Ahmed Abdelrafee

MD Julio Nunez

MD Luis Cano

PhD Marc-Antoine Allard

MD PhD Gabriella Pittau

MD Oriana Ciacio

MD Chady Salloum

MD Antonio Sa Cunha

MD Denis Castaing

MD Daniel Azoulay

PhD Daniel Cherqui

PhD René Adam

MD PhD Eric Vibert

MD PhD Nicolas Golse
email: nicolasgolse@me.com

Predictors of unresectability after portal vein embolization for centrally located cholangiocarcinoma

Keywords: cholangiocarcinoma, portal vein embolization, sarcopenia, score, resection, complications CLCC: centrally-located cholangiocarcinoma ERCP: endoscopic retrograde cholangio-pancreatography FLR: future liver remnant IHCC: intrahepatic cholangiocarcinoma MDCT: multidetector computed tomography MRI: magnetic resonance imaging NLR: neutrophil-to-lymphocyte ratio OS: Overall survival PET: positron emission tomography PHCC: perihilar cholangiocarcinoma platelet-to-lymphocyte ratio PMA: psoas muscle area PMI: psoas muscle index PVE: portal vein embolization ROC: receiver operator characteristic

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

inexpensive parameters should be considered in all patients with CLCC scheduled to undergo PVE.

INTRODUCTION

Cholangiocarcinoma (CC) is a malignancy originating from the epithelial lining of the biliary ducts, representing 3% of all gastrointestinal malignancies [START_REF] Lazaridis | cholangiocarcinoma. gastroenteology[END_REF][START_REF] Kambakamba | Perihilar cholangiocarcinoma: paradigms of surgical management[END_REF] . Centrally-located cholangiocarcinomas (CLCC) include both perihilar cholangiocarcinoma (PHCC) and centrallylocated intrahepatic cholangiocarcinomas (IHCC). PHCC, the most common subtype accounting for 60-70% of CC [START_REF] Deoliveira | Advances in liver surgery for cholangiocarcinoma[END_REF] , develops from the right and/or left hepatic ducts or common bile duct near the hilum [START_REF] Bismuth | Intrahepatic cholangioenteric anastomosis in carcinoma of the hilus of the liver[END_REF] . These neoplasms remain a challenging entity with a poor prognosis [START_REF] Bismuth | Intrahepatic cholangioenteric anastomosis in carcinoma of the hilus of the liver[END_REF] . Radical resection is the only possible chance to achieve long-term survival [START_REF] Rassam | Modern work-up and extended resection in perihilar cholangiocarcinoma: the AMC experience[END_REF] , but, at the time of laparotomy, only 40-70% of patients have resectable disease [START_REF] Coelen | Diagnostic accuracy of staging laparoscopy for detecting metastasized or locally advanced perihilar cholangiocarcinoma: a systematic review and meta-analysis[END_REF] . Resection of PHCC often requires a major hepatectomy with extrahepatic bile duct resection, hepatic pedicle lymphadenectomy, and sometimes vascular reconstruction [START_REF] Neuhaus | Surgical management of proximal bile duct cancer: extended right lobe resection increases resectability and radicality[END_REF] to obtain negative surgical margins [START_REF] Esnaola | Evaluation and management of intrahepatic and extrahepatic cholangiocarcinoma[END_REF] . These procedures, however, remain associated with a substantial risk of postoperative morbidity and mortality [START_REF] Coelen | External Validation of the Estimation of Physiologic Ability and Surgical Stress (E-PASS) Risk Model to Predict Operative Risk in Perihilar Cholangiocarcinoma[END_REF] .

The major concern with radical resection in potentially resectable CLCC is the small future liver remnant (FLR), increasing the risk of post-hepatectomy liver failure which is the most common cause of postoperative mortality [START_REF] Alvarez | Natural history of portal vein embolization before liver resection: a 23-year analysis of intention-to-treat results[END_REF] . For this reason, Makuuchi et al. [START_REF] Makuuchi | Preoperative transcatheter of the portal venous branch for patients receiving extended lobectomy due to the bile duct carcinoma[END_REF] were the first to introduce portal vein embolization (PVE) before hepatic resection. PVE is the gold standard strategy to increase FLR volume, because the hyertrophy after PVE permits safer major liver resection with a decreased risk of liver failure and death [START_REF] Clavien | Strategies for Safer Liver Surgery and Partial Liver Transplantation[END_REF] . PVE is indicated when the FLR volume is less than 20-30% of the total liver volume or less than 0.5% of the body weight ratio (BWR) in normal liver [START_REF] Kishi | Three hundred and one consecutive extended right hepatectomies: evaluation of outcome based on systematic liver volumetry[END_REF][START_REF] Truant | Remnant liver volume to body weight ratio > or =0.5%: A new cut-off to estimate postoperative risks after extended resection in noncirrhotic liver[END_REF] . Specific cut-offs in case of cholestatic livers, however, are not clearly defined. Resectability rates after PVE before major hepatectomy ranging from 58% to 100% have been reported in the literature, [START_REF] Madoff | Transhepatic Portal Vein Embolization: Anatomy, Indications, and Technical Considerations[END_REF][START_REF] Abdalla | Extended hepatectomy in patients with hepatobiliary malignancies with and without preoperative portal vein embolization[END_REF] . Many studies reported the reasons for unresectability after PVE under an intention-to-treat analysis, but predictors of unresectability after PVE for CLCC have not been clarified in the literature. These predictors could have a major impact on the decision-making process, particularly in borderline patients.

The aim of this study was therefore, to assess predictors of unresectability after PVE for CLCC in an intention-to treat analysis.

METHODS

Study design and population

We reviewed all patients who underwent PVE before planned major hepatectomy (≥3 segments) for potentially resectable CLCC between January 2000 and December 2018, whether the the patients finally underwent hepatectomy or not. CLCC included PHCC and IHCC extending to the first-order portal pedicle branches and requiring major hepatectomy. We merged both entities, because patients with centrally-located IHCC usually require a major hepatectomy with biliary reconstruction (± vascular reconstruction) as for PHCC. Patients with gallbladder carcinoma, IHCC not extending to the hilum, and distal CC (not requiring hepatectomy) were excluded. All patients were evaluated by our institutional multidisciplinary tumor board. Patients were scheduled for PVE if they had insufficient FLR volume, in normal liver, <30% of total liver volume and/or <0.5% of BWR [START_REF] Truant | Remnant liver volume to body weight ratio > or =0.5%: A new cut-off to estimate postoperative risks after extended resection in noncirrhotic liver[END_REF] , and empirically in cholestatic liver, <40% of TLV or <0.8% of BWR [START_REF] Alvarez | Natural history of portal vein embolization before liver resection: a 23-year analysis of intention-to-treat results[END_REF] .

Preoperative radiologic assessment of resectability

Comprehensive radiologic assessment was done for all patients before any biliary drainage.

Before 2010, all patients were evaluated by multi-detector, computed tomography (MDCT) and CT angiography to evaluate the relation of the tumor to the hepatic artery and portal vein, and on demand in few cases, with magnetic resonance imaging (MRI) and a positron emission tomography (PET) scan for detection of extrahepatic disease (because MRI and PET scan were not available in our center). Starting from the year 2010, all patients were assessed by MDCT, MRI, magnetic resonance cholangiopancreatography (MRCP), and PET scan.

Data collection

The demographic and preoperative nutritional parameters of patients, as well as laboratory investigations including a complete blood count, liver function tests, and tumor markers were all collected prior to PVE. Patients with jaundice underwent biliary drainage of FLR by endoscopic retrograde cholangio-pancreatography (ERCP) as first-line treatment, and it was usually accompanied by endoscopic biopsy (brush cytology) of the tumor, or percutaneous transhepatic drainage in the event of failure. The total number of drainage procedures, the side of the liver drained, post-drainage complications, and the degree to which serum bilirubin levels decreased after drainage were also recorded.

