Protocol for Systematic Review and Meta-analysis of Olive oil and Non-Alcoholic Fatty Liver Disease (NAFLD) Authors: Georgios Tsamos MD*, Georgios Kalopitas MD,MSc, Kleo Evripidou, Andreas Vadarlis MD,MSc, Christina Antza MD,PhD , Georgios Germanidis MD,PhD, Michail ChourdakisMD,MPH, PhD Contact: *Corresponding author. Tel.: +30 6979741222 E-mail address: tsamgeor@gmail.com Introduction: Nonalcoholic fatty liver disease (NAFLD) is considered as the most common chronic liver disease, with an approximately global prevalence of up to 25%. [1] It is defined as the presence of >5% of hepatic steatosis (HS), in the absence of excessive alcohol consumption, other causes of secondary liver steatosis or other causes of chronic liver disease. [1]. Clinically NAFLD is mostly reported in obese, insulin resistant patients, as a part of the metabolic syndrome (MetS). The disease progression can lead to a range of disorders, varying from non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH) to severe fibrosis and cirrhosis. Olive oil is widely known for its beneficial health effect, mainly as a part and key nutritional component of the Mediterranean diet (MeD). Olive oil has many constituents, with particular attention recently been paid to phenolic compounds, largely due to their antioxidant effect [2], but also to its anti-inflammatory activity [3][4]. Its consumption either individually or as a part of dietary treatments, is associated with the prevention of metabolic disorders [5], that probably lead to the pathogenesis as well as the progression of NAFLD. Recently, results from randomized controlled trials that have been conducted, show an improvement on liver steatosis and hepatic enzymes with the consumption of olive oil. Objectives: The aim of this study is to systematically review the current literature and conduct a meta-analysis about the efficacy of olive oil consumption in patients with Nonalcoholic fatty liver disease (NAFLD). Methods: The study protocol is conducted according to the Preferred Reporting Items for Systematic reviews and Meta-analyses (PRISMA) statement. [6] 1.Study eligibility criteria: Types of studies: This study will include only Randomized Controlled Trials with duration of intervention of 8 weeks or more, published in English language and irrespective of publication status or date. Inclusion criteria: The following inclusion criteria where considered: -Patients older than 18 years old -Patients diagnosed with NAFLD with imaging (liver ultrasonography, Magnetic Resonance Imaging or Computed Tomography) or by liver biopsy, with daily alcohol intake less than 30 g/day for men and less than 20g/day for women. (7) -Studies with dietary intake of olive oil. -Studies comparing olive oil with other type of fat and/or with a specific diet. Exclusion criteria: -Patients with a history of alcohol abuse. -Pregnancy -Patients with other causes of steatosis or chronic liver diseases (such as hepatitis B and/or C, autoimmune hepatitis, Wilson’s disease and others) -Studies without regular intake of olive oil, namely single-dose interventions. -Patients being treated with hepatotoxic medicine. -Non-English language studies. -Patients that during the last 6 months, have either started/changed an antidiabetic or an antilipidemic medication, or have started during the last 6 months therapy with vitamin E for NAFLD. Intervention: The role of a rich in olive oil diet, on liver related and anthropometric outcomes in patients with NAFLD. Comparator: Dietary plans with lower than the mean olive oil consumption or other specific diets without olive oil, will be compared with the intervention group (diets rich in olive oil) in patients with NAFLD. Outcomes: Primary outcomes: Changes in pre and after treatment values in ALT. Secondary outcomes: Changes in BMI, liver enzymes, lipid profiles parameters (Total cholesterol, HDL-C, LDL-C, TG), anthropometric characteristics (waist circumference etc.), fasting glucose/insulin, HOMA-IR and liver histology parameters when available. 2.Information sources and search strategy: All eligible studies will be searched by two authors through the online databases of Pubmed, Scopus and Central using an advanced search strategy using key concepts of the research question: Olive oil administration in patients with NAFLD. Clinical trials.gov will be searched also for more evidence. For each key concept, appropriate free-text words, and Medical Subject Headings (MESH) will be created using all the possible synonyms and associated words. The above will be combined using appropriate Boolean logic operators: AND, OR, and NOT. Grey literature will be searched to find the theses related to the study subject, also electronic databases including ProQuest and Scopus will be used in addition to contacting the experts. Furthermore, relevant conference papers and proceedings will be searched, and electronic databases will be used in addition to the expert information obtained. These references will be searched manually. Finally, a search in PROSPERO will be performed in order to avoid a duplicate review. Below there is an indicative analytical search strategy for PUBMED (table 1). PUBMED Search Strategy (Table 1): #1 NAFLD #2 NASH #3 Nonalcoholic fatty liver disease #4 Nonalcoholic steatohepatitis #5 Non-alcoholic fatty liver disease #6 Non-alcoholic steatohepatitis #7"Non-alcoholic Fatty Liver Disease"[Mesh] #8 "Fatty Liver"[Mesh] #9 liver and (fatty or steatosis or steatoses) #10 #1 OR #2 OR #3 OR #4 OR #5 OR #6 OR #7 OR #8 OR #9 #11 Olive oil #12 "Olive oil"[Mesh] #13 "Olive oil" [tw] #14 EVOO #15 "EVOO"[tiab] #16 Extra virgin olive oil #17 "Extra virgin olive oil"[Mesh] #18 VOO #19 Olive oils #20 Polyphenol #21 Polyphenols #22 Phenol #23 Phytochemical * #24 HPOO #25 High-polyphenol #26 LPOO #27 "Polyphenol"[Mesh] #28 Refined olive oil OR ROO #29 FVOO #30 (#11 OR #12 OR #13 OR #14 OR #15 OR #16 OR #17 OR #18 OR #19 OR #20 OR #21 OR #22 OR #23 OR #24 OR #25 OR #26 OR #27 OR #28 OR #29) #32 #10 AND #30 3.Study Selection: Studies identified from the search in the electronic medical databases will be imported into reference manager software. After removal of duplicates, the remaining eligible studies will be screened by two independent reviewers in two phases, firstly on level of title and abstract and then on full text screening. The screening will be conducted with the help of online screening software. Disagreements arising between the two reviewers will be resolved with the help of a third senior reviewer. For grey literature, we will search LILACS, DANS EASY Archive, dissertations/thesis and reports. Then search process will be presented in a PRISMA flow chart. 4.Data collection/extraction process and data items: Data will be extracted by two independent reviewers using a standardized data abstraction form, developed according to the sequence of variables required from the primary studies. Disagreements in data abstraction between the two reviewers will be resolved by a third independent reviewer. Data will be extracted on the following: author’s first name, publication date, location (country in which the research was conducted), duration of therapy, sample size, therapy group (olive oil), treatment regimen (specific olive oil supplementation and dosage when available), treatment outcomes, secondary data. Authors will be conducted to obtain any missing data relevant to the analysis. 5.Risk of bias Two independent reviewers will assess the quality of the eligible studies using the revised Cochraine’s Collaboration Risk of Bias Tool 2.0 (RoB2). [8]Any disagreements will be resolved with the help of a third senior reviewer. Individual studies will be classified at low, some concerns or high risk of bias. 6.Dealing with missing outcome data: Contact and request of the first authors will be made through mails to provide any missing outcome data, perform sensitivity analysis to assess the robustness of meta-analytic results, and discuss the potential impact of missing data on the review findings. 7.Statistical analysis and data synthesis strategy: Review Manager and statistical program SPSS 28 will be used for all the statistical analyses during the synthesis of the review. Data synthesis will be performed with random-effects or fixed-effects models. The statistically significant difference value is set when P<0.05. We will use weighted mean difference and 95% confidence interval (95% CI) for continuous data and relative risk (RR) and 95% CI for dichotomous data. The Cochrane chi-square test will be used to estimate the statistical heterogeneity and I2 statistics to estimate the degree of heterogeneity. Heterogeneity will be considered low, moderate, and high when the values are below 25%, between 25% and 75%, and above 75%, respectively. If necessary, subgroup analyses will be conducted to reduce heterogeneity. 8.Assessment of publication bias: The first strategy to deal with the publication bias is performing the most inclusive search in the search stage of the study including those unpublished (grey literature). Also, funnel plots will be used to assess potential reporting bias and a non-significant study effect. Begg’s test and Egger’s test will also be performed, and significant results (p>0.1) shall suggest a publication bias. Where publication bias exists, Duval and Tweedie non-parametric ‘trim and fill’ analysis will be performed to formalize use of funnel plot, estimate the number and outcome of missing studies, and adjust for missing studies. Funding Sources None Conflicts of interest None Language English Country Greece Literature-Citations: [1] Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016 Jul; 64(1):73-84. [2] D. Turck, J.L. Bresson, B. Burlingame, et al. Guidance for the scientific requirements for health claims related to antioxidants, oxidative damage and cardiovascular health: (Revision 1) EFSA J, 16 (2018), pp. 1-21 [3] S. Cicerale, L. Lucas, R. Keast, G. Gazzani, M. Grusak Antimicrobial, antioxidant and anti-inflammatory phenolic activities in extra virgin olive oil Curr Opin Biotechnol, 23 (2011), pp. 129-135 [4] A. Camargo, A. Rangel-Zuñiga, C. Haro, E.R. Meza-Miranda, P. Peña-Orihuela, M.E. Meneses, et al. Olive oil phenolic compounds decrease the postprandial inflammatory response by reducing postprandial plasma lipopolysaccharide levels Food Chem, 162 (2014), pp. 161-171 [5] De Santis, Stefania et al. Extra Virgin Olive Oil: Lesson from Nutrigenomics. Nutrients vol. 11,9 2085. 4 Sep. 2019, doi:10.3390/nu11092085] [6] Moher D, Liberati A, Tetzlaff J, Altman DG. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. Annals of internal medicine. 2009;151(4):264-9, w64 [7] European Association for the Study of the Liver (EASL) European Association for the Study of Diabetes (EASD); European Association for the Study of Obesity (EASO). EASL-EASD-EASO Clinical Practice Guidelines for the management of non-alcoholic fatty liver disease. J Hepatol. 2016; 64:1388–1402. [8] Sterne JAC, Savović J, Page MJ, Elbers RG, Blencowe NS, Boutron I, et al. RoB 2: a revised tool for assessing risk of bias in randomised trials. BMJ. 2019 Aug;366:l4898.