Ludovic Geelhand De Merxem

Daniela Vuluga

Laurence Lecamp

A Biobased Self-Healing Thermoset Coating with a Dynamic Photosensitive Molecule

Keywords: Thermoset, photo-healing, disulfide, sustainable resource, vegetable oil

A biobased photo-healable thermoset coating has been developed from 4 components in only 2 steps via a solvent-free process involving an epoxy/amine reaction. Its composition includes epoxidized linseed oil and a dynamic molecule (4,4'-diaminodiphenyl disulfide) which, under UV irradiation, can be photolyzed reversibly. Compared to an analogous material without disulfide bond, this coating exhibits a lower crosslinking density and a higher elongation at break. Importantly, a 100 μm depth scratch has been significantly repaired under UV light: a photo-healable coating having both thermoplastic and thermosetting properties has thus been elaborated.

INTRODUCTION

The need to find alternatives to the non-renewable resources used in polymer chemistry has gradually become obvious. In this context, vegetable oils can be considered as a very interesting 2 option: in addition to the renewable aspect, their low cost is an important argument. These are mostly composed of triglycerides, i.e. esterification products of glycerol with mostly unsaturated fatty acids -that represent 95% of the total weight of triglycerides-and their composition is characteristic of each vegetable oil. Many studies have been conducted on such oils to develop new materials [START_REF] Sharma | Addition polymers from natural oils -A review[END_REF][START_REF] Adekunle | A review of vegetable oil-based polymers: synthesis and applications[END_REF][START_REF] Gandini | Progress of polymers from renewable resources: furans, vegetable oils, and polysaccharides[END_REF][START_REF] Bobade | Bio-based thermosetting resins for future generation: A review[END_REF] but few products are available on the market. Moreover, bio-based raw materials are rarely usable directly after harvesting or extraction, and long processes are often required to transform them into molecular building blocks. In this context, highly unsaturated oils are an exception since their native form has been used directly in coatings for a very long time, or after chemical modification (epoxidation, maleinisation, acrylation ...). [START_REF] Gandini | Progress of polymers from renewable resources: furans, vegetable oils, and polysaccharides[END_REF][START_REF] Zovi | A solventless synthesis process of new UV-curable materials based on linseed oil[END_REF][START_REF] Zhao | Photoinitiated thiol-epoxy addition for the preparation of photoinduced self-healing fatty coatings[END_REF][START_REF] Samarth | Modified vegetable oil-based additives as a future polymeric material -A review[END_REF] Unfortunately, these materials have a major drawback: they exhibit high crosslinking, so that they are difficult to process or modify once they have been damaged.

On another hand, self-repairing materials are a class of intelligent materials that have the ability to repair damages caused by mechanical wear and tear over time. The initiation of cracks and other types of damage at a microscopic level modify the properties of the materials (polymer, ceramic, etc.). Materials that can intrinsically correct the damage have a higher long service life, with, ideally, no reduction in efficiency over time due to degradation. For a material to qualify as self-healable, it is required that the healing process occurs without human intervention. The first application in coatings has been described by White [START_REF] White | Autonomic healing of polymer composites[END_REF] and involves a selfrepairing agent based on a microencapsulated monomer that, once released by cracking, polymerizes. While this method is currently the only one commercially available in the field of coatings, it is unfortunately a 'one shot' self-repair with strong limitations on lifespan. [START_REF] Pratama | Room temperature self-healing thermoset based on the Diels-Alder reaction[END_REF] Other self-healing methods activated by external stimuli (electrical, thermal, UV) have been developed. [START_REF] Odarczenko | Sunlight-activated self-healing polymer coatings[END_REF] In theory, this type of self-repair can take place ad infinitum when resting on reversible interactions such as metal-ligand coordination, hydrogen bonding, ionic interactions, ... [11][START_REF] Du | Diels-Alder-based crosslinked self-healing polyurethane/urea from polymeric methylene diphenyl diisocyanate[END_REF][START_REF] Chen | New thermally remendable highly cross-linked polymeric materials[END_REF][START_REF] Zeng | Bio-based furan polymers with self-healing ability[END_REF][START_REF] Tian | Optimization of thermal remendability of epoxy via blending[END_REF][START_REF] Yang | Self-healing polymeric materials[END_REF][START_REF] Chatani | The power of light in polymer science: photochemical processes to manipulate polymer formation, structure, and properties[END_REF][START_REF] Habault | Light-triggered self-healing and shape-memory polymers[END_REF][START_REF] Hillewaere | Fifteen chemistries for autonomous external self-healing polymers and composites[END_REF][START_REF] Hughes | Light-Healable Epoxy Polymer Networks via Anthracene Dimer Scission of Diamine Crosslinker[END_REF] To date, the wide majority of developed materials exhibiting such properties rests upon mobility of the molecules within the materials, such as in thermoplastics or gels. The challenge on coating preparation is still to have thermosetting materials that can self-heal. For this, the coatings must exhibit both thermoplastic and thermosetting features, an interesting path lying on the use of a dynamic molecule that reversibly cleaves.

