Théo Grilot

Sarah Jean

Elisabeth Faassen

Zouher Amzil

Emilien Mineaud
email: emilie.lance@univ-reims.fr

Luc Brient

Véronique Savar

Elise Robert

Alexandra Lepoutre

J Hervieux

Usability of the bivalves Dreissena polymorpha and Anodonta anatina for a biosurvey of the neurotoxin BMAA in freshwater ecosystems

Keywords: β-Methylamino-L-alanine, bioaccumulation, Dreissena polymorpha, Anodonta anatina, biosurvey

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

Introduction

The environmental neurotoxin β-methylamino-L-alanine (BMAA) is of growing concern because it may be involved in the development of the amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), a human neurodegenerative pathology [START_REF] Murch | Occurrence of β-methylamino-L-alanine (BMAA) in. ALS/PDC patients from Guam[END_REF][START_REF] Nunn | 50 Years of Research on Α-Amino-Β-Methylaminopropionic Acid (Β-Methylaminoalanine)[END_REF]. Even if no etiological link has been demonstrated so far [START_REF] Delcourt | Cellular and Molecular Aspects of the β-N-Methylamino-l-alanine (BMAA) Mode of Action within the Neurodegenerative Pathway: Facts and Controversy[END_REF], laboratory experiments have shown that this non-proteinogenic amino acid can induce : (i) excitotoxicity mediated by glutamate receptors in the presence of bicarbonate at physiological concentrations [START_REF] Weiss | Beta-N-methylamino-L-alanine neurotoxicity: requirement for bicarbonate as a cofactor[END_REF], (ii) dysregulation of the cellular protein homeostasis and a potent interaction with neuromelanin, which could lead to long-lasting neurotoxic activity [START_REF] Frøyset | Quantitative proteomics analysis of zebrafish exposed to sub-lethal dosages of β-methyl-amino-L-alanine (BMAA)[END_REF]Karlsson et al., 2009a[START_REF] Karlsson | Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA[END_REF], and (iii) inhibition of the cysteine/glutamate antiporter, leading to potential oxidative stress [START_REF] Lobner | Mechanisms of beta-N-methylamino-L-alanine induced neurotoxicity. Amyotrophic lateral sclerosis : official publication of the World Federation of[END_REF][START_REF] Pierozan | The environmental neurotoxin β-N-methylamino-L-alanine inhibits melatonin synthesis in primary pinealocytes and a rat model[END_REF]. Moreover, this toxin may also act as a developmental toxin potentially linked to neurodegeneration [START_REF] Karlsson | Intracellular fibril formation, calcification, and enrichment of chaperones, cytoskeletal, and intermediate filament proteins in the adult hippocampus CA1 following neonatal exposure to the nonprotein amino acid BMAA[END_REF](Karlsson et al., , 2009b;;[START_REF] Pierozan | Mitotically heritable effects of BMAA on striatal neural stem cell proliferation and differentiation[END_REF]. The onset of the ALS-PDC pathology is thought to occur following long-term chronic exposure via the consumption of BMAA contaminated food [START_REF] Banack | Biomagnification of cycad neurotoxins in flying foxes: Implications for ALS-PDC in Guam[END_REF], or after short exposure during embryo development as observed with rats embryonic neural stem cells [START_REF] Pierozan | Mitotically heritable effects of BMAA on striatal neural stem cell proliferation and differentiation[END_REF]. BMAA accumulates in various seafoods (mussels, crustaceans, shark fins) (Lance et al., 2018). Marine bivalve 'molluscs have even been qualified as the most BMAAcontaminated food source for humans, and blue mussels sampled along the French coasts systematically contained BMAA (Réveillon et al., 2016b;[START_REF] Lance | Occurrence of β-N-methylamino-lalanine (BMAA) and Isomers in Aquatic Environments and Aquatic Food Sources for Humans[END_REF]. However, its occurrence is not solely restricted to marine ecosystems, as it has been found in the tissues of fish from a Swedish Lake [START_REF] Lage | Biotransfer of β-Nmethylamino-L-alanine (BMAA) in a eutrophicated freshwater lake[END_REF]. BMAA producers have not been fully characterized. Initial BMAA quantification in cyanobacteria suggested that the neurotoxin may be produced by most of the freshwater cyanobacterial genus [START_REF] Cox | Diverse taxa of cyanobacteria produce beta-N-methylamino-L-alanine, a neurotoxic amino acid[END_REF]. However, the wide production of BMAA by freshwater cyanobacteria was not confirmed using more selective analytical methods (e.g., LC-MS/MS). Up to now, BMAA has been demonstrated to be produced by few cyanobacterial species (Nostoc sp., Leptolyngbya sp.), and also by diatoms (Chaetoceros sp., Phaeodactylum tricornutum) and marine dinoflagellates (Heterocapsa triquetra, Gymnodinium catenatum) [START_REF] Faassen | Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine ) and DAB (α-,γ-diaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms[END_REF][START_REF] Monteiro | Screening of BMAA-producing cyanobacteria in cultured isolates and in in situ blooms[END_REF]Réveillon et al., 2016b[START_REF] Réveillon | β-Nmethylamino-l-alanine (BMAA) and isomers: Distribution in different food web compartments of Thau lagoon, French Mediterranean Sea[END_REF].

Phytoplankton proliferations in fresh waters often occur in reservoirs used for recreational activities or drinking water. BMAA may therefore represent a threat to populations using water supply or living nearby contaminated lakes. In addition to the consumption of contaminated freshwater fish, human contamination may also occur through inhalation of aerosolized toxins or accidental ingestion of lake water [START_REF] Caller | A cluster of amyotrophic lateral sclerosis in New Hampshire: A possible role for toxic cyanobacteria blooms[END_REF]. A follow-up of BMAA in fresh waters might be useful to assess a potential risk of human exposure because phytoplankton proliferations are expected to be enhanced by increasing eutrophication and global warming [START_REF] Hudnell | Cyanobacterial harmful algal blooms: state of the science and research needs[END_REF]. As very little is known about the bioavailability of BMAA in fresh waters, quantification of its dissolved form (in the medium) or intracellular form (in the phytoplankton biomass) may be required. However, quantification of dissolved BMAA in water remains a challenge because of the hydrophilic nature of this small amino acid compound and the potential co-existence of seven isomers: DAB (2,4-diaminobutyric acid), BAMA (β-amino-N-methyl-alanine), N-2(aminoethyl)glycine, DABA (2,3-diaminobutyric acid), 3,4-diaminobutyric acid, AEG (3-amino-2-(aminomethyl)-propanoic acid), and 2,3diamino-2-methylpropanoic acid [START_REF] Jiang | Selective LC-MS/MS method for the identification of BMAA from its isomers in biological samples[END_REF]Faassen, 2014). Concerning BMAA quantification in the phytoplankton biomass, very little reliable data is currently available because highly selective analytical methods are required [START_REF] Lance | Occurrence of β-N-methylamino-lalanine (BMAA) and Isomers in Aquatic Environments and Aquatic Food Sources for Humans[END_REF]. Such data of intracellular BMAA concentrations in water range from 0.3 µg/L measured in a Canadian lake [START_REF] Roy-Lachapelle | Determination of BMAA and three alkaloid cyanotoxins in lake water using dansyl chloride derivatization and highresolution mass spectrometry[END_REF], to 39.6 µg/L in an American lake [START_REF] Al-Sammak | Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants[END_REF], or are expressed by phytoplankton biomass and vary from 2.3 ng/g [START_REF] Jonasson | Transfer of a cyanobacterial neurotoxin within a temperate aquatic ecosystem suggests pathways for human exposure[END_REF] to 42 µg/g [START_REF] Faassen | Determination of the neurotoxins BMAA (β-N-methylamino-L-alanine ) and DAB (α-,γ-diaminobutyric acid) by LC-MSMS in Dutch urban waters with cyanobacterial blooms[END_REF]. However, BMAA analysis in the phytoplankton biomass may not be sufficient to assess the overall contamination levels of an ecosystem for the two following reasons: i) BMAA production is not steady, it varies over time and may be influenced by environmental parameters such as nitrogen availability [START_REF] Scott | Environmental modulation of microcystin and β-N-methylamino-l-alanine as a function of nitrogen availability[END_REF], ii) phytoplankton communities are prone to display considerable short-term spatiotemporal variability [START_REF] Deng | Horizontal migration of algal patches associated with cyanobacterial blooms in an eutrophic shallow lake[END_REF][START_REF] Qi | Diurnal changes of cyanobacteria blooms in Taihu Lake as derived from GOCI observations[END_REF][START_REF] Salmaso | Basic Guide to Detection and Monitoring of Potentially Toxic Cyanobacteria[END_REF]. Therefore, the use of biological integrative tools such as sentinel bivalve species may be relevant to evaluate the presence of BMAA in the environment.

Freshwater bivalves can be used as bioindicators of metals, microplastics, organochlorine contaminants and parasites [START_REF] Baqar | Organochlorine contaminants in freshwater mussels; occurrence, bioaccumulation pattern, spatio-temporal distribution and human health risk assessment from the tributaries of River Ravi, Pakistan[END_REF][START_REF] Bourgeault | Lessons from a transplantation of zebra mussels into a small urban river: An integrated ecotoxicological assessment[END_REF][START_REF] Camusso | Use of freshwater mussel Dreissena polymorpha to assess trace metal pollution in the lower River Po (Italy)[END_REF][START_REF] Ladeiro | Bioaccumulation of human waterborne protozoa by zebra mussel (Dreissena polymorpha): Interest for water biomonitoring[END_REF][START_REF] Su | Using the Asian clam as an indicator of microplastic pollution in freshwater ecosystems[END_REF][START_REF] Zuykov | Bivalve mollusks in metal pollution studies: From bioaccumulation to biomonitoring[END_REF]. They can reveal the presence of the cyanotoxins microcystins (MC) in their medium, in the laboratory [START_REF] Lauceri | Coupling a bio-accumulator organism and MALDI-TOF MS: an early warning detection system for microcystins in water bodies[END_REF] and in situ, even when water analysis did not reveal it, in relation with the great spatio-temporal variability of the producers [START_REF] Preece | Ecotoxicology and Environmental Safety Transfer of microcystin from freshwater lakes to Puget Sound , WA and toxin accumulation in marine mussels ( Mytilus trossulus )[END_REF]. There is yet no data regarding BMAA concentrations in freshwater bivalves in situ, but laboratory experiments have shown that freshwater mussels bioaccumulate BMAA. The toxin was found in tissues of the mussels Anodonta cygnea and Dreissena polymorpha after exposure to 100 µg of radiolabelled BMAA/L for 24 and 48 h [START_REF] Downing | The fate of the cyanobacterial toxin β-N-methylamino-l-alanine in freshwater mussels[END_REF]. One of our previous studies showed that BMAA spread and accumulated throughout D. polymorpha soft tissues without a specific target organ, with similar kinetics of accumulation and elimination for all analysed fractions (total BMAA, free BMAA, BMAA associated with high-and low-molecular-weight polypeptides), when exposed to 2.5 µg BMAA/day for 21 days, followed by 21 days of depuration (Lepoutre et al., in press). Therefore, these bivalves could be used as potential bioindicators of the presence of BMAA in fresh water by quantifying total BMAA in one organ or in the entire body. The two species D. polymorpha and A. anatina were selected for this purpose. D. polymorpha has long been used in biomonitoring studies and has been suggested to represent the freshwater counterpart of the marine mussel Mytilus in ecotoxicological studies [START_REF] Binelli | Does zebra mussel (Dreissena polymorpha) represent the freshwater counterpart of Mytilus in ecotoxicological studies? A critical review[END_REF]. This sessile bivalve lives mostly on hard substrates and is present in a wide range of habitats, from freshwater lakes and rivers to brackish estuaries [START_REF] Van Damme | Dreissena polymorpha. The IUCN Red List of Threatened Species[END_REF]. However, the use of this species as a sentinel is restricted because D. polymorpha is an invasive species [START_REF] Van Damme | Dreissena polymorpha. The IUCN Red List of Threatened Species[END_REF]. Those restrictions may not apply to A. anatina, a vagile bivalve living unattached on soft substrates of flowing streams and standing waters [START_REF] Lopes-Lima | Cellular and subcellular distribution of metals in molluscs[END_REF].

