Charlène Brochard
email: charlene.brochard@chu-rennes.fr

Marion Chambaz

Alain Ropert

Alexandre Merlini L'héritier

Timothée Wallenhorst

Guillaume Bouguen

Laurent Siproudhis

Quality of life in 1870 patients with constipation and/or fecal incontinence : constipation should not be underestimated

Keywords: quality of life, faecal incontinence, constipation Conception and design of the study: CB, LS Acquisition of data: CB, MC, AML, AR, TW, LS Analysis

Background: Quality of life is increasingly seen as important, but remains difficult to assess in patients with functional anorectal complaints.

Objective: We aimed to quantify quality of life and to analyse the symptomatic descriptors associated with a poor outcome in patients with faecal incontinence (FI) and/or constipation.

Methods:

The characteristics of the patients, data from self-administered questionnaires and from physical examinations were evaluated prospectively for all cases of functional anorectal disease over a period of thirteen years. Functional anorectal disease included faecal incontinence (FI) and/or constipation. Patients with scores in the lowest quartile of the Gastrointestinal Quality of Life Index (GIQLI) were considered to have suffered severe alterations to their quality of life, and were compared with the other patients.

Results:

In total, 1870 patients with functional anorectal disease were included (470 with a severely altered quality of life (GIQLI<70)). Constipation predominated (1212/1870; 65.1%) and severe FI was frequent (761/1870; 40.9%). Severely altered quality of life was significantly associated with constipation (p= 0.0001), urinary urgency and incontinence (p=0.0001), depression (p=0.001), diabetes (p=0.0224), severe FI (p= 0.0001), neurological disease (p= 0.0138) and liquid stools (p= 0.0002) in multivariate analysis.

Conclusion:

Several treatable factors are associated to an impaired quality of life in patients with functional anorectal disorders. Intervention studies are mandatory (stool consistency and frequency).

INTRODUCTION

Health-related quality-of-life (HRQoL) studies have come to the fore in recent years, and provide a new dimension for analysing the impact of both disease and treatment. In cases of functional disease, it is particularly difficult to evaluate quality of life, because the dysfunction relates partly to social and psychological factors. Constipation and faecal incontinence (FI) were found to have a negative impact on quality of life [START_REF] Parés | Prevalence of faecal incontinence and analysis of its impact on quality of life and mental health[END_REF][START_REF] Sailer | Quality of life in patients with benign anorectal disorders[END_REF]. Many studies have evaluated the impact of FI on quality of life [START_REF] Markland | Factors impacting quality of life in women with fecal incontinence[END_REF][START_REF] Bedard | Relationship between symptoms and quality of life in fecal incontinence[END_REF][START_REF] Bharucha | Symptoms and quality of life in community women with fecal incontinence[END_REF][START_REF] Damon | Clinical characteristics and quality of life in a cohort of 621 patients with faecal incontinence[END_REF][START_REF] Rockwood | Incontinence severity and QOL scales for fecal incontinence[END_REF][START_REF] Smith | Factors associated with quality of life among women with fecal incontinence[END_REF], but fewer data are available concerning the impact of constipation on quality of life [START_REF] Sailer | Quality of life in patients with benign anorectal disorders[END_REF][START_REF] Damon | Prevalence of anal incontinence in adults and impact on quality-of-life[END_REF][START_REF] Kovacic | A multicenter study on childhood constipation and fecal incontinence: effects on quality of life[END_REF][START_REF] Klages | Empirically Derived Patterns of Pain, Stooling, and Incontinence and Their Relations to Health-Related Quality of Life Among Youth With Chronic Constipation[END_REF][START_REF] Neri | Constipation severity is associated with productivity losses and healthcare utilization in patients with chronic constipation[END_REF]. FI and constipation frequently occur together, but the association of these two conditions is rarely considered [START_REF] Sailer | Quality of life in patients with benign anorectal disorders[END_REF].

The Gastrointestinal Quality of Life Index (GIQLI) [START_REF] Eypasch | Gastrointestinal Quality of Life Index: development, validation and application of a new instrument[END_REF] is a validated tool for assessing HRQoL in clinical studies of patients with gastrointestinal disease. of its principal strength is that can be used to evaluate HRQoL in different types of functional digestive complaints. It is easy to perform and yields a quantified global score. This tool has already been used in patients with anorectal disorders [START_REF] Sailer | Quality of life in patients with benign anorectal disorders[END_REF][START_REF] Damon | Clinical characteristics and quality of life in a cohort of 621 patients with faecal incontinence[END_REF][START_REF] Damon | Clinical characteristics and quality of life in a cohort of 621 patients with faecal incontinence[END_REF][START_REF] Brochard | Prospective cohort study of phenotypic variation based on an anal sphincter function in adults with fecal incontinence[END_REF].

The aims of this study were: (i) to describe the characteristics and quality of life of patients with functional anorectal disease (faecal incontinence (FI) and/or constipation) and (ii) to identify the factors associated with changes to HRQoL in this population.

MATERIALS AND METHODS

Study Population

All patients referred to our tertiary unit (Rennes University Hospital, France) for an anorectal complaint between 2005 and 2016 were consecutively recruited and prospectively included in a registry (Fondamentum, CNIL no. 1412467).

The patients included in this registry were excluded from the study if they were pregnant or had a history of colon, rectal or anal cancer, or of anal or rectal stricture. Self-administered questionnaires and physical examination data were prospectively recorded in a database. Age, sex, height, weight, medical history (including diabetes, neurological disease and depression) and surgical history (including haemorrhoidectomy, anal surgery, cholecystectomy, and surgery for anterior or posterior colopocele) were recorded. Symptoms were recorded as previously described [START_REF] Favreau | Underlying functional bowel disorder may explain patient dissatisfaction after haemorrhoidal surgery[END_REF]. The questionnaire focused on the main anorectal complaints (incontinence, constipation, dyschezia, Bristol stool scale, number of stools per week, symptom duration). Dyschezia was defined by sensation of blockage at defecation, sensation of incomplete evacuation or straining during defecation. Urge and passive FI were differentiated. Urgency was associated with a perceived urge to defaecate and passive FI with the stool loss without being aware of it. FI was evaluated with the validated Cleveland Clinic Incontinence Score (CCIS 0-20) [START_REF] Longstreth | Functional bowel disorders[END_REF]. Constipation was assessed with the validated Knowles-Eccersley-Scott Symptom Constipation Score (KESS) [START_REF] Knowles | Linear discriminant analysis of symptoms in patients with chronic constipation: validation of a new scoring system (KESS)[END_REF]. Quality of life was quantified with a validated scale for gastrointestinal complaints (Gastrointestinal Quality of Life Index (GIQLI)) [START_REF] Eypasch | Gastrointestinal Quality of Life Index: development, validation and application of a new instrument[END_REF] and the Urinary Distress Inventory scale (UDI) for urinary incontinence, as previously descrived in studies of faecal incontinence cohorts [START_REF] Uebersax | Short forms to assess life quality and symptom distress for urinary incontinence in women: the Incontinence Impact Questionnaire and the Urogenital Distress Inventory. Continence Program for Women Research Group[END_REF]. Functional anorectal disease included faecal incontinence (FI) and/or constipation in this study.

Clinical dyssynergic defaecation and pelvic floor disorders were assessed by physical examination. Internal intussusception and high-grade prolapse were defined as an intra-anal or exteriorised intussusception of the rectal wall during straining.

Until 2016, French legislation does not require the written consent for this type of study and the CNIL declaration was sufficient.

Definitions

FI was defined as a CCIS > 5 and severe FI was defined as a CCIS ≥ 9 [START_REF] Rothbarth | What is the impact of fecal incontinence on quality of life?[END_REF][START_REF] Irvine | Usual therapy improves perianal Crohn's disease as measured by a new disease activity index. McMaster IBD Study Group[END_REF]. Significant constipation was defined as a KESS ≥ 10 [START_REF] Knowles | Linear discriminant analysis of symptoms in patients with chronic constipation: validation of a new scoring system (KESS)[END_REF]. Patients with both severe FI and severe constipation were considered to have a "mixed" condition. Liquid stools were defined as Bristol stool score of 5, 6 or 7 [START_REF] Lewis | Stool form scale as a useful guide to intestinal transit time[END_REF].

We chose to define altered quality of life on the basis of GIQLI score because the GIQLI is a validated scale for gastrointestinal complaints. A key objective of the study was to identify the factors associated with a severely altered quality of life in our population. We defined patients with scores for this scale in the lowest quartile as having a severely altered quality of life. We compared these patients with the other patients.

Statistical analysis. Quantitative data are expressed as means (interquartile ranges [IQRs:

25% and 75%]). Categorical variables are presented as totals and percentages of the cohort.

Qualitative variables are expressed as positive values. The upper limit of the lowest quartile for GIQLI score was 71. Below this threshold, patients were considered to have a severely altered quality of life. We compared the patients with a severely altered quality of life (GIQLI< 71) with the other patients (GIQLI > 71), in t-tests for quantitative variables and Chi-squared tests for categorical variables. For each analysis, a p-value < 0.05 was considered to be statistically significant. Items with p<0.05 in the univariate analysis were integrated into a binary logistic regression model for multivariate analysis. If a univariate analysis included several significant mutually dependent variables, only one of these variables was included in the multivariate analysis. Qualitative variables were preferred when possible. Optimal cut-off values were obtained by optimising the Youden index from an area under receiver operating characteristic (AUROC) curve analysis. The results are shown as odd ratios (ORs) with 95% CIs. Statistical analyses were performed with JMP Pro Software, version 9.0.2 (SAS Institute Inc., Cary, NC, USA).