In this context, disulfide compounds appear as an attractive repair system. Indeed, the homolytic cleavage of the S-S bond occurs naturally at room temperature and the resulting thiyl radicals can then be involved in metathesis reactions with other disulfide bridges (Scheme 1). [START_REF] Abaskharon | Direct measurement of the tryptophan-mediated photocleavage kinetics of a protein disulfide bond[END_REF][START_REF] Lu | Steady-state and time-resolved studies on photoinduced disulfide bond cleavage using aniline as an electron donor[END_REF][START_REF] Klepel | Dynamic covalent chemistry in aqueous solution by photoinduced radical disulfide metathesis[END_REF] However, this exchange reaction is very slow in materials in solid state. Although several chemical or physical actions (adding a basic catalyst, working in solution, under high pressure or in ball mill, increasing the temperature …) can enhanced the reaction kinetics, [START_REF] Sobczak | Dynamic covalent chemistry under high pressure: a new route to disulfide materials[END_REF] this limitation explains why most of examples of healing materials involving disulfide bridge exchanges are elastomers. [START_REF] Michal | Inherently photohealable and thermal shapememory polydisulfide networks[END_REF][START_REF] Lei | Room-temperature self-healable and remoldable cross-linked polymer based on the dynamic exchange of disulfide bonds[END_REF][START_REF] Xu | A novel self-healing polyurethane based on disulfide bonds[END_REF][START_REF] Wang | UV-triggered thiol-disulfide exchange reaction towards tailored biodegradable hydrogels[END_REF][30][START_REF] Zhang | Trapping dynamic disulfide bonds in the hard segments of thermoplastic polyurethane elastomers[END_REF][START_REF] Ohishi | Insertion metathesis depolymerization of aromatic disulfide-containing dynamic covalent polymers under weak intensity photoirradiation[END_REF][START_REF] Matxain | Design of new disulfide-based organic compounds for the improvement of self-healing materials[END_REF][START_REF] Yuan | Highly stretchable, recyclable and fast room temperature self-healable biobased elastomers using polycondensation[END_REF][START_REF] Rekondo | Catalyst-free room temperature self-healing elastomers based on aromatic disulfide metathesis[END_REF][START_REF] Chen | Rapid stress relaxation and moderate temperature of malleability enabled by the synergy of disulfide metathesis and carboxylate transesterification in epoxy vitrimers[END_REF] To the best of our knowledge, only two papers deal with thermosetting materials in which the reversible cleavage of disulfide bridges has been used to give the materials a thermoplastic behavior allowing their processing and recycling at temperatures above 200°C. [START_REF] Ruiz De Luzuriaga | Epoxy resin with exchangeable disulfide crosslinks to obtain reprocessable, reparaible and recyclable fiber-reinforced thermoset composites[END_REF][START_REF] Ocando | Dynamic network based on eugenol-derived epoxy as promising sustainable thermoset materials[END_REF] In this work, we report a thermoset material based on vegetable oil that can self-repair after surface damage by dynamic exchange of disulfide bridges by means of a relatively low temperature process involving UV radiation. 4,4'-Diaminodiphenyl disulfide was chosen as a photo-repair molecule. This symmetrical compound offers the great advantage of giving recombination products identical to the initial molecule (avoiding undesirable non-photolyzable products), thus guaranteeing a priori constant reversibility over time.

Scheme 1. Schematic representation of cleavage and metathesis within the material.

As an easily affordable and cost-effective biobased source, epoxidized linseed oil was selected to design the target material through an epoxy/amine pathway. Whereas materials based on vegetable oils often afford elastomer networks, the original integration of aromatic structures within the network, that we propose herein, should lead to materials with higher Tg and a strengthened thermoplastic feature.

Worthnotingly, a synthesis process in bulk, and without catalyst, was privileged.

The main part of this paper is in two sections. First, an experimental protocol for the elaboration of a photo-healable material from the aforementioned compounds was developed. In a second instance, the photo-repairing ability was investigated and characterized by tensile tests, and the results were compared to those obtained on a reference material with similar chemical structure but without disulfide bond.

EXPERIMENTAL 2.1 Materials

Epoxidized linseed oil (ELO), kindly provided by Traquisa, was filtrated to remove suspended particles before use. Trimethylolpropane triglycidylether (TMPTGE, Sigma Aldrich), 4,4'diaminodiphenyl disulfide (DADS, TCI), 4,4'-methylenedianiline (MDA, TCI), Priamine ® 1074 (Croda) were used as supplied. Chemical formula and physicochemical data of these compounds are collected in Scheme 2 and Table S1, respectively. Scheme 2. Chemical formula of reactants.

Methods

1 H NMR analyses were carried out on a 300 MHz Bruker spectrometer. IR spectra were measured on a Bruker Vertex 70 FTIR spectrometer in ATR mode (ATR MK II Golden Gate, Specac, Eurolabo). The ATR diamond crystal is integrated in a heating plate allowing temperatures up to 300°C. The disappearance of reactive groups was followed at 905 cm -1 (C-O deformation band of asymmetric epoxy group), 800 cm -1 (C-O deformation band of symmetric epoxy group), and at 1620 cm -1 (N-H stretching of aromatic primary amine) and 1540 cm -1 (N-H stretching of aliphatic primary amine). A reference band was chosen between 2750 and 3100 cm - 1 (C-H stretching of aliphatic group). Conversion of epoxy or amine functions was calculated as follows:

Conversion % = A A - A A A A × 100
where A 0 and A t are the area of the absorption bands at t = 0 and t, respectively.

Molecular weight and polydispersity determinations were made using of a Varian PL-GPC50 calibrated with a polymethylmethacrylate standards and equipped with a 5 µm PLgel guard column and a 5 µm Mixed-C analysis column.

Differential scanning calorimetry (DSC) analysis and thermogravimetric analysis (TGA) were performed under nitrogen atmosphere at a 10 °C.min -1 heating rate using TA Instruments devices, DSC Q2000 and TGA Q800, respectively. Dynamic mechanical analysis (DMA) was carried out using a TA Instruments DMA Q800.

Analyses were performed at a 10 °C.min -1 heating rate with frequency and strain rate set at 1 Hz and 5 µm, respectively. The average molar mass between crosslinks (M c ) was determined using the Flory's law:

G = φ RTρ M
where R is the constant of perfect gases, ρ is the material density, G' is the storage modulus measured at a temperature T on the rubbery plateau, ϕ is an empirical factor related to conversion and usually equal to 1. Measurements were repeated on 5 different specimens.

UV-visible spectra were collected using a Varian Cary 50 spectrometer. Solutions at 10 -4 mol.L -1 in methanol were analyzed. Films absorption was measured using another device with an integrating sphere (Scheme S1). The polychromatic radiation came from a 500 W Hg-Xe lamp (Hamamatsu LC8). When required, emission spectrum was limited to the 300-450 nm range thanks to an interference filter. UV lamp irradiance and radiation transmitted by samples were measured between 300 and 800 nm using an Ocean Optics USB2000 spectrometer.

Material elaboration

Photo-healable material. In a reactor A, a mixture of 52 wt-% TMPTGE and 48 wt-% DADS was heated 40 min at 100 °C under mechanical stirring until 67% conversion was reached. In a reactor B, a mixture of 41 wt-% ELO and 59 wt-% Priamine ® 1074 was heated 30 min at 140 °C under mechanical stirring until 35% conversion was reached. After cooling at 70-80 °C, 50 wt-% of reactor A and 50 wt-% of reactor B were mixed under mechanical stirring. The resulting formulation was spread on the silicone support at 80°C by means of a threaded bar. Spreading was repeated at regular time intervals for 15 minutes. After peeling, the film was kept at 80°C for 3 hours before a final phase of 100 h at 100 °C for full reaction.