We also studied markers of the inflammatory response. i.e. the neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) and focusing specifically on previously reported cut-off values for NLR and PLR [START_REF] Chen | The elevated preoperative neutrophil-to-lymphocyte ratio predicts poor prognosis in intrahepatic cholangiocarcinoma patients undergoing hepatectomy[END_REF][START_REF] Kitano | Effects of Preoperative Neutrophil-to-Lymphocyte and Platelet-to-Lymphocyte Ratios on Survival in Patients with Extrahepatic Cholangiocarcinoma[END_REF][START_REF] Lin | Elevated neutrophil-to-lymphocyte ratio is an independent poor prognostic factor in patients with intrahepatic cholangiocarcinoma[END_REF][START_REF] Dumitrascu | Major hepatectomies for perihilar cholangiocarcinoma: Predictors for clinically relevant postoperative complications using the International Study Group of Liver Surgery definitions[END_REF][START_REF] Lee | Neutrophil-lymphocyte ratio predicts survival in patients with advanced cholangiocarcinoma on chemotherapy[END_REF][START_REF] Beal | Elevated NLR in gallbladder cancer and cholangiocarcinomamaking bad cancers even worse: results from the US Extrahepatic Biliary Malignancy Consortium[END_REF] . Preoperative parameters of nutritional assessment were analyzed using previously published cut-off values that included markers for muscle mass depletion (sarcopenia):

• Psoas muscle index (PMI): psoas muscle area (PMA) measured on the axial section of a CT through the third lumbar vertebra, normalized by height squared [START_REF] Golse | A new definition of sarcopenia in patients with cirrhosis undergoing liver transplantation[END_REF] with cut-off value of 500 mm 2 /m 2 24 .

• PMA normalized by body surface area (BSA) [START_REF] Durand | Prognostic value of muscle atrophy in cirrhosis using psoas muscle thickness on computed tomography[END_REF] .

All these variables were evaluated before PVE.

Technical details regarding the PVE procedure and post-PVE assessment

All PVE procedures were performed by experienced hepatobiliary surgeons. Different approaches were used (either percutaneous transhepatic or ileocolic (ELSEVIER HAVE THEM EXPLAIN WHAT THEY MEAN BY ILEOCOLIC), depending on technical feasibility and the surgeon's preference. A control portal venography was performed, then the portal branches supplying the diseased side of liver were catheterized selectively, a guidewire was introduced, and embolization was initiated. The embolic agents used included n-butylcyanoacrylate mixed with ethiodized oil or absolute ethanol. We did not embolize the portal branch of segment IV in cases of planned extended right hepatectomy. Total venous deprivation (PVE and hepatic vein occlusion) was not performed during this study.

Daily clinical evaluations and liver function tests were ensured to enable the rapid detection of post-PVE complications. Volumetric assessments were made regarding total liver volume and FLR, both before and then 3 or 4 weeks after PVE, based on volumetric CT reconstructions. If the FLR reached a sufficient volume, the patient was scheduled for liver surgery. Otherwise, provided that all branches of the portal vein supplying the part of liver to be resected were correctly occluded on CT images, a further volumetric assessment was performed after 4 weeks.

When the targeted branches of the portal vein were still permeable or partially embolized, a supplementary PVE was scheduled. PVE was considered to have failed when there was an insufficient increase in FLR volume or contralateral embolization of non-targeted portal vein branches in the FLR. Post-PVE follow-up also included subsequent CTs enabling evaluation of the tumor and potential detection of any local progression or distant metastases.

Operative exploration and assessment of resectability

During operative exploration by laparotomy or laparoscopy, all patients were evaluated for distant metastases in the form of peritoneal nodules, malignant ascites, liver metastases, distant lymph node metastases or locally advanced tumor which precluded resection. In the event of nonresectability (defined as non-radical resection or insufficient FLR), patients were referred for palliative chemotherapy or were assessed for their eligibility for liver transplantation according to the Mayo clinic protocol [START_REF] Heimbach | Liver transplantation for unresectable perihilar cholangiocarcinoma[END_REF] (and included in the non-resected group for our study because of the intention-to-treat analysis). No further procedures were performed during this exploratory surgery.

The whole cohort was divided into two groups: a resected group which included patients who underwent liver resection, and an unresected group including the patients who did not undergo operative exploration or underwent exploration alone (no resection). Overall survival (OS) was calculated as the interval between the first outpatient visit and death or the last follow-up visit.

The primary endpoint was the detection of predictors of unresectability after PVE for CLCC in an intention-to-treat analysis based on pre-PVE parameters whatever the reasons of unresectability.

This study was approved by the Institutional Review Board for our institution.

Statistical analysis

Categorical variables are described using absolute numbers with percentages. Continuous variables are expressed as medians and ranges. The Chi-square or Fisher exact test was used to compare categorical variables, with Student's t test or the Mann-Whitney U test for continuous variables. A stepwise logistic regression test was used to detect predictors of unresectability.

After univariate analysis, variables with P value <0.1 were included in the multivariate analysis.

A P value ≤0.05 was considered to be statistically significant. A prognostic model based on the predictors thus identified (multivariate analysis) was built, and the probability of unresectability was calculated. For each variable considered in the composite model, a receiver operator characteristic (ROC) curve enabled identification of the best cut-off value (Youden index). The discriminatory power of this model (+1 point when the cut-off point was reached) in predicting unresectability was presented as an AUROC (area under the ROC) and its associated 95% confidence interval. The Kaplan-Meier test was applied for survival analysis and the difference in survival between the groups was compared using a log rank test. All statistical analyses were performed using SPSS statistical software, version 23.0 (SPSS, Inc., Chicago, IL).

RESULTS

Patient characteristics

A total 88 patients were included, 56 (64%) who underwent liver resection after PVE (resected group) and 32 (36%) in the unresected group. Eleven patients (13%) did not undergo operati e l exploration because of the failure of PVE (n=6; 55%), tumor progression detected on post-PVE CT images (n=3; 27%), severe septicemia, septic shock after ERCP leading to multi-organ failure and death (n=1; 9%), and a contraindication to anesthesia (n=1; 9%) (Fig 1). Seventy-seven patients (88%) underwent operative exploration to assess resectability, 21 of whom (24%) were considered to be unresectable. The most frequent reason for unresectability was tumor progression (86%), whether it was intrahepatic or extrahepatic, followed by cirrhotic liver with early signs of portal hypertension and ascites that were not detected in the preoperative imaging (10%); finally, no mass found in the hilar region with a negative frozen section and normal cholangiogram in one patient. The tumor progression was most often found intra-operatively (n=18), mainly in patients diagnosed before 2010 (n=12) due to limited availability of modern imaging in this period.

Patterns of tumor progression a-Before operative exploration

Three patients (4%) with tumor progression were detected before operative exploration on post-PVE imaging. One patient each developed lung and left suprarenal gland metastases, bone metastasis, and intra-hepatic metastases associated with aorto-caval lymph node metastases diagnosed by PET scan.

b-During operative exploration

Tumor progression was detected intra-operatively in 18 patients (21%) and included local direct extension (n=7), intrahepatic metastases (n=4), and extrahepatic metastases (n=7) in the form of peritoneal metastases (n=5) and LN metastases (n=2).

The demographic, clinical, and pathologic characteristics of both patient groups are summarized in Table 1. Unresected patients displayed a somewhat greater body mass index (BMI) (25.5 vs. 23.7 kg/m 2 ; p=0.049). As for nutritional parameters (sarcopenia), a greater proportion of patients in the unresected group had a PMI <500 mm 2 /m 2 than in the resected group (84% vs 56%; p=0.018, respectively). The PMA/BSA ratio was much less in the unresected group (618 vs. 753 mm 2 /m 2 ; p=0.033). As shown in Table 2, there were more patients with hypoalbuminemia (<35 g/L) and PLR>185 in the unresected group than in the resected group (p=0.046 and p=0.02, respectively).

Preoperative biliary drainage and PVE

There were no differences regarding data on biliary drainage, which were comparable in terms of the number of drainage procedures, the side of the stented bile duct, and post-procedure complications (Table 3). Nine patients (10%) did not undergo biliary drainage before PVE, two of whom were in the unresected group. In relation to PVE, 56 patients (73%) underwent the first biliary drainage (BD) before PVE, 17 patients (22%) underwent biliary drainage and PVE during the same hospitalization, and 3 patients (4%) underwent biliary drainage after PVE before operative exploration. The median interval between the first drainage and the time of operative exploration was greater in unresected patients (90 vs 71 days; p=0.093). Ten patients required a single additional session of PVE to achieve complete occlusion of the targeted branches of the portal vein (seven and three patients in the resected and unresected groups, respectively; p=0.657). The median interval between PVE and operative intervention in the resected group was 55 days, range (9-323). Note that in one case we observed a long interval between PVE and resection (323 days), because the patient had developed metastases to the hepatic pedicle and celiac LN during the intial post-PVE operative exploration after which he received chemotherapy (GEMOX) and multiple sets of biliary drainage. Then, after re-evaluation, there was a very good response to chemotherapy allowing for complete resection of the tumor and lymphadenectomy. Among the 77 patients who underwent exploration, nine (12%) needed a complementary PVE session due to insufficient FLR leading to a greater interval (> 55 days) until the time of operative exploration.