The objectives of this study were to evaluate if D. polymorpha and A. anatina could reflect the levels and variations of BMAA concentrations in water and may therefore be suitable bioindicators of BMAA occurrence in aquatic environments. Due to the lack of a known microorganism that could produce BMAA steadily in laboratory conditions, A. anatina and D. polymorpha were exposed to 1, 10 and 50 µg of dissolved BMAA/L for 21 days, followed by 42 days of depuration in clean water. After 0, 7, 14, and 21 days of exposure and 1, 7, 14, 21, and 42 days of depuration, mussels were sampled to quantify total BMAA in tissues. Preliminary experiments showed that D. polymorpha was smaller than A. anatina (2 ± 0.3 cm vs. 6.5 ± 0.05 cm) with an approximately 12-times lower fresh weight (184.4 ± 9.7 mg FW vs. 2,171.7 ± 107.1 mg FW). Whole D. polymorpha were sampled to quantify BMAA. However, concerning A. anatina, the quantity of tissues required to perform the analysis represented less than 1% of the whole body (dry weight), so only one of the accumulating organs (the digestive gland) was sampled to avoid large inter-individual variability. Results are discussed in terms of dynamics of accumulation and elimination of the neurotoxin, and relevance of the use of each species as a bioindicator of the presence of BMAA in fresh water.

Materials and methods

Mussel acclimation

. Anodonta anatina

Mussels measuring 58.3 ± 5.6 mm were provided by Univers aquatique (Sartrouville, France), and were acclimated by groups of 50 at 14 ± 2°C in the dark in aerated 15-L tanks containing Cristalline® (Saint Yorre, France) spring water for two weeks.

During this phase, they were fed twice a week with 3.7 x 10 7 cells of Chlorella vulgaris/individual/day (Greensea, Mèze, France). Algal density was measured with an optical microscope (Primovert, Zeiss, Oberkochen, Germany) and KOVA® slides (Kova slide, VWR, Fontenay-sous-Bois, France).

1.2 . Dreissena polymorpha D. polymorpha were collected at the Lac-du-Der-Chantecoq (48°36'07.7"N; 4°44'37.0"E) in June 2016. In the laboratory, they were gently cleaned, sorted according to size (25 ± 2 mm), and placed in aerated tanks containing Cristalline® (Saint Yorre, France) spring water. As they originated from the field, they were gradually acclimated from lake temperature to 14 ± 2°C in the dark for up to seven weeks. During this phase, they were fed as described for A. anatina, with a ratio of 2 x 10 6 cells of Chlorella vulgaris/individual/day.

Experimental design

Preliminary experiments were performed to reach an equivalence in available BMAA per milligram of soft tissues (FW) of each species. As whole D. polymorpha fresh weight was 12 times lower than whole A. anatina fresh weight (184.4 ± 9.7 mg vs. 2,171.7 ± 107.1 mg), the density of mussels per litre was adjusted to one A. anatina/L and 12 D. polymorpha/L. Three days before the experiment, mussels were no longer fed and randomly dispatched in six aerated 40-L tanks containing 30.5 L of spring water. Before adding BMAA in the tanks, negative controls were taken by sampling 5 digestive glands of A. anatina and 2 pools of 3 whole D. polymorpha. Exposure was conducted in duplicate (two tanks per BMAA concentration for each species). A total of 61 A. anatina and 192 D. polymorpha were used for each concentration. Both species were exposed for 21 days to 1, 10 or 50 µg of dissolved BMAA/L (L-BMAA hydrochloride B-107, Sigma-Aldrich®, Saint-Louis, MO, USA), daily added in the tanks. The mussels were not fed throughout the experiment. The water was renewed every three days. At the end of the exposure period, the mussels were transferred into new tanks containing only clear water to study depuration for 42 days. Mussels were randomly collected and sacrificed after 0, 1, 7, 14, and 21 days of exposure and after 1, 7, 14, 21, and 42 days of depuration. The water volume was reduced after each sampling to keep the same mussel:water ratio over time. Samplings consisted of 4 pools of 3 whole D. polymorpha and, because of their size, 5 digestive glands of A. anatina per concentration. Tissues were kept in Eppendorf® tubes (Eppendorf, Hamburg, Germany), frozen in liquid nitrogen and stored at -80°C. Then they were freeze-dried and ground with a Mixer Mill MM400 (Retsch, Haan, Germany) using 4 beads, 4 min of beating at 30 Hz.

Total BMAA extraction and analysis by tandem mass spectrometry

Several fractions of BMAA can be analysed in tissues, requiring each a different extraction procedure [START_REF] Faassen | A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction[END_REF]. BMAA can accumulate in its free form ("free BMAA") when it is extracted with polar solvents. But some BMAAs can be associated to unknown compounds that can stay in solution, suggesting a low molecular weight of the BMAA-molecule complex ("soluble bound BMAA"), while some BMAAs can be bound in the precipitate, suggesting a heavier weight ("precipitated bound BMAA"). To reach a global understanding of BMAA concentrations in tissues, we analysed the total concentrations of BMAA ("total BMAA") encompassing free, soluble bound and protein-bound BMAA.

Total BMAA was extracted as described in [START_REF] Faassen | Extraction and LC-MS/MS Analysis of Underivatised BMAA[END_REF]. Briefly, 1 mg of tissues were spiked with 40 µl of D3BMAA in 20 mmol/L HCl, an internal standard, then dried under vacuum. After adding 30 µL of HCl 6 M, tissues were hydrolysed approximately at 0.7 mbar for 20 h at 105°C in an Eldex® hydrolysis workstation (Eldex, Napa, CA, USA). Then they were dried, resuspended twice in 500 µL of a 67:33:0.1 ACN:water:formic acid mix, and transferred into spin filter tubes and centrifuged. The underivatized extracts were analysed with a UHPLC-MS/MS system 1290 Infinity II connected with a 6490C triple quadrupole mass spectrometer (Agilent, Santa Clara, CA, USA). The method was slightly modified from the underivatized BMAA analysis described in [START_REF] Faassen | A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction[END_REF]: we injected 5 µL, and the compounds were separated on a HILIC column (ZIC®HILIC, 150 x 2.1 mm, 5 µm, 200 Å, Merck, Darmstadt, Germany) set at 40 °C. The mobile phase consisted of acetonitrile with 0.1% formic acid (A) and MilliQ-water with 0.1% formic acid (B). The initial conditions were 5% B for 2 minutes, followed by a gradient from 2 to 4 minutes to 35% B, from 4 to 8 minutes to 45% B, and up to 16 minutes at 45% B. Between 16 and 17 minutes, B was decreased to 5%, and this was held for another 5 minutes. The mass spectrometer was used in the positive mode with a gas flow of 12 mL/min, a source temperature of 230 °C, a nebulizer pressure of 40 psi, a sheath gas temperature of 200 °C, a sheath gas flow of 12 L/min, and a capillary voltage of 2.5 kV. The compounds were analysed in multi reaction mode (MRM) using nitrogen as the collision gas. BMAA was monitored based on the m/z transitions 119.1 > 76.2, 119.1 > 88.1 and 119.1 > 102.1 using collision energies of 9, 9 and 5 V and a fragmentor voltage of 73 V. Transitions for DAB were m/z 119.1 > 101.1 and 119.1 > 74.2 using collision energies of 5 and 13 V and a fragmentor voltage of 68 V. D3BMAA was monitored based on the m/z transitions 122.1 > 76.2, 122.1 > 88.1 and 122.1 > 105.1 using collision energies of 9, 9 and 5 V and a fragmentor voltage of 75 V. Masshunter B 08.02 (Agilent, Santa Clara,CA, USA) was used for data acquisition and analysis.

A 20% relative deviation from the average ion ratios in the standards was allowed in the samples. Furthermore, the BMAA retention time was verified by D3BMAA retention time. BMAA was quantified against an external calibration curve, and the concentrations in each sample were corrected for the signal intensity of the internal standard. DAB and AEG were not quantified, but only included in the analysis to ensure that there was no co-elution with BMAA.

In addition, in our study, LOD was defined as the lowest concentration at which the quantifier and the qualifier ions all had a signal to noise (S/N) ratio > 3. LOQ was defined as the lowest concentration at which the qualifier ions had a S/N ratio > 3 and the quantifier ion had a S/N >10. As the intensity of the quantifier was clearly higher than that of both qualifiers, the criteria for LOQ were met at the same concentration as the LOD. For both sample types (D. polymorpha and A. anatina), the LOQ (and LOD) was approximately 1 microgram/gram. Apparent recovery (extraction recovery plus matrix effect during ionisation) was determined for each sample by adding deuterated BMAA before extraction. Average apparent recovery for D. polymorpha was 81% (stdev 18, n=125). For A. anatina, the average apparent recovery was 47% (stdev 14, n=184).

Data treatment and statistics

A cumulative percentage of BMAA accumulation (cum % acc) was calculated for each sampling time, using the total BMAA concentration in tissues (µg/g DW) converted into µg/g FW using pre-established values from preliminary experiments, the tissue weights, and the cumulative quantities of BMAA to which mussels were exposed.

cum % acc =

Total BMAA in tissues μg/g on day n × tissue weight g Cumulative quantity of BMAA available per mussel in water between days 1 and n μg × 100

Half-lives of BMAA in A. anatina and D. polymorpha were calculated for each concentration using [START_REF] Rutherford | LX. Radioactive Change[END_REF] formula:

& '/( = ln 2 × 42 ln + ,(' |+ ./( CE21 and CD42 were the mean BMAA concentrations in mussels exposed to BMAA after 21 days of exposure and 42 days of depuration, respectively. Polynomial regression functions were obtained using Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA).

Statistical analyses were performed with Statistica (Version 8, Statsoft, Tulsa, USA, 2007). Pearson's correlation coefficient r at a significance level of p < 0.05 was used to determine the correlation between variables. Normality was studied with a Shapiro-Wilk test, and the homogeneity of variances was studied with a Levene test. The comparison of multiple independent samples was done with Bonferroni-corrected Kruskal-Wallis tests. Pairwise comparisons of independent samples were done using Mann-Whitney tests.