Reference material. In a reactor C, a mixture of 60 wt-% TMPTGE and 40 wt-% MDA was heated 22 min at 95 °C under mechanical stirring until 67% conversion was reached. Reactors B and C were then mixed at 70-80 °C in the same weight proportion (50/50) and the elaboration of the final film was carried out in the same way as for the photo-healable one.

Photo-healing

Each tensile specimen was notched perpendicular to its main axis of symmetry and over its entire width (4.4 mm) using a home-made notching machine. The notch was 100 µm deep and 50 µm wide. Photo-healing was carried out using a 350 W Hg lamp (Oriel). The light intensity was measured at the sample level using a radiometer (Vilber Intraspec Oriel VLX-3W) fitted with a 254, 312 or 365 nm cell (Bioblock Scientific). This one was set to 5.8 mW.cm -2 at 254 nm, 142.4 mW.cm -2 at 312 nm and 137.3 mW.cm -2 at 365 nm. Each specimen, damaged or not, was placed on a thermostatically controlled metal platform at 100 °C and exposed to UV radiation for 1 to 3 h. No mechanical action was taken to bring the edges of the damage closer together.

Tensile tests were performed on 5 different specimens similarly treated. The recovery percentage of elongation at break was calculated according to the following formula:

Recovery % = ε ) * + , * , * , -ε ) * + , ε ) * + , -ε ) * + , × 100

RESULTS AND DISCUSSION

Epoxidized linseed oil (ELO) and 4,4'-diaminodiphenyl disulfide (DADS) were the main raw materials selected for use in the composition of the target photo-healable material. Two other molecules were added to the previous composition in order to modulate the medium reactivity as well as crosslinking density and absorption ability of the final photo-healable coating:

trimethylolpropane triglycidylether (TMPTGE) and a biobased aliphatic diamine (Priamine ® 1074). At last, DADS was replaced by 4,4'-methylenediamine (MDA) to synthesize a reference material without disulfide bond.

In order to impart healing properties to a thermoset material, it must be possible to transform it into a thermoplastic during the repair time and for it to regain its thermosetting character after repair. [START_REF] Kloxin | Covalent adaptable networks: smart, reconfigurable and responsive network systems[END_REF] This thermoplastic behavior can be obtained by including reversible bonds in the tridimensional structure of the thermoset network. Besides, no significant repair can take place if the exchange dynamic between macromolecular chains is not sufficient within the polymer network. [START_REF] Cromwell | Malleable and self-healing covalent polymer networks through tunable dynamic boronic ester bonds[END_REF][START_REF] Amamoto | Self-healing of covalently crosslinked polymers by reshuffling thiuram disulfide moieties in air under visible light[END_REF] This means that a repairable polymer network must ideally have sufficient reversible bonds to provide chain mobility. According to these considerations, the strategy developed for elaborating the photo-healable material consisted in synthesizing a polymer network that ideally contains a reversible link in each mesh. To achieve this objective, it was decided to systematically work at the stoichiometry in epoxy and amine functions. 1. Compositions of the studied formulations (stoichiometry in epoxy and amine functions).

Formulation

Kinetic investigation

A kinetic study was first performed to characterize the relative reactivity of the five selected reagents. Five bicomponent formulations (1 to 5 in Table 1) were prepared and heated to initiate the epoxy-amine reaction without catalyst. Conversion of each reactive group was monitored in real time by FTIR spectroscopy (Figure S3) at various reaction temperatures. Thus, the monomers envisaged to elaborate the photo-healable biobased material exhibit very different reactivity according to the nature of their functional groups. In addition, the temperature dependence of the reaction speed is highly variable from one couple of monomers to another.

This kinetic study led us to the conclusion that elaborating this material by a one-step process will lead to heterogeneous materials. Indeed, if these monomers are heated together in a same device, the two more reactive monomers (i.e. TMPTGE and Priamine® 1074 -Scheme 3)

quickly react together in a first time and the two less reactive molecules (i.e. ELO and DADS -Scheme 3) slowly react in a second time, leading to the formation of two interpenetrating networks instead of a single network. Hence, a two-step process was preferred. In this process, 3) will first react together in separate reactors before to be mixed in a second step. Scheme 3. Relative reaction speed of each bicomponent system.

Elaboration and characterization of materials

As described in the experimental section, three reactors A-C of bicomponent formulations (reactor A: 52 wt-% TMPTGE + 48 wt-% DADS; reactor B: 42 wt-% ELO + 58 wt-% Priamine ® 1074; reactor C: 60 wt-% TMPTGE + 40 wt-% MDA) were heated until an adequate viscosity for film application and nearly identical residual function rates were achieved. Thus, the heating duration of reactor A was set to 40 min at 100 °C, leading to a 67% conversion (measured by FTIR spectroscopy) and 1.18 meq.g -1 of residual epoxy functions. The same conversion and residual epoxy functions amount were obtained after only 22 min at 95 °C for reactor C. In the case of reactor B, a 30 min heating at 140 °C was necessary to reach 35% conversion, which corresponds to 1.44 meq.g -1 of residual epoxy groups.

Reactors A and B for the photo-healable material, on the one hand, reactors B and C for the reference material, on the other hand, were then hot-mixed in identical mass proportions before 16 being cast on a silicone support. Formulations were then spread out with threaded bar and oven dried up to the maximum progress of the reaction as indicated by the stagnation of the glass transition temperature of the material (Figure S9). 250 µm thick yellow films were thus obtained (Figure 3). These films are fold-resistant and have a certain ability to stretch. Physicochemical properties of these films were then characterized. For comparison, films from bicomponent systems were also prepared and characterized (Table 2). Thermogravimetric curves are presented in Supporting Information (Figure S10). Whatever their compositions, films exhibit very low polar components. In contrast, the dispersive component is higher for the photo-repairable film compared to the reference, which can be attributed to the presence of DADS. These data as well as the low polarity of the materials are characteristic of hydrophobic surfaces. As far as the thermal stability of the films is concerned, the lower degradation temperature of the photo-healable material compared to the reference material can be attributed to the presence of disulfide groups whose cleavage is known to be sensitive to temperature. 21,[START_REF] Sobczak | Dynamic covalent chemistry under high pressure: a new route to disulfide materials[END_REF][START_REF] Chen | Rapid stress relaxation and moderate temperature of malleability enabled by the synergy of disulfide metathesis and carboxylate transesterification in epoxy vitrimers[END_REF][START_REF] Ruiz De Luzuriaga | Epoxy resin with exchangeable disulfide crosslinks to obtain reprocessable, reparaible and recyclable fiber-reinforced thermoset composites[END_REF] Besides, Tg and pendulum hardness values of the photo- Tg values lower than room temperature (5°C) and therefore a soft character. Indeed, the long aliphatic chains separating the two amine functions of Priamine ® 1074 ultimately induce lower crosslinked materials compared to DADS which, moreover, bears two aromatic rings. Finally, it is noteworthy that the Tg value of the photo-healable material is consistent with that calculated by Gordon-Taylor's law, indicating a perfectly homogeneous network.