Complications after PVE

Eighteen of the 88 patients experienced complications after PVE (21%). Major complications (Dindo-lavien ≥ IIIa) occurred in four patients in the resected group and two in the unresected group. In the resected group, two perihepatic collections and one liver abscess required percutaneous drainage, while one case of hematoma in the mesentery of the right colon leading to severe ischemia required right hemicolectomy. In the unresected group, one patient developed liver failure with encephalopathy associated with pneumonia and sepsis which required management on the intensive care unit, and another experienced a pneumothorax requiring the insertion of a pleural catheter.

Failure of PVE

PVE failed in nine patients (28%) in the unresected group: four patients with erratic embolization (two with embolization of the portal branches of the FLR, one with incomplete occlusion of the portal branches in the diseased part of the liver that would be resected, and one who presented with both events), two with portal vein thrombosis of the FLR, one with insufficient hypertrophy of FLR, one lost to follow-up after PVE, and one with severe portal hypertension contraindicating PVE. Three of these 9 patients achieved unsatisfactory hypertrophy with borderline FLRV and underwent exploration and finally were not resected due to advanced disease (n=2) and cirrhosis with ascites (n=1). In the resected group, two patients presented with erratic embolization; the first was prepared for extended right hepatectomy and developed thrombosis of the proximal part of the left portal vein of the FLR while the right anterior sectorial portal branch was still permeable with insufficient hypertrophy (FLR/BWR=0.4%); therefor this patient underwent an additional PVE; in the second case, accidental embolization of the contralateral side occurred, but sufficient hypertrophy was achieved (FLR/BWR=0.64).

Operative and post-operative outcomes in the resected group

There were 79% and 21% of right (± extended) and left extended hepatectomies, respectively as shown in Table 4. Eighteen patients (32%) underwent portal vein reconstruction, while arterial resection was performed in 5 (9%) cases. Two patients underwent resection under venovenous bypass. The 90-day post-resection mortality rate was 16% (9 patients). The most common cause of mortality was post hepatectomy liver failure(defined according 50/50 criteria 27 ) in 6 patients (11%) followed by infectious complications with septicemia and septic shock in 3 patients (5%) due to biliary fistula (n=2), and leakage from distal entero-enteric anastomosis with peritonitis (n=1). There was no difference in the post-PVE FLR volume between survivors and early death patients (p=0.592). Median OS was greater in the resected than in the unresected group (30.2 vs. 17.2 months, respectively; p=0.119).

Management of unresected patients

Three patients (9%) in the unresected group underwent liver transplantation according to the Mayo clinic protocol [START_REF] Heimbach | Liver transplantation for unresectable perihilar cholangiocarcinoma[END_REF] , two of whom had long overall survival (89 and 139 months), 19 patients (59%) received palliative chemotherapy, palliative care (n=8), and 2 patients were lost follow up.

Recurrent cholangitis in all patients was managed by repeated biliary stents either by endoscopic (n=26) or percutaneous approaches (n=5); one patient was managed by hepatico-enterostomy (Roux-en-Y). One patient underwent right hemicolectomy due to intestinal obstruction because of of peritoneal carcinomatosis.

Long-term outcomes following PVE

The median follow-up of the entire cohort was 21.2 [0.8 -142.2] months. Resected patients had better OS than those not resected (1-y, 3-y, 5-y OS=70%, 578%, and 25% vs. 75%, 22%, 18%; p=0.006) as shown in Figure 2. Sarcopenia did not associate with any difference in OS between the resected and unresected groups (median OS of 15.4 months and 21.9 months, respectively; p=0.783). There was no difference in OS between the PHCC and IHCC (p=0.359), while IHCC had worse DFS than PHCC (p=0.018) in the resected group.

Predictors of unresectability after PVE

Univariate and multivariate analyses of the predictors of unresectability after PVE are detailed in Table 5. Univariate analysis found that PMI <500 mm 2 /m 2 , a low PMA/BSA, a low serum albumin level, and PLR>185 were associated with a greater rate of unresectability. PMI <500 mm 2 /m 2 (OR=6.030; p=0.040; 95% CI 1.218-37.981), high BMI (OR=1.346; p=0.023; 95% CI 1.041-1.741), and low serum albumin levels (OR=1.793; p=0.007; 95% CI 0.669-0.939) were independent predictors of unresectabilty after PVE under multivariate analysis.

Using ROC curve analyses to predict unresectability, the best cut-off point for albumin was 33 g/L (AUC=0.723; p=0.001; 95% CI 0.605 -0.841), while for BMI it was 24.8 kg/m 2 (AUC=0.620; p=0.020; 95% CI 0.529 -0.770). In addition, we built a score using these three independent predictors of unresectability (PMI< 500 mm 2 /m 2 , albumin ≤ 33 mg/L, and BMI ≥ 24.8 kg/m 2 ). We stratified this score over 0 -3 points, with one point for each factor present. The incidence of unresectability with scores of 0, 1, 2, and 3, were 0%, 5%, 54%, and 64%, respectively (p <0.001). This score achieved very good discrimination with respect to unresectability (AUC=0.817), as shown in Figure 3.

In univariate analysis of predictive factors of PHLF and 90-day mortality in the resected group, PMI <500 mm 2 /m 2 , serum albumin ≤33 mg/L, and BMI ≥24.8 kg/ m2 were not significant predictors.

DISCUSSION

This is the first study to have demonstrated that a low PMI, high BMI, and low serum albumin level are independent predictors of unresectabilty after PVE. These findings highlight the negative impact of sarcopenia as an important risk factor for unresectability after PVE for CLCC. This simple score proposed, available at the first consultation, can discriminate accurately (AUC=0.817) the chance of resectability.

Sarcopenia is defined as a syndrome characterized by a progressive and generalized loss of skeletal muscle mass and strength [START_REF] Baeyens | European Working Group on Sarcopenia in Older People. Sarcopenia: European consensus on definition and diagnosis: Report of the European Working Group on Sarcopenia in Older People[END_REF][START_REF] Hahn | The role of sarcopenia in patients with intrahepatic cholangiocarcinoma: Prognostic marker or hyped parameter?[END_REF] and is associated with several cancers [START_REF] Voron | Sarcopenia Impacts on Short-and Long-term Results of Hepatectomy for Hepatocellular Carcinoma[END_REF][START_REF] Miyamoto | Sarcopenia is a Negative Prognostic Factor After Curative Resection of Colorectal Cancer[END_REF] . Preoperative sarcopenia is a poor prognostic factor after hepatic, biliary, or pancreatic surgery [START_REF] Okumura | Impact of Skeletal Muscle Mass, Muscle Quality, and Visceral Adiposity on Outcomes Following Resection of Intrahepatic Cholangiocarcinoma[END_REF][START_REF] Zhang | Clinical significance of sarcopenia in the treatment of patients with primary hepatic malignancies, a systematic review and meta-analysis[END_REF] .In contrast, obesity represents a substantial risk factor for many health disorders, including cholangiocarcinomas [START_REF] Calle | Overweight, obesity, and mortality from cancer in a prospectively studied cohort of U.S. adults[END_REF][START_REF] Jing | Diabetes mellitus and increased risk of cholangiocarcinoma: a metaanalysis[END_REF] , and is associated with poor outcomes in patients with hepatocellular carcinoma [START_REF] Fujiwara | Sarcopenia, intramuscular fat deposition, and visceral adiposity independently predict the outcomes of hepatocellular carcinoma[END_REF] . The combined condition of sarcopenia and obesity, or sarcopenic obesity, has been described as a prognostic factor for poor outcomes in the case of solid tumors [START_REF] Prado | Prevalence and clinical implications of sarcopenic obesity in patients with solid tumours of the respiratory and gastrointestinal tracts: a populationbased study[END_REF] , but not until now in the setting of CLCC assessment.