RESULTS

Population

From 2005 to 2016, 1870 patients with faecal incontinence (FI) and/or constipation were included in the registry and their data were recorded in a prospective database. The characteristics of the population are shown in Table 1. Mean GIQLI score was 86.7 (23.2).

Based on the distribution of GIQLI scores in our population, 470 of 1870 (25%) patients had a severely altered quality of life (GIQLI<71). These patients were compared with the remaining 1400 patients with GIQLI >71. FI was reported by 1212 of the 1870 patients (65.1%) and was severe in 761 of the 1870 patients (40.9%). Constipation was considered significant in 1398 (74.5%) patients. Among the 658 patients with "isolated "significant constipation, 322 (48.9%) had IBS-C. Overall, 450 of the 1870 (24.1%) patients had a "mixed" condition combining both FI and constipation. Among the 470 patients with severely altered quality of life, 137 (29.1%) had constipation, 74 (15.7%) had FI and 259 (55.1%) had both. In total, 1077 patients self-reported FI: 366 (34.0%) experienced urgency, 473 (43.9%) described passive FI and 238 (22.1%) had a mixed form of FI. There was no significant difference in age or sex ratio between patients with and without severely altered quality of life. Those with a severely altered quality of life had a higher body mass index (BMI). Overall 120 of 951 patients (12.6%) were obese (BMI>30). Diabetes, neurological disease, depression and a history of colpocele surgery were more frequent in patients with a severely altered quality of life. Patients with a severely altered quality of life were more likely to have urinary incontinence.

Anorectal complaints

Patients with a severely altered quality of life had significantly higher scores for FI and constipation. The association of severe FI with severe constipation was more frequent in patients with a severely altered quality of life (182/470 (38.7%) vs 268/1400 (19.1%), p=0.0001). In terms of clinical characteristics, patients with a severely altered quality of life had stools that were more frequent and more liquid. By contrast, there was not difference between the two groups in terms of the presence of clinical dyssynergic defaecation and rectal prolapse.

Factors associated with a severely altered quality of life

According to receiver-operator characteristic (ROC) curves (data not shown), the optimal cutoff value for UDI was 8. In a multivariate analysis model including history of diabetes, neurological disease, depression, cholecystectomy, prior surgery for pelvic floor disorders, liquid stools, CCIS ≥ 9, KESS ≥ 10, and UDI score >8, the factors significantly associated with a severely altered quality of life were history of diabetes, neurological disease, depression, liquid stools, severe FI, constipation and a UDI score >8.

DISCUSSION

This study highlights the high prevalence of constipation (74.5%) and of an association of severe FI and constipation (24.1%) in our population. Patients with a severely altered quality of life reported higher severities in assessments of both FI and constipation. These symptoms had at least as strong an impact on quality of life as neurological disease and depression.

The main strengths of this work are the large sample size, and the prospective and systematic quantification of FI, constipation and urinary disorders in a proctology unit. The data were recorded in a prospective database, according to recommended classifications and the results obtained for validated scales [START_REF] Eypasch | Gastrointestinal Quality of Life Index: development, validation and application of a new instrument[END_REF][START_REF] Knowles | Linear discriminant analysis of symptoms in patients with chronic constipation: validation of a new scoring system (KESS)[END_REF][START_REF] Lewis | Stool form scale as a useful guide to intestinal transit time[END_REF]. However, care is required in the interpretation of our results: recruitment at a tertiary centre specialising in these fields may lead to the selection of the patients with the most severe complaints. In addition, despite the adequate assessment of most patients in the registry, some data, particularly for anthropometric data or treatments were missing and not all patients had anorectal studies. Finally, specific questionnaire for the constipation as PAC-QOL [START_REF] Marquis | Development and validation of the Patient Assessment of Constipation Quality of Life questionnaire[END_REF] and for the FI as FIQL [START_REF] Rullier | Validation of the French version of the Fecal Incontinence Quality-of-Life (FIQL) scale[END_REF] were not used and that is a limitation. However, it is important to note that FIQL is validated in French [START_REF] Rullier | Validation of the French version of the Fecal Incontinence Quality-of-Life (FIQL) scale[END_REF] such as GIQLI but PAC-QOL is not.

It is not surprising to find that comorbid conditions, such as depression and neurological disorders, are associated with a decrease in QoL. Anorectal complaints may increase the impact of such conditions. It has already been suggested that HRQoL is associated with depression in patients with FI [START_REF] Smith | Factors associated with quality of life among women with fecal incontinence[END_REF][START_REF] Damon | Clinical characteristics and quality of life in a cohort of 621 patients with faecal incontinence[END_REF][START_REF] Menees | Factors associated with symptom severity in women presenting with fecal incontinence[END_REF]. Anorectal complaints are frequently observed in patients with neurological disease are generally involve both FI and constipation [START_REF] Choung | Chronic constipation and co-morbidities: A prospective population-based nested case-control study[END_REF]. These problems are known to reduce the patient's quality of life and to cause anxiety [START_REF] Coggrave | Management of faecal incontinence and constipation in adults with central neurological diseases[END_REF]. The items "depression" and "neurological disease" (e.g. diabetes) are very difficult to modify because they are chronic diseases causing permanent damage. However, physicians must take into account as the impact of the patients' backgrounds on their quality of life, in addition to their complaints.

Interestingly, Qol was more strongly linked to constipation than to FI. Very few studies of quality of life in patients with anorectal disorders have included evaluations of constipation [START_REF] Sailer | Quality of life in patients with benign anorectal disorders[END_REF][START_REF] Damon | Clinical characteristics and quality of life in a cohort of 621 patients with faecal incontinence[END_REF][START_REF] Kovacic | A multicenter study on childhood constipation and fecal incontinence: effects on quality of life[END_REF][START_REF] Damon | Clinical characteristics and quality of life in a cohort of 621 patients with faecal incontinence[END_REF]. However, in one cohort of patients with various anorectal disorders, the authors [START_REF] Sailer | Quality of life in patients with benign anorectal disorders[END_REF] suggested that patients with severe constipation or with both severe constipation and FI had a poor quality of life. These findings are consistent with our data for a population of patients with anorectal disorders. Two paediatric studies [START_REF] Kovacic | A multicenter study on childhood constipation and fecal incontinence: effects on quality of life[END_REF][START_REF] Klages | Empirically Derived Patterns of Pain, Stooling, and Incontinence and Their Relations to Health-Related Quality of Life Among Youth With Chronic Constipation[END_REF] have shown that children with functional constipation and FI are at particular risk of developing specific HRQoL problems, such as illness-related activity limitations, psychosocial issues, disease burden and worry, and family conflict. These results suggest that the management of patients with poor HRQoL and anorectal disorders should involve the detection and treatment of constipation. It would be interesting to follow our population to assess the efficacy of the treatments.

Finally, both stool consistency and stool frequency were major factors associated with HRQoL in our study. However, conflicting results have been obtained for these factors in previous studies. Some studies have reported that hard stools have a stronger negative impact on HRQoL than liquid stools [START_REF] Markland | Factors impacting quality of life in women with fecal incontinence[END_REF], but stool consistency was not evaluated with the validated Bristol Stool Form Scale [START_REF] Lewis | Stool form scale as a useful guide to intestinal transit time[END_REF]. Other studies focusing on FI have reported that liquid stools have a more negative impact on general QoL than solid stools [START_REF] Boreham | Anal incontinence in women presenting for gynecologic care: prevalence, risk factors, and impact upon quality of life[END_REF]. In our study, liquid stools were associated with a poor HRQol, as were constipation and severe FI. A liquid consistency of the stools may be linked to severe constipation (stercoral diarrhoea) and severe FI. Taken together, these results highlight the need for better therapeutic control of stool consistency.

In conclusion, this study quantified the link between poor QoL and constipation and/or FI.

By contrast to constitutive comorbid conditions (diabetes, neurological disorders, depression), it may be possible to use simple therapeutic approaches to improve some components (stool consistency, straining at stool, restraining) in patients with a combination of constipation and faecal incontinence (a quarter of the study population). A prospective longitudinal study would be useful, for analysis of a step-by-step therapeutic approach to Qol. This comprehensive approach would make it easier to determine the strategies to be given priority in multimodal treatments.

CB received lecture fees from IPSEN GB received lecture fees from AbbVie Laboratories, Ferring and MSD Pharma LS received lecture fees from Ferring and Takeda

Funding: None

Author contributions:


B La Levasseur

Scola

Michel Hosny

Emeline Baptiste

Anthony Levasseur

MD Bernard La Scola
email: bernard.la-scola@univ-amu.fr

E Hosny

A Baptiste

Scola ) La

with the occurrence of necrotizing enterocolitis in preterm neonates

Keywords: Necrotizing enterocolitis, Clostridium neonatale, rpoB-based specific quantitative real-time polymerase chain reaction, core-genome phylogeny, clonal lineage, whole genome Necrotizing enterocolitis, Clostridium neonatale, rpoB-based specific quantitative real-time polymerase chain reaction, core-genome phylogeny, clonal lineage

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

Introduction

Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in neonatal intensive care units (NICUs), especially for very low birth weight born (VLBW) [START_REF] Hosny | Updating on gut microbiota and its relationship with the occurrence of necrotizing enterocolitis[END_REF]. Over the past decade, most studies have focused on intestinal immaturity, feeding strategies and the composition of the gut microbiota, as NEC is recognized as a multifactorial disease [START_REF] Hosny | Updating on gut microbiota and its relationship with the occurrence of necrotizing enterocolitis[END_REF][START_REF] Cassir | Gut microbiota and the pathogenesis of necrotizing enterocolitis in preterm neonates[END_REF].

