Several studies have demonstrated that bariatric surgery is effective for inducing weight loss in obese patients. In addition, the effects of this surgery on multiple associated alterations are well known, including changes in the secretion and activity of hormones involved in appetite regulation, satiety, and energy expenditure, as well as alterations in the gut microbiota composition. However, in cases of severe obesity, recent data have challenged the prevailing view, as bacterial species associated with low microbial richness (prior to surgery) appear to change only marginally after bariatric surgery, despite significant metabolic improvements. Our objective is to examine whether gut microbiota and gastrointestinal peptides are further impaired in severe obesity and, additionally, to explore how the microbiota relates to metabolic profile or sex, as well as whether bariatric surgery may differentially correct obesity-related intestinal microbial features. To this end, we propose a prospective, interventional, translational clinical study involving a cohort of 60 obese patients (BMI \> 35 kg/m²) undergoing laparoscopic gastric bypass surgery. Patients will be grouped according to their degree of obesity to assess potential baseline differences and to evaluate the efficacy of the intervention. Furthermore, we will investigate whether these parameters differ according to metabolic profile or sex. Body composition and nutritional status will be assessed, along with cardiovascular risk factors and comorbidities (hypertension, obstructive sleep apnea syndrome, dyslipidemia, type 2 diabetes mellitus, and insulin resistance). Gastrointestinal hormones (ghrelin, GIP, GLP-1, PYY, CCK, and leptin) will be measured in serum using Luminex XMAP technology. The content and diversity of the gut microbiota will be analyzed (16S rRNA amplicon sequencing and shotgun metagenomic sequencing using Illumina MiSeq technology) in stool samples collected before and 6-12 months after surgery. Additionally, individualized dietary follow-up and assessment of participants' quality of life will be conducted.

1. Introduction Obesity is a chronic disease with a major impact on health and a high economic burden. Its prevalence in the global population has increased from 5% in 1975 to 13% in 2014. Based on these figures, one fifth of the world's population is expected to be obese by 2025. Moreover, obesity is recognized as the most important risk factor for the development of type 2 diabetes mellitus (T2DM). Accordingly, a parallel rise in the prevalence of T2DM has been observed, currently affecting 9% of the world's population, with an estimated increase to 12% by 2025. The metabolic alterations associated with obesity increase the risk of cardiovascular diseases, including coronary heart disease and myocardial infarction. At present, bariatric surgery is the recommended treatment for weight reduction and improved glycemic control in obese patients, particularly in those who fail to achieve adequate results through medical therapy and lifestyle modification. Among the various surgical techniques available, laparoscopic gastric bypass (LGB)-which drastically reorganizes the gastrointestinal tract architecture and combines intestinal malabsorption with food intake restriction-is the most recognized and widely performed procedure in Europe for the treatment of morbid obesity. Recently, meta-analyses comparing metabolic outcomes across different bariatric procedures have been published; however, these are based on a small number of randomized controlled trials and mainly on studies with significant limitations, such as retrospective design, inclusion of heterogeneous cohorts with a wide range of body mass index and diabetes duration, and short follow-up periods. Most of these studies indicate comparable short-term outcomes; nevertheless, the STAMPEDE trial demonstrated superior metabolic results with LGB after three years. Conversely, studies involving strictly malabsorptive procedures have shown higher rates of T2DM remission and a lower risk of disease recurrence. A recent analysis of large, heterogeneous clinical cohorts identified shorter diabetes duration, lower baseline fasting glucose levels, and procedures diverting gastric contents distally into the small intestine-rather than restrictive procedures-as independent predictors of higher T2DM remission rates after two years of follow-up. It remains unclear whether this is a consequence of greater weight loss achieved through these procedures or whether other factors are involved, particularly intestinal hormone dynamics. Intestinal bypass surgery excluding the duodenum leads to increased secretion of incretin hormones, specifically glucagon-like peptide 1 (GLP-1) and the orexigenic peptide YY (PYY), due to rapid gastric emptying and the early arrival of undigested food in the ileum. The exaggerated GLP-1 secretion is considered one of the main mechanisms underlying improved β-cell function in T2DM after surgery. However, some studies have questioned the role of GLP-1 as the primary factor responsible for