Material composition (w/w)

DMA analyses show that photo-repairable and reference films have approximately identical G' modules in the glassy state. In contrast, it can be noted that the rubbery modulus is two times greater for the reference material. This results in an average molar mass between crosslinks (Mc) almost twice as large for the photo-repairable material compared to the reference. DADS and MDA having a very close chemical structure with a chain length nearly identical, this difference can be attributed to the presence of the disulfide bridge in the photo-repairable material. Indeed, as illustrated on Scheme 4, the cleavage of the S-S bond leads to an apparent Mc higher than it would be if the bond did not cleave. Finally, tensile tests show that the photo-repairable film has a higher elongation at break and a lower tensile stress at break than the reference. This indicates a lower crosslinking density in the DADS-containing material. This also fully corroborates the DMA results and confirms the dynamic effect of the S-S bridges.

Photo-healing

Photo-healing could occur provided that the material is able to absorb the energy required for the S-S bond photolysis. Among the reagents present in composition of the photo-repairable film, DADS is mainly responsible for the absorption of UV radiation in the wavelength range between 250 and 400 nm (Figure S11). Specifically, for DADS, the effective wavelength range for the S-S bridge cleavage is between 250 and 320 nm. [START_REF] Fan | Wavelength-controlled dynamic metathesis: a light-driven exchange reaction between disulfide and diselenide bonds[END_REF] The absorption of the photo-repairable film was characterized using an integrating sphere, as described in the experimental section. Two series of measurements were carried out (Figure 4).

In the first one, the spectral irradiance was measured over the entire wavelength range of the lamp, in the presence or not of a 200 µm thick film of the photo-repairable material (Figure 4a). In the second experiment, the wavelength range was limited to 300-450 nm by using an interference filter (Figure 4b). As expected, the intensity of the transmitted radiation decreases as a result of its absorption by the film. However, this decrease is not similar in both cases. Thus, if we look at the 365 nm band, which is not efficient for the photolysis of S-S bridges, 35% of the unfiltered radiation is transmitted by the film as opposed to only 22% of the filtered radiation for which effective wavelengths for the S-S bridges photolysis are suppressed or strongly attenuated. It can then be assumed that in the absence of filter, part of the disulfide bridges has been photocleaved, whereas in the presence of filter, no photocleavage has occurred. One can assume that this transmission increase can be related to the transition from S-S bonds to S• radicals. Indeed, a thiol (or thyil) molecule exhibits lower absorbance than its disulfide dimer, as shown on Figure S12. This increase of transmission after S-S bridges photolysis is analogous to a photobleaching phenomenon that may be of particular interest for photo-repair. From the curves obtained on Figure 4b, absorbance (A) of the non-photolyzed material can be determined for each wavelength as the logarithm of the ratio of the incident (I0) and transmitted (It) intensities. Thus, for the three main emission wavelengths of the Hg-Xe lamp, A = 0.65 at 365 nm, A = 0.36 at 335 nm and A = 0.22 at 312 nm. From these values and considering for simplification that εC is invariant during reaction (and equal to 3250 m -1 at 365 nm, 1800 m -1 at 335 nm and 1100 m -1 at 312 nm), the evolution of the transmitted and incident intensities ratio can be predicted as a function of material thickness (l) using the Beer-Lambert's law: Damaged or not, specimens were therefore exposed to UV radiation under different conditions.

I I = 10 ./01
Irradiation time and intensity were optimized in order to limit a blackening phenomenon observed on films containing disulfide bridges, which can be attributed to photooxidation. [START_REF] Lemoine | Vieillissement de la matie re organique[END_REF] Photo-healing properties were then characterized by means of tensile tests. Figure 6a depicts the tensile curves obtained on undamaged (blank), damaged (scratched), and finally damaged and UV exposed test specimens of photo-healable films. Analyses were also carried out on undamaged specimens exposed to UV radiation at 100°C and on damaged specimens subjected to heat treatment alone (Figure 6b). It can be noted that the slight increase in tensile stress at break for scratched material compared to virgin material can probably be explained by a slight variation in the cross section of the specimens. break and toughness obtained in the same conditions are reported in Supporting Information (Figure S13). Compared to blank film, the damage of photo-repairable film is responsible for a decrease of almost 46% in elongation at break, 38% in tensile stress at break and 60% in toughness. For the reference, the consequences of the damage are even more pronounced with a loss of 80% of the elongation at break, 60% of the tensile stress at break and 90% of the toughness. Besides, it is noteworthy that heat treatment alone has almost no influence on these values. Thus, no healing of the photo-repairable material occurred by simple heating, i.e. no creep of material could take place, as expected for a thermoset material. It can also be noted that, after 3 h of UV at 100 °C, the elongation at break of the undamaged photo-repairable material was reduced by 14% compared to the initial value whereas the tensile stress at break was increased by 22%. A visual observation of the samples after irradiation shows a stiffening of their surface by photooxidation of disulfide bridges, which can probably explain these variations. Finally, Figure 6 clearly put forward the beneficial effect of UV exposure. Indeed, the longer the irradiation time, the higher the elongation recovery. After 3 hours of irradiation, 42% of the elongation lost after damage was recovered. The photolysis of the disulfide bridges thus allowed to increase the mobility of the macromolecular chains and to favor the involvement of thiyl radicals in metathesis reactions as illustrated on Scheme 1. This resulted in the promotion of the creep of the material and gave it a thermoplastic behavior. The same treatment performed on the reference film did not highlight this behavior, confirming the essential role of disulfide bridges for repair.

