (28.4) 95% CI (6.6 to 50.3), p<0.05), while there was no heterogeneity in doses<800 mg. Given the stratification factor of doses<500 mg/day, statistically significant heterogeneity was observed in both oxidative/antioxidative markers of GPx (SMD (−1.6) 95% CI (−5.01 to 1.61), p<0.001) and CAT (SMD (−0.6) 95% CI (−3.4 to 2.09), p<0.001), while in the subgroup analysis stratified by doses≥500 mg/day, no significant association was observed in both GPx and CAT (online supplementary figure 2). In subgroup analysis concerning duration of treatment in target population, statistically significant heterogeneity was emerged in the results of the overall SMD of SOD in duration <60 days (SMD (7.68), (95% CI 1.76 to 13.59), p=0.01) (I2=97.9%, p<0.05) but no duration ≥60 while, the results of the pooled SMD of CAT with duration <120 was (SMD (−0.07) 95 % CI (−3.3 to 2.04), p<0.001) (I2=97.5%, p<0.001)) that showed a high significant heterogeneity. There was no statistically significant heterogeneity in oxidative markers of TAC (I2=89%, p=0.2) (online supplementary figure 3).

Table 2

Evaluation of the effect of resveratrol supplementation on markers of oxidative stress using subgroup analysis

Taken together, these findings suggest that there is a direct association between alterations in circulating levels of oxidative/antioxidative markers following consumption of resveratrol supplementation. Towards this end, in this meta-analysis, subgroup analysis demonstrated that resveratrol supplementation with dose ranged between 500 and 800 mg/day along with duration of treatment greater than 60 days changed the oxidative/antioxidative status with alteration in circulating levels of oxidative/antioxidative markers.

Discussion

Summary of the main results

Resveratrol is a non-flavonoid polyphenol compound which lowers the increased levels of oxidative stress through the scavenging of ROS, as shown in a number of in vitro and in vivo studies.29–32 To our knowledge, this is the first meta-analysis that comprehensively summarises the effect of resveratrol supplementation on circulating oxidative markers. In summary, this study revealed that resveratrol supplementation significantly increased TAC level, while, it did not support any significant effect on oxidative markers of SOD, CAT and GPx. There was statistically significant heterogeneity among the trials for SOD (I2=97.7%, p<0.001), TAC (I2=84.2%, p<0.001), GPx (I2=94.3%, p<0.001) and CAT (I2=94.5%, p<0.001). Publication bias was assessed by funnel plot as graphically and the Begg and the Egger test as statistically. The funnel plot had a symmetrical distribution (figure 3) and no obvious publication bias also observed for SOD, TAC and CAT, except for GPx with the Egger, s-weighted regression intercept (p=0.002) (figure 4). Subgroup analyses showed that the pooled effects of resveratrol on markers of oxidative stress were affected by resveratrol dose, duration of intervention and type of sample. As resveratrol could significantly increase SOD level in trails with duration of the intervention <60 days and dose of resveratrol ≥800 mg/day.

A variety of enzymatic and no enzymatic antioxidants that present in human serum serve to counterbalance the effect of oxidants. When the levels of SOD as an enzymatic antioxidant involves in the conversion of superoxide ion to hydrogen peroxide is depleted, the superoxide formed will cause direct cellular damage.33 Although SOD could be used as a biomarker of oxidative stress, no single component of serum antioxidant complex could represent the protective efficiency of blood against oxidative stress, probably because of interactions that occur in vivo among different antioxidant compounds. Hence, TAC considers the cumulative effect of all antioxidants present in the blood.34 Measurement of TAC of blood is believed to be a useful indicator of the ability of antioxidants present in the blood to protect against oxidative damage of cellular components. Most studies consistently reported the increased levels of SOD and TAC. Ali Mahmood et al 35 showed that 1000 mg twice daily of resveratrol supplementation for 8 weeks markedly increases SOD. Apostolidou et al 36 reported a significant increase in TAC in normocholesterolemic individuals after resveratrol consumption. In another study, Kumar et al 37 showed that 250 mg/day of resveratrol consumption for 6 months significantly increases SOD in T2D patients. However, Chachay et al 38 have reported that supplementation of 3000 mg of resveratrol does not have any beneficial effect on SOD and TAC in patients with NAFLD. In addition, De Groote et al 39 provided an important evidence that 150 mg/day of resveratrol for 28 days has no effects on SOD and TAC in healthy obese subjects. In the present study, our pooled analysis showed that SOD and TAC levels are increased compared with placebo although they were not statistically significant. This may be due to the heterogeneity and/or a small number of studies evaluating these factors in the included publications. The endogenous enzymatic antioxidant is composed of two key enzymes: CAT and GPx, which convert hydrogen peroxide into hydrogen oxide (water) and oxygen.40 Therefore, CAT and GPx may play a major role in first-line antioxidant defense against the toxic effects of ROS and can be used as oxidative stress markers.41