Results

BMAA was found in four out of five unexposed digestive glands of A. anatina (Fig. 1), at a mean concentration of 2.95 ± 0.96 µg BMAA/g DW. This concentration did not significantly differ from the one quantified in A. anatina exposed to 1 µg BMAA/L (Mann-Whitney test, p=0.50), whose concentration ranged from the detection limit (on day 7 of depuration) to 3.35 ± 1.73 µg BMAA/g DW (on day 14 of exposure). The BMAA concentration in A. anatina digestive glands started to differ significantly from the controls after 14 days of exposure to 10 µg BMAA/L, and after 7 days of exposure to 50 µg BMAA/L (Mann-Whitney test, p<0.05). The maximum concentration of BMAA in A. anatina digestive glands was observed on the last day of the 3-week exposure period (7.65 ± 0.96 µg BMAA/g DW in mussels exposed to 10 µg/L, and 49.64 ± 11.74 µg BMAA/g DW in mussels exposed to 50 µg/L). Throughout the exposure period, the BMAA concentration in the digestive glands of A. anatina exposed to 10 µg BMAA/L was significantly higher than in the digestive glands of A. anatina exposed to 1 µg BMAA/L (Mann-Whitney test, p<0.01). The BMAA concentration was also significantly higher in A. anatina exposed to 50 µg BMAA/L than in those exposed to 10 µg BMAA/L (Mann-Whitney test, p<0.01) considering the entire exposure period. The BMAA concentration in A. anatina digestive glands was correlated with the length of exposure to the toxin only in mussels exposed to 50 µg BMAA/L (Pearson's r = 0.74, p<0.01).

During the depuration period, BMAA elimination from A. anatina digestive glands was consistent, but not complete as BMAA was still detected after 42 days spent in clean water (0.39 ± 0.39 µg BMAA/g DW in mussels exposed to 1 µg/L, 0.48 ± 0.48 µg BMAA/g DW in mussels exposed to 10 µg/L, and 2.52 ± 0.27 µg BMAA/g DW in mussels exposed to 50 µg/L). Elimination was significant in A. anatina exposed to 10 and 50 µg BMAA/L (Mann-Whitney test, p<0.01) as BMAA concentrations decreased by 94 and 95 % between the last day of exposure and day 42 of depuration, respectively. The BMAA contents observed during the depuration period were similar between the treatments whatever the exposure concentration (Fig. 1). Moreover, the BMAA contents in all exposed mussels rapidly decreased during the depuration period down to levels similar to the one in the controls before the experiment, except on day 1 of depuration in the mussels exposed to 50 µg BMAA/L; the BMAA concentration in these mussels remained significantly higher than in the controls (Mann-Whitney test, p<0.01).

As opposed to A. anatina digestive glands, no BMAA was detected in unexposed D. polymorpha (Fig. 2). BMAA concentrations in D. polymorpha exposed to 1 µg BMAA/L ranged from 2.0 ± 1.2 µg BMAA/g DW on day 42 of depuration to 4.3 ± 0.2 µg BMAA/g DW on day 14 of exposure. BMAA was detected from the first day of exposure in whole D. polymorpha exposed to 10 (4.6 ± 0.3 µg BMAA/g DW) and 50 µg BMAA/L (19.9 ± 4.0 µg BMAA/g DW). The maximum BMAA concentrations in D. polymorpha exposed to 10 µg BMAA/L were observed on day 14 of exposure (22.6 ± 0.6 µg BMAA/g DW) and were the same one week later, on the last day of exposure (21.5 ± 2.4 µg BMAA/g DW). In D. polymorpha exposed to 50 µg BMAA/L, the maximum BMAA accumulation was observed on the last day of exposure (236.9 ± 22.9 µg BMAA/g DW). During the exposure period, BMAA concentrations in mussels exposed to 10 µg BMAA/L were significantly higher than in those exposed to 1 µg BMAA/L. Similarly, BMAA concentrations in mussels exposed to 50 µg BMAA/L were significantly higher (Mann-Whitney test, p<0.01) than in those exposed to 10 µg BMAA/L. The calculation of the correlation between BMAA concentrations in whole D. polymorpha and the duration of exposure showed that BMAA accumulation was correlated with time in mussels exposed to 10 (Pearson's r = 0.75, p = 0.001) and 50 µg BMAA/L (Pearson's r = 0.89, p = 0.001), but not in those exposed to 1 µg BMAA/L (Pearson's r = 0.49, p > 0.05). During the depuration period, BMAA was only partially eliminated as it was still found after 42 days spent in clean water in mussels previously exposed to 10 µg BMAA/L (5.2 ± 1.8 µg BMAA/g DW) and 50 µg BMAA/L (28.5 ± 10.4 µg BMAA/g DW). The elimination of BMAA was significant (Mann-Whitney test, p<0.05), with decreases of 76 and 88 % of the BMAA content in tissues between the last day of exposure and the 42 nd day of depuration, respectively. During the depuration period, the BMAA concentrations in mussels exposed to 10 µg/L were (i) significantly higher than those in mussels exposed to 1 µg BMAA/L (Mann-Whitney test, p<0.01), and (ii) significantly lower (Mann-Whitney test, p<0.01) than in mussels exposed to 50 µg BMAA/L. The BMAA concentration was negatively correlated with time during the depuration period in D. polymorpha exposed to 10 (Pearson's r = -0.73, p < 0.001) and 50 µg BMAA/L (Pearson's r = -0.78, p < 0.001), as well as in mussels exposed to 1 µg BMAA/L (Pearson's r = -0.53, p = 0.016).

We compared BMAA concentrations, expressed in µg/g DW, measured in A. anatina digestive glands and in whole D. polymorpha. Whole D. polymorpha accumulated BMAA at significantly higher concentrations, i.e., 1.7 (during exposure to 1 µg BMAA/L) to 18.8 (during depuration after exposure to 50 µg BMAA/L) times more than A. anatina digestive glands, under the same exposure and depuration conditions (Table 1).

Third-degree polynomial regression was chosen to observe BMAA dynamics in tissues during exposure because it yielded better results of fitting than linear regression. Those regression lines were not fitted to BMAA dynamics in A. anatina exposed to 1 µg BMAA/L (r²=0.18), but were correlated with BMAA concentrations in A. anatina exposed to 10 and 50 µg BMAA/L (r² = 0.92 and 0.98, respectively). However, polynomial regressions were fitted to BMAA dynamics in whole D. polymorpha (r²>0.9 for all exposure concentrations). The BMAA half-life values evaluated during the 42-day depuration period were higher in whole D. polymorpha (13.8 and 20.4 days for mussels exposed to 10 and 50 µg BMAA/L, respectively), than in A. anatina digestive glands (9.8 and 10.5 days for mussels exposed to 10 and 50 µg BMAA/L, respectively). The predictive BMAA concentrations in A. anatina digestive glands, calculated for each day of depuration using the respective halflives, were correlated with the observed values for each concentration (Pearson's r = 0.99 and p<0.01 for 10 µg BMAA/L and r = 0.94 and p<0.05 for 50 µg BMAA/L). Therefore, the mean half-life of BMAA in this matrix was used to estimate the BMAA concentration in mussels before their 2-week acclimation based on the values observed in the controls at the beginning of exposure.

The hypothetical kinetics of BMAA elimination by digestive glands of A. anatina containing 2.95 ± 0.96 µg BMAA/g DW in their tissues (concentration observed in unexposed mussels) over three weeks were compared with the BMAA concentrations measured in mussels exposed to 1 µg BMAA/L (Fig. 3). While the BMAA concentrations in those two conditions were similar on the first day of exposure (observed concentration: 2.57 ± 1.33 µg BMAA/g DW, estimated BMAA concentration: 2.80 µg BMAA/g DW), differences seemed to appear later. After 7 days, the BMAA concentration measured in A. anatina exposed to 1 µg BMAA/L was twice as much as the estimated concentration with no BMAA added in the medium. The difference remained roughly the same after 21 days of exposure, when the observed concentration was 1.66 ± 0.49 µg BMAA/g DW, and the estimated concentration was 0.70 µg BMAA/g DW. This suggests BMAA accumulation by the digestive glands of A. anatina exposed to 1 µg BMAA/L. The calculation of the cumulated percentage of BMAA accumulation (cum % acc, Fig. 4) in each model showed no significant variation over time (Kruskal-Wallis test, p>0.05). However, the cum % acc tended to decrease between day 1 and 7 of exposure in A. anatina digestive glands and whole D. polymorpha exposed to 1 and 10 µg BMAA/L.

Discussion

The freshwater bivalves A. anatina and D. polymorpha were exposed to dissolved BMAA to assess their potential use as tools for biomonitoring the contamination of aquatic environments by BMAA.

Because of the difference in size (average difference factor of 2) and in soft tissue weight (average difference factor of 12) between the two bivalve species, only one organ was selected to analyse BMAA concentrations in the larger species A. anatina. Total BMAA was assessed from 1 mg of freeze-dried tissue, representing less than 1% of the dry mass of a whole A. anatina, which is unlikely to be representative of the entire organism. It is admittedly preferable to take only one organ to limit inter-sample variability and to propose a simple tool for multi-risk assessment of water contamination by cyanotoxins. Therefore, we collected the digestive gland, which is the target organ of oligo-peptide cyanotoxins of interest such as MC and nodularins in bivalves [START_REF] Chen | Seasonal dynamics of the hepatotoxic microcystins in various organs of four freshwater bivalves from the large eutrophic Lake Taihu of subtropical China and the risk to human consumption[END_REF][START_REF] Kankaanpää | Accumulation and depuration of cyanobacterial toxin nodularin and biomarker responses in the mussel Mytilus edulis[END_REF]. However, D. polymorpha is smaller, so that the sampling of its digestive gland would have required 6 to 10 individuals to pool in order to have one replicate of analyse. As this strategy would have required too many individuals for long-term monitoring in multiple sites, we analysed BMAA concentrations in whole D. polymorpha.