Approaches of gut microbiota composition determination was performed using molecular-based tools, such as 16S rRNA pyrosequencing, shotgun metagenomic sequencing and specific quantitative real-time polymerase chain reaction [START_REF] Hosny | Updating on gut microbiota and its relationship with the occurrence of necrotizing enterocolitis[END_REF][START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF][START_REF] Ward | Metagenomic sequencing with strain-level resolution implicates uropathogenic E. coli in necrotizing enterocolitis and mortality in preterm infants[END_REF]. These tools have been used to describe dysbiosis-linked alterations in neonates' gut microbiota. In addition, several bacterial species have been specifically associated with NEC including K. pneumoniae, E. cloacae, Uropathogenic Escherichia coli, Clostridium butyricum and Clostridium neonatale [START_REF] Hosny | Updating on gut microbiota and its relationship with the occurrence of necrotizing enterocolitis[END_REF][START_REF] Ward | Metagenomic sequencing with strain-level resolution implicates uropathogenic E. coli in necrotizing enterocolitis and mortality in preterm infants[END_REF][START_REF] Boccia | Nosocomial necrotising enterocolitis outbreaks:epidemiology and control measures[END_REF][START_REF] Smith | Community analysis of bacteria colonizing intestinal tissue of neonates with necrotizing enterocolitis[END_REF][START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF].This latter was first isolated in the context of epidemic NEC cases in a Canadian NICU [START_REF] Alfa | An outbreak of necrotizing enterocolitis associated with a novel Clostridium species in a neonatal intensive care unit[END_REF]. Pulsedfield gel electrophoresis identified similar banding profile from blood and stool culture isolates [START_REF] Alfa | An outbreak of necrotizing enterocolitis associated with a novel Clostridium species in a neonatal intensive care unit[END_REF]. Later, Roze et al. reported correlations between nutritional strategies and gut microbiota composition, where C. neonatale was identified from first stool of preterm neonates with NEC [START_REF] Roze | Nutritional strategies and gut microbiota composition as risk factors for necrotizing enterocolitis in very-preterm infants[END_REF] and its genome sequence was determined [START_REF] Benamar | Genome sequence of a Clostridium neonatale strain isolated in a Canadian neonatal intensive care unit[END_REF][START_REF] Bernard | Clostridium neonatale sp. nov. linked to necrotizing enterocolitis in neonates and a clarification of species assignable to the genus Clostridium (Prazmowski 1880) emend. Lawson and Rainey[END_REF]. A causal relationship between the presence of C. neonatale in stool samples and NEC has not yet been clearly demonstrated, but it warrants further studies. Based on a cohort of neonates and controls enrolled to study the involvement of C. butyricum with NEC [START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF][START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF], we evaluated the association of C. neonatale in the occurrence of NEC. This hypothesis was developed by studying the frequency of C. neonatale in the neonates' stools using a specific rpoB-based quantitative real-time polymerase chain reaction (qPCR), and then comparing the strains isolated by genomic sequencing.

Materials and Methods

Study design and patients

The study was validated by agreements from the ethics committee of the "Institut Fédératif de Recherche, IFR48" and the "Institut Hospitalo-Universitaire, IHU-2017-007". Written approval was obtained from the parents of all patients [START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF]. One hundred and fifty-nine stool samples were collected from preterm neonates treated in five neonatal intensive care units (NICUs), whose parents consented to the study. Enrolled samples were obtained from our previous cohort studies, that included 88 patients with NEC and 71 healthy controls [START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF][START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF]. None of the patients were subjected to probiotic therapy. Stool samples were collected, if possible, on the day of symptoms onset and stored at -80°C. Patients and controls were assorted by sex, gestational age (under 37 gestational weeks), birth weight, days of life, feeding strategies, mode of delivery and previous antibiotic therapy (Table 1). Routine microbiological analysis was negative for all samples.

Genome sequencing, assembly and annotation of Clostridium neonatale isolates

Five C. neonatale strains, all from Marseille, isolated in a previous work were sequenced and analyzed [START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF]. Four were NEC-associated isolates (NEC25, NEC26, NEC32, NEC86) and one was from a control neonate (C25). Strains were cultured on 5% Columbia sheep blood agar (Becton Dickinson®, USA) at 37°C for 48 hours under anaerobic conditions before DNA extraction on EZ1 DNA Tissue Kit (QIAGEN, Germany). Genomic DNA was sequenced on MiSeq Technology (Illumina, Inc., San Diego, CA) using the paired-end applications. NEC86 strain was additionally sequenced by MinION technology (Nanopore, Oxford, UK) [START_REF] Lu | Oxford Nanopore MinION sequencing and genome assembly[END_REF][START_REF] Hosny | Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium[END_REF].

SPAdes software was used for NEC86 reads assembly obtained from both sequencing methods [START_REF] Bankevich | SPAdes: 15 A new genome assembly algorithm and its applications to single-cell sequencing[END_REF]. Reads of the other C. neonatale genomes, were mapped against NEC86 dataset using the CLC genomics workbench 7 (Qiagen Inc., Valencia, CA). Coding DNA sequences (CDS) and annotation were procured using Prokka software [START_REF] Seemann | Prokka: Rapid prokaryotic genome annotation[END_REF]. Finally, sequenced genomes were deposited to the European Molecular Biology Laboratory -European Bioinformatics Institute (EMBL-EBI) under accession numbers: NEC25 (UWJD01000001-01000003), NEC26 (UICU01000001-01000003), NEC32 (UICW01000001-01000003), NEC86 (UICR01000001-01000003), C25 (UICQ01000001-01000003) (Table 2). Further analysis and comparison was conducted by introducing genome sequences from two strains isolated from a Canadian NICU (LCDC99A005: GCA_001458595.1 and LCDC99A006: GCA_002553455.1) [START_REF] Alfa | An outbreak of necrotizing enterocolitis associated with a novel Clostridium species in a neonatal intensive care unit[END_REF][START_REF] Bernard | Clostridium neonatale sp. nov. linked to necrotizing enterocolitis in neonates and a clarification of species assignable to the genus Clostridium (Prazmowski 1880) emend. Lawson and Rainey[END_REF].

Design of specific qPCR for Clostridium neonatale detection

As previously performed for the detection of C. butyricum [START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF], we designed a C. neonatale specific qPCR targeting the rpoB gene. In a first time, we verified that the rpoB gene was presented in unique copy within C. neonatale genomes (see above). In a second time, these sequences were aligned with those of 4 pathogenic species using MEGA7 software [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets[END_REF]. Other Clostridium genomes included in this analysis were C. butyricum E4 str. BoNT E BL5262 (GCA_000182605.1), Clostridium botulinum A str. (GCA_000017025.1), Clostridium perfringens ATCC 13124 (GCA_000013285.1) and Clostridioides (Clostridium) difficile QCD-66c26 (GCA_000003215.1) as an outgroup at the class level (Clostridia). Primers and probe were designed to detect specifically C. neonatale rpoB gene using Primer3 software [START_REF] Untergasser | Primer3new capabilities and interfaces[END_REF].

Finally, a 151 nucleotides rpoB sequence was selected: this region is homogenous among C. neonatale strains and highly heterogenic compared to other Clostridia. This molecular construction was validated by a Maximum-Likelihood phylogenetic analysis generated by MEGA7 software (Figure 1) [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for bigger datasets[END_REF]. LightCycler ® 480 Probes Master (Roche, Germany) was used to achieve qPCR reactions. The optimized 20 µL qPCR mix contained: master mix (10 µL), primers (0.5 µL, 20 nM), probe (0.5 µL, 5 nM), water (3.5 µL) and 5 µL of DNA. The qPCR cycling protocol started with one cycle of 50°C for 2 minutes, followed by one activation cycle (95°C for 5 min) and 40 amplification cycles (95°C for 1 second; Tm= 60°C for 30 seconds).

Tubes were deposed in a thermal cycler CFX96 Touch™ (Bio-rad ® , France). Sequences of the qPCR system were designed as follows: forward primer: AATAGTTGATAAAGAACACGGTAGAGC, reverse primer: TAGCGGTTCTTTTGCTTGAG and probe (FAM-TGCAGATGAAGAAGATCAGTTTTT-TAMRA) (Supplementary Figure S1). First, the qPCR system was tested on a collection of 79 bacterial species associated with the gut microbiota, including five locally isolated C. neonatale and 24 Clostridium species (Supplementary Table S1). Total genomic DNA was extracted directly from stool samples using the NucleoSpin Tissue Kit (Macherey-Nagel, Hoerdt, France) as previously described [START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF].

