ABSTRACT: Setting: DNA methylation is an epigenetic mechanism through which environmental factors including obesity influence health. Obesity is a major modifiable risk factor for many common diseases including cardiovascular diseases and cancer. Obesity- 5 induced metabolic stress and inflammation are key mechanisms that affect disease risk and which may result from changes in methylation of metabolic and inflammatory genes. Objectives: This review aims to report the effects of weight loss induced by bariatric surgery (BS) on DNA methylation in adults with obesity focusing on changes in 10 metabolic and inflammatory genes. Methods: A systematic review was performed using Medline, EMBASE and Scopus, to identify studies in adult humans that reported DNA methylation following BS. Results: Out of 15996 screened titles, 15 intervention studies were identified, all of which reported significantly lower body mass index (BMI) post-surgery. DNA 15 methylation was assessed in five different tissues (blood=7 studies, adipose tissues =4, skeletal muscle =2, liver and spermatozoa). Twelve studies reported significant changes in DNA methylation after BS. Meta-analysis showed that BS increased methylation of PDK4 loci in skeletal muscle and blood in two studies while the effects of BS on IL6 methylation levels in blood were inconsistent. BS had no overall effect 20 on LINE1 or PPARGC1 methylation. Conclusion: The current evidence supports the reversibility of DNA methylation at specific loci in response to BS-induced weight loss. These changes are consistent with improved metabolic and inflammatory profiles of patients after BS. However, the 1 evidence regarding the effects of BS on DNA methylation in humans is limited and 25 inconsistent, which makes it difficult to combine and compare data across studies. Key words: Bariatric surgery, DNA methylation, obesity, inflammation 30 35 40 2 Introduction: 45 Obesity is a major modifiable, and preventable, risk factor for many common diseases including cardiovascular diseases and cancer1. Obesity increases disease risk by multiple mechanisms, including increased metabolic stress and chronic inflammation. Obesity-induced inflammation is orchestrated by metabolic cells, results in local expression of inflammatory mediators and creates a proinflammatory 50 tissue environment that is maintained in the long-term.2 This dysregulated metabolism is characterised by abnormal glucose metabolism, dyslipidemia and insulin resistance which subsequently increase the inflammatory response3. These effects lead to endothelial dysfunction and atherosclerosis, increasing the risk of cardiovascular diseases4. In addition, these mechanisms may underpin the greater 55 cancer risk in those with obesity.3 DNA methylation is an epigenetic mechanism through which obesity may influence disease risk. In humans, DNA is methylated by the addition of a methyl group to the 5’ position on cytosine (C) residues in CpG dinucleotides and is a key element in the regulation of gene expression5. Abnormal patterns of DNA methylation result in 60 reduced DNA integrity, changes in gene expression and mutations6. Patterns of DNA methylation respond to many environmental factors, including dietary interventions and weight loss7. Bariatric surgery (BS) is an effective therapy which induces long-term weight loss and improves comorbidities in obese patients.8 BS induces remission from type 2 65 diabetes (T2D) in a large proportion of initially obese patients, lowers risk of cardiometabolic disease9 and lowers incident cancer risk including breast, endometrial and colorectal cancers10. However, whilst these changes are associated 3 with decreased systemic and adipose tissue inflammation11, the underlying molecular mechanisms remain unresolved. 70 This systematic review reports the effects of weight loss induced by BS on DNA methylation in adults with obesity, aiming to: i) synthesize the evidence for the relationships between weight loss and corresponding changes in DNA methylation and ii) establish the links between these changes and specific metabolic and inflammatory genetic loci. 75 Methods: The systematic review is reported following the PRISMA checklist and flowchart12 (Supplementary Figure1). The systematic review was registered with PROSPERO (CRD42018112261). 