In the present series, we showed that 84% of patients in the unresected group had sarcopenia with PMI <500 mm 2 /m 2 and a greater BMI than the resected group. There was no difference in OS between resected and unresected sarcopenic patients (p=0.783), although resected sarcopenic patients tended to present a poorer OS (three unresected patients were transplanted, thus increasing the OS of the whole subgroup). In a retrospective study of Coelen et al. [START_REF] Coelen | Preoperative computed tomography assessment of skeletal muscle mass is valuable in predicting outcomes following hepatectomy for perihilar cholangiocarcinoma[END_REF] showed that low skeletal muscle mass (<46.8 cm 2 /m 2 in males and <39.1 cm 2 /m 2 in females) was an independent predictor of early postoperative mortality (p=0.009) and poor OS (HR=2.02; p=0.020; 95% CI 1.12 -3.65), while in our study, a low PMI (<500 mm 2 /m 2 ) was not a predictor for 90-day mortality and poor OS (p=0.201). Van Vugt et al. [START_REF] Van Vugt | Low Skeletal Muscle Density Is Associated with Early Death in Patients with Perihilar Cholangiocarcinoma Regardless of Subsequent Treatment[END_REF] showed that low skeletal muscle mass did not indicate a difference in OS in any treatment group (resected, laparotomy without resection, initially unresectable), and the same finding was shown in the present study. To date, no reports have assessed the predictive effect of sarcopenia on the resectability of patients.

A high PLR has been reported as affecting the development of sarcopenia [START_REF] Liaw | Higher Platelet-to-Lymphocyte Ratio Increased the Risk of Sarcopenia in the Community-Dwelling Older Adults[END_REF][START_REF] Kitano | Sarcopenia Affects Systemic and Local Immune System and Impacts Postoperative Outcome in Patients with Extrahepatic Cholangiocarcinoma[END_REF] and promoting tumor progression and hematogenous metastases [START_REF] Balkwill | Inflammation and cancer: back to Virchow?[END_REF][START_REF] Grivennikov | Immunity, inflammation, and cancer[END_REF] . In our study, a PLR >185 was a risk factor for unresectability under univariate analysis (p=0.023) but did not reach a level of significance under multivariate analysis (p=0.072).

Previous studies have reported that lower serum albumin levels were a poor prognostic indicator of survival in several cancers regardless of therapeutic management [START_REF] Gupta | Pretreatment serum albumin as a predictor of cancer survival: a systematic review of the epidemiological literature[END_REF][START_REF] Lai | Low preoperative serum albumin in colon cancer: a risk factor for poor outcome[END_REF] . In the study by Waghray et al. [START_REF] Waghray | Serum albumin predicts survival in patients with hilar cholangiocarcinoma[END_REF] , a serum albumin level <30 g/L was identified as an independent predictor of OS after resection of hilar cholangiocarcinoma (HR 0.31; 95% CI 0.14-0.70). During our study, serum albumin levels (≤ 33g/L) were found to be an independent risk factor for unresectability after PVE. This observation could be explained by cancer cachexia and an inflammatory response resulting in an increase in the catabolic state, a loss of vital body proteins, and changes to immune status with tumor progression [START_REF] Waghray | Serum albumin predicts survival in patients with hilar cholangiocarcinoma[END_REF] .

The aforementioned results of recent studies have demonstrated the importance of perioperative nutritional therapy and regular exercise in preventing frailty, improving skeletal muscle mass and physical function and achieving an improvement in OS [START_REF] Kaido | Impact of sarcopenia on survival in patients undergoing living donor liver transplantation[END_REF][START_REF] Landi | Exercise as a remedy for sarcopenia[END_REF] . The preoperative immunonutrition was reported to have improved the outcome of liver resection for cancer [START_REF] Ciacio | Interest of preoperative immunonutrition in liver resection for cancer: study protocol of the PROPILS trial, a multicenter randomized controlled phase IV trial[END_REF] .

Improving nutritional status in icteric patients remains somewhat theoretic and is quite complicated and unreliable. In fact, most CLCC patients in our study presented with jaundice, abdominal pain, and anorexia, resulting in multi-parametric malnutrition with low serum albumin levels. Because we have shown that unresectability after PVE was not affected by biliary drainage before PVE but was only predicted by nutritional parameters, and in the knowledge that the nutritional status of patients is closely related to biliary excretion in the digestive tract, patients should be drained internally before PVE in order to improve their nutritional parameters.

As a consequence, we propose that the early detection and treatment of sarcopenia, obesity, and malnutrition using different strategies may improve the resectability rate. For example, high risk patients (score 3) should receive an effective preoperative rehabilitation program with specific nutritional therapy to improve their nutritional status, sufficient biliary drainage, and reevaluation of the surgical strategy to gain time before tumor progression without losing the chance of curative resection,although their score predicts high unresectability rate. In the view of shortening the delay before operation and consequently the risk of cancer progression and reducing post-PVE complications (21%), one could propose in the rare cases where both types of hepatectomy are indicated -i.e. right extended or left extended sides-recommending a leftextended hepatectomy without PVE (often possible) rather than a right-extended hepatectomy that requires complex preparation; although this strategy is surgically more demanding, it may offer more rapid surgical exploration.

Finally, the 90-day mortality rate (16%) in the resected group of our series was comparable to that reported in recent western studies (17.6% & 16.2%) [START_REF] Oltohf | External biliary drainage following major liver resection for perihilar cholangiocarcinoma: impact on development of liver failure and biliary leakage[END_REF][START_REF] Filmann | Mortality after liver surgery in Germany[END_REF] but was greater than that reported in the meta-analysis by Franken et al. 52 of patients in Western centers. These results were poorer than those reported in the Eastern centers. Note that as a tertiary reference center, we often accept "marginal indication" patients contra-indicated by other teams (Bismuth type IV in 17 patients for example), and that might be the explanation for observed high postoperative mortality observed.

The principal strength of this study was its innovative subject, because it is the first study to have identified predictors of unresectability after PVE for CLCC. Our findings support previously published variables in comparable situations with clear cut-off points, thus reflecting the validity of our statistical analysis. The identified predictors of unresectability are simple, objective, and easily applicable to in the clinical practice of all teams. We were also able to

propose a new score with high predictive value that could have a major impact on detecting highrisk patients in terms of their unresectability after PVE, thus modifying the treatment strategies implemented to improve outcomes.

The present study had several limitations. First, it was retrospective in design. Second, it was conducted at a single center on a relatively small patient cohort, so the results should be interpreted with caution. Third, the patients enrolled in the study were operated on over a long

Figure 1 .Figure 2 .Figure 3 .Figure 1 .Figure 2 .

 12312 Figure 1. Treatment flow chart of the study population and the causes of unresectability after PVE

Figure 3 .

 3 Figure 3. Receiver operator characteristic (ROC) curve analysis of our composite risk score to predict the unresectability after PVE

Table 2 .

 2 Preoperative laboratory assessment all continuous variables are expressed in median[range]. ALAT: alanine aminotransferase. ASAT: aspartate aminotransferase. GGT:gamma glutamyl transpeptidase. INR:international normalized ratio. CEA: Carcinoembryonic antigen. CA19-9: cancer antigen. ICG: indocyanine green. NLR: neutrophil-tolymphocyte ratio. PLR: platelet-to-lymphocyte ratio.

	Variables*	Resected (N=56)	Unresected (N=32)	P value
	Hemoglobin (g/dl)	12.5 [8.3 -15.4]	11.9 [8.2 -14.3]	0.162
	Platelets (×1000)	295 [158 -648]	333 [145 -803]	0.490
	Bilirubin (µmol/L)
	-	Total	31.2 [5 -568]	38 [7 -390]	0.145
	-	Direct	18.5 [2 -185]	28 [1 -335]	0.366
	ALAT (U/L)	66 [12 -542]	68 [21 -397]	0.748
	ASAT (U/L)	55 [16 -309]	63 [24 -347]	0.323
	GGT (U/L)	345 [26 -2112]	462.5 [32 -1910]	0.245
	Prothrombin time	91 [60 -100]	81.5 [58 -100]	0.016
	INR		1.06 [0.89 -1.47]	1.15 [0.96 -1.5]	0.014
	Albumin (g/L)	34.7 [22.4 -41.8]	30.2 [20 -37.8]	0.002
	Albumin (g/L)
		< 35	28 (56%)	22 (79%)	0.046
		≥ 35	22 (44%)	6 (21%)
	C-reactive protein (mg/L)	9.1 [1 -163]	33 [4.7 -87.7]	0.027
	Creatinine (µmol/L)	71.5 [46 -137]	72 [42 -117]	0.630
	CEA (µg/L)	2.4 [0.5 -45.9]	2.2 [1 -29.9]	0.862
	CA19-9 (kU/L)	75 [0.8 -10631.2]	176 [2 -4867]	0.155
	ICG %	11 [1.4 -38.1]	8.9 [3 -61]	0.308
	NLR		2.9 [1.06 -9.74]	3.12 [0.9 -13.96]	1.000
	NLR
	-	> 2.8	29 (56%)	17 (61%)	0.670
	-	> 3	24 (46%)	14 (50%)	0.742
	-	≥ 5	11 (21%)	6 (21%)	0.977
	-	≥ 2.49	37 (71%)	18 (64%)	0.527
	-	> 3.3	22 (42%)	13 (46%)	0.723
	PLR		181.38 [79 -419.7]	217.7 [49 -863.4]	0.054
	PLR
	-	> 185	25 (48%)	21 (75%)	0.020
	-	≤ 185	27 ( 2 5 %)	7 (25%)

*

of strategy to avoid PVE, when possible, could also be advocated. The external validation of these results is now required.