Statistical analysis

Statistical analysis was performed using SPSS® statistics 2016 (IBM, NY, USA). Mean and standard deviation were used to describe continuous variables. Percentage and number of events were used for quantitative variables. Student t-test or Mann-Whitney U test were used to perform two-group comparisons for quantitative variables. The chi-square (Mantel-Haenszel) test was used to perform two-group comparisons for qualitative variables, or the Fisher exact test was used when the expected count was less than 5. A multivariate analysis using logistic regression was performed to identify independent risk factors for NEC. Variables clinically relevant and associated (p < 0.10) with NEC in univariate analysis were used in the model. A p value of < 0.05 was considered statistically significant.

Draft genome-based and core-genome phylogeny

For draft genome-based phylogeny, only mapped genomes were included. Scaffolds were concatenated, and aligned using the MAFFT software [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: Improvements in performance and usability[END_REF]. For core-genome analysis, a dataset comprising seven genomes were generated. Orthologous proteins were obtained from ProteinOrtho software using the following parameters: identity 50%, coverage 60% and E-value 1.10 -10 . CDSs of core-genes were inferred from the pan-genome, then concatenated and aligned using a Python script. The phylogenetic trees were generated using the maximum-likelihood method within PhyML [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF] and edited by TreeGraph 2 software [START_REF] Stöver | TreeGraph 2: Combining and visualizing evidence from different phylogenetic analyses[END_REF]. BLAST was used to identify hemolysin (A, B, C), beta-hemolysin sequences, dlt operon (dlt A, B, C, D) and the Clusters of Orthologous Groups (COG) identification (E-value 1e -03 , coverage 0,7 and identity percent 30% for this later).

Results

Evaluation of patients features and Clostridium neonatale specific rpoB-based qPCR system

There was no statistically significant difference between NEC and control groups regarding gestational age, days of life, sex ratio, birth weight and feeding strategies. Only vaginal delivery was significantly more prevalent in controls than in NEC (37/71 [52%]; vs 32/88 [36%]; p < 0.05). This statistical analysis improved the rationality of clinical features between NEC and healthy controls. Specificity of primers and probe were validated due to positive amplification for all C. neonatale strains (5/5) and the absence of amplification for other bacterial strains tested isolated from gut microbiota (0/74). This result excluded cross-reaction between C. neonatale qPCR system and bacterial species inhabited within the gut microbiota. C. neonatale was more frequently detected in stools from preterm neonates with NEC than in controls (respectively 30/88 [34%]; vs. 9/71 [13%]; p = 0.003). The presence of C. neonatale in stool samples was significantly associated with the occurrence of NEC. Statistical results are summarized in Table 1. Means of C. neonatale qPCR cycle thresholds (Ct) were statistically non-significant between NEC and controls (26.9 and 25.4 respectively, p value = 0.5292, unpaired student t test), suggesting that the density of C. neonatale is similar in both cohorts. The prevalence of C. neonatale was also evaluated with that of C. butyricum reported in Hosny et al. [START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF]. In brief, the prevalence of C. butyricum, evaluated by both culture and qPCR, were assessed from each cohort and compared with C. neonatale frequency of detection (present work). In the majority of NEC cases, C. neonatale was frequently detected in association with C. butyricum when compared to controls (respectively 20/30, [66.7%] vs 1/9, [11.1%], p=0.003) (Supplementary Table S2).

Genomic analysis of Clostridium neonatale

Final draft genomes of C. neonatale isolates sequenced herein consist of 3 scaffolds. The average length of included genomes was 4,431,047 bp, NEC86 had the longest genome length (4,739,641 bp) and NEC26 the shortest one (4,282,644 bp). GC content varied between 28.4% (LCDC99A006) and 28.9% (NEC26). Predicted CDSs ranged between 3,795 genes (LCDC99A005) and 4,188 genes (NEC86) with genes average of 3,945. General features of these genomes are summarized in Table 2. The draft genome-based phylogeny of C. neonatale isolates enabled the identification of three clusters, revealing clonality among isolates from the same NICU by following: (1) strain isolated from NICU-1 (LCDC99A005), (2) strain isolated from NICU-2 (NEC86) and (3) strains isolated from NICU-3 (NEC25, NEC26, NEC32 and C25) (Figure 2A). Furthermore, core-genome analysis distinguished the same clustering, where the strict pangenome consisted of 5,150 genes, including 3,157 core-genes (Figure 2B). Predicted COG categories were homogenous in all C. neonatale genomes. The COGs encoding carbohydrate metabolism and transport, and therefore general functional prediction (G and R), were over-represented, on the contrary, no treatment or modification of RNA, and therefore the structure and dynamics of chromatin were identified (A and B) (Supplementary Figure S2 and Table S3).

Identification of virulent and unique genes

Comparative analyses identified hemolysin-encoding genes in all strains as follows: hemolysin A (coverage: 100%, identity: 84% to 92%), two protein sequences encoded for hemolysin B (coverage: 100%, identity: 44% to 99%) for the first protein and (coverage: 99%, identity: 76% to 77%) for the second one, hemolysin C (coverage: 96%, identity: 81%) and betahemolysin (coverage: 100%, identity: 92%). We also identified genes encoding for the secretion of C. difficile toxin A and toxin B. Moreover, sequences of dlt operon (dlt A, B, C, D) were not detected in all draft genomes of C. neonatale (Supplementary Table S4). Unique genes also existed and mostly represented by hypothetical proteins, as follows: NEC25 (1/4, 25%), NEC26 (77/113, 68%), NEC32 (28/39, 72%), C25 (2/3, 67%), NEC86 (139/212, 65.5%) and LCDC99A005 (31/59, 52.5%) LCDC99A006 (Supplementary Table S5).

Discussion

Dysbiosis remains a main risk factor inducing the establishment of NEC, by promoting the translocation of pathogenic bacteria. Specific microorganisms were involved; the predominance of γ-proteobacteria, generating excessive inflammatory response and irregular colonization by strictly anaerobic bacteria, including Clostridia [START_REF] Hosny | Updating on gut microbiota and its relationship with the occurrence of necrotizing enterocolitis[END_REF]. Multidisciplinary approaches suspected the link between this class and the occurrence of NEC, especially C. butyricum, C. neonatale and C. perfringens [START_REF] Hosny | Updating on gut microbiota and its relationship with the occurrence of necrotizing enterocolitis[END_REF][START_REF] Zhao-Fleming | Are anaerobes a major, underappreciated cause of necrotizing infections?[END_REF]. To date, C. butyricum is the unique Clostridium species among the cases of Clostridia-associated NEC clearly described in correlation with NEC [START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF][START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF][START_REF] Benamar | Multi-spacer typing as an effective method to distinguish the clonal lineage of Clostridium butyricum strains isolated from stool samples during a series of necrotizing enterocolitis cases[END_REF]. However, several studies have already reported the involvement of C. neonatale in NEC disease [START_REF] Hosny | Updating on gut microbiota and its relationship with the occurrence of necrotizing enterocolitis[END_REF][START_REF] Alfa | An outbreak of necrotizing enterocolitis associated with a novel Clostridium species in a neonatal intensive care unit[END_REF]. Using a culture-based strategy, we observed that the prevalence of C. neonatale in stool samples from patients with NEC was not significantly different from that of controls (respectively 4/88 [4.5%]; vs. 1/71 [1.14%]; p = 0.26) [START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF]. During the review process of this previous work, a reviewer proposed that this discrepancy could be due to the use of heat-shock-based protocol. If it is supposed to kill all bacteria species except those spore-forming like C. neonatale, we could not exclude that the method skips the isolation of in vivo non-spore-form of Clostridia species. Therefore, we decided to investigate our cohort using C. neonatale specific rpoB-based qPCR.

With a strong phylogeny and taxonomy index, rpoB is a conserved gene whose efficacy for the identification of fastidious microorganisms directly from samples has been previously demonstrated [START_REF] Adékambi | The rpoB gene as a tool for clinical microbiologists[END_REF][START_REF] Fenollar | Molecular genetic methods for the diagnosis of fastidious microorganisms[END_REF] and which was used for the heterogeneous detection of BoNT producing Clostridia [START_REF] Fach | Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum[END_REF] and C. butyricum directly from stool samples [START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF]. A significant frequency of C. neonatale in stools from NEC was detected compared to controls (respectively 34% vs 13%, p = 0.003). In contrast with the study conducted by Rozé et al., in which the authors reported links between feeding strategies and NEC and the abundance of C. neonatale in NEC patients [START_REF] Roze | Nutritional strategies and gut microbiota composition as risk factors for necrotizing enterocolitis in very-preterm infants[END_REF], the feeding strategy was herein not associated with the development of NEC. The only correlation identified was the vaginal delivery mode, a feature already observed in NEC cases [START_REF] Cassir | Gut microbiota and the pathogenesis of necrotizing enterocolitis in preterm neonates[END_REF]. Beside C. neonatale and C. butyricum, several NEC outbreaks have been described to be associated with other bacterial species [START_REF] Hosny | Updating on gut microbiota and its relationship with the occurrence of necrotizing enterocolitis[END_REF][START_REF] Cassir | Gut microbiota and the pathogenesis of necrotizing enterocolitis in preterm neonates[END_REF]. A case report described toxin-producing C. perfringens, where NEC severity was independent from the α-toxin concentration [START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: Diagnosis, clinical course, and role of alpha toxin[END_REF]. Also, nosocomial colonization by Klebsiella pneumoniae type 26 and antibiotic typing was used to specify strain characteristics [START_REF] Hill | Nosocomial colonization with Klebsiella, type 26, in a neonatal intensive-care unit associated with an outbreak of sepsis, meningitis, and necrotizing enterocolitis[END_REF]. Furthermore, genetically similar clones of Enterobacter sakazakii were distinguished from powdered milk formula and neonates with NEC [START_REF] Acker | Outbreak of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formula[END_REF]. Few other bacterial species were suspected in association with NEC like UPEC and C. paraputrificum [START_REF] Ward | Metagenomic sequencing with strain-level resolution implicates uropathogenic E. coli in necrotizing enterocolitis and mortality in preterm infants[END_REF][START_REF] Ferraris | Clostridia in premature neonates' gut: Incidence, antibiotic susceptibility, and perinatal determinants influencing colonization[END_REF].