diabetes remission. Additionally, exclusion of the gastric fundus results in reduced secretion of the orexigenic hormone ghrelin, leading to appetite suppression and increased insulin secretion. Indeed, both mechanisms have been described in observational studies following LGB and are associated with improved glucose homeostasis. Furthermore, recent studies suggest that the gut microbiota may play an important role in metabolic regulation and in the modulation of both local and systemic inflammatory responses. Obesity and T2DM are associated with changes in the abundance and composition of intestinal bacteria, as well as in their biological diversity. The exact mechanisms through which the gut microbiota influences metabolic traits remain unclear, but they may involve fermentation products of dietary fiber, particularly short-chain fatty acids (SCFAs: butyrate, propionate, and acetate), which affect energy homeostasis. The recent discovery of receptors across a wide range of cell types and tissues for which SCFAs appear to be natural ligands further highlights their potential role in gut-microbiota-metabolism interactions. Another relevant mechanism of direct microbiota influence on metabolic regulation may lie in its connection with the onset and progression of subclinical inflammation, which contributes to the development of insulin resistance and T2DM. It has been proposed that obesity and T2DM are associated with increased translocation of bacterial lipopolysaccharides into the circulation, subsequently triggering innate immune activation within adipose tissue. While modulation of the gut microbiota has been shown to effectively prevent obesity and improve metabolic status in rodent models of obesity and T2DM, human data supporting such effects in obese diabetic patients are far less conclusive. A recent study exploring the impact of fecal microbiota transplantation from lean donors demonstrated moderate improvements in insulin sensitivity in male recipients with metabolic syndrome six-but not eighteen-weeks after transplantation. Therefore, the gut microbiota appears to be a relevant contributor and is likely a key factor bridging lifestyle and host biology. To date, intestinal dysbiosis has been identified in overweight and moderate obesity, as evidenced by substantial changes in gut microbiota composition (enrichment or depletion of specific bacterial groups) and reduced microbial gene richness, both associated with metabolic disturbances such as insulin resistance, low-grade inflammation, and adipocyte hypertrophy. However, gut microbiota characteristics remain poorly explored in extreme forms of obesity, despite the increasing prevalence of severe (BMI \> 35 kg/m²) and morbid obesity (BMI \> 40 kg/m²) worldwide, currently affecting 2.3% of men and 5% of women, respectively. Although some individuals with severe obesity remain metabolically healthy, in general, reaching a BMI \> 35 kg/m² significantly increases the risk of chronic disorders. Moreover, metabolically healthy obese individuals often develop metabolic alterations and comorbidities over time. Despite the well-documented improvements in metabolism and inflammation after bariatric surgery-and its association with gut microbiota changes-recent studies indicate that reduced microbial richness and compositional alterations are linked to adverse metabolic outcomes in overweight or moderate obesity, but remain poorly characterized in severe obesity. Furthermore, emerging evidence suggests that gut microbiota composition does not fully recover following bariatric surgery. In addition, Santos-Marcos et al. recently reported sex-related differences in gut microbiota composition, as well as variations among women in different hormonal stages, which may underlie the sexual dimorphism observed in the incidence of metabolic diseases and related comorbidities. Bariatric surgery is an effective intervention that promotes weight loss and improves glycemic control. In addition to the positive modulation of bile acids and intestinal hormones, this surgery induces profound changes in the composition of the gut microbiota, an important contributor to the health effects mediated by bariatric procedures. However, in cases of severe obesity, recent data have challenged the prevailing view, as bacterial species associated with low microbial richness (prior to surgery) appear to change only marginally after bariatric surgery despite substantial metabolic improvements. To test the validity or inconsistency of this premise, we established a comparative analysis of metabolic parameters and risk factors in obese individuals undergoing surgical weight-loss intervention. Furthermore, the expression and identification of peripheral biomarkers, such as gastrointestinal peptides, may enable better characterization of obesity phenotypes and a more precise diagnosis, thereby improving the treatment and follow-up of obesity on an individualized basis. In addition, we propose to explore whether alterations in gut microbiota are closely related to metabolic abnormalities and whether weight-loss intervention through bariatric surgery may determine a differential response in gut microbial diversity and metabolic biomarkers across different degrees and phenotypes of obesity, as well as between sexes. 