We detected BMAA at low concentrations (3.3 ± 1.6 µg BMAA/g DW) in the digestive glands of unexposed A. anatina. However, as no BMAA was detected in unexposed D. polymorpha, it is unlikely that the concentration measured in A. anatina digestive glands was a background noise related to matrix interference. It is also unlikely that BMAA was a constitutive molecule of the bivalve tissue or a secondary product of the extraction procedure. Indeed, no BMAA was found in the digestive glands of unexposed A. anatina caged in situ, originated from the same location as the ones used in this study (Lepoutre et al., unpublished data). Therefore, commercially available A. anatina might have accumulated BMAA during farming, probably through the ingestion of phytoplankton producers. Despite the two-week acclimation period, all of the initial BMAA content may not have been completely eliminated from their tissues. It is known that BMAA depuration can take long, as it was still found in M. edulis maintained two months in clean water [START_REF] Reveillon | BMAA et cyanotoxines : microalgues productrices et niveaux d'accumulation dans les organismes marins[END_REF]. Moreover, BMAA was still found in all soft tissues after an exposure of D. polymorpha for 21 days to an equivalent of 2.5 µg BMAA/day, followed by 21 days in clean water (Lepoutre et al., in press). We applied the calculated BMAA half-life in A. anatina digestive glands (obtained from the kinetics of BMAA contents observed during the depuration period) to the BMAA contents observed in the controls on day 0 of exposure. We estimated that the BMAA concentration in this tissue may have been around 8.26 µg/g DW upon their reception in the laboratory, before the two-week acclimation period. Using the estimated elimination of BMAA by the controls, we hypothesized that A. anatina digestive glands were potentially able to accumulate the neurotoxin when exposed to 1 µg BMAA/L. However, further analyses would be required to investigate when digestive glands of A. anatina could reveal the presence of 1 µg BMAA/L. The low and quite constant BMAA levels, with no kinetics of accumulation or elimination, in both A. anatina and D. polymorpha exposed to 1 µg BMAA/L may be attributed to gradual and partial elimination of BMAA from tissues slightly after accumulation, involving detoxification mechanisms that are as yet unknown [START_REF] Downing | The fate of the cyanobacterial toxin β-N-methylamino-l-alanine in freshwater mussels[END_REF]. Therefore, it appears that the threshold for whole D. polymorpha and A.

anatina digestive glands to reflect environmental contamination of the medium may be around or slightly above 1 µg of dissolved BMAA/L. Freshwater mussels like Dreisenids can clearly take up small dissolved organic molecules [START_REF] Baines | Dissolved organic matter and persistence of the invasive zebra mussel (Dreissena polymorpha) under low food conditions[END_REF], but they are primarily filter feeders consuming nutritious particles present in the water. Assuming that most of the BMAA present in situ is in particulate form (e.g., inside phytoplankton cells), the relatively high filtration rates of the mussels per gill area unit (1.2-1.3 mL min -1 cm -2 for A. anatina and 1.4-1.9 mL min -1 cm -2 for D. polymorpha; [START_REF] Kryger | Filtration rate capacities in 6 species of European freshwater bivalves[END_REF], suggest that the limit of detection of environmental BMAA using bivalves could be lower. To determine this, further investigations are required with either BMAA-loaded green algae as feed or with a natural stable BMAA producer.

The decrease in accumulation capacities (cum % acc) of total BMAA by the two mussels exposed to 1 and 10 µg BMAA/L between early exposure (day 1) and the remaining exposure times may be explained by three mechanisms. First, some detoxification mechanisms may have been activated between days 1 and 7 of exposure in both species and then remained constantly activated. In mussels exposed to 50 µg BMMA/L, those mechanisms were probably triggered during the first 24h of exposure, explaining the constant cum % acc observed in both species. During the depuration period, BMAA elimination was partial but significant in whole D. polymorpha: 82 % of the BMAA content quantified at the end of the exposure period had been eliminated after 42 days of depuration. As BMAA is a hydrophilic compound, the biotransformation of the free fraction by the enzyme glutathione-Stransferase (GST) is not expected [START_REF] Downing | The fate of the cyanobacterial toxin β-N-methylamino-l-alanine in freshwater mussels[END_REF]. [START_REF] Contardo-Jara | Response of antioxidant and biotransformation systems of selected freshwater mussels ( Dreissena polymorpha , Anadonta cygnea , Unio tumidus , and Corbicula javanicus ) to the cyanobacterial neurotoxin β-N-methylamino-Lalanine[END_REF] exposed A. cygnea and D. polymorpha to 100 µg BMAA/L for 48h; they measured no variation of the activity of this enzyme during these 48h. However, BMAA was found in bivalves mainly associated with molecules that can either be soluble (soluble bound BMAA) or found in precipitates (precipitated bound BMAA) after the use of organic solvents (Réveillon et al., 2016a;[START_REF] Salomonsson | Seafood sold in Sweden contains BMAA: A study of free and total concentrations with UHPLC-MS/MS and dansyl chloride derivatization[END_REF]. The molecules to which BMAA may be associated are still unknown, but their precursors could be heavier than BMAA (i.e., >300 k vs 118.1 Da) [START_REF] Rosén | BMAA detected as neither free nor protein bound amino acid in blue mussels[END_REF], and therefore could be detoxified by enzymes such as GST. The determination of the accumulation kinetics and of the activity of detoxification enzymes at a fine temporal scale remains to be investigated.

Second, in A. anatina digestive gland, some of the BMAAs measured during the first day of exposure may have been part of the pre-contamination observed in the controls. This could potentially explain the higher cum % acc observed in the early days of exposure than in the rest of the exposure period. However, cum % acc values were also higher during the first day of exposure than on other sampling days in whole D. polymorpha although no BMAA was detected in unexposed D. polymorpha. Moreover, as BMAA was measured only in A. anatina digestive gland, it is possible that after a first accumulation in the digestive gland BMAA was further distributed to other tissues through the haemolymph during the intoxication process. Pollutants can be mobile once taken up by bivalves as they may bind to haemocytes or to dissolved haemoproteins [START_REF] Beyer | Blue mussels ( Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: A review[END_REF]Marigómez et al., 2002). We previously demonstrated that BMAA was present in the haemolymph of D. polymorpha, and further in all organs, and associated to negative cytotoxic and genotoxic effects on haemocytes [START_REF] Lepoutre | How the neurotoxin β-N-methylamino-L-alanine accumulates in bivalves: kinetics and distribution of the different accumulation fractions among organs[END_REF]Lepoutre et al., in press).

Finally, the health status of both species might have been impaired by BMAA. The neurotoxin is known to form an analogue of glutamate when bicarbonate is present at physiological concentrations [START_REF] Weiss | Beta-N-methylamino-L-alanine neurotoxicity: requirement for bicarbonate as a cofactor[END_REF]. Glutamate is a potential neurotransmitter in bivalves [START_REF] Bone | The role of l-glutamate in neuromuscular transmission in some molluscs[END_REF][START_REF] Vitellaro-Zuccarello | Immunocytochemical demonstration of neurotransmitters in the nerve plexuses of the foot and the anterior byssus retractor muscle of the mussel, Mytilus galloprovincialis[END_REF]. Therefore, BMAA may have altered their physiology, but not enough to impair their survival as no mortality was observed for either species throughout the experiment.

The higher BMAA concentration in whole D. polymorpha than in A. anatina digestive glands (up to 223.5 ± 48.5 vs. 38.3 ± 8.0 µg BMAA/g DW when exposed to 50 µg BMAA/L) was expected. A study of BMAA distribution in D. polymorpha organs showed that BMAA diffused through all tissues, and that the digestive gland could represent 4.2 ± 1.2 % of the total BMAA in the whole body when considering the haemolymph, and 12.3 ± 3.5 % when considering only soft tissues (Lepoutre et al., in press). This could also explain why the cumulated percentages of accumulation measured in D. polymorpha were higher than in A. anatina (overall means of 6.4 ± 0.6 and 0.6 ± 0.3 % when exposed to 10 and 50 µg BMAA/L, respectively). However, BMAA concentrations in A. anatina digestive glands were overall 7.3 times higher than 4% of the measured BMAA concentration in whole D. polymorpha, and 2.4 times higher than 12% of the measured BMAA concentration in whole D. polymorpha. This suggests greater accumulation capacities of BMAA by A. anatina than D. polymorpha. For instance, microcystin concentrations in whole A. anatina were higher than in whole D. polymorpha sampled in northern Latvian lakes the difference was attributed to D. polymorpha selective feeding and strong microcystin biotransformation [START_REF] Barda | Bioaccumulation of hepatotoxins -A considerable risk in the Latvian environment[END_REF].

Relevance of the use of the two bivalves as bioindicators of the presence of

BMAA in fresh water

The bivalves A. anatina and D. polymorpha were exposed to environmental concentrations of BMAA in fresh water, i.e., from 0.3 to 39.6 µg/L [START_REF] Al-Sammak | Co-occurrence of the cyanotoxins BMAA, DABA and anatoxin-a in Nebraska reservoirs, fish, and aquatic plants[END_REF][START_REF] Roy-Lachapelle | Determination of BMAA and three alkaloid cyanotoxins in lake water using dansyl chloride derivatization and highresolution mass spectrometry[END_REF]. The results suggest that BMAA accumulation may be time-and concentration-dependent in whole D. polymopha. Therefore, whole D. polymorpha could be suitable to potentially reflect BMAA contamination levels in water. As total BMAA was detected from day 1 of exposure in whole D. polymorpha exposed from 1 to 50 µg BMAA/L, it analysis in this mussel could be used as an early warning of the onset of a freshwater contamination. Regarding A. anatina digestive gland, BMAA accumulation was concentration-dependent during exposure and correlated with time during the exposure to 50 µg BMAA/L. However, using A.

anatina digestive gland may be less relevant to reveal low contamination levels of water by BMAA because i) very little BMAA present in the medium was accumulated in tissues (0.6 ± 0.3 %), and ii) accumulation was only significant compared to the controls from day 14 at 10 µg/L. Because of the relatively slow BMAA elimination by digestive glands of A. anatina and whole D. polymorpha, the emphasis should be laid on the monitoring strategy. To analyse the presence of BMAA within a specific time-frame, active monitoring using cages with unexposed mussels seems to be more appropriate than passive monitoring, based on one-time grab samples. This technique also makes it possible to use unexposed individuals, and thus to control the length of exposure and also to use sized animals with the same filtering capacities [START_REF] Phillips | Use of bio-indicators in monitoring conservative contaminants: Programme design imperatives[END_REF]Salazar andSalazar, 2006, 1996;[START_REF] Sylvester | Filtration rates of the invasive pest bivalve Limnoperna fortunei as a function of Size and Temperature[END_REF]. Active monitoring also allows for a better characterization of BMAA accumulation by freshwater mussels, and therefore a comparison of the contamination levels of different sites. Similarly, to analyse the dynamics of BMAA in water within a small time-frame, it appears more relevant to cage mussels for a short time. This will avoid overestimating the environmental contamination if a proliferation of BMAA-producing organisms occurs while some of the BMAA accumulated during a previous contamination event is still present in sentinel species.

Modelling tools may be considered to establish a link between the BMAA content in those potential bioindicator species caged in situ and environmental contamination. The development of an accumulation model would require additional exposure periods to characterise the kinetics of BMAA in A. anatina and D. polymorpha by integrating the potential influence of environmental factors (e.g., resource availability, temperature) on their filtration rate and their accumulation and detoxification capacities [START_REF] Englund | Valve movement of the freshwater mussel Anodonta anatina: A reciprocal transplant experiment between lake and river[END_REF][START_REF] Reeders | Zebra mussels (Dreissena polymorpha): a new perspective for water quality management[END_REF][START_REF] Sylvester | Filtration rates of the invasive pest bivalve Limnoperna fortunei as a function of Size and Temperature[END_REF]. Such additional investigations are required to use bivalves as sentinel species because the kinetics of accumulation has an impact on how to extrapolate environmental contamination. Additionally, data from active biomonitoring of BMAA in areas with contrasting levels of contamination may implement this approach.

Conclusion

Whole D. polymorpha would be suitable as a bioindicator of the presence of BMAA in fresh water because they can reveal the presence of 10 µg/L BMAA within one day and because BMAA concentrations in their tissues are correlated with water BMAA concentrations. However, further investigations are required at finer temporal and concentration scales. The significant but incomplete elimination of BMAA from D. polymorpha tissues and from A. anatina digestive glands after 42 days of depuration shows that both models may not be suitable to rapidly reveal the end of a water contamination event. Therefore, active caging of regularly renewed uncontaminated mussels would be more suitable to characterise the current BMAA risk in water, whereas passive samplings may overestimate it in case of a previous contamination event. Moreover, as we do not know whether BMAA is dissolved in water or remains inside producers, information regarding BMAA occurrence and bioavailability in fresh water is needed.