The use of WGS allowed us to further compare and characterize isolates even if the number of strains and available genomes was limited. This study disclosed genetic similarity between strains isolated from the same NICU. Through phylogenetic analysis, we identified three distinct clusters, highlighted by their geographical area of isolation, suggesting the same clone of C. neonatale spreading in the same NICU-3 as we previously observed with C. butyricum [START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF]. This is in agreement with the discovery of this species as it was firstly isolated from a Canadian NICU (strains LCDC) and clonality between isolates was proved by PFGE from stool and blood cultures [START_REF] Alfa | An outbreak of necrotizing enterocolitis associated with a novel Clostridium species in a neonatal intensive care unit[END_REF]. Geographic and temporal clustering of NEC were described in several studies with various etiologies. Temporal clustering was reported by Faustini et al. in diverse NICUs [START_REF] Faustini | An epidemic of gastroenteritis and mild necrotizing enterocolitis in two neonatal units of a University Hospital in Rome, Italy[END_REF]. The similarity between control (C25) and NEC-associated C. neonatale in cluster [START_REF] Cassir | Clostridium butyricum strains and dysbiosis linked to necrotizing enterocolitis in preterm neonates[END_REF] presupposes the existence of asymptomatic carriage. This same kind of mechanism was described in case of C. difficile-associated pseudomembranous colitis [START_REF] Eyre | Diverse sources of C. difficile infection identified on whole-genome sequencing[END_REF] and we suggested a similar mechanism for C. butyricum in neonates [START_REF] Hosny | Multidisciplinary evaluation of Clostridium butyricum clonality isolated from preterm neonates with necrotizing enterocolitis in South France between 2009 and 2017[END_REF]. It should be noted that there appears to be a strong association between C. neonatale and neonates/children as it is a species that has never been isolated from adults in our microbiota studies [START_REF] Lagier | Culturing the human microbiota and culturomics[END_REF]. On the contrary, C. neonatale has been identified by sequencing of 16S rRNA in the microbiota of young children with a strong association of developing asthma [START_REF] Stiemsma | Shifts in Lachnospira and Clostridium sp. in the 3-month stool microbiome are associated with preschool age asthma[END_REF].

If the association of Clostridia with NEC was increasingly reported, the pathogeny of this disease remains elusive in spite production of toxins is usually suggested, as in the case of C. difficile infections (CDI) [START_REF] Stiemsma | Shifts in Lachnospira and Clostridium sp. in the 3-month stool microbiome are associated with preschool age asthma[END_REF]. Herein, genes encoding for the secretion of bacterial toxins were predicted, especially hemolysin and C. difficile toxin A/B (TcdA/B). These later, is a leading cause of CDI, where the pathogenic mechanism was the consequence of TcdA (enterotoxin) and TcdB (cytotoxin) productions inducing colonic tissue damage [START_REF] Fach | Development of real-time PCR tests for detecting botulinum neurotoxins A, B, E, F producing Clostridium botulinum, Clostridium baratii and Clostridium butyricum[END_REF][START_REF] Dittmar | Necrotizing enterocolitis of the neonate with Clostridium perfringens: Diagnosis, clinical course, and role of alpha toxin[END_REF]. The idea of toxin-mediated disease rather than invasive-mediated disease is supported by the work of Heida et al. [34]. Furthermore, hemolysins sequences with highly conserved domain found in a toxin of Brachyspira hyodysenteriae that disclosed cytolytic effect on several cell lines [START_REF] Hosny | Description of Clostridium phoceensis sp. nov., a new species within the genus Clostridium[END_REF]. Cassir et al. identified cytotoxic activity of C. butyricum supernatant on Jurkat cells [START_REF] Ward | Metagenomic sequencing with strain-level resolution implicates uropathogenic E. coli in necrotizing enterocolitis and mortality in preterm infants[END_REF].

In conclusion, this study highlights the association between C. neonatale and NEC as the possible existence NEC-associated geographic clones. Further genomic analysis is required on a larger number of sequenced genomes.

Table 1 : Factors associated with the occurrence of necrotizing enterocolitis when compared with control preterm neonates

 1

	338
		NEC	Controls	Univariate analysis Multivariate analysis
		(n=88)	(n=71)
	Factors			p value	OR [95% CI]	p value
		No. (%)	No. (%)
	Gestational age +/-SD (days)	28.36 (+/-2.82)	28.24 (+/-2.93)	0.7864	NA	NA
	Days of life +/-SD (days)	25.99 (+/-13.16)	23.99 (+/-11.61)	0.3166	NA	NA
	Male sex	49 (55.7)	43 (60.5)	0.594	NA	NA
	Birth weight mean +/-SD (g)	1179 (+/-380)	1174 (+/-432)	0.950	NA	NA
	Very low birth weight (≤1500g) 69 (78.4)	58 (81.7)	0.617	NA	NA
	Pasteurized breast milk	59 (67)	49 (69)	0.372	NA	NA
	Formula fed	9 (10)	13 (18)	0.139	NA	NA
	Vaginal delivery	32 (36)	37 (52)	0.023	2.3 [1.2-4.5]	0.018
	Antibiotics before collection	64 (73)	40 (56)	0.092	1.9 [0.9-3.8]	0.07
	C. neonatale q-PCR positive	30 (34)	9 (13)	0.003	3.7 [1.6-8.8]	0.003
	SD = standard deviation; NA = not applicable.

339

Table 2 : Characteristics of Clostridium neonatale draft genomes

 2

	340
					GC
	Location, date	Sequencing	Scaffolds	Genome length
	Isolate				content	ORFs number	Accession number
	of isolation	technique	number	(base pair)
					(%)
	LCDC
	NICU-1, 2000	Mate-pair	3	4,304,459	28.6	3,795	GCA_001458595.1
	99A005
	LCDC
	NICU-1, 2000	NA	58	4,658,596	28.4	4,139	GCA_002553455.1
	99A006
	NICU-3,
	NEC25	Paired-end	3	4,352,344	28.7	3,883	UWJD01000001-01000003
	December 2011
	NICU-3, July
	NEC26	Paired-end	3	4,282,644	28.9	3,840	UICU01000001-01000003
	2012
	NICU-3,
	NEC32	Paired-end	3	4,321,416	28.7	3,859	UICW01000001-01000003
	December 2012
	NICU-2,	Paired-end,
	NEC86		3	4,739,641	28.6	4,188	UICR01000001-01000003
	December 2010	Nanopore

Acknowledgments

We are grateful to the medical teams at Assistance publique -Hôpitaux de Marseille (AP-HM), CHU Nîmes, CHU Montpellier and CHU Nice for providing the stool samples. We thank the genomic and biobank platforms at the Institut Hospitalo-Universitaire (IHU) -Méditerranée Infection for technical assistance.

Funding

This work was supported by the French Government under the "Investissements d'avenir" program managed by the Agence Nationale de la Recherche (ANR), [reference: Méditerranée-Infection 10-IAHU-03], by Région Provence-Alpes-Côte d'Azur and European funding FEDER PRIMI. M. Hosny was supported by Fondation de Coopération Scientifique, Méditerranée-Infection [Infectiopôle-Sud 2015].

Conflict of interest

None of the authors have any conflicts of interest to report.


Emma Vecchioli1

Alexandra Benachi2

Isabelle Monier2

Affiliations

Emma Vecchioli
email: emma.vecchioli@hotmail.fr

Facteurs associés à la réussite d'une tentative voie basse après une césarienne : une étude observationnelle rétrospective monocentrique

Keywords: previous caesarean delivery, trial of labor, risk factors

Objective : To identify the maternal, obstetrical and neonatal factors associated with the success of trial of labor after one previous cesarean delivery.

Methods : An observational retrospective study was conducted between January 1st, 2013 and October 26th, 2016 in a level-III university maternity unit. We included 337 women with a history of one cesarean birth with a trial of labor and a singleton liveborn fetus in cephalic presentation at least 37 weeks of gestation.

Results : 226 women (67.1%) had a vaginal birth. Factors associated with the success of trial of labor were a history of a vaginal birth (OR=3.5 [1.8-7.0]) and a vaginal birth after the previous cesarean (OR=3.8 [1.5-9.2]). Factors associated with a caesarean section after onset of labor were obesity ≥30 kg/m² (OR=0.3 [0.2-0.6]), no epidural anesthesia (OR=0.2 [0.1-0.4]), induction of labor (OR=0.5 [0.3-0.8]), a delivery at 41 weeks or after (OR=0.5 [0.3-0.9]) and having a male fetus (OR=0.6 [0.4-0.9]). Maternal age, the existence of pregnancy complications, the use of oxytocine during labor and birthweight were not associated with the mode of delivery.