CONCLUSION

In summary, a biobased photo-healing coating that exhibits thermoplastic and thermosetting properties has been developed. This coating is easily prepared from four components in only two steps through an epoxy/amine reaction and a solvent-free process. Its composition includes epoxidized linseed oil and 4,4'-diaminodiphenyl disulfide, a dynamic molecule whose reversible cleavage can be accelerated under UV irradiation. The obtained material contains ca. 50 wt-% of biobased compounds and 24 wt-% of photosensitive molecule. Compared to an analogous material without disulfide bond, the coating exhibits a lower crosslinking density and a higher elongation at break, due to the cleavage of disulfide molecules. This cleavage was intensified by UV light and a significant repair of a 100 μm depth scratch has been achieved: a 42% increase of tensile strength has been observed in 3 hours of UV irradiation at 100°C. Unfortunately, this long duration of UV irradiation induced a photooxidation of the material surface. This process might therefore be more interesting for repairing less deeper scratches by means of lower radiation intensity or shorter UV exposition. This work paves however the way for the development of novel classes of sustainable biobased thermosetting coatings.

Corresponding Authors

*E-mail: daniela.vuluga@insa-rouen.fr *E-mail: laurence.lecamp@insa-rouen.fr

  Contact angles were determined by a GBX Instruments goniometer (Digidrop ASE). Water, ethyleneglycol and diiodomethane were used as analysis solvents. Polar and dispersive components were calculated using the Owens-Wendt method (detailed in Supporting Information). Results are mean values of ten measurements on different parts of the sample surface. Pendulum hardness (Persoz and König) was measured at room temperature with an automatic pendulum tester (Labomat Essor). Mechanical tensile tests were carried out on a Zwick/Roell Z030 equipped with a 500 N force sensor and a thermostatically controlled chamber for a temperature range of -80 to 200°C. Samples of calibrated thickness (around 250 µm) were analyzed at 31.8°C and in accordance with ISO 527-2 2012 type 1 BB: tensile test piece of 50 mm length x 4.4 mm width, initial distance between jaws: 40 mm, test speed: 5 mm.min -1 .

Figure 1 shows a representative example of kinetic curves obtained for formulation 1 (

 1 Figure1shows a representative example of kinetic curves obtained for formulation 1

Figure 1 .Figure 2 .

 12 Figure 1. Evolution of (a) amine and (b) epoxy conversion versus temperature for formulation 1

  bicomponent formulations with intermediate reactivity (i.e. TMPTGE and DADS (or MDA for the reference material), on one hand, and ELO and Priamine® 1074, on the other hand -Scheme

Figure 3 .

 3 Figure 3. Appearance of the photo-repairable film.

  healable film are in between those of the two-component materials based on DADS and Priamine ® 1074. If the presence of DADS (TMPTGE + DADS) leads to materials with a high Tg (70°C) and a rigid character, materials made from Priamine ® 1074 (ELO + Priamine ® 1074) have

Scheme 4 .

 4 Scheme 4. Schematic representation of the effect of disulfide bridge cleavage on Mc.

Figure 4 .

 4 Figure 4. Spectral distribution of radiation in the absence and presence of photo-repairable film:

Figure 5

 5 Figure 5 presents this evolution for the three aforementioned wavelengths. This figure shows

Figure 5 .

 5 Figure 5. Wavelength-dependent radiation penetration profile of photo-repairable film.

Figure 6 .

 6 Figure 6. Tensile curves obtained on specimens of photo-repairable material after various

Figure 7 .

 7 Figure 7. Elongation at break and percentage recovery of materials according to treatment.

  Photo-healable film Reference film Recovery (%)

Table

		TMPTGE	DADS	MDA	ELO	Priamine ® 1074
		wt %	mol %	wt %	mol %	wt %	mol %	wt %	mol %	wt %	mol %
	1 (reactor A)	52	47	48	53	-		-		-
	2 (reactor B)	-		-		-		42	29	58	71
	3 (reactor C)	60	50	-		40	50	-		-
	4	35	49	-		-		-		65	51
	5	-		39	71	-		61	29	-

mol -1 ) e

					Surface properties a
		δ d (mN.m -1 )	δ p (mN.m -1 )	γ (mN.m -1 )	Polarity (%)	Tg (°C) b	Td (°C) c
	TMPTGE + DADS (52/48) (A)	32.6		3.7	36.3	10		70	233
	ELO + Priamine ® 1074 (42/58) (B)	19.3		2.2	21.5	10		5	205
	Photo-healable material (50A/50B)	36.8		3.6	40.4	9		38	242
	Reference material (50B/50C)	19.3		3.7	23.0	16		46	275
						Elongation	Stress at	Pendulum hardness (s)
		On the glassy	On the rubbery		at break	break	Persoz	König
		plateau			plateau		(%) f	(MPa) f	(12°)	(6°)
	TMPTGE + DADS (52/48) (A)	1555 (313 K)		3.08 (413 K)	1368	n.d.	n.d.	316.6	138.9
	ELO + Priamine ® 1074 (42/58) (B) 1369 (243 K)		2.53 (343 K)	1072	66	0.5	43.4	24.3
	Photo-healable material (50A/50B) 1678 (273 K)		3.25 (373 K)	1061	177	3.7	86.4	49.2
	Reference material (50B/50C)	1608 (273 K)		6.32 (393 K)	566	70	12.3	81.1	43.7

a calculated from contact angle measurements; b measured by DMA at the onset of storage modulus G'; c measured by TGA at 5% weight loss Material composition (w/w) Storage modulus G' (MPa) d Mc (g.d measured by DMA; e calculated from rubbery plateau's modulus measured by DMA; f measured by tensile test; n.d.: not determined (brittle material)

Table 2 .

 2 Physicochemical properties of synthesized films.

Acknowledgements

The authors gratefully acknowledge the Région Haute-Normandie for financial support. Authors also thank the European Union for financial support (Smart Surf Project). Europe is committed in Haute-Normandie with the European Regional Development Fund. This manuscript is a tribute to the 50-year anniversary of the French Polymer Group (Groupe Français des Polymères -GFP).