2. Materials and methods 2.1. Study Design and Participants A prospective, interventional, comparative, and translational clinical study was designed. To achieve the proposed objectives, 60 obese patients scheduled to undergo bariatric surgery in the Department of General and Digestive Surgery at the same hospital will be recruited. Patients will be sequentially enrolled as they attend the Obesity Unit of the Endocrinology Department at Hospital Universitario Doctor Peset until the required number is reached and provided they meet the inclusion and exclusion criteria described below. * Inclusion criteria: patients with a body mass index (BMI) greater than 40 kg/m² (or \> 35 kg/m² with minimum one obesity comorbidity), aged between 18 and 65 years, and with a known duration of obesity exceeding five years. * Exclusion criteria: due to the nature of the study, patients with acute or chronic inflammatory diseases, established hepatic or renal insufficiency (defined as transaminase levels ±2 SD from the mean and estimated glomerular filtration rate \[CKD-EPI formula\] \>60), neoplasic diseases, or secondary causes of obesity (e.g., hypothyroidism, Cushing's syndrome) will be excluded. Before participating, all patients-or their legal representatives-will sign an informed consent form after receiving a detailed explanation of the study objectives and having all questions answered. The study will adhere to the ethical principles of the Declaration of Helsinki and its subsequent amendments and will receive approval from the Hospital Ethics Committee. All medical information and data collected during the study will be kept confidential in accordance with the national laws. To assess the influence of adiposity degree, patients will be classified into three groups according to BMI: 1) Severe obesity: BMI 35-40 kg/m², 2) Morbid obesity: BMI 40-50 kg/m², 3) Extreme obesity: BMI \>50 kg/m². To evaluate the influence of metabolic profile, patients will also be divided into two groups: metabolically healthy obese (MHO) and metabolically abnormal obese (MAO), adjusted for age and sex. According to previously established criteria, MAO individuals are defined as having at least two of the following cardiometabolic alterations: 1) Fasting plasma glucose ≥100 mg/dL or use of antidiabetic medication; 2) Systolic blood pressure (SBP) ≥130 mmHg and/or diastolic blood pressure (DBP) ≥85 mmHg, or use of antihypertensive medication; 3) Triglyceride concentrations ≥150 mg/dL; 4) HDL-cholesterol \<40 mg/dL in men or \<50 mg/dL in women, or use of lipid-lowering drugs. Conversely, MHO individuals are defined as those who do not meet any of the above criteria. In addition, sex-related differences will also be analyzed. Prior to surgery, the dietitian-nutritionist will implement a dietary intervention consisting of a hypocaloric diet individualized according to the patient's nutritional requirements and adjusted for obesity. The dietary plan will be qualitatively complete, adhering to the following macronutrient distribution: 55% carbohydrates, 30% fats, and 15% proteins. During the two weeks preceding the metabolic surgery procedure, a very low-calorie diet will be administered using Optisource® sachets, divided into three daily intakes. The post-bariatric surgery dietary intervention will be carried out progressively, aiming at weight loss and metabolic normalization. Follow-up visits will occur at 1, 6, and 12 months, taking into account individual tolerance and nutritional requirements. 2.2. Data collection and analysis All subjects will undergo an initial evaluation, including nutritional status assessment and cardiovascular risk factors and comorbidities (hypertension, obstructive sleep apnea, dyslipidemia), as well as evaluation to rule out secondary causes of obesity. Throughout the study, both at baseline and during post-surgical follow-ups, patients will undergo analytical assessments, body composition and basal metabolism evaluation, microbiota studies, and gastrointestinal hormone determination. The obtained results will be analyzed using SPSS 17.0. For categorical variables, the distribution of frequencies and total percentage of cases will be evaluated. For quantitative variables, the mean, range, and standard deviation will be analyzed. The Kolmogorov-Smirnov test will be performed to determine whether quantitative variables follow a normal distribution. Differences in metabolic variables between groups (sex, metabolic profile) will be analyzed using an unpaired t-test or the Mann-Whitney U test, and for obesity grades, an ANOVA will be performed. To evaluate changes after surgical intervention, a repeated measures ANOVA or paired t-test will be conducted. Finally, correlations will be assessed using Pearson or Spearman bivariate correlation. Additionally, logistic regression analyses will be performed to develop predictive models for specific groups.