COI/ DISCLOSURE

The authors of this manuscript have no conflicts of interest to disclose.

FUNDING/ SUPPORT

The authors of this manuscript have no any financial support to disclose.


Hassane Zouhal

Reza Bagheri

Damoon Ashtary-Larky

Alexei Wong

Raoua Triki

Anthony C Hackney

Ismail Laher

Abderraouf Ben Abderrahman

Abderraouf Ben

Effects of Ramadan intermittent fasting on inflammatory and biochemical biomarkers in males with obesity

Keywords: Obese, overweight, inflammation, biochemical biomarkers, Ramadan, fasting

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Obesity is a key health risk factor characterized by an excessive accumulation of pro-inflammatory cytokines such as C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-6 [START_REF] Bagheri | Effects of green tea extract supplementation and endurance training on irisin, proinflammatory cytokines, and adiponectin concentrations in overweight middle-aged men[END_REF][START_REF] Bagheri | Does green tea extract enhance the anti-inflammatory effects of exercise on fat loss?[END_REF]. CRP and TNF-α are considered important specific biomarkers of vascular inflammation as well as being predictors of atherosclerosis [START_REF] Blake | High sensitivity C-reactive protein for predicting cardiovascular disease: an inflammatory hypothesis[END_REF][START_REF] Sedger | TNF and TNF-receptors: From mediators of cell death and inflammation to therapeutic giants-past, present and future[END_REF], while IL-6 has intrinsic pro-inflammatory activity and increases TNF-α concentrations [START_REF] Yudkin | Inflammation, obesity, stress and coronary heart disease: is interleukin-6 the link?[END_REF].

Adipose tissue is the main source of these cytokines [START_REF] Coppack | Pro-inflammatory cytokines and adipose tissue[END_REF], making it important to implement different therapies to ameliorate excess adiposity and abnormal pro-inflammatory cytokine concentrations [START_REF] Wisse | The inflammatory syndrome: the role of adipose tissue cytokines in metabolic disorders linked to obesity[END_REF]; such strategies are likely to reduce the risk of developing metabolic syndrome in populations with obesity [START_REF] Coppack | Pro-inflammatory cytokines and adipose tissue[END_REF].

Intermittent fasting (IF) is a frequently used strategy to decrease excess adiposity and circulating proinflammatory cytokines while improving overall health [START_REF] De Cabo | Effects of Intermittent Fasting on Health, Aging, and Disease[END_REF][START_REF] Mo'ez Al-Islam | Impact of diurnal intermittent fasting during Ramadan on inflammatory and oxidative stress markers in healthy people: Systematic review and meta-analysis[END_REF]. A common approach for undertaking IF involves severe caloric restriction (75-90% of energy needs) for 1-2 days per week. Another form of IF is Ramadan IF (RIF), which is routinely practiced by over 1.8 billion adherents on an annual basis where they undertake one month of RIF in observance of the Islamic obligation (Ramadan) involving abstinence from food and liquids from dawn to sunset [START_REF] Ziaee | The changes of metabolic profile and weight during Ramadan fasting[END_REF]. Ramadan is the ninth month of the Islamic lunar calendar and fasting during this month represents one of the five pillars of Islam. The duration of the fasting period commonly lasts from 10 to 18 hours and varies according to the season and geographical location [START_REF] Cherif | Effects of intermittent fasting, caloric restriction, and Ramadan intermittent fasting on cognitive performance at rest and during exercise in adults[END_REF][START_REF] Issaoui | Effect of Ramadan fasting: Association with time of day on time-motion, technical aspect and psychophysiological response to simulated karate competition in young amateur competitors[END_REF]. The lunar year is 11 days shorter than the Gregorian year; therefore, Ramadan occurs in a different season every 9 years [START_REF] Bahammam | Does Ramadan fasting affect sleep? International journal of clinical practice[END_REF]. The season during which Ramadan occurs affects the duration of each fast because fasting hours are longer in summer than in winter. Ramadan is an extensively studied example of a timed dietary approach to IF [START_REF] Michalsen | Fasting therapy for treating and preventing disease-current state of evidence[END_REF][START_REF] Zouhal | Exercise training and fasting: current insights[END_REF].

Several studies reported decreases in body weight following RIF [START_REF] Alsubheen | The effect of intermittent fasting during Ramadan on energy expenditure and substrate oxidation in healthy men[END_REF]. For instance, a meta-analysis of 35 studies by [START_REF] Sadeghirad | Islamic fasting and weight loss: a systematic review and meta-analysis[END_REF] assessed body weight changes in fasting individuals during and after RIF reported fairly small but significant weight loss (-1.24 kg) in both genders, with most of the weight loss being regained within a few weeks after Ramadan [START_REF] Sadeghirad | Islamic fasting and weight loss: a systematic review and meta-analysis[END_REF]. Another meta-analysis of 21 studies that analyzed the effects of RIF on body weight reported a slight but significant reduction in body weight in men, with no significant changes observed in women [START_REF] Kul | Does Ramadan fasting alter body weight and blood lipids and fasting blood glucose in a healthy population? A meta-analysis[END_REF]. A more recent meta-analysis of 70 studies (∼3,000 participants) in individuals classified as overweight and obese, demonstrated significant decreases in body fat percentage. This reduction was not observed in normal-weight participants [START_REF] Fernando | Effect of Ramadan fasting on weight and body composition in healthy non-athlete adults: a systematic review and meta-analysis[END_REF]. Nevertheless, body weight and composition returned to near pre-Ramadan levels after 2-5 weeks of follow-up. Moreover, other studies failed to observe significant changes in body weight during RIF [START_REF] Latiri | The effects of Ramadan fasting on the spirometric data of healthy adult males[END_REF], while some even described weight gain during this period [START_REF] Meckel | The effect of the Ramadan fast on physical performance and dietary habits in adolescent soccer players[END_REF]. The inconsistency in these findings can be explained by varied eating routines, socioeconomic status, differences in the number of fasting hours, ethnicity, gender, and the health status and medical history of participants [START_REF] Sadeghirad | Islamic fasting and weight loss: a systematic review and meta-analysis[END_REF][START_REF] Aliasghari | The effects of Ramadan fasting on body composition, blood pressure, glucose metabolism, and markers of inflammation in NAFLD patients: an observational trial[END_REF][START_REF] Mazidi | The effect of Ramadan fasting on cardiometabolic risk factors and anthropometrics parameters: a systematic review[END_REF].

Other than changes in body weight and composition, several health benefits have been credited to RIF including improvements in blood pressure and brain function [START_REF] Fernando | Effect of Ramadan fasting on weight and body composition in healthy non-athlete adults: a systematic review and meta-analysis[END_REF]. However, data on the effects of RIF on proinflammatory biomarkers is equivocal, with some studies reporting benefits [START_REF] Ünalacak | Effects of Ramadan fasting on biochemical and hematological parameters and cytokines in healthy and obese individuals[END_REF], while others showing no changes [START_REF] Ibrahim | Effect of Ramadan fasting on markers of oxidative stress and serum biochemical markers of cellular damage in healthy subjects[END_REF][START_REF] Harder-Lauridsen | Ramadan model of intermittent fasting for 28 d had no major effect on body composition, glucose metabolism, or cognitive functions in healthy lean men[END_REF]. This discrepancy in results may be related to participants' characteristics and study design as well as the length of fasting which differs from one geographical location to the other and from one year to the other [START_REF] Bahammam | Does Ramadan fasting affect sleep? International journal of clinical practice[END_REF][START_REF] Bahammam | Recent Evidence on the Impact of Ramadan Diurnal Intermittent Fasting, Mealtime, and Circadian Rhythm on Cardiometabolic Risk: A Review[END_REF]. Moreover, to the best of authors' knowledge, only a few studies have explored the effects of RIF on inflammatory biomarkers in individuals with obesity, but with inconclusive findings due to a lack of control groups [START_REF] Ünalacak | Effects of Ramadan fasting on biochemical and hematological parameters and cytokines in healthy and obese individuals[END_REF][START_REF] Faris | Impact of Ramadan intermittent fasting on oxidative stress measured by urinary 15--isoprostane[END_REF]. Many studies on RIF measured a limited number of pro-inflammatory and biochemical biomarkers but only before and immediately after RIF, with no measurements of biomarkers during and or for a period after RIF. Thus, we chose to evaluate the effects of RIF on inflammatory (CRP, IL-6, and TNF-α) and biochemical biomarkers [aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin, lactate dehydrogenase (LDH), urea, and creatinine] in males with obesity. We measured these biomarkers at four-time points: 24 hours before the start of Ramadan (T0), on the 15th day of Ramadan (T1), the day after the end of Ramadan (T2) and 21 days after the end of Ramadan (T3). We hypothesized that RIF would improve circulating pro-inflammatory cytokines and biochemical biomarkers in middle aged males with obesity across all the measured times points.