80 Search strategy and screening: The databases, Embase, Scopus and Medline, were searched from inception until January 2019 by using the following search terms: ( ( (methylat*) OR methylation [Mesh] OR dna methylation [Mesh] ) AND ( ( Surg*) OR Surgery [Mesh] OR Bariatric Surgery [Mesh] ) ). Other databases that were searched included: Prospero, 85 Cochrane library, ClinicalTrial.gov and International clinical trials registry platform (WHO) for relevant protocols of clinical trials and systematic reviews that addressed DNA methylation and bariatric surgery. Articles were screened against the pre-set inclusion criteria (PICOS): a) Population: adult human beings (≥16 years old); b) Intervention: bariatric surgical interventions or 90 procedures; c) Comparator: healthy control group, other bariatric interventions, and 4 other interventions aiming for weight loss including dietary and physical exercise; d) Outcome: DNA methylation measured using any technique (global or locus specific) as a primary or secondary outcome, assessed before and after the intervention; e) Study design: any observational or intervention study, randomized or non- 95 randomized. Studies that recruited patients who had a history of, or were undergoing active treatment for, specific diseases (e.g. cancer) or patients with hereditary genetic disorders were excluded because of the likelihood that such conditions or therapies would confound the intervention effects. Titles and abstracts were screened by two independent reviewers (KE and FCM). 100 Neither of the reviewers was blind to the journal titles or to the study authors or institutions. Following screening of the titles and abstracts, full texts were reviewed to ensure eligibility for inclusion. Comparisons were made between the results of the two reviewers. Any discrepancy between their decisions regarding inclusion in the study was resolved by a third reviewer (JCM). 105 Data extraction, narrative synthesis and meta-analysis: The following data were collected using a pre-tested standard form: year of publication; study design; health or disease status of participants; number of participants; BMI of participants before and after intervention; nature of bariatric 110 intervention; duration of follow up; any other pre-procedure intervention; nature of other interventions; sample site; DNA methylation assessment method (including genomic loci, where appropriate), and DNA methylation levels of participants pre- and post- intervention, with measures of variance and level of significance. These 5 data were recorded using Microsoft® Excel 2017 which was used to synthesize 115 descriptive statistics and summary tables to support the narrative synthesis. Eligible studies were included in a meta-analysis conducted using the Review Manager software (v5.3, The Cochrane Collaboration, 2014) and intervention effects were quantified using a random effects model (due to heterogeneity) and standardized mean difference (due to the different methods used to quantify DNA 120 methylation). The quality of the included studies was assessed using the Newcastle- Ottawa Scale (NOS). Heterogeneity between studies was assessed using the Chi2 statistic (expressed as p value) and I2 statistics (expressed as percentage) using Review Manager v5.3. 125 Results: The PRISMA flowchart12 (Supplementary Figure 1) summarizes the outcomes of the search strategy. Out of 15996 screened titles, 15 studies were included. Of these studies, two were cross sectional and eight were cohort, three of which did not include a control group. None of the studies was a randomized controlled trial (RCT) 130 (Table 1). Two BS procedures were applied in the included studies: Roux-en-Y Gastric Bypass (RYGB, n=15) and Sleeve Gastrectomy (SG, n=3). In the fifteen studies, 312 obese patients underwent BS (range 6 – 120 patients, median =11) with an average follow up of 10.1 months (range 6 – 24 months). Mean BMI dropped from 45.9 kg/m2 (42.1- 135 50.9) to 32.8 kg/m2 (25.7-36.4) after BS. Only three studies13–15 reported mean BMI below the obesity cutoff (30) at ≥ 12-month follow-up. DNA methylation was 6 assessed in five different tissues: blood (n=7), adipose tissues (n=4), skeletal muscles (n=2), liver (n=1) and spermatozoa (n=1) (Table1). 140 Effects of bariatric surgery on DNA methylation in blood: Five studies investigated the effects of BS on DNA methylation at specific genomic loci in blood. Kirchner et al.13 followed up 7 patients who had undergone RYGB and for whom mean BMI was 27.3 kg/m2 after 12-month follow-up. Methylation of PDK4 (Pyruvate Dehydrogenase Kinase 4) (involved in metabolic homeostasis), IL1-B 145 (Interleukin 1 beta), IL6 (Interleukin 6) and TNF (Tumor Necrosis Factor) (inflammatory genes) in whole blood was significantly higher at the end of the 12- month follow-up compared with pre-surgery levels. In addition, methylation of IL1-B, IL6 and TNF and PPARGC1A (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha gene) was lower immediately after surgery (two days), indicating 150 that the effects of acute stress by BS on the inflammatory process may mediated through these hypomethylated inflammatory genes. Nicoletti et al16 investigated the effects of BS on the methylation