S Ruiz

D Concordet

T Lanot

B Georges

P Goudy

S Baklouti

C Mane

E Losha

H Vinour

D Rousset

M Lavit

V Minville

J-M Conil

P Gandia

Stéphanie Docteur

Ruiz

Hydroxychloroquine lung pharmacokinetics in critically ill patients with COVID-19

Keywords: COVID-19, hydroxychloroquine, plasma drug monitoring, bronchoalveolar lavage (BAL)

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

Introduction

Based on the in vitro work carried out against SARS-CoV-2 and preliminary clinical data, hydroxychloroquine is currently being used in the management of COVID-19 patients [START_REF] Gautret | Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial[END_REF][START_REF] Chen | A pilot study of hydroxychloroquine in treatment of patients with moderate COVID-19[END_REF].

Hydroxychloroquine may have an antiviral action through three main mechanisms: (1) viral entry prevention, (2) impairment of viral replication and (3) pleiotropic action on the human immune system through immuno-modulating activity [START_REF] Oscanoa | A pharmacological perspective of Chloroquine in SARS-CoV-2 infection[END_REF]. The various in vitro studies have shown that the EC50 of hydroxychloroquine ranged from 0.72 to 4.4 µM (i.e. 0.241 to 1.4 mg/l) at 48 hours and 72 hours post-infection, respectively [START_REF] Oscanoa | A pharmacological perspective of Chloroquine in SARS-CoV-2 infection[END_REF][START_REF] Maisonnasse | Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates[END_REF][START_REF] Yao | In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)[END_REF]. Pending the results of robust clinical trials and due to the lack of pharmacokinetic-pharmacodynamic information in COVID-19 patients, and in accordance with the National French Team, AC43-ANRS/STP-SFPT, different dosage regimens were applied in Toulouse University Hospital (France) (200 mg x 3 / day; 400 mg x 2 on day 1 then 200 mg x 3 / day; 400 mg x 2 on day 1 then 400 mg / day; and at least for ICU patients, 600 mg x 2 on day 1 then 400 mg/day) in order to reach pharmacokinetic equilibrium as quickly as possible [START_REF] Le | Rationale of a loading dose initiation for hydroxychloroquine treatment in COVID-19 infection in the DisCoVeRy trial[END_REF].

On 2 April 2020, the French Ministry of Health imposed a dosage regimen identical to the one used in systemic lupus erythematosus (SLE; no loading dose and 200 mg x 3/day) for patients treated outside the context of a clinical trial.

Regardless of the dosage regimen, plasma concentration was monitored in order to evaluate individual drug exposure. It has been proven that the plasma concentrations measured in COVID-19 patients tended to be lower than the values reported in SLE patients, in particular for the standard regimen of "200 mg x 3/day" [START_REF] Martin-Blondel | Hydroxychloroquine in COVID-19 patients: what still needs to be known about the kinetics[END_REF]. These preliminary results suggest that hydroxychloroquine concentrations are unlikely to be adequately predicted using the pharmacokinetic models derived from patients receiving hydroxychloroquine for SLE or rheumatoid arthritis treatment [START_REF] Perinel | Towards Optimization of Hydroxychloroquine Dosing in Intensive Care Unit COVID-19 Patients[END_REF].

As the apparent volume of distribution of hydroxychloroquine is so large in volunteers as well as in malaria patients (≈ 5 000 l for the blood volume of distribution and ≈ 40 000 l for the plasma volume, at steady state) [START_REF] Tett | A dose-ranging study of the pharmacokinetics of hydroxy-chloroquine following intravenous administration to healthy volunteers[END_REF][START_REF] Lim | Pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against malaria caused by Plasmodium vivax[END_REF], it can be suggested that the hydroxychloroquine gets trapped in the red cells and granulocytes [START_REF] Tett | A dose-ranging study of the pharmacokinetics of hydroxy-chloroquine following intravenous administration to healthy volunteers[END_REF][START_REF] Ma | Chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases[END_REF][START_REF] Nosál | Distribution of chloroquine in human blood fractions. Methods and findings in experimental and clinical pharmacology[END_REF] and probably in various tissues [START_REF] Lim | Pharmacokinetics of hydroxychloroquine and its clinical implications in chemoprophylaxis against malaria caused by Plasmodium vivax[END_REF]. It could be assumed that the same occurs for COVID-19 patients. Consequently, it is natural to question the concentration of hydroxychloroquine at the infectious site (i.e. the lung) [START_REF] Wang | Lung tissue distribution of drugs as a key factor for COVID-19 treatment[END_REF].

Unfortunately, this information is not available. A lung biopsy is the most informative approach. Even if a biopsy is a mixture of both intra-and extra-cellular matrices usually homogenised so as to determine a mean concentration [START_REF] Mouton | Tissue concentrations: do we ever learn?[END_REF], hydroxychloroquine gets trapped in the cells suggesting this drug is more likely present in the cells rather than outside.

However, this option would be highly intrusive and an alternative approach would be to evaluate the hydroxychloroquine concentration in lung epithelial lining fluid (ELF) at the bedside of intensive care unit (ICU) patients [START_REF] Mimoz | Steady-state trough serum and epithelial lining fluid concentrations of teicoplanin 12 mg/kg per day in patients with ventilator-associated pneumonia[END_REF][START_REF] Boselli | Pharmacokinetics and lung concentrations of ertapenem in patients with ventilator-associated pneumonia[END_REF].

Drug concentration in ELF can be inferred based on the concentration of hydroxychloroquine in the bronchoalveolar lavage (BAL) fluid and the concentration of urea both in the plasma and the BAL fluid [START_REF] Kiem | Interpretation of antibiotic concentration ratios measured in epithelial lining fluid[END_REF]. This method is not new and is usually applied to explore the lung diffusion of antibiotics in ICU patients. Consequently, when available, BAL fluid can be used as a kind of "quality control" used to obtain information on the degree of impregnation in the lung for a short period (10-15 days) of treatment.

The aim of our study was to evaluate hydroxychloroquine in the lung's ELF in COVID-19 patients in order to estimate the level of lung exposure.

Patients and Methods

This study is a retrospective, observational, multicentre, pharmacokinetic study of hydroxychloroquine in critically ill patients. We included all intubated COVID-19 patients treated with crushed hydroxychloroquine tablets, regardless of the dosage administered by nasogastric tube.

According to the guidelines established by the French National AC43-ANRS/STP-SFPT Team in March/April 2020, blood samples were collected at different time points (from 48h to 192h) during clinical management of COVID-19 patients after hydroxychloroquine initiation and 30 minutes before drug administration (i.e. trough concentration). In plasma, the steady state is supposed to be reached in 48 hours [START_REF] Morita | Population Pharmacokinetics of Hydroxychloroquine in Japanese Patients With Cutaneous or Systemic Lupus Erythematosus[END_REF].