Conclusion :

An evaluation of antenatal and obstetrical factors is needed to estimate the probability of success rate of trial of labor after a history of one cesarean birth and to avoid the risk of uterine rupture.

Résultats : Au total, 226 femmes (67,1%) ont accouché par voie basse. Les facteurs associés à un succès de tentative voie basse après césarienne étaient l'antécédent d'accouchement par voie basse (OR=3,5 [START_REF] Blondel | Trends in perinatal health in France from 1995 to 2010. Results from the French National Perinatal Surveys[END_REF],0]) et si celui-ci a eu lieu après la césarienne (OR=3,8 [START_REF] Blondel | Trends in perinatal health in France from 1995 to 2010. Results from the French National Perinatal Surveys[END_REF][START_REF] Ricbourg-Schneider | Contre le déclenchement des utérus cicatriciels[END_REF][START_REF] Velemir | Pour le déclenchement sur utérus cicatriciel[END_REF][START_REF] Sentilhes | Déclenchement sur utérus cicatriciel : une option raisonnable ? Gynécologie Obstétrique[END_REF][START_REF] Beucher | Bénéfices et risques maternels de la tentative de voie basse comparée à la césarienne programmée en cas d'antécédent de césarienne[END_REF][START_REF] Lopez | Bénéfices et risques néonataux de la tentative de voie basse comparée à la césarienne programmée en cas d'antécédent de césarienne[END_REF][START_REF] Blondel | Les naissances en 2010 et leur évolution en[END_REF]).

Les facteurs associés à un échec d'accouchement par voie basse étaient l'obésité ≥30 kg/m² (OR=0,3 [0,2-0,6]), l'absence d'analgésie péridurale (OR=0,2 [0,1-0,4]), le déclenchement artificiel du travail (OR=0,5 [0,3-0,8]), un accouchement ≥41 SA (OR=0,5 [0,3-0,9]) et avoir un enfant de sexe masculin (OR=0,6 [0,4-0,9]). L'âge maternel, l'existence d'une pathologie pendant la grossesse, l'utilisation d'oxytocine pendant le travail et le poids de naissance n'étaient pas associés à la voie d'accouchement. Nous avons également exclu les utérus sains (n=7821), les utérus multicicatriciels (n=17), les utérus unicicatriciels dont l'origine était une myomectomie ou une autre chirurgie utérine (n=15) et les femmes avec un utérus unicicatriciel pour lesquelles une césarienne était programmée (n=116). Au total, la population d'étude était constituée de 337 femmes.

Conclusion

Recueil des données

Les variables étudiées ont été recueillies à partir de la base de données informatisées utilisée

Déclaration d'intérêts

Les auteurs n'ont aucun conflit d'intérêt à déclarer.

18.

Cameron

1 RésuméObjectif:

 1 Identifier les facteurs maternels, obstétricaux et néonataux associés à la réussite d'une tentative voie basse après césarienne. Méthodes : Nous avons réalisé une étude observationnelle rétrospective entre le 1 er janvier 2013 et le 26 octobre 2016 dans une maternité universitaire de type 3. La population d'étude incluait 337 femmes porteuses d'un utérus unicicatriciel dont l'origine était une césarienne, avec une tentative de voie basse et un enfant unique né vivant en présentation céphalique à 37 SA ou plus.

:

  La décision de la voie d'accouchement en cas d'utérus unicicatriciel nécessite une évaluation minutieuse des facteurs anténataux et obstétricaux afin d'estimer la probabilité de réussite d'un accouchement par voie basse et d'éviter les risques associés à cette voie d'accouchement, notamment le risque de rupture utérine. Mots-clés : utérus unicicatriciel ; tentative voie basse après césarienne ; facteurs de réussite Introduction En France, près d'1/5 ème des naissances a lieu après une césarienne soit environ 160 000 naissances annuellement.(1,2) La fréquence des césariennes a augmenté régulièrement depuis 20 ans : elle était de 15,9% en 1995 et de 21,0% en 2010.(1) En 2016, cette fréquence était stable par rapport à 2010 avec 20,4% de césariennes et 16,2% des femmes avaient un antécédent d'une césarienne.(3) L'utérus cicatriciel est une situation fréquente en obstétrique mais qui demeure une préoccupation importante pour les équipes médicales. En effet, l'utérus cicatriciel est un facteur de risque important de rupture utérine, définit par l'ouverture complète du muscle utérin, intrapéritonéale, avec ou sans rupture des membranes.(4) L'utérus cicatriciel est aussi un facteur de risque d'anomalie d'insertion placentaire (placenta praevia, placenta accreta etc.) et de complications hémorragiques (transfusion sanguine et hystérectomie d'hémostase).(4) Le mode de début de travail et d'accouchement en cas d'utérus cicatriciel sont des questions débattues dans la littérature.(5-9) La tentative voie basse après césarienne (TVBAC) expose principalement au risque de rupture utérine. Néanmoins, ce risque est relativement faible, compris entre 0,2 à 0,8% en cas d'utérus unicicatriciel.(10,11) Quant à la césarienne programmée après césarienne (CPAC), elle est associée à une augmentation du risque de complications hémorragiques, infectieuses et thromboemboliques.(4) En France, la Haute Autorité de Santé (HAS) et le Collège National des Gynécologues et Obstétriciens Français (CNGOF) recommandent une tentative voie basse après césarienne en dehors de toute contreindication (cicatrice corporéale, antécédent de rupture utérine, etc.) mais encouragent une sélection prudente des femmes éligibles à la voie basse.(4,12) Cette sélection repose sur une évaluation des facteurs maternels et obstétricaux en anténatal et une estimation des bénéfices et des risques d'une tentative voie basse par rapport à une CPAC.(4,8-9) L'objectif de cette étude était d'identifier les facteurs maternels, obstétricaux et néonataux associés à la réussite d'un accouchement par voie basse en cas de tentative de voie basse après césarienne. Population et méthodes Population d'étude Nous avons réalisé une étude observationnelle rétrospective et monocentrique entre le 1 er janvier 2013 et le 26 octobre 2016 au centre hospitalier universitaire Antoine Béclère (Clamart). Cette maternité de type 3 réalisait environ 3 000 naissances annuellement pendant la période de l'étude. La population d'étude incluait les femmes porteuses d'un utérus unicicatriciel avec une tentative de voie basse ayant accouché à 37 semaines d'aménorrhée (SA) ou plus d'un enfant unique né vivant en présentation céphalique. Ont été exclues les naissances <37 SA (n=1575), les grossesses multiples (n=552), les présentations non céphaliques (n=1103), les interruptions médicales de grossesse et les morts foetales (n=307).