Materials and Methods

Participants

Fifty-one males were recruited from the students and staff of a regional university, by direct contact as well as via social networks. The inclusion criteria were as follows: body mass index of 30-40 kg/m 2 , no use of drugs or alcohol, lack of regular exercise for at least 6 months before the study, no history of renal, hepatic, cardiovascular diseases or diabetes, and no physical injuries or mobility concerns. The exclusion criteria were as follows: diabetes, cognitive disorders, cardiovascular diseases including uncontrolled hypertension, cancer, liver or renal disease, chronic pulmonary disease, physical disability, or any other contraindications. Moreover, those with evidence of clinical depression were excluded from the study as antidepressant medications can alter proinflammatory cytokine concentrations [START_REF] Kenis | Effects of antidepressants on the production of cytokines[END_REF][START_REF] Schmidt | Cytokine levels in depressed and non-depressed subjects, and masking effects of obesity[END_REF]. Thirty-two eligible males with obesity provided informed consent before participating in the study. A physician using a medical health/history questionnaire as well as the Physical Activity Readiness Questionnaire (PAR-Q) evaluated all these criteria. The study was approved by the Ethical Committee on Human Research of the University of Rennes 2, and the University of la Manouba, Tunisia, and all procedures were carried out in accordance with the Helsinki Declaration.

Study Design

This study was conducted during Ramadan 2019 (from May 5 th to June 3 th ) in Tunis, Tunisia. The fasting duration was 15-16 hours per day when the mean daily temperature was approximately 24°C. Participants were allocated (simple randomization) to either an experimental group (EG, n=16, who declared that they would fast for the entire month of Ramadan) or to a control group (CG, n=16, who declared that they would not fast during Ramadan) (Figure 1). The EG completed their fasting rituals for the duration of Ramadan (30 days) while the CG continued with their normal daily habits. Participants were fully familiarized with the tests prior to all procedures. Anthropometric measurements and blood samples were collected at four-time points: 24 hours before the start of Ramadan (T0), on the 15th day of Ramadan (T1), the day after the end of Ramadan (T2), and 21 days after the end of Ramadan (T3). All measurements were obtained at the same time of day (within ~1 hour).

Dietary habits

All participants were asked to record their normal nutritional habits during the experiment; none of the participants reported using dietary supplements. Specifically, participants were asked to complete a two-day food diary prior to each blood sampling. The amount of nutrients consumed was calculated using the method described by [START_REF] Mccance | McCance and Widdowson's the composition of foods[END_REF] [START_REF] Mccance | McCance and Widdowson's the composition of foods[END_REF]. Subjects in the CG were instructed to not alter their dietary habits during Ramadan. Other than RIF, no other dietary changes occurred in the EG group.

Blood sampling and analysis

Participants were instructed to comply with the following conditions before each blood sample was obtained: 1) avoid using medications for at least 72 h before a blood draw, 2) avoid any strenuous exercise for at least 72 h before a blood draw, and 3) maintain the same diet for 48 hours prior to each blood draw. Blood was collected from an antecubital vein in the morning (between 8 and 9 a.m.) after an overnight fast. Blood sampling and analysis were performed at the laboratory of Biological Analysis, Principal Military Hospital (Tunis, Tunisia).

Samples were collected in two pre-cooled 4.9-mL EDTA vacuettes (Klap activator model). Each whole blood sample was analyzed for a complete blood cell count using an automated cell counter (Luna-II TM , Biosystems, France). Hematocrit (Ht) and hemoglobin concentrations ([Hb]) were determined. Plasma volume variation (ΔPV) was calculated according to the formula proposed by [START_REF] Dill | Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration[END_REF] [START_REF] Dill | Calculation of percentage changes in volumes of blood, plasma, and red cells in dehydration[END_REF]:

ΔPV (%) = 100 ×[([Hb]a/[Hb]b) × [(1-Htb) / (1-Hta)] -100]
where 'a' and 'b' refer to fasting state values for Ht and [Hb] before and after training, respectively.

One EDTA vacuette containing freshly drawn blood was immediately centrifuged at 1500 × g for 10 min at 4 °C (Heraeus Multifuge X3R, Thermo Scientific, Loughborough, UK). The plasma supernatant was then dispensed into separate 2-mL cryovials and stored at -80 °C for later analysis of inflammation (CRP, IL-6, and TNFα) and biochemical (ALT, AST, bilirubin, LDH, urea, and creatinine) biomarkers.

Plasma concentrations of IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA) using human quantikine ELISA kits. The sensitivity was 5.5 pg/mL for TNF-α and 0.7 pg/mL for IL-6 measurements. The activity of CRP was determined using the immunoturbidimetry method (Tina-quant CRP; Boehringer Mannheim, Germany). The intra-assay coefficient of variation for the CRP kit was 1.7%. Activities of AST, ALT, bilirubin, LDH, urea, and creatinine were determined using a multiparametric analyzer (Konelab 30 TM , Thermo Electron Corporation). The intra-assay and inter-assay coefficient of variation for these biochemical parameters were ˂10 % and ˂12%, respectively. The activity of LDH was determined by applying the enzymatic rate method (IFCC). The intra-assay coefficient of variation for the LDH assay was 1.1%.

Anthropometric measurements

Body weight were measured on a digital scale (Lumbar, China) with a precision of 0.1 kg. The height was measured with a medical sampler (Race Industrialization, China) with a precision of 0.1 cm. Body mass index was calculated as kg/m 2 . Waist circumference was measured as the smallest circumference on the waist area (typically 2.5 cm above the navel) and hip circumference was measured around the widest part of the hips. The waist-hip ratio was calculated using the participant's waist and hip circumferences. Body fat percentage was evaluated using a multi-frequency bioelectrical impedance device (Inbody 720, South Korea) according to the manufacturer's recommendations.

Statistical analysis

An a priori power analysis (expected SD of residuals = 2 pg/ml for IL-6, desired power = 0.80, and alpha error = 0.05) was computed to estimate a statistically significant group-by-time-interaction effect for our primary outcome (IL-6) [START_REF] Bagheri | Effects of upper-body, lower-body, or combined resistance training on the ratio of follistatin and myostatin in middle-aged men[END_REF]. The analysis revealed a sample size of n = 10.8 per group. To account for potential dropouts, 16 participants were included in each of the two study groups.

Data are presented as mean ± standard deviation (M±SD). After confirming the normality of data distribution using the Shapiro-Wilk test, differences within and between groups were calculated using a two-way analysis of variance (ANOVA) with repeated measures. A Newman-Keul's post hoc test was calculated. Correlation between parameters were assessed using Pearson's product-moment correlation coefficient (r) if the group x time interactions were significant. Effect sizes (ES) were determined from ANOVA output by converting partial etasquared to Cohen's d. Within-group ES were computed using the following equation: ES = (mean post -mean pre)/SD. ES were considered trivial (< 0.2), small (0.2-0.6), moderate (0.6-1.2), large (1.2-2.0) and very large (2.0-4.0) [START_REF] Hopkins | Progressive statistics for studies in sports medicine and exercise science[END_REF]. The level of significance was set at p<0.05. All statistical analyses were computed using SPSS for Windows, version 24.0 (SPSS Inc., USA).

Results

We screened 41 males with obesity during Ramadan 2019 (from May 5 th to June 3 th ) in Tunis, Tunisia. Only 36 qualified for baseline evaluation and were subsequently randomized to either the EC or the CG groups; 2 participants in the EC group dropped out of the study as they did not fast for all the days of Ramadan. In the CG group, two participants dropped out as they were unable to maintain their normal daily dietary habits (fasted 5 to 7 days during this month). No harms or unintended effects were reported by participants. Data are presented for the 28 participants that successfully completed our intervention [(EG, n=14, age: 24± 3.4 y, height: 173 ± 6 cm), (CG, n=14, age: 23.8 ± 3.8 y, height: 171 ± 3.8 cm)] (Figure 1). All remaining participants reported a 100% adherence to study procedures. There were no differences in anthropometric and hormonal measurements between the two groups at baseline. Macronutrient values of EG and CG recorded at T0, T1, T2, and T3 are described in Table 1. There were no differences in macronutrient intakes between the two groups at any stage of the study.