As part of our standard practice for monitoring patients with acute respiratory distress syndrome (ARDS) who are at high risk for infectious complications, and in this particular context of COVID-19, a mini-bronchoalveolar lavage (BAL) (twice 20 ml of physiological saline) was systematically performed 7 days (+/-2 days) after treatment initiation or in the event of a new respiratory degradation, for microbiological monitoring purposes (bacteriology, mycology and viral replication of SARS-CoV-2). A leftover volume of approximately 500 µl remained after these microbiological investigations, which was used to determine the hydroxychloroquine concentration. BAL sampling was carried out between two dose administrations, with no specific time imposed. Except for one case, only the BALs for which plasma determinations were performed within 48 hours before or after collection were included.

Hydroxychloroquine in plasma and BAL fluid concentrations were determined using a chromatographic analytical method, validated as per FDA guidelines. All BAL sample preparations included a protein precipitation and a virus inactivation step in a methanol solution. The HCQ dosage method presents a lower limit of quantitation (LLOQ) of 0.05 mg/l in the plasma and 0.01 mg/l in the BAL fluid, an upper limit of quantitation (ULOQ) of 2 mg/l in the plasma and the BAL fluid, and an intra-and inter-day variability of <4% and <10% respectively. When plasma concentrations were <LOQ, the value was set to 0.025 mg/L (i.e. half the LLOQ). The plasma samples were stored at +4°C before analysis, for a period of 24h maximum. The BAL fluid samples were stored at -80°C before analysis, for a period of 30 days maximum. Previous studies have shown that hydroxychloroquine was stable in the whole blood under these conditions [START_REF] Chhonker | Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC-ESI-MS/MS: An application for pharmacokinetic studies[END_REF]. As whole blood is a complex matrix, in which xenobiotics tend to be less stable than other biological fluids, we considered that hydroxychloroquine was also stable in plasma and BAL fluid in these conditions. Urea is used as an endogenous marker of ELF because urea, a small and relatively nonpolar molecule, can freely travel across membranes to reach the outer surfaces of alveoli. The concentration of urea in ELF (Urea ELF) is considered to be same as in the serum urea (Urea serum) concentration, implying complete distribution. Therefore, the volume of ELF (VELF) is adjusted for excess exogenous water using the following equation: VELF / VBAL = Urea BAL / Urea plasma Knowing (1) the concentration of hydroxychloroquine measured in the BAL (HCQBAL), [START_REF] Chen | A pilot study of hydroxychloroquine in treatment of patients with moderate COVID-19[END_REF] the volume of BAL collected (VBAL), the estimated ELF volume (VELF), it is then possible to determine the concentration of hydroxychloroquine in ELF (HCQELF) using the following formula: HCQELF = VBAL/VELF x HCQBAL= (Urea plasma / Urea BAL) x HCQBAL Plasma urea levels were determined using an automated enzymatic method, validated as per FDA guidelines. This method presents a LLOQ of 0.5 mmol/l, an ULOQ of 15 mmol/l and an intra-and inter-day variability < 2% and <3%, respectively. Safety practices require a greater level of caution when handling respiratory specimen from SARS-CoV-2 positive patients [START_REF] Iwen | Safety Considerations in the Laboratory Testing of Specimens Suspected or Known to Contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)[END_REF]. Thus, urea concentrations in BAL was assayed using a gas chromatography -a mass spectrometry method which included a protein precipitation and virus inactivation step in a methanol solution. A LLOQ of 0.1 mmol/l, and a ULOQ of 20 mmol/l were achieved.

Precision assays showed an intra-day variability < 9% and an inter-day variability < 10%. BAL fluid samples were stored at -80°C before analysis, for a period of 60 days maximum.

Previous studies have shown that urea was stable in serum in these conditions [START_REF] Brinc | Longterm stability of biochemical markers in pediatric serum specimens stored at -80 °C: a CALIPER Substudy[END_REF].

Continuous data was expressed as median (25th-75th percentiles) and categorical variables in numbers (percentages). The relationship between the plasma and ELF concentrations and the other parameters was assessed by simple linear regression. The analysis was performed using the MedCalc®15 statistical software program (Ostend, Belgium). p < 0.05 was considered to be statistically significant.

This study is entered in the Toulouse University Hospital register of retrospective studies (registration number: RnIPH 2020-33) and is covered by MR-004 (CNIL number: 2206723 v 0). This study was approved by Toulouse University Hospital and ethical requirements were entirely respected.

Results

Population

Twenty-eight hydroxychloroquine plasma and BAL fluid concentrations from 22 patients were measured (Table 1). The median patient age was of 60 [interquartile range (IQR) 53-70] years, and 91% of the patients were male. The median body mass index (BMI) was of 28 [IQR [START_REF] Inciardi | Cardiac Involvement in a Patient With Coronavirus Disease 2019 (COVID-19)[END_REF][START_REF] Devaux | New insights on the antiviral effects of chloroquine against coronavirus: what to expect for COVID-19?[END_REF][START_REF] Gladue | In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection[END_REF][START_REF] Group | Effect of Hydroxychloroquine in Hospitalized Patients with Covid-19[END_REF][30][31] kg/m 2 . The median SAPS II and SOFA scores pertaining to the included patients were of 37 [IQR 32-46] and 6 [IQR 3-7] respectively, indicating a critically ill patient population.

Hydroxychloroquine trough concentrations

The values of the BAL fluid hydroxychloroquine concentration were determined 7 to 12 days after treatment initiation. For one point, the time from blood collection to BAL was 9.8 days, but the plasma concentration was at stead-state.

The median hydroxychloroquine plasma concentrations were of 0.09 [0.06; 0.14] mg/l and 0.07 [0.05; 0.08] mg/l for 400 mg x 1/day and 200 mg x 3/day, respectively. The median hydroxychloroquine ELF concentrations were of 3.74 [1.10; 7.26] mg/l and 1.81 [1.20; 7.25] mg/l for 400 mg x 1/day and 200 mg x 3/day, respectively. The median ratio of ELF/plasma concentrations were of 40.0 [7.3; 162.7] and 21.2 [18.4; 109.5] for 400 mg x 1/day and 200 mg x 3/day, respectively (Table 2).