  dans la maternité d'étude. Les caractéristiques sociodémographiques étudiées incluaient l'âge maternel, la parité et le fait de vivre en couple. Les caractéristiques anthropométriques étaient le poids habituel de la femme avant la grossesse et sa taille ce qui a permis de calculer l'indice de masse corporelle (IMC) avant la grossesse. Les facteurs médicaux recueillis étaient les antécédents obstétricaux et gynécologiques (la gestité, la parité et l'antécédent d'accouchement par voie basse après césarienne) et la survenue d'une pathologie pendant la grossesse. Enfin, nous avons recueilli les caractéristiques liées à l'accouchement (le mode de début de travail, l'utilisation d'oxytocine pendant le travail et le type d'analgésie) et les caractéristiques néonatales (l'âge gestationnel à la naissance, le poids de naissance et le sexe).Stratégie d'analyseTout d'abord, nous avons décrit les caractéristiques maternelles et néonatales de la population d'étude. Nous avons ensuite comparé les facteurs maternels, obstétricaux et néonataux associés à la réussite d'une tentative voie basse après césarienne. Ces comparaisons ont été réalisées à l'aide des tests du Chi 2, Fisher ou Yates. Nous avons également mesuré l'association entre les facteurs étudiés et la tentative voie basse après césarienne en estimant des odds ratios (OR) bruts avec leurs intervalles de confiance à 95%. Les analyses ont été réalisées à l'aide du logiciel Excel et Stata 13.0.Résultats L'ensemble des caractéristiques de la population d'étude est décrit dans le tableau 1. La majorité des femmes (62,3%) avaient entre 25 et 34 ans, et moins de 5% avaient moins de 25 ans ou plus de 40 ans. La proportion de femmes obèses était de 17,3% et 56,4% des femmes étaient en couple. Concernant les antécédents obstétricaux, 77,7% des femmes étaient primipares (avaient déjà eu un enfant), et 15,1% ont déjà eu 2 enfants. Parmi les 337 femmes, 22,0% ont déjà accouché par voie basse et 13,6% ont accouché par voie basse après l'antécédent de césarienne. Concernant la grossesse actuelle, près de la moitié des femmes (49,3%) ont présenté une pathologie pendant la grossesse. Le diabète représentait 38% des pathologies contre 6,6% pour les pathologies hypertensives et 3,0% pour la cholestase gravidique. L'âge gestationnel moyen était de 39,8 SA et la proportion de nouveau-nés de poids inférieur à 2 500 grammes était de 2,7% et de 4,1% pour ceux dont le poids était supérieur ou égal à 4 000 grammes.Le tableau 2 présente les facteurs maternels et obstétricaux associés à la réussite d'un accouchement par voie vaginale. Parmi les 337 femmes porteuses d'un utérus unicicatriciel, 67,1% d'entre elles ont accouché par voie basse. Un IMC ≥30kg/m² avant la grossesse (OR=0,3 [0,2-0,6]), l'absence d'analgésie péridurale pendant le travail (OR=0,2 [0,1-0,4]) et le déclenchement du travail (OR=0,5 [0,3-0,8]) étaient associés à une probabilité moindre d'accoucher par voie basse. A l'inverse, 85,1% des femmes avec un antécédent d'accouchement par voie basse ont accouché par voie basse alors qu'elles étaient 62,0% parmi celles sans antécédent d'accouchement par voie vaginale (p<0,001). L'OR associé à un antécédent d'accouchement par voie basse était de 3,5 [1,8-7,0]. De même, 87,0% des femmes dont l'accouchement par voie basse a eu lieu après l'antécédent de césarienne ont accouché par voie basse contre 63,9% en cas d'accouchement par voie basse avant l'antécédent de césarienne (OR= 3,8 [1,5-9,2]). En revanche, aucune association n'a été mise en évidence avec l'âge maternel, l'existence d'une pathologie pendant la grossesse et l'utilisation d'oxytocine pendant le travail. Le tableau 3 présente les facteurs néonataux associés à la réussite d'une tentative de voie basse. La grossesse prolongée (41 SA ou plus) était associée à plus d'échec de tentative de voie basse (OR=0,5 [0,3-0,9]). Le sexe masculin était également associé à une probabilité moindre d'accoucher par voie basse par rapport aux enfants de sexe féminin (60,9% versus 73,2%, OR=0,6 [0,4-0,9]). Enfin, la moitié des enfants de 4000g ou plus sont nés par voie basse alors qu'ils étaient 70,7% parmi ceux de poids compris entre 3000 et 3499g. Cette différence n'était pas statistiquement significative mais il faut noter qu'il n'y avait que 14 enfants dans la classe de poids de naissance de plus de 4000g. Discussion Dans notre population d'étude constituée de 337 femmes porteuses d'un utérus unicicatriciel avec tentative de voie basse, le taux d'accouchement par voie basse était de 67,1%. Nous avons montré que les facteurs associés à une réussite de voie basse étaient un antécédent d'accouchement par voie basse notamment s'il a lieu après l'antécédent de césarienne, un début de travail spontané et le recours à une analgésie péridurale pendant le travail. En revanche, nous n'avons pas trouvé de lien entre la voie d'accouchement et l'âge maternel, l'existence d'une pathologie de la grossesse, l'utilisation d'oxytocine pendant le travail ainsi que le poids de naissance. Le caractère monocentrique et la taille de notre population ne nous ont probablement pas permis d'obtenir des résultats significatifs concernant les facteurs précédemment cités et controversés dans la littérature. Par ailleurs, nous avons choisi d'étudier le poids de naissance comme un proxy de l'estimation de poids foetal et de le considérer comme un facteur anténatal. La marge d'erreur entre l'estimation de poids foetal et le poids de naissance peut expliquer notre absence de significativité pour ce critère. En revanche, le caractère monocentrique de notre étude a permis de limiter les différences de pratiques et de protocoles qui auraient pu être observées si nous avions réalisé une étude multicentrique. Nous avons fait le choix de débuter l'étude en 2013 à la suite des recommandations du CNGOF sur l'accouchement en cas d'utérus cicatriciel publiées en 2012 pour limiter les biais pouvant être liés aux évolutions des pratiques en raison de la mise en place de ces recommandations. La restriction de notre population à certaines situations (siège, grossesse multiple, utérus multicicatriciel, prématurité) a permis d'identifier les facteurs indépendamment de l'impact qu'auraient eu ces situations sur les résultats. Enfin, nous avons recueilli de nombreuses variables dans notre étude, ce qui nous a permis d'identifier une majorité des facteurs étudiés dans la littérature. Nos résultats sont concordants avec ceux d'une étude prospective multicentrique menée dans 19 centres aux Etats-Unis.(13) Cette étude a comparé 10 690 femmes avec un succès d'accouchement par voie basse à 3 839 femmes avec un échec de TVBAC. L'OR associé à l'antécédent d'accouchement par voie basse était de 3,9 [3,6-4,3], de 4,4 [2,9-6,7] pour l'antécédent d'accouchement par voie basse après l'antécédent de césarienne et de 1,6 [1,5-1,8] pour la mise en travail spontané. A l'inverse, un IMC ≥30 kg/m², le déclenchement artificiel du travail, un accouchement à 41 SA ou plus et le sexe masculin étaient associés à plus d'échec d'accouchement par voie basse. Cette même étude avait montré que la probabilité d'accouchement par voie basse était plus faible en cas d'IMC ≥30kg/m² (OR=0,5 [0,5-0,6]) et de déclenchement (OR=0,5 [0,4-0,5]). Dans une autre étude rétrospective écossaise qui s'est intéressée à 23 286 tentatives d'accouchement par voie basse, une diminution significative du succès d'accoucher par voie basse était liée à l'âge gestationnel supérieur à 41 SA (OR=1,3 [1,2-1,4]) et au sexe masculin (OR=1,2 [1,1-1,3]).(14) Nous n'avons pas montré d'association entre le succès d'une TVBAC et l'âge maternel, l'existence d'une pathologie pendant la grossesse, l'utilisation d'oxytocine pendant le travail et le poids de naissance. Les études rapportent des résultats conflictuels concernant l'association du mode d'accouchement après césarienne et l'existence d'une pathologie de la grossesse.(13,15-16) Une méta-analyse de 10 études a montré que l'âge maternel supérieur à 40 ans était associé à une diminution de la probabilité d'accoucher par voie basse (OR=0,5 [0,3-0,9]).(13) L'étude écossaise précédemment citée montrait également que l'augmentation de l'âge maternel augmentait le risque d'accoucher par césarienne (OR=1,2 [1,2-1,3]).(14) L'utilisation d'oxytocine au cours du travail était également liée à une diminution de la probabilité d'accoucher par voie basse dans une méta-analyse regroupant 4 études américaines (OR=0,5 [0,3-0,8]).(17) Enfin, concernant le poids de naissance, une étude prospective multicentrique américaine observait qu'un poids de naissance ≥4 000g (OR=0,6 [0,5-0,6]) diminuait la probabilité d'accoucher par voie basse.(18) La décision de la voie d'accouchement après césarienne est une discussion qui doit être menée avec le couple afin de leur exposer les bénéfices ainsi que les risques associés à chaque voie d'accouchement (CPAC ou TVBAC). Elle doit tenir compte des contre-indications à une voie basse après une césarienne qui sont une cicatrice corporéale, un antécédent de rupture utérine et une estimation du poids foetal supérieure à 4 500 grammes et des facteurs maternels et obstétricaux identifiés en anténatal. En 2012, le CNGOF privilégie la tentative voie basse pour les femmes ne cumulant pas les facteurs de risque.(19) Cette décision peut être rediscutée à tout moment du travail. En effet, si un ensemble d'arguments s'accumule en faveur d'un échec d'une tentative voie basse, une césarienne pourra être envisagée principalement pour d'éviter le risque de rupture utérine. Conclusion Dans notre étude, deux tiers des femmes porteuses d'un utérus unicicatriciel ont accouché par voie basse. L'antécédent d'accouchement par voie basse notamment s'il a eu lieu après l'antécédent de césarienne et la mise en travail spontané étaient associés à un succès d'une TVBAC. A l'inverse, un IMC ≥30 kg/m², un déclenchement, un âge gestationnel supérieur ou égal à 41 SA et le sexe masculin foetal étaient liés à plus d'échec d'accouchement par voie basse. La sélection des femmes porteuses d'un utérus unicicatriciel éligibles à une TVBAC doit être menée avec minutie par les équipes médicales, en concertation avec les couples, pour accepter une tentative de voie basse aux femmes avec des facteurs anténataux favorables à un succès d'une voie basse. Néanmoins, elle ne doit pas minimiser le risque de rupture utérine et de césarienne. Enfin, la décision de la voie d'accouchement peut être rediscutée devant l'apparition au cours du travail de facteurs associés à un échec d'accouchement par voie basse.

  CA, Roberts CL, Peat B. Predictors of labor and vaginal birth after cesarean section. Int J Gynaecol Obstet Off Organ Int Fed Gynaecol Obstet. juin 2004;85(3):267-9. Tableau 3. Facteurs néonataux associés à la réussite d'une tentative de voie basse après césarienne

	Tableau 1. Description de la population d'étude Tableau 2. Facteurs maternels et obstétricaux associés à la réussite d'une tentative de voie basse
	après césarienne					Effectifs
	19. Total N Gallot D, Delabaere A, Desvignes F, Vago C, Accoceberry M, Lémery D. Quelles sont n(%) ou moy ± ds Total Réussite voie Total Réussite voie basse basse 337 N n(%) p-value OR [IC95%] N n(%) p-value OR [IC95%]
	les recommandations d'organisation et d'information en cas de proposition de tentative de Caractéristiques maternelles Total N 337 226 (67,1) Total N 337 226 (67,1)
	Age (années) Age (années) Age gestationnel (SA) voie basse pour utérus cicatriciel ? J Gynecol Obstet Biol Reprod, déc 2012; 41(8), 782-787 18-24 9 (2,7) 18-24 9 6 (66,7) 0,996 1,0 [0,2-4,1] 37-38 61 41 (67,2) 0,084 0,8 [0,5-1,6]
	25-34 25-34 39-40	210 205	140 (66,7) 145 (70,7)		210 (62,3)	Ref Ref
	35-39 35-39 41+	100 71	68 (68,0) 40 (56,3)		100 (29,7) 1,1 [0,6-1,8] 0,5 [0,3-0,9]
	40+ 40+	18	12 (66,7)			18 (5,3) 1,0 [0,4-2,8]
	IMC (kg/m²) <18,5 18,5-24,9 25,0-29,9 30+ Poids de naissance (grammes) IMC (kg/m²) <18,5 <2500 18,5-24,9 2500-2999 25,0-29,9 3000-3499 30+ 3500-3999 ≥4000 ATCD d'accouchement voie basse En couple Oui Sexe	13 9 176 54 87 157 58 103 14 74	10 (76,9) 5 (55,6) 129 (73,3) 42 (77,8) 58 (66,7) 111 (70,7) 28 (48,3) 61 (59,2) 7 (50,0) 63 (85,1)	13 (3,9) 176 (52,7) 87 (26,0) 58 (17,4) 0,5 [0,1-2,0] 1,2 [0,3-4,6] 1,5 [0,7-3,0] Ref Ref 0,7 [0,4-1,3] 0,6 [0,4-1,0] 0,3 [0,2-0,6] 0,4 [0,1-1,2] <0,001 0,005 0,054 3,5 [1,8-7,0]
	Oui Non Masculin	263 169	163 (62,0) 103 (60,9)	0,017	190 (56,4) 0,6 [0,4-0,9] Ref
	Non ATCD d'accouchement voie basse Féminin	168	123 (73,2)		147 (43,6)	Ref
	après césarienne
	Parité Oui	46	40 (87,0)	0,004		3,8 [1,5-9,2]
	1 Non	291	186 (63,9)		262 (77,7)	Ref
	2 3+ Pathologie de la grossesse Oui	166	105 (63,3)	0,143		51 (15,1) 24 (7,2) 0,7 [0,5-1,1]
	ATCD* d'accouchement voie basse Non	171	121 (70,8)			74 (22,0)	Ref
	ATCD d'accouchement voie basse après césarienne Analgésie péridurale Oui	298	214 (71,8)	<0,001	46 (13,6)	Ref
	Pathologie de la grossesse Non	39	12 (30,8)			0,2 [0,1-0,4]
	Pathologies hypertensives Mode de début de travail					11 (3,3)
	Diabète Déclenché	109	62 (56,9)	0,003		63 (18,7) 0,5 [0,3-0,8]
	Cholestase gravidique Spontané	228	164 (71,9)			2 (0,6)	Ref
	Autres pathologies** Utilisation d'oxytocine					90 (26,7)
	Oui Caractéristiques néonatales Age gestationnel (SA) Non	187 150	124 (66,3) 102 (68,0)	0,743		Ref 1,1 [0,7-1,7] 39,8 ± 1,1
	Poids (grammes)			3335,0 ± 404,0
	<2500					9 (2,7)
	2500-2999					54 (16,0)
	3000-3499				157 (46,6)
	3500-3999				103 (30,6)
	≥4000					14 (4,1)
	Sexe
	Masculin				169 (50,1)

* ATCD : antécédent

Remerciements

Nous remercions Anne Chantry, sage-femme épidémiologiste et enseignante à l'école de Baudelocque pour ses conseils, ainsi que Mélanie Guignard, sage-femme du PMSI, pour sa disponibilité sans qui nous n'aurions pu obtenir les données nécessaires à l'étude.


Dr Nadia Benmoussa

Coordonnées Auteur

Scientific integrity applied to human remains samples research

En tant que chercheur quelle est notre légitimité à prélever ou à échantillonner, détruire ou révéler une vérité biologique de ces objets dont la propriété est incertaine ou qui sont « mal acquis » car utilisés sans l'autorisation du sujet à des fins scientifiques ? Ces restes humains s'inscrivent également dans un contexte muséal qui au delà du caractère scientifique relèvent d'un patrimoine. Il est donc souvent difficile de faire la distinction entre une pièce muséographique et une personne ou corps-mort. Comment répondre aux exigences de la science sans dénaturer ces pièces muséographiques ? L'intérêt scientifique de ces recherches n'a plus à être démontré, elles paraissent alors légitimes, si elles sont menées dans l'intérêt du « Bien Commun ». Pour le jeune chercheur moderne préoccupé par l'intégrité de son travail scientifique, il devient nécessaire d'adopter une ligne de conduite spécifique à ces recherches.

L'absence de statut propre à ces objets de recherche, expose d'autant plus au risque de manquement [START_REF] Cadot | Les restes humains : une gageure pour les musées ?[END_REF]. L'absence de cadre induit également immanquablement d'une diminution de la crédibilité de ces recherches par rapport à la société. Les travaux menés dans ce domaine de recherche même s'ils sont effectués sur un corps humain ne peuvent pas répondre aux mêmes exigences que la recherche sur vivant. D'une part, car les enjeux ne sont pas les mêmes et d'autres part, car il est parfois difficile de normaliser des recherches qui traitent de pièces uniques ou exceptionnelles. Il est important de mettre en oeuvre des moyens d'éducation et de prévention des différents acteurs: archéologues, médecins, chercheurs, conservateurs de musée dans le but de limiter les manquements en matière d'intégrité scientifique. Dans ce domaine de recherche la création d'un comité consultatif pourrait apporter des réponses aux demandes de prélèvements permettant de guider les différentes procédures, notamment sur les questions éthiques.

Les comités d'éthique des recherches effectuées sur les vivants ont été créés dans les années 80 et ils ont pour mission de donner des avis sur les questions éthiques soulevées par les progrès de la connaissance dans les domaines de la biologie, de la médecine et de la santé. Il est maintenant obligatoire d'avoir l'avis de ces comités pour toutes les études prospectives et récemment sur les études rétrospectives. Il a donc été considéré que les études même tournées vers ce qui a déjà été fait, donc le passé, devaient être aussi coordonnées par ces comités. Donc les recherches sur les restes humains peuvent elles aussi considérées comme des études rétrospectives et donc nécessiter aussi l'aval d'un comité consultatif pour guider leur réalisation.

L'intégrité scientifique est définie comme l'ensemble des règles et valeurs qui doivent régir l'activité de recherche pour en garantir le caractère honnête et scientifiquement rigoureux[START_REF] Winter | Defining Scientific Integrity[END_REF].La recherche intègre est devenue une nécessité pour tout chercheur, elle fonde le contrat entre la société et la science. Depuis plusieurs années il existe une prise de conscience au sein de la communauté scientifique multipliant les congrès, rapports, publications, jusqu'à la création de poste de référents sur l'intégrité scientifique au sein des universités[START_REF] Corvol | Bilan et propositions de mise en oeuvre de la charte nationale d'intégrité scientifique[END_REF].L'intégrité scientifique est maintenant prise en compte de façon sérieuse. La controverse autour de la recherche sur la tête du roi Henri IV est au coeur de ces débats[START_REF] Delorme | La mauvaise tête de Henri IV: contre-enquête sur une prétendue découverte[END_REF][START_REF] Charlier | Multidisciplinary medical identification of a French king's head (Henri IV)[END_REF]. Une équipe scientifique propose un ensemble d'arguments médico-historiques pour assigner une tête à celle d'Henri IV: datation au carbone 14, reconstruction faciale etc. Il y aura une querelle d'experts, matérialisée par des échanges d'articles. Au-delà de la controverse scientifique, l'intégrité de la première équipe a été interrogée notamment sur la présence de "faux auteurs" ou "auteurs de convenance" dans la première publication du British Journal of Medicine.Cette controverse montre que le sujet de l'intégrité scientifique est au coeur des débats sur la recherche, c'est une arme puissante et qu'il incombe à l'équipe de recherche de fournir la preuve de cette intégrité par tous les moyens disponibles. Dans l'article de Ross et al., une équipe scientifique apporte la preuve de la présence du virus de l'hépatite B chez une momie du 16 ème siècle alors qu'une étude faite 30 ans plus tôt apportait la preuve d'une infection par le virus de la variole[START_REF] Ross | The paradox of HBV evolution as revealed from a 16th century mummy[END_REF]. Les mêmes méthodes scientifiques ont été réalisées pour rechercher le virus de la variole mais elles n'ont pas été reproductibles. S'agit il d'un manque d'intégrité de la première équipe ? Existe t-il une falsification des résultats ? Il est difficile d'apporter des réponses claires car les méthodes d'analyses ont évolué et les prélèvements n'ont pas été réalisés au même endroit. Les auteurs du nouvel article n'émettent pas de jugement sur la méthodologie mise en place ou sur l'intégrité de l'équipe car il peut s'agir tout simplement d'une inconduite scientifique par manque de preuve ou d'un manque de puissance méthodologique lié aux procédures de l'époque.Les problématiques de recherche sur les prélèvements humains sont nombreuses. Tout d'abord la nature de ces prélèvements, issus de corps humain, interpelle sur les limites autorisées de ces prélèvements. Nous ne pouvons pas dénaturer ou détruire ces collections dans le simple but scientifique. Jusqu'ou est-il permis d'aller ? Comment est-il donc possible de conserver l'intégrité de ces pièces tout en réalisant des prélèvements à visées scientifiques ?