Anthropometric and body composition characteristics of participants are presented in Table 2. Participants in the EG experienced significant decreases in body weight (-2.7%, p = 0.002, d = 1.12), body mass index (-2.8%, p = 0.006, d = 0.73), body fat percentage (-4.2%, p = 0.008, d = 1.23), and waist-hip ratio (-6.2%, p = 0.005, d = 1.19) over during Ramadan, with no changes recorded in the CG.

Inflammatory and biochemical markers are shown in Table 3 and4 Concentrations of AST (p=0.27, η 2 = 0.05), ALT (p=0.42, η 2 =0.01), bilirubin (p=0.40, η 2 =0.06), creatinine (p=0.48, η 2 =0.03), CRP (p = 0.3; η 2 =0.02), LDH (p=0.66, η 2 =0.03), and urea (p=0.32, η 2 =0.05) did not differ between the two study groups over time Some significant correlations were noted between anthropometric changes and inflammatory markers (Table 5).

The Δ of TNF-α correlated with Δ of body weight (r=-0.649, p=0.008) and with Δ of BMI (r=-0.683, p=0.007).

Discussion

The main finding of our study is RIF improves body composition and inflammatory biomarkers in middle aged males with obesity without harmful effects on their hepatic or renal function. The improvements in body composition (weight, body mass index, body fat percentage, and waist-hip ratio) occurred later in Ramadan (30day) but not in the middle of Ramadan and remained unchanged for 21 days following RIF. Furthermore, decreases in IL-6 and TNF-α occurred in the middle of Ramadan and remained unchanged until the end of Ramadan, and 21 days thereafter. To the best of our knowledge, our study is the first to assess the effects of RIF on body composition, inflammatory, and biochemical biomarkers at four different time points: 24 hours before the start of Ramadan, on the 15th day of Ramadan, the day after the end of Ramadan, and 21 days after the end of Ramadan.

Obesity increases pro-inflammatory cytokines and acute-phase proteins such as TNF-α, IL-6, or CRP [START_REF] De Heredia | Obesity, inflammation and the immune system[END_REF].

Weight loss by caloric-restriction is a key recommendation for decreasing the complications of obesity [START_REF] Ashtary-Larky | Rapid weight loss vs. slow weight loss: which is more effective on body composition and metabolic risk factors[END_REF].

Hence, RIF as a form of intermittent fasting, (caloric restrictions involving a restricted feeding period based on religious and spiritual traditions) can decrease systemic inflammation in individuals with obesity and chronic diseases [START_REF] Mo'ez Al-Islam | Impact of diurnal intermittent fasting during Ramadan on inflammatory and oxidative stress markers in healthy people: Systematic review and meta-analysis[END_REF][START_REF] Ünalacak | Effects of Ramadan fasting on biochemical and hematological parameters and cytokines in healthy and obese individuals[END_REF]. In agreement with our results, a previous systematic review and meta-analysis reported that TNF-α and IL-6 were the most impacted biomarkers following RIF [START_REF] Mo'ez Al-Islam | Impact of diurnal intermittent fasting during Ramadan on inflammatory and oxidative stress markers in healthy people: Systematic review and meta-analysis[END_REF], while another study reported that RIF reduced circulating concentrations of the pro-inflammatory cytokines (IL-6 and TNF-α) before, during, and after Ramadan in overweight participants [START_REF] Kacimi | Intermittent fasting during Ramadan attenuates proinflammatory cytokines and immune cells in healthy subjects[END_REF]. The concentrations of IL-6 and TNF-α on day 15 of Ramadan were lower compared to pre-Ramadan concentrations [START_REF] Faris | Impact of Ramadan intermittent fasting on oxidative stress measured by urinary 15--isoprostane[END_REF]. A study by Unalacak et al. reported that RIF reduced TNF-α (but not CRP) concentrations in healthy and individuals with obesity [START_REF] Ünalacak | Effects of Ramadan fasting on biochemical and hematological parameters and cytokines in healthy and obese individuals[END_REF]. Animal studies suggest that IF inhibits the nuclear factor κB (NF-κB) signaling pathway [START_REF] Fann | Intermittent fasting attenuates inflammasome activity in ischemic stroke[END_REF]. NF-κB is an important pro-inflammatory transcriptional regulator that regulates the expression of pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α.

Weight loss following RIF improves the inflammatory biomarkers in adipose tissue by reducing inflammationrelated gene expression in adipocytes and macrophages [START_REF] Clément | Weight loss regulates inflammation-related genes in white adipose tissue of obese subjects[END_REF]. Calorie restriction and weight loss decreases activation of NF-κB, a major pro-inflammatory signaling pathway that regulates the expression of proinflammatory cytokines such as IL-6, and TNF-α [START_REF] Harvey | Calorie restriction decreases murine and human pancreatic tumor cell growth, nuclear factor-κB activation, and inflammation-related gene expression in an insulin-like growth factor-1-dependent manner[END_REF]. Weight loss decreases CRP concentrations [START_REF] Selvin | The effect of weight loss on C-reactive protein: a systematic review[END_REF]. On the other hand, the most of the cytokines produced by adipose tissue originates from non-fat cells, of which macrophages are key components [START_REF] Fain | Release of interleukins and other inflammatory cytokines by human adipose tissue is enhanced in obesity and primarily due to the nonfat cells[END_REF]. Weight loss lowers macrophage infiltration of adipose tissue; macrophages are mainly derived from monocytes [START_REF] Bastard | Recent advances in the relationship between obesity, inflammation, and insulin resistance[END_REF]. Therefore, it is likely that improving inflammatory biomarkers by decreasing cytokine concentrations may be related to a decreased number of monocytes following RIF-induced weight and fat loss [START_REF] Kacimi | Intermittent fasting during Ramadan attenuates proinflammatory cytokines and immune cells in healthy subjects[END_REF].

The reduction in the circulating concentrations of pro-inflammatory cytokines during RIF could also be attributed to decreased oxidative stress [START_REF] Ibrahim | Effect of Ramadan fasting on markers of oxidative stress and serum biochemical markers of cellular damage in healthy subjects[END_REF]. RIF improved the gene expression of antioxidant defense enzyme systems including mitochondrial transcription factor A (TFAM), superoxide dismutase (SOD2), and nuclear factor erythroid 2-related factor 2 (Nrf2) [START_REF] Madkour | Ramadan diurnal intermittent fasting modulates SOD2, TFAM, Nrf2, and sirtuins (SIRT1, SIRT3) gene expressions in subjects with overweight and obesity. diabetes research and clinical practice[END_REF]. These changes could also explain the reduction in circulating proinflammatory cytokines at the end of Ramadan as reported in previous studies [START_REF] Mo'ez Al-Islam | Impact of diurnal intermittent fasting during Ramadan on inflammatory and oxidative stress markers in healthy people: Systematic review and meta-analysis[END_REF][START_REF] Madkour | Ramadan diurnal intermittent fasting modulates SOD2, TFAM, Nrf2, and sirtuins (SIRT1, SIRT3) gene expressions in subjects with overweight and obesity. diabetes research and clinical practice[END_REF] and our study. Moreover, decreased reactive oxygen species could also alter the activation of NF-kB signaling during RIF [START_REF] Bubici | Mutual cross-talk between reactive oxygen species and nuclear factor-kappa B: molecular basis and biological significance[END_REF]. Another possible mechanism for the anti-inflammatory effects of RIF could be related to its association with mental health and mood improvement since mental health is also associated with improvement in systemic inflammation [START_REF] Kaplan | The emerging field of nutritional mental health: Inflammation, the microbiome, oxidative stress, and mitochondrial function[END_REF].

The results of our study indicate a positive correlation between weight loss, BMI, and TNF-α concentrations following RIF. Some potential mechanisms that could account for weight and BMI decreases associated with lower circulating TNF-α concentrations involve calorie restriction following RIF [START_REF] Chung | The inflammation hypothesis of aging: molecular modulation by calorie restriction[END_REF]. Findings from animal and human studies suggest that dietary restrictions which causes weight loss are related to improvement in TNF-α concentrations and gene expression [START_REF] Cordero | Leptin and TNF-alpha promoter methylation levels measured by MSP could predict the response to a low-calorie diet[END_REF][START_REF] Spaulding | Calorie restriction inhibits the agerelated dysregulation of the cytokines TNF-α and IL-6 in C3B10RF1 mice[END_REF] that may be mediated by NF-kB signaling [START_REF] Ye | Regulation of energy metabolism by inflammation: a feedback response in obesity and calorie restriction[END_REF].