Figure 1 :

 1 Figure 1: Mean ± SE of total BMAA concentrations in digestive glands of A. anatina exposed to 1 (A), 10 (B) and 50 (C) µg/L of dissolved BMAA, and in controls (E0), during the exposure period (E, grey background) and the depuration period (D, white background), n=5. *, significant difference in the BMAA concentrations in tissues between two dates (Mann-Whitney test, p<0.01). , significant difference in BMAA concentrations with the controls (Mann-Whitney test, p<0.05). Polynomial regressions during the exposure period are represented by black dotted lines, and BMAA half-life curves by grey dotted lines.

Figure 2 :

 2 Figure 2: Mean ± SE of total BMAA concentrations in whole D. polymorpha exposed to 1 (A), 10 (B) and 50 (C) µg/L of dissolved BMAA, during the exposure period (E, grey background) and the depuration period (D, white background), n=4. *, significant difference in the BMAA concentrations in tissues between two dates (Mann-Whitney test, p<0.05). Polynomial regression lines calculated during the exposure period are represented by black dotted lines, and the BMAA half-life curve by grey dotted lines.

Figure 3 :

 3 Figure 3: Predicted BMAA elimination by A. anatina estimated from BMAA concentrations measured in the controls (2.95 ± 0.96 µg BMAA/g DW) over 21 days using BMAA half-life (grey bars) compared to observed BMAA concentrations in mussels exposed to 1 µg BMAA/L (black bars) in A. anatina digestive glands throughout the exposure period.

Figure 4 :

 4 Figure 4: Cumulated percentage of BMAA accumulation (cum % acc) over time during the exposure period in digestive glands of A. anatina (A) and in the whole body of D. polymorpha (B) exposed to 1 (white boxes), 10 (grey boxes) and 50 (black boxes) µg BMAA/L. Boxplots indicate first and third quartiles of the observations, whiskers indicate minimum and maximum values, ○ indicate outliers, and the median is indicated by a square.

Table 1 : Mean difference factor between BMAA concentrations in whole D. polymorpha compared to A. anatina digestive glands during exposure to 1, 10 and 50 µg BMAA/L and the depuration period. Tabulated p-values for Mann-Whitney tests comparing BMAA concentrations in whole D. polymorpha and A. anatina digestive glands are given in brackets.

 1

		Exposure	Depuration
	1 µg BMAA/L	1.7 (p = 0.009)	4.5 (p<0.001)
	10 µg BMAA/L	2.7 (p=0.003)	6.0 (p<0.001)
	50 µg BMAA/L	4.9 (p<0.001)	18.8 (p<0.001)

Acknowledgments

This work was partly financed by the French National Research Program for Environmental and Occupational Health of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES 2015/1/191), and by the Eole scholarship from the Réseau Franco-Néerlandais de l'enseignement supérieur et de la recherche (RFN) and the Nuffic (Netherlands Organization for International Co-operation in Higher Education).

Declaration of interest

The authors declare no conflict of interest.


Maela Le

MD Maela Le Lous
email: maela.le.lous@chu-rennes.fr

Ombeline Simon

Linda Lassel

Vincent Lavoue

Pierre Jannin

Hybrid simulation for obstetrics training A systematic review

Keywords: Simulation, Hybrid, Patient-Actor, Obstetrics, Medical Education, Training, Immersion, Non Technical Skills

Introduction

Validity and benefits of simulation medical training are now well admitted [START_REF] Cook | How much evidence does it take? A cumulative meta-analysis of outcomes of simulation-based education[END_REF] . In France, it is recommended to use simulation at every level of training to prevent from medical errors 2 .

Alinier et al.'s classified gradually the different types of simulation-based training as following: 0. Written simulation ; 1. Three dimensional models ; 2. Screen-based simulator computer ; 3.

Standardized patients (SP) or Hybrid simulation ; 4-5. High Fidelity mannequins [START_REF] Alinier | A typology of educationally focused medical simulation tools[END_REF] .

Hybrid simulation is defined as the use of two or more simulation modalities within the same simulation session. Typically, a task trainer or partial simulator is realistically affixed to a standardized patient (SP), allowing training and assessment of both technical and communication skills. Hybrid simulation may be particularly interesting in the field of obstetrics. Indeed, obstetricians have to operate/deliver the patient and at the same time to communicate efficiently with her. During labor and delivery, most procedures occur in conscious women, often accompanied with close family members, all of them having an important emotional charge. The professionals need to correctly managed the emergency and take into consideration the needs and concerns of both patient and her partner, in a very short time. The interaction with a "real" patient combined with hands-on clinical practice may reflect the complexities associated with birth.

The Kirkpatrick pyramid was used to rank medical education studies depending on the main outcome in four levels: 1-student satisfaction; 2-progress in theoretical and practical knowledge outside the clinic ; 3-benefit on clinical skills or behavior ; and 4-impact or benefit for the patient [START_REF] Kirkpatrick | Evaluating Training Programs: The Four Levels: Easyread Edition[END_REF] .

We sought to review the literature and investigate the current level of evidence about the clinical benefits of hybrid simulation training in the field of obstetrics according to the Kirkpatrick Hierarchy.

Methods

This review was planned, conducted, and reported in adherence with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) standards of quality [START_REF] Moher | Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement[END_REF] .

A search of scientific publications was conducted in PubMed, using the keywords:

-Obstetrics AND Medical Education AND (Standardized patient OR Hybrid simulation).

We searched MEDLINE and ClinicalTrials.gov. Eligible studies had to be published in English after year 2000 and to investigate the use of hybrid simulation in the specific field of obstetrics to health profession learners at any stage (initial and continuous) in training. Systematic reviews were not included.

Based on Kirkpatrick's hierarchy, we included studies reporting the level of the learning outcome, and we specified if non-technical aspects were considered. For each study, we notified if technical skills or non-technical skills were investigated.

The date of last searched was 2018, November the 12 th .

Study Selection

A total of 155 studies were screened, from which 10 articles were selected from the title and the abstract in PubMed. Four articles were excluded afterwards because they were dealing with high fidelity simulation, and one article was excluded because it was describing a simulation scenario, with no evaluation. Six more articles were added from the bibliographies (Figure 1 -Article flow chart). No additional article was added from ClinicalTrials.gov.

A total of 11 articles were analyzed, presented in Table 1.

We distinguished the articles addressing technical skills from those addressing nontechnical skills as well as the involved studied skill(s).

Results

Using Hybrid simulation to improve Technical skills

Improving preeclampsia management

In a randomized study by Fisher et al., hybrid simulation was used for eclampsia training in 38 obstetrics and gynecology (OB/GYN) residents [START_REF] Fisher | Resident training for eclampsia and magnesium toxicity management: simulation or traditional lecture?[END_REF] . They involved a standardized patient with a display of simulated maternal vital signs and fetal heart rate monitoring. This study compared hybrid simulation alone, traditional lecture alone, and a combination of both. Three months later, they assessed student competency through an "eclampsia drill score" between the 3 groups, during a simulation-based evaluation.

Postintervention management scores were significantly better in the "simulation-based intervention" groups compared with the "lecture only" group. Significantly higher residents in the "Simulation group" group knew the correct dose and route of calcium gluconate administration compared with the "Lecture Only" (73% vs 25%; p=0.05). Their results demonstrated that simulation-based training was superior to traditional lecture alone for teaching crucial technical skills to manage eclampsia, in a simulated environment. Skills transfer to patients was not investigated in this study (Kirkpatrick level 2).

In a randomized study by Ellis et al., hybrid simulation in local hospitals was compared to high-fidelity simulation in simulation centers [START_REF] Ellis | Hospital, simulation center, and teamwork training for eclampsia management: a randomized controlled trial[END_REF] . A total of 140 junior, senior obstetricians, and midwifes participated to this training about eclampsia management. The evaluation was based on a simulated scenario with a patient-actor. Two assessors independently reviewed all video recordings of the eclampsia scenario, and scored the competency thanks to an "eclampsia checklist".

It appeared that both types of simulation training, hybrid and high-fidelity, were associated with improved management of eclampsia: better completion of tasks in a reduced time. Eclampsia training in a regional simulation center with high-fidelity mannikins seemed to confer no advantage over local training with hybrid simulation (basic mannequins and patient-actor) in terms of management scores (Kirkpatrick level 2). Again, in this study, the level of immersion of the participants for both simulation modalities was not investigated, neither were non-technical skills.

Shoulder Dystocia Management

In another randomized study by Crofts et al., a severe shoulder dystocia was simulated in a hybrid simulation scenario. A PROMPT Birthing Trainer (Limbs and Things Ltd, Bristol, UK) was integrated with a patient-actor, and compared to a high fidelity mannequin scenario [START_REF] Crofts | Training for shoulder dystocia: a trial of simulation using low-fidelity and high-fidelity mannequins[END_REF] .

Participants (45 doctors, 95 midwives) were taken individually into a delivery room, given a standardized description of the scenario, and asked to complete the shoulder dystocia delivery.

The simulation was continued until either delivery of the posterior arm of the baby, the participant chose to stop, or 5 minutes had elapsed.

The management of the simulated shoulder dystocia was assessed using the following outcome criteria: 1) success or failure of delivery, 2) the head-to-body delivery time, 3) performance of appropriate actions, 4) force applied, and 5) communication.

Both types of training were associated with improved performance. They highlighted in both groups a better use of basic maneuvers (81% to 95%), more successful deliveries (43% to 83%), and a better communication with the patient (57 to 83%), comparing pre-and posttraining. Comparing both types of simulations, the high-fidelity training was associated with a higher successful delivery rate than training with hybrid simulation: 94% versus 72% (P=0.002).

The same team conducted a 6-12 months follow-up study after this training. For the majority of staff, a 40-minute shoulder dystocia training session was sufficient to gain and sustain the skills required to manage shoulder dystocia. One year after the first evaluation, 94% of participants who had been able to achieve delivery could still do so 9 .

In a level 4 Kirkpatrick study, published in 2008, the same team in Bristol compared the management and the neonatal outcomes of births complicated by shoulder dystocia before and after the introduction of shoulder dystocia hybrid training [START_REF] Draycott | Improving neonatal outcome through practical shoulder dystocia training[END_REF] . They found a significant reduction in neonatal injury after shoulder dystocia from 30/324 (9.3%) to 6/262 (2.3%) with a relative risk of 0.25 [CI95% 0.11-0.57], thanks to hybrid training.

The same team showed in another study published in 2016 a decade after the introduction of training, that there were no cases of brachial plexus injury lasting over 12 months in 562 cases of shoulder dystocia, since hybrid training had been introduced [START_REF] Crofts | Prevention of brachial plexus injury-12 years of shoulder dystocia training: an interrupted time-series study[END_REF] .