Some reports suggest that the levels of CAT and GPx enzyme activities may decrease in a diabetic and metabolic syndrome (Mets) patients.42–44 Also decreased CAT activities were found in patients with various types of cancer, including lymphoma, breast cancer, head and neck cancer, gynaecological and urological cancer.45 It was shown that higher antioxidant enzyme activities are associated with greater protection against oxidative injury.46 The resveratrol as an antioxidant was found to increase GPx activities in renal tissues obtained from type 1 diabetes mellitus-induced rats.47 Additionally, resveratrol can increase activities of SOD and CAT in streptozotocin-induced diabetic kidneys.48 The activation of antioxidant enzyme may possibly due to the ROS scavenging activity of resveratrol.49 Furthermore, resveratrol could regulate the activity and expression of antioxidant enzymes such as SOD.50 However, its effect on oxidative stress and antioxidant enzymes is controversial. Kumar et al reported that resveratrol in a dose of 250 mg/day for 6 months in patients with T2D significantly increases the activities of CAT.49 Macedo et al, who used resveratrol treatment for 90 days (100 mg/day) in military firefighters, found that erythrocytes GPx activity was reduced by resveratrol supplementation compared with the placebo group but erythrocytes CAT activity was not altered.51 Saldanha et al in a RCT evaluated the effect of resveratrol (500 mg /day) in 20 non-dialysed CKD patients and found there is no significant difference in GPx and CAT after resveratrol supplementation.52 A similar result has been reported by Seyyedebrahimi et al. They suggested that resveratrol supplementation (800 mg/day) for 2 months does not affect the levels of CAT activity in diabetic patients.24 Also in the study by Asghari et al, daily dose of 600 mg of resveratrol for 12 weeks did not significantly affect the GPx activity in patients with NAFLD.53 In this study, we found no significant evidence for the effect of resveratrol supplementation on SOD, GPx and CAT. Though the inhibitory effects of resveratrol on GPx and CAT are unclear, but, the overall effect of resveratrol supplementation on these factors in this meta-analysis is somewhat unexpected. Due to the strong link between SOD, TAC, CAT and GPx, if resveratrol supplementation is beneficial in increasing TAC level, we expected to observe a increasing influence of resveratrol supplementation on SOD and CAT levels. It remains unclear why we did not find a preventive effect of resveratrol on GPx level; however, it is likely that a relatively small number of pooled participants provide not enough statistical power to estimate the intervention effect. There are several strengths in our systematic review and meta-analysis. First, our search strategy was very detailed and spanned multiple databases, second, according to the results of Beggs and Egger tests as statistically and funnel plot as graphically, we did not find a significant publication bias among included studies except for GPx that it is associated with small sample size and low number of included studies in this analysis. Third, we performed a subgroup analysis to explore the possible sources of the heterogeneity. Fourth, the selected studies were conducted in different countries, which made the results more generalisable. Fifth, we conducted a quality assessment of included studies by Jadad score. Our systematic review and meta-analysis had some limitations. First, a total of 12 studies with 290 cases and 285 placebos, which were pooled in this meta-analysis, decreases the statistical power of the data analyses and subsequently lower reliable estimation. Second, there was a substantial heterogeneity between studies, although we used random-effects models to combine the overall results and performed subgroup analysis to explore the sources of heterogeneity according to our results in this meta-analysis. Third, due to the low number of included studies in the meta-analysis, we did not find statistically significant effect of resveratrol supplementation on oxidative markers. Thus, future intervention studies are needed to authenticate the beneficial effect of resveratrol supplementation in the prevention of oxidative stress.

Conclusion

Collectively, overall results of this study showed that resveratrol consumption could increase circulating level of TAC, although it did not significantly change the levels of oxidative markers of SOD, CAT and GPx. Further studies are required to validate the effect of consumption of resveratrol supplementation on levels of oxidative/antioxidative markers.

Main messages

  • Oxidative stress plays an important role in the onset and progression of various diseases such as cardiovascular comorbidities, cancers and type 2 diabetes.

  • In this systematic review and meta-analysis of 13 randomised controlled trials, there was no significant effect of resveratrol supplementation on superoxide dismutase, catalyse and glutathione peroxidase.

  • Our meta-analysis supports the benefit of resveratrol supplementation on total antioxidant capacity.

Current research questions

  • Have clinical trials well demonstrated the effect of resveratrol supplementation on oxidative stress markers?

  • Based on this meta-analysis, can resveratrol supplement change oxidative markers?

  • Is it possible that resveratrol acts as a new compound for the improvement of oxidative/antioxidative imbalance?

Acknowledgments

We thank Department of clinical Biochemistry, Tehran University of Medical Sciences.

References

Footnotes

  • Contributors MK, GP, ML and HK conceptualised and designed the research. RM critically reviewed the manuscript and approved the final manuscript as submitted. MK, GP, ML, HK and HH wrote the draft of this paper, data collection, statistical analyses and data interpretation.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Patient consent for publication Not required.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available in a public, open access repository. There are no data in this work. Data are available upon reasonable request. Data may be obtained from a third party and are not publicly available. No data are available. All data relevant to the study are included in the article or uploaded as supplementary information.