Our study suggests that RIF is a relatively convenient means of achieving weight loss over a short period, and therefore may be a useful tool for weight loss strategies [START_REF] Varady | Alternate day fasting for weight loss in normal weight and overweight subjects: a randomized controlled trial[END_REF]. However, the effects of RIF on body weight is controversial. Although RIF decreases meal frequency, some studies reported either increased energy intake [START_REF] Gharbi | Food intake during and outside Ramadan[END_REF] or comparable energy intake before, during, and after RIF occurrences [START_REF] Beltaifa | Food intake, and anthropometrical and biological parameters in adult Tunisians during Ramadan[END_REF]. However, a meta-analysis of thirtyfive studies suggests that RIF causes significant (1.24 kg) weight loss [START_REF] Sadeghirad | Islamic fasting and weight loss: a systematic review and meta-analysis[END_REF]. This analysis also indicated that most of the weight loss was regained within a few weeks after RIF [START_REF] Sadeghirad | Islamic fasting and weight loss: a systematic review and meta-analysis[END_REF]. Our study indicates that RIF resulted in a significant weight loss at days 15 (-0.6 kg) and 30 (-3kg or 3.1%). However, 17% (not statistically significant) of the weight loss was regained within 21 days after the end of RIF.

We report a 2.1% decrease in body fat percentage, which can lower carbohydrate oxidation and increase fat oxidation during RIF [START_REF] El Ati | Increased fat oxidation during Ramadan fasting in healthy women: an adaptative mechanism for body-weight maintenance[END_REF]. Decreases in body fat percentage are generally anticipated during RIF [START_REF] Fernando | Effect of Ramadan fasting on weight and body composition in healthy non-athlete adults: a systematic review and meta-analysis[END_REF], but this is not always the case as some studies report no changes in body fat percentage [START_REF] Ongsara | The effect of Ramadan fasting on biochemical parameters in healthy Thai subjects[END_REF], while others reported non-significant changes [START_REF] Norouzy | Effect of fasting in R amadan on body composition and nutritional intake: a prospective study[END_REF]. These discrepancies may be related to the different characteristics of the participants in terms of age, sex, dietary intake, physical activity, and the number of hours of fasting.

Previous studies demonstrated that RIF did not adversely affect the liver and renal function in individuals with obesity [START_REF] Ünalacak | Effects of Ramadan fasting on biochemical and hematological parameters and cytokines in healthy and obese individuals[END_REF][START_REF] Sadiya | Effect of Ramadan fasting on metabolic markers, body composition, and dietary intake in Emiratis of Ajman (UAE) with metabolic syndrome. Diabetes, metabolic syndrome and obesity: targets and therapy[END_REF][START_REF] Asegaonkara | Effect of Ramadan fasting on renal function markers in healthy adults from Aurangaba[END_REF] which we confirmed, but other studies offered different conclusions. For instance, Nasiri et al. reported that RIF increased ALT, AST, ALP, and bilirubin concentrations, although these changes were within the normal range and non-significant, but nonetheless, the authors offered caution about such changes [START_REF] Nasiri | Effect of Ramadan fast on liver function tests[END_REF]. Our findings indicate no differences in renal and liver biomarkers during and after RIF, which is supported by most studies on liver and renal biomarkers following RIF [START_REF] Asegaonkara | Effect of Ramadan fasting on renal function markers in healthy adults from Aurangaba[END_REF].

Our study has some limitations. Although physical activity levels usually decrease during RIF [START_REF] Ziaee | The changes of metabolic profile and weight during Ramadan fasting[END_REF], we did not measure the levels of physical activity before, during, and after RIF. Physical activity influences inflammatory and biochemical biomarkers. Additionally, we performed a per-protocol analysis which may have biased our findings. Although this type of analysis better reflects the effects of intervention when performed optimally, the clinical applicability of this per-protocol effect is limited if the effect in the intervention compliers differs substantially from the effect in other cohorts [START_REF]Intention to treat analysis and per protocol analysis: complementary information[END_REF].

Our study indicates that RIF improves body composition and biomarkers of systemic inflammation, and that these changes persisted for three weeks after Ramadan. Moreover, RIF did not adversely impact liver and renal biomarkers, suggesting the approach is not only efficacious but safe to perform.

  , respectively. The EG experienced significant decreases in IL-6 (p = 0.02, d = 1.49) and TNF-α (p = 0.019, d = 0.78) concentrations over time.

Table 1 :

 1 Macronutrient values (mean ± SD) of the experimental group (EG, n=14) and control group (CG, n=14) measured 24 hours before the start of Ramadan (T0), on the 15th day of Ramadan (T1), the day after the end of Ramadan (T2) and 21 days after the end of Ramadan (T3). Data are expressed as means ± SD.

	Macronutrients	Group	T0	T1	T2	T3
	Protein	EG	17 ± 10	20 ± 8	19 ± 6	19 ± 9
	(% kcal)	CG	17 ± 2	18 ± 4	19 ± 8	19 ± 6
	Carbohydrate	EG	48 ± 7	46 ±7	45 ± 7	47 ± 11
	(% kcal)	CG	47 ± 7	46 ± 9	46 ± 8	47 ± 8
	Fat (% kcal)	EG CG	35 ± 3 36 ± 3	34 ± 4 36 ± 3	36 ± 9 35 ± 8	36 ± 6 34 ±5

Table 2 :

 2 Anthropometric and body composition characteristics (mean ± SD) of the experimental group (EG, n=14) and control group (CG, n=14) measured 24 hours before the start of Ramadan (T0), on the 15th day of Ramadan (T1), the day after the end of Ramadan (T2) and 21 days after the end of Ramadan (T3).

	Variables	Group	Phases

Table 3 .

 3 Inflammation markers of the experimental group (EG, n=14) and control group (CG, n=14) measured 24 hours before the start of Ramadan (T0), on the 15th day of Ramadan (T1), the day after the end of Ramadan (T2) and 21 days after the end of Ramadan (T3).

	Markers	Group		Phases			p values (ES)
			T0	T1	T2	T3	Time	Group	Group x Time
	CRP (mg/L)	EG CG	18.02 ± 1.7 $ 17.6 ± 1.7	18.03 ± 1.7 $ 17.9 ± 2.1	18.2 ± 1.8 $ 17.7 ± 2.2 $	18.7 ± 1.7 *#^ 18.3 ± 2.5 ^	0.002 (0.66)	0.6 (0.008)	0.3 (0.13)
	IL-6 (pg/mL)	EG CG	49.1 ± 4.2 #^$ 48.2 ± 4.8	46.2 ± 3.5 * 47.9 ± 5.1	44 ± 3.1 * 47.4 ± 5.5	45.5 ± 3.2 * 48 ± 5.1	0.001 (0.5)	0.2 (0.04)	0.006 (0.39)
	TNF-α (pg/mL)	EG CG	4485.7 ± 575 # 4192.8 ± 880 #^	4078.5 ± 629 * 4021.4 ± 893 *	4028.5 ± 610.7 4000 ± 842.1 *$	3971.4 ± 693.2 4157.1 ± 809.2 ^	0.001 (0.53)	0.86 (0.001)	0.024 (0.32)
	Data are expressed as mean ± SD.
	CRP, high-sensitivity C-reactive protein; IL-6, Interleukin 6; TNF-α, tumor necrosis factor alpha. * Significant from T0. # Significant from T1, ^
	Significant from T2, $ Significant from T3.

Table 4 :

 4 Biochemical markers (AST, ALT, Bilirubin, LDH, Urea and creatinine) (means ± SD) of the experimental group (EG) and control group (CG) measured 24 hours before the start of Ramadan (T0), on the 15th day of Ramadan (T1), the day after the end of Ramadan (T2) and 21 days after the end of Ramadan (T3).Data are expressed as mean ± SD. AST, aspartate Aminotransferase; ALT, Alanine Aminotransferase; LDH, Lactate Dehydrogenase.* Significant from T0. # Significant from T1, ^ Significant from T2, $ Significant from T3. Figure 1. Flow diagram of study design and participant allocation.

	Variables	Group	Phases

Acknowledgements

We are thankful for the participants that took part in this project.

Conflict of interest

The authors declare no conflict of interest.