Cord Prolapse Management

In a study by Siassakos et al., the impact of the introduction of hybrid simulation team training was measured. They compared the improvements in the management of cord prolapse before and after the implementation of hybrid simulation training [START_REF] Siassakos | Retrospective cohort study of diagnosis-delivery interval with umbilical cord prolapse: the effect of team training[END_REF] .

Umbilical cord prolapse is an acute obstetrical emergency which occurs when the umbilical cord becomes compressed as it lies ahead or alongside the fetal presenting part, occasioning fetal hypoxia, and requiring rapid identification and intervention [START_REF] Gibbons | Umbilical cord prolapse--changing patterns and improved outcomes: a retrospective cohort study[END_REF] . They conducted simulation training with a patient-actor, and a cushion to imitate a pregnant abdomen, a model of a baby with its umbilical cord, and a mock perineum. The scenario was run in a labor room and stopped when teams decide to move the woman to theatre for a caesarean section.

Afterwards, in all actual cases of cord prolapse, they measured before and after team training, the diagnosis-delivery interval (DDI), the proportion of caesarean section (CS) in whom actions were taken to reduce cord compression, and neonatal outcomes (rate of low Apgar scores; rate of admission to neonatal intensive care unit).

They showed that after training, there was a statistically significant reduction of the diagnosis -delivery time interval from 25 to 14.5 minutes (P < 0.001). In other words, hybrid simulation training permitted teams to go faster in a situation of cord prolapse (Kirkpatrick level 3). Post-training, there was also a significant increase in the proportion of caesarean sections where recommended actions to reduce cord compression had been performed (from 34.78 to 82.35%, P = 0.003). However, they did not find significant differences in neonatal outcome. The reduction in the rate of neonates with low Apgar scores (from 6.45 to 0% p=0.3) and in the rate of neonate admission to intensive care (38.46 to 22.22%) was not statistically significant (p=0.2).

Using hybrid simulation to improve Non-Technical skills:

Improving communication

In a study by Crofts et al., about shoulder dystocia hybrid training, there was no difference in the post-training communication according to the type of mannequin used (81% of good communication in the low fidelity versus 84% in the high-fidelity group, p=0.697).

However, those trained on the high-fidelity mannequin were significantly less likely to have called for pediatric support (P=.003) [START_REF] Crofts | Training for shoulder dystocia: a trial of simulation using low-fidelity and high-fidelity mannequins[END_REF] . commands were directed to specific individuals, tasks were more likely to be acknowledged and performed [START_REF] Siassakos | Content analysis of team communication in an obstetric emergency scenario[END_REF] .

In another study by

In another study by the same author, 24 medical students were trained first with highfidelity simulation teaching and then a refresher session for which the same students were randomly allocated to either a "tuto" or "hybrid simulation". Afterwards, they were asked to deliver a baby with simulated shoulder dystocia in a hybrid scenario, with communication measured with a perception score rated by the actress herself. Primary outcome measure was their communication score. The percentage of participants with good communication scores (4/5) was also significantly higher for the hybrid simulation group (74% HYB versus 22% SGT p=0.03). They concluded that hybrid simulation improves students' communication skills [START_REF] Siassakos | Exploratory randomized controlled trial of hybrid obstetric simulation training for undergraduate students[END_REF] .

Improving patient perception of safety

In another study by Crofts et al, pre and post training perception of safety, communication, and respect were rated by the patient actor himself. They compared the improvements in those items between hybrid simulation and high-fidelity computerized mannikin. The rating was done by a five-point Likert scale with the following affirmations : Communication : ''I felt well informed due to good communication'' ; Respect: ''I felt I was treated with respect at all times'', Safety: ''I felt safe at all times''. It appeared that both trainings improved outcomes, but during simulated post-partum hemorrhage, safety and communication scores were significantly higher for teams trained locally with a patient-actor compared with teams trained at the simulation center using a computerized patient manikin (safety p = 0.048, communication p = 0.035) [START_REF] Crofts | Patient-actor perception of care: a comparison of obstetric emergency training using manikins and patient-actors[END_REF] .

In another study by Siassakos, hybrid simulation was used to determine patient's perception of safety. A total of 108 professionals were video recorded during management of a patient-actor with a simulated obstetrical emergency. The trained patient-actor scored the quality of interaction with the staff. They showed important variation in staff interaction with the patient, some teams not exchanging a single word and others striving to interact with the patient-actor. This shows that although technical skills are precisely guided by algorithms, communication is not standardized at all. In that study, most teams failed to communicate efficiently with the patient-actor. There was a significant correlation between patient-actor perceptions of communication, respect, and safety and individual behaviors. The patient-actor perception of safety was better when the content of the communication episodes included certain items of information (nature of the emergency, immediate treatment, cause of the emergency, condition of baby, non-verbal staff interaction episodes, number and duration of the communication episodes) [START_REF] Siassakos | Team communication with patient actors: findings from a multisite simulation study[END_REF] .

Discussion

This review shows that considering technical aspects of obstetrical emergencies management, hybrid simulation training is as efficient as high-fidelity training, which are superior than traditional lecture or written classes. However, non-technical aspects need more investigation, as well as immersion evaluation.

Most of obstetrical emergencies have been taught by hybrid simulation already: shoulder dystocia (0.1% of births in France), pre-eclampsia (between 0.5 to 3%), post-partum hemorrhage (5%), and cord prolapse (0.1%) 18 . For shoulder dystocia, Kirkpatrick level 4 evidence exists, as it has been proven that hybrid simulation training reduces the rate of brachial plexus injuries, with a sustained effect [START_REF] Crofts | Prevention of brachial plexus injury-12 years of shoulder dystocia training: an interrupted time-series study[END_REF] . For cord prolapse, it has been shown that team effectiveness is improved, with a reduce Diagnosis-to-Delivery Interval (Kirkpatrick level 3).

For pre-eclampsia and post-partum hemorrhage, it has been proved that trained teams have a better knowledge of drug doses in a simulated situation (Kirkpatrick level 2), but no benefit in terms of maternal mortality has been yet proven.

It is to notice that, although it is a quite frequent (0.19%) and potentially harmful obstetrical situation, no data exists about unplanned home delivery simulation training. It has been shown that in the case of unplanned home deliveries, neonatal morbimortality is higher 1920 . In most countries no obstetrical team are dedicated to unplanned home deliveries, and these situations are under the responsibility of emergency teams. As they are not well trained for deliveries, hybrid simulation could also be interesting for them.

It seems that hybrid simulation is more often associated to in situ simulation while high fidelity scenario, are more often taking place in dedicated simulation centers. This could be because of the facilities of transportation of birthing trainers compared to high fidelity computerized mannikins, which are heavy and need connection with a computer. Simulation centers are expensive; 'on-site' clinical simulations might be an acceptable alternative for the study of teamwork. Familiarity with the environment, equipment and personnel might improve psychological and environmental fidelity and could be an advantage over unfamiliar simulation centers, particularly for team training. Of course, difficulties encountered with he uses hybrid simulation are the cost and the training of patient-actors.

All types of simulation training seem to improve non-technical skills as well. However, whether hybrid simulation is more efficient than high-fidelity simulation to teach non-technical skills is not well established. Training with patient-actors may offer some advantages to teach better communication, as suggested by Crofts et al.'s study in post-partum hemorrhage scenario [START_REF] Crofts | Patient-actor perception of care: a comparison of obstetric emergency training using manikins and patient-actors[END_REF] .

In a delivery room, good communication with the mother and the father has been identified as a key point [START_REF] Siassakos | Team communication with patient actors: findings from a multisite simulation study[END_REF] . Unfortunately, a significant number of women are dissatisfied of their delivery, as attested by public debates about obstetrical violence, particularly if they experienced an emergency [START_REF] Uotila | Traumatic experience with vacuum extraction--influence of personal preparation, physiology, and treatment during labor[END_REF] . As post hoc debriefing may be insufficient after traumatic medical birth [START_REF] Rowan | Postnatal debriefing interventions to prevent maternal mental health problems after birth: exploring the gap between the evidence and UK policy and practice[END_REF] , a good opportunity to prevent dissatisfaction would be a more efficient communication during the acute event. In a systematic review, factors associated with dissatisfaction around birth were: the amount of support from caregivers, the quality of the caregiver-patient relationship, and their involvement in decision making, and all of these can be taught by simulation. These factors appeared to be so important for women satisfaction that they override the influences of age, socioeconomic status, ethnicity, childbirth preparation, pain, immobility, or medical interventions [START_REF] Hodnett | Pain and women's satisfaction with the experience of childbirth: a systematic review[END_REF] .

Hybrid simulation may also be associated with better learner satisfaction, as the interaction may be more realistic. Construct validity evidence is necessary for all types of simulationtraining, including hybrid simulation. The level of immersion of the participants is of primary importance for the learning adherence [START_REF] Engström | The impact of contextualization on immersion in healthcare simulation[END_REF] . If the simulated scenario is not immersive enough, learners can be refrained from giving the best of themselves, affecting both technical and nontechnical skills. Whether hybrid simulation affects immersion positively is unknown.

Conclusion

With this review we sought to gather all information about hybrid simulation in the field of obstetrics. There is evidence that hybrid simulation is an effective tool to teach both technical and non-technical skills training and should be expanded. However, construct validity evidence studies are needed. Whether hybrid simulation offers better training for communication and offers better immersion than high-fidelity simulations for learners remains to be investigated.

  Siassakos et al., audio-video recordings of eight postpartum hemorrhage hybrid simulations were collected and communication patterns were analyzed. Two of the four teams received extra training in specific teamwork behaviors the other half received only clinical training. Communication patterns in a new simulated situation were analyzed. There was a reduction in the total number of communications after training. Teams that received additional teamwork training used more directed commands after training. When

Table 1 .Figure 1 .

 11 Figure 1. Flow Chart

Figure 1 .

 1 Figure 1. Flow Chart

Table 1 .

 1 Literature overview of obstetric hybrid simulation

Acknowledgements

We thank Felicity Neilson for English editing

Funding

No funding


MD a ; Judicael Hotton

MD Meriem Koual

MD Marie Gosset

MD Lea Rossi

MD b ; Myriam Delomenie

MD, PhD Charlotte Ngo

MD, PhD Fabrice Lecuru

MD, PhD Anne-Sophie Bats
email: anne-sophie.bats@aphp.fr

Outcomes of robotic surgery for endometrial cancer in elderly women

Keywords: Elderly, Endometrial neoplasm, Robotic-assisted laparoscopy

Introduction: Few data have been reported on robot-assisted surgery in elderly. The objectives were to compare feasibility, complication data, and survival of patients under and upper the age of 70 who are managed for endometrial cancer by robot-assisted laparoscopy.

Materials and

Methods: This is a retrospective comparative single-center study including patients treated between January 2007 and December 2016. Patients were divided into 2 groups: less than 70 years and greater than or equal to 70 years. The primary endpoint was the rate of complications. The secondary endpoints were conversion rate and follow-up.

Results: 148 patients were included: 86 under 70 (group A) and 62 aged 70 and over (group B). More adhesiolysis was performed in group B (p<.01); the pelvic and para-aortic lymph node dissection rates were not different between both groups (p=.2 and p=.9). The operating times were significantly longer in group B (220.1 vs. 234.4 minutes, p=.02). The conversion rate was similar between the 2 groups (p=.7). The tumors were endometrioid adenocarcinomas for 77.9 and 66.7% respectively (p=.2), with grade 3 tumors more represented in older patients (24.4% vs. 48.4%, p<.01). There were more tumors at high risk of recurrence after 70 years (33.7 vs. 45.2%, p=.04). No significant difference was found for postoperative complications. There was no difference in overall survival (p=.7) or progression-free survival (p=.2). Undertreated women rate was similar in both groups (p=.1).

Conclusion:

Robotic surgery appears feasible and reproducible and could bring a benefit and allow optimal surgery without increasing the morbidity in the management of endometrial cancers whatever the age is.

Introduction

Endometrial cancer is the most common gynecological cancer worldwide after cervix cancer with incidence in 2018 about 382,069 new cases for 89,829 deaths [START_REF] Bray | GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA[END_REF]. It most often affects elderly women, with an average age of 68 years. With the aging of the population in developed countries, the incidence of the disease will continue to increase [START_REF] Morice | Endometrial cancer[END_REF].

A lot of studies have shown that high age is an independent factor of poor prognosis in women with endometrial cancer [START_REF] Bourgin | Endometrial cancer in elderly women: Which disease, which surgical management? A systematic review of the literature[END_REF][START_REF] Koual | Endometrial cancer in the elderly: does age influence surgical treatments, outcomes, and prognosis?[END_REF]. In addition, elderly women have more comorbidities, more advanced and aggressive lesions requiring extensive surgical resection [START_REF] Zeng | Outcome of robotic surgery for endometrial cancer as a function of patient age[END_REF][START_REF] Uccella | Laparoscopic vs. open treatment of endometrial cancer in the elderly and very elderly: An age-stratified multicenter study on 1606 women[END_REF]. They are therefore at greater risk of immediate and late postoperative complications, which explains that they have less aggressive surgical treatment and are less eligible for adjuvant treatment [START_REF] Zeng | Outcome of robotic surgery for endometrial cancer as a function of patient age[END_REF][START_REF] Eggemann | Management of elderly women with endometrial cancer[END_REF]. Optimization of surgical treatment and staging would seem to improve their prognosis [START_REF] Colombo | ESMO-ESGO-ESTRO Endometrial Consensus Conference Working Group, ESMO-ESGO-ESTRO Consensus Conference on Endometrial Cancer: Diagnosis, Treatment and Follow-up[END_REF][START_REF] Rauh-Hain | Management for Elderly Women With Advanced-Stage, High-Grade Endometrial Cancer[END_REF].

Standard surgical treatment for endometrial cancers includes total hysterectomy with bilateral salpingo-oophorectomy [START_REF] Morice | Endometrial cancer[END_REF]. In the presence of high-risk tumors, more complex procedures are performed including pelvic and para-aortic lymph node dissections ± omentectomy [START_REF] Colombo | ESMO-ESGO-ESTRO Endometrial Consensus Conference Working Group, ESMO-ESGO-ESTRO Consensus Conference on Endometrial Cancer: Diagnosis, Treatment and Follow-up[END_REF].

Although robot-assisted surgery has been evaluated for many years in the management of this pathology, few data have been reported in the elderly population [START_REF] Lewis | Participation of patients 65 years of age or older in cancer clinical trials[END_REF].

The objectives of this study were to compare the feasibility, the per-and post-operative data, and the survival of patients under and upper the age of 70 who are managed for endometrial cancer by robot-assisted laparoscopy.

2.Materials and Methods

2.1.Patients

All patients with histologically proven endometrial cancer who received minimally invasive robot-assisted laparoscopic surgical management were included. The characteristics were obtained from the medical databases and the anonymous data were recorded on a computerized database. The collection was conducted between January 2007 to December 2016 within the Georges Pompidou European Hospital in Paris.

The entire cohort was divided into 2 groups according to their age: less than 70 years (Group A) and greater than or equal to 70 years (Group B).

2.2.Surgical procedure

The first version of the Da Vinci Surgical System was used up to 2012, then the Da Vinci Si Surgical System (Intuitive Surgical Inc. Sunnyvale, California, USA). All patients underwent a total hysterectomy and bilateral salpingo-oophorectomy. Since 2010, in accordance with the recommendations of the National Cancer Institute (INCa), pelvic and para-aortic lymphadenectomy have been performed only in patients with endometrial cancer at high risk (type 1 histology stage IB grade 3, type 2 histology) [START_REF] Colombo | ESMO-ESGO-ESTRO Endometrial Consensus Conference Working Group, ESMO-ESGO-ESTRO Consensus Conference on Endometrial Cancer: Diagnosis, Treatment and Follow-up[END_REF]. Para-aortic lymphadenectomy was performed by trans-or extra-peritoneal approach. Patients with early-stage endometrial cancer (low risk (type 1 histology stage IA grade 1-2) and intermediate-stage endometrial cancer (type 1 histology stage IA grade 3 and IB grade 1-2)) underwent pelvic sentinel node biopsy.

2.3.Data collection

Demographic characteristics (age, body mass index, comorbidities such as hypertension, diabetes, history of abdominal surgery), surgical (operating time, operating theatre time, type of lymph node dissection), histology (histological type, FIGO (International Federation of Gynecology-Obstetrics) and ESMO (European Society for Medical Oncology) classifications) and follow-up (early and late complications, recurrence or death) have been recorded and compared between the 2 groups [START_REF] Colombo | ESMO-ESGO-ESTRO Endometrial Consensus Conference Working Group, ESMO-ESGO-ESTRO Consensus Conference on Endometrial Cancer: Diagnosis, Treatment and Follow-up[END_REF][START_REF] Lewin | Comparative performance of the 2009 international Federation of gynecology and obstetrics' staging system for uterine corpus cancer[END_REF].

The primary endpoint was the rate of per and post-operative complications and the secondary endpoints were the rate of conversion in laparotomy and oncologic follow-up.

Postoperative complications were classified according to the Clavien-Dindo classification [START_REF] Dindo | Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey[END_REF].

The length of stay was defined as the number of nights in hospitalization. Adequate treatment for each patient was noted. Patients were considered undertreated if optimal treatment was not realized. It was also noted that stadification surgery was performed, and adjuvant treatments with chemotherapy, external beam radiotherapy and brachytherapy.

Recurrence was defined as the re-emergence of the disease locally or by regional or distal metastatic evolution and confirmed by histological sampling or imaging. The recurrence site and the period between surgery and the date of the first recurrence were recorded. Overall survival was defined as the time between surgery and latest news date or death, and progression-free survival as the time between surgery and the diagnosis of the evolution of the disease. Data on alive patients were censored at the last follow-up visit.

2.4.Statistical analysis

The results were expressed as mean ± standard deviation or median [ranks]. The frequencies were presented in percentage. We compared all the data using accurate Chi-2 or Fisher tests for categorical or ordinal variables and Student's t-test analysis for continuous variables. P values below .05 were considered statistically significant. All analyzes were performed using the statistical software R version 3.2.4 [START_REF] Core | R: A language and environment for statistical computing[END_REF].

3.Results

During the study period, 385 patients were treated for an endometrial cancer: 82 by laparotomy; 155 by laparoscopy; 148 by robotic-assisted procedures. Robotic surgery was first-line treatment approach according to its accessibility. Among 148 patients underwent robotic surgery for endometrial cancer, 86 were under 70 years and were included in group A and 62 were aged 70 and over and constituted the group B.

3.1.Population characteristics

The demographic and clinical characteristics were summarized in Table 1. The mean age between the 2 groups was significantly different (group A: 60 ± 6.8 years vs. group B: 77 ± 5.5 years, p<.01). The 2 groups were comparable in terms of Body Mass Index (BMI), parity and history of abdominal surgery. Patients over 70 years had more comorbidities: they had more Eastern Cooperative Oncology Group (ECOG) performance status 1 and 2 (p=.02), hypertension (p=.01) and had more often taken hormone replacement therapy (p=.02). The main circumstance of discovery was the occurrence of postmenopausal bleeding (89.9% on average for both groups).

3.2.Surgical procedures

Surgical characteristics were summarized in Table 2. All patients underwent total hysterectomy with bilateral salpingo-oophorectomy in robotic-assisted laparoscopy.

Interventions took place under general anesthesia, in Tredelenburg position. A double docking might be required in case of para-aortic lymphadenectomy. More adhesiolysis were performed in group B (13.9% vs. 35.4%, p<.01). Sentinel lymph node procedure was performed in 55.8% of patients under age 70 versus 74.2% of elderly patients (p=.02). Pelvic lymph node dissection was performed in 47 (54.7%) in group A versus 29 (46.8%) in group B without difference between groups (p=.2). Thirty-four patients underwent para-aortic lymph node dissection and rates were not different between the groups (25.6% vs. 19.4%, p=.9). Four para-aortic lymphadenectomies were performed retroperitoneally in group A versus tree in group B. The operating times were significantly longer in group B (220.1 vs. 234.4 minutes, p=.02). Eleven patients in group A benefited from a reintervention for restaging against 4 in group B (p=NS). The hospitalization time was similar between groups (6.5 days, p=.8).

3.3.Tumor characteristics (Table 3)

Tumors were endometrioid adenocarcinomas for 77.9% and 67.7% respectively (p=.2), with grade 3 tumors more represented in older patients (29.1% vs. 43.5%, p<.01). There were more tumors with high risk of recurrence after 70 years (33.7 vs. 45.2%, p=.04). There was no significant difference in tumor stage, histology, histological type, or lymphovascular space invasion (LVSI).

3.4.Surgical complications (Table 4)

The conversion rate was similar between the 2 groups (5.8 vs. 3.2%, p=.7). In patients over 70 years, the 2 conversions in laparotomy were secondary to multiple adhesions. In the other group, 4 conversions in laparotomy and 1 conversion in laparoscopy were recorded for technical difficulties (2 of which were due to poor tolerance of an exaggerated Trendelenburg position).

The rates of intraoperative complications were also comparable (2.3% vs. 3.2%, p=1). A sutured bladder and a sutured rectal serous wound were noted in group A. In the second group, there was a rectal serous and a vascular wound during para-aortic dissection.

No significant difference was found for early and late postoperative complications according to the Clavien-Dindo classification (p=.8). In group B, there were 2 early complications (ureteral wound and evisceration on umbilical trocar port) requiring surgical revision. Other early complications required medical treatment only (3 pulmonary embolism, 1 occlusive syndrome and 1 wall hematoma). Late complications were secondary to pelvic lymphoceles (3), urinary sepsis and ureteral stenosis. In group A, early complications included 3 cases of sepsis (1 pyelonephritis, one wall abscess and one infected lymphocele), 2 pulmonary emboli and 1 hemoperitoneum and 1 pneumothorax. In late complications, there were 5 cases of pelvic lymphoceles and 1 lymphedema of the right lower limb and 1 case of pulmonary embolism, 1 hernia. When comparing comorbidities by procedure type, patients over 70 years with total hysterectomy and pelvic and para-aortic lymphadenectomies had more major complications (Clavien Dindo III and above) than patients under 70 years (p=.01). Complications were similar whether patients had a total hysterectomy with (p=.3) or without pelvic lymphadenectomy (p=.8).

3.5.Adjuvant treatments (Table 5)

There was no difference in terms of adjuvant treatments between the two groups.

Chemotherapy was given in 25.8% of patients in group B against 30.2% (p=.5) in group A.

Radiation (External Beam RadioTherapy (EBRT) and brachytherapy respectively) were similar too (p=.2) with 35.5% and 50% of the cases in group B, compared to 39.5% and 43% in the group A. Among the elderly (group B), 21% did not receive the indicated treatment compared to 11.6% in the youngest patients (group A), with no significant difference between the two groups (p=.1)

3.6.Follow-up (Table 5)

The mean follow-up time in group A was 643.1 days compared to 550.4 days in group B.

Mean time between surgery and relapse was 635.3 days for patients under 70 years and 501.3 days for patients over 70 years of age. There was no difference in overall survival (p=.7) as in progression-free survival between the 2 groups (p=.2) (figure 1).

4.Discussion

Surgery is crucial for the treatment of endometrial cancers. The minimally invasive pathway demonstrated superiority over laparotomy in terms of morbidities, length of stay, quality of life and recovery [START_REF] Guy | Comparative outcomes in older and younger women undergoing laparotomy or robotic surgical staging for endometrial cancer[END_REF]. The majority of randomized trials included low-risk patients. It has no impact on recurrence or survival [START_REF] Walker | Laparoscopy compared with laparotomy for comprehensive surgical staging of uterine cancer: Gynecologic Oncology Group Study LAP2[END_REF]. With the rise of robotic surgery, many benefits have been demonstrated, especially in obese population [START_REF] Ran | Comparison of robotic surgery with laparoscopy and laparotomy for treatment of endometrial cancer: a meta-analysis[END_REF].

Elderly care is proving to be a real public health problem. The incidence of endometrial cancer increase with age and tumors have worse prognosis [START_REF] Uccella | Laparoscopic vs. open treatment of endometrial cancer in the elderly and very elderly: An age-stratified multicenter study on 1606 women[END_REF][START_REF] Dumas | Improving outcomes for older women with gynaecological malignancies[END_REF]. It has been estimated that 20% of older women waited at least 1 year between the first symptoms and the specialized consultation [START_REF] Bourgin | Impact of age on surgical staging and approaches (laparotomy, laparoscopy and robotic surgery) in endometrial cancer management[END_REF]. Due to a number of significant comorbidities, elderly women with endometrial cancer are also challenging for their surgical management. These patients have more often indications of additional intraoperative treatments such as extensive lymphadenectomies because of more several lesions [START_REF] Rauh-Hain | Management for Elderly Women With Advanced-Stage, High-Grade Endometrial Cancer[END_REF][START_REF] Clark | Adjuvant Treatment and Clinical Trials in Elderly Patients With Endometrial Cancer: A Time for Change?[END_REF][START_REF] Aloisi | Is Robotic-Assisted Surgery Safe in the Elderly Population? An Analysis of Gynecologic Procedures in Patients ≥ 65 Years Old[END_REF]. In this work, older patients had more comorbidities. We did not show any statistical difference in terms of complications, nor conversion rate.

There is a higher rate of postoperative complications in elderly women such as episodes of occlusion, local infection or perioperative cardiac events [START_REF] Clark | Adjuvant Treatment and Clinical Trials in Elderly Patients With Endometrial Cancer: A Time for Change?[END_REF]. Benefits of robotic surgery were a significant reduction in complication rates, bleeding, and length of hospital stay [START_REF] Guy | Comparative outcomes in older and younger women undergoing laparotomy or robotic surgical staging for endometrial cancer[END_REF][START_REF] Lavoue | Impact of robotics on the outcome of elderly patients with endometrial cancer[END_REF][START_REF] Lavoué | Benefits of robotic surgery for elderly patients with endometrial carcinoma[END_REF][START_REF] Gallotta | Robotic Surgery in Elderly and Very Elderly Gynecologic Cancer Patients[END_REF].

Our complication rate was acceptable at 11.5% (12.9% in group B versus 10.5% in group A). This rate is consistent with the literature data described regardless of age [START_REF] Kueck | Laparoscopic technology for the treatment of endometrial cancer[END_REF]. Eddib et al. as Vaknin et al. compared surgical results in patients undergoing robotic hysterectomy and demonstrated that advanced age did not appear to be associated with increased risk of morbidity or adverse perioperative outcomes [START_REF] Eddib | Impact of Age on Surgical Outcomes after Robot Assisted Laparoscopic Hysterectomies[END_REF][START_REF] Vaknin | Outcome and quality of life in a prospective cohort of the first 100 robotic surgeries for endometrial cancer, with focus on elderly patients[END_REF].

Our data confirm the data available in the literature in support of the safety of a robotic surgical approach for the treatment of elderly patients with endometrial cancer, in terms of preoperative and postoperative morbidity rates, except the duration of hospitalization (table 6).

In our study, length of hospital stay was similar in both groups (6.5 days, p=.7) whereas several studies have found a shorter average length of hospital stay [START_REF] Guy | Comparative outcomes in older and younger women undergoing laparotomy or robotic surgical staging for endometrial cancer[END_REF][START_REF] Lavoué | Benefits of robotic surgery for elderly patients with endometrial carcinoma[END_REF][START_REF] Eddib | Impact of Age on Surgical Outcomes after Robot Assisted Laparoscopic Hysterectomies[END_REF]. This can be explained by the advanced age of our patients who, for a quarter of them, wanted postoperative recovery.

Elderly patients develop more aggressive tumors associated with a poor prognosis [START_REF] Lavoue | Impact of robotics on the outcome of elderly patients with endometrial cancer[END_REF][START_REF] Vaknin | Outcome and quality of life in a prospective cohort of the first 100 robotic surgeries for endometrial cancer, with focus on elderly patients[END_REF].

They are most often non-endometrioid tumors, associated with vascular emboli. Their disease is diagnosed at a later stage; in the case of endometrioid tumor, they are most often grade 3 tumors [START_REF] Lavoué | Benefits of robotic surgery for elderly patients with endometrial carcinoma[END_REF][START_REF] Poupon | Management and Survival of Elderly and Very Elderly Patients with Endometrial Cancer: An Age-Stratified Study of 1228 Women from the FRANCOGYN Group[END_REF]. Our data found significantly more grade 3, advanced, and patients at high risk of recurrence.

Even in the absence of significance, it was found that the number of pelvic and para-aortic lymph node dissections performed was similar in the 2 groups with rates of 54.7% versus 46.8% for pelvic procedures, and 25.6% versus 19.4% for para-aortic procedures, respectively (p=.5). On the other hand, the number of undertreated patients was similar in both groups and concerns 13 elderly patients (21%) and 10 younger patients (11.6%). These were mainly lymph node staging. The main reasons for not receiving the indicated treatment were a reduced ECOG or medical/surgical contraindications. These data are in contradiction with the histological characteristics of tumors. They should have benefited from a greater number of lymph node staging, according to ESMO recommendations [START_REF] Colombo | ESMO-ESGO-ESTRO Endometrial Consensus Conference Working Group, ESMO-ESGO-ESTRO Consensus Conference on Endometrial Cancer: Diagnosis, Treatment and Follow-up[END_REF][START_REF] Lewin | Comparative performance of the 2009 international Federation of gynecology and obstetrics' staging system for uterine corpus cancer[END_REF]. These data are in contrast with the findings of Eggeman et al, and Poupon et al, studies, which found that elderly were undertreated, had fewer lymphadenectomies and fewer adjuvant treatments [START_REF] Eggemann | Management of elderly women with endometrial cancer[END_REF][START_REF] Poupon | Management and Survival of Elderly and Very Elderly Patients with Endometrial Cancer: An Age-Stratified Study of 1228 Women from the FRANCOGYN Group[END_REF].

In our cohort, 9 patients had recurrence during follow-up; one of the patients in group A did not benefit from para-aortic dissection while she belonged to the group at high risk of recurrence and 2 patients in group B were concerned. This can be explained by a difference between the pre-and post-operative data, sometimes explaining an underprocessing or treatment not recommended beforehand [START_REF] Robinson | Redefining geriatric preoperative assessment using frailty, disability and co-morbidity[END_REF].

Complete lymph node dissection increases operating time and complications [START_REF] Turrentine | Surgical risk factors, morbidity, and mortality in elderly patients[END_REF]. But there is evidence that para-aortic lymphadenectomy improves prognosis in this population [START_REF] Todo | Survival effect of para-aortic lymphadenectomy in endometrial cancer (SEPAL study): a retrospective cohort analysis[END_REF]. Older age should not be a contraindication to lymphadenectomy. These data allow us to highlight the interest of the sentinel lymph node in this population [START_REF] Raimond | Impact of sentinel lymph node biopsy on the therapeutic management of early-stage endometrial cancer: Results of a retrospective multicenter study[END_REF]. Paradoxically, it is less realized in the older subject [START_REF] Ballester | Detection rate and diagnostic accuracy of sentinel-node biopsy in early stage endometrial cancer: a prospective multicentre study (SENTI-ENDO)[END_REF]. In our study, the sentinel lymph node procedure was significantly more successful (p=.02) in elderly patients in group B (74.2%) than in group B (55.8%).

Decreased survival secondary to poorer prognosis in balance with major complications secondary to complete surgery in patients with more comorbidities makes limit treatments [START_REF] Eggemann | Management of elderly women with endometrial cancer[END_REF]. These data are confirmed, especially since the patients are over 70 years old; age beyond this limit becomes an independent prognostic factor [START_REF] Dumas | Improving outcomes for older women with gynaecological malignancies[END_REF]. Only half of older women will have the recommended treatment [START_REF] Uccella | Laparoscopic vs. open treatment of endometrial cancer in the elderly and very elderly: An age-stratified multicenter study on 1606 women[END_REF][START_REF] Bourgin | Impact of age on surgical staging and approaches (laparotomy, laparoscopy and robotic surgery) in endometrial cancer management[END_REF][START_REF] Clark | Adjuvant Treatment and Clinical Trials in Elderly Patients With Endometrial Cancer: A Time for Change?[END_REF].

The limitations of our study are related to missing data on the assessment of overall patient satisfaction and quality of life. Other authors have shown that overall satisfaction with robotic procedure is very high regardless of age [START_REF] Lavoué | Benefits of robotic surgery for elderly patients with endometrial carcinoma[END_REF]. A notable limitation of this study is also the lack of systematic onco-geriatric evaluation to consider the vulnerabilities of each patient (G8 score,…). Although this study is retrospective with a reduced effect, it positively assesses the feasibility of robotic surgery in staging surgery for endometrial cancer for the elderly patient, which is poorly represented in clinical studies.

5.Conclusion

Robotic surgery appears feasible and reproducible and can be proposed for the management of endometrial cancers regardless of age. Older patients have more aggressive and higher-risk tumors for which robotic surgery could provide a benefit and allow optimal surgery without increasing morbidity. New specific guidelines are needed for elderly patients with endometrial cancer.

Figure 1 .

 1 Figure 1. Survival for elderly women with endometrial cancer. A) Progression-Free Survival (PFS) and B) Overall Survival (OS) depending on patient age.

Acknowledgements

None

was not required.

Disclosure

The authors declare that there are no conflicts of interest and nothing to disclose.