The relationship between ELF and the plasma hydroxychloroquine concentration is presented in Figure 1.

No relationship was observed between the measured hydroxychloroquine concentrations and the biological parameters characterising renal and hepatic functions (Supplementary Table 1).

Discussion

As previously reported for many anti-infective drugs used to treat pulmonary infections, ELF concentration gives information on the intra-and extracellular lung exposure [START_REF] Mimoz | Steady-state trough serum and epithelial lining fluid concentrations of teicoplanin 12 mg/kg per day in patients with ventilator-associated pneumonia[END_REF][START_REF] Boselli | Pharmacokinetics and lung concentrations of ertapenem in patients with ventilator-associated pneumonia[END_REF][START_REF] Boselli | Steady-state plasma and intrapulmonary concentrations of piperacillin/tazobactam 4 g/0.5 g administered to critically ill patients with severe nosocomial pneumonia[END_REF].

However, this approach is essentially reserved for clinical research as the therapeutic monitoring of anti-infective drugs in ELF is determined by practical and organisational constraints. Firstly, performing a BAL requires that the operator be trained and that the patient be stable enough to tolerate the serum injection in such way that it is only exceptionally carried out on non-intubated patients. Secondly, drug quantification has to be performed in an unconventional matrix (i.e. BAL) with a very sensitive analytical method (i.e. more often LC-MS/MS). In the special case of COVID-19 patients, the BAL is contaminated by SARS-CoV-2, thus imposing a specific and time consuming pre-analytical process.

We were able to gather all these conditions in order to assess whether all ELF concentrations are higher than plasma concentrations, despite the variability of ELF values. The significant variability in ELF concentrations may be explained in part by the BAL sampling. In fact, even if the injection volume were standardised (2 x 20 ml), the dwell time and the aspiration pressure cannot be strictly identical [START_REF] Rodvold | Penetration of anti-infective agents into pulmonary epithelial lining fluid: focus on antibacterial agents[END_REF]. Cells can also be part of the ELF, especially macrophages, and may be lysed when measuring the drug concentration. Depending on their quantity, the lysis of these cells may induce an increase in the hydroxychloroquine concentration [START_REF] Ma | Chloroquine analogues in drug discovery: new directions of uses, mechanisms of actions and toxic manifestations from malaria to multifarious diseases[END_REF][START_REF] Kiem | Interpretation of antibiotic concentration ratios measured in epithelial lining fluid[END_REF]. As no measurement of the cell burden in the BAL sample was performed due to insufficient BAL volumes available for pharmacokinetic exploration, this lack of information has to be considered as a limitation of our study. Indeed, the cell burden in the BAL sample is likely associated with the ELF hydroxychloroquine concentration (i.e. the more the cells, the higher the concentrations).

Collecting blood and BAL samples at a different moment (day and/or time) and the potential post-dose discrepancy between the blood sample and the collection of BAL, could appear as limitating the interpretation of the ELF/plasma concentrations ratios. However, hydroxychloroquine presents a large volume of distribution with deep compartments (i.e. lung, spleen, melanin-containing tissues… [START_REF] Oscanoa | A pharmacological perspective of Chloroquine in SARS-CoV-2 infection[END_REF] leading to different kinetic profiles in the plasma and lung tissue [START_REF] Toutain | Plasma terminal half-life[END_REF]. Indeed, half-life elimination is likely to be short in the plasma of COVID-19 patients [START_REF] Martin-Blondel | Hydroxychloroquine in COVID-19 patients: what still needs to be known about the kinetics[END_REF][START_REF] Morita | Population Pharmacokinetics of Hydroxychloroquine in Japanese Patients With Cutaneous or Systemic Lupus Erythematosus[END_REF] as opposed to deep compartments. The blood samples have always been taken at steady state while the BALs could be collected always after the plasma concentration peak. Indeed, the staff in charge of carrying out the BALs was warned of this constraint. Furthermore, a flat kinetic profile was expected in the lung tissue [START_REF] Mouton | Tissue concentrations: do we ever learn?[END_REF]. As a consequence, it seems reasonable to suppose that ELF/plasma concentrations ratios do not change between administrations, once the steady state has been reached. In fact, the ideal solution would consist in determining the area under the time-concentration curve (AUC) in both plasma and BAL matrices at steady state and to calculate the AUC ratio. This option is not feasible and not ethical for critically ill patients presenting ARDS because multiple BALs would alter the gas exchanges between alveoli and capillaries. It would worsen PaO2/FiO2 ratios (the ratio of arterial oxygen partial pressure (PaO2 in mmHg) to fractional inspired oxygen (FiO2 expressed as a fraction)).

However, as our data was retrospectively collected from a small population of ICU patients, one limitation of our study is that the inter-individual variability of the plasma and ELF hydroxychloroquine concentrations likely under/overestimates the actual inter-individual value.

Our results show that hydroxychloroquine concentrations in the lung are higher than in the ELF. Passage from the blood compartment to the ELF involves passing through the pulmonary epithelial cells (i.e. prime target for the replication of SARS-CoV-2 [START_REF] Kiem | Interpretation of antibiotic concentration ratios measured in epithelial lining fluid[END_REF][START_REF] Bao | The pathogenicity of SARS-CoV-2 in hACE2 transgenic mice[END_REF][START_REF] Inciardi | Cardiac Involvement in a Patient With Coronavirus Disease 2019 (COVID-19)[END_REF]) in which hydroxychloroquine is most likely accumulated with pharmacokinetic hysteresis.

Hydroxychloroquine's mechanism of action is poorly elucidated, but includes, among others, the increase in endolysosomal pH necessary for viral fusion. The initial fusion between the viral and the cellular membranes (e.g. lung epithelial cells) requires an interaction between the surface proteins of the two partners, and this interaction can only take place under particular acidic conditions, through the phenomenon of endocytosis. The inability to obtain the ideal pH can block this process, and it is probably through this means that hydroxychloroquine may act. Other properties may be involved: modification in the glycosylation of angiotensin converting enzyme-2, the receptor that SARS-CoV-2 uses to enter the cells and/or posttranslational modification of some viral proteins [START_REF] Devaux | New insights on the antiviral effects of chloroquine against coronavirus: what to expect for COVID-19?[END_REF]. In addition, viral invasion may also trigger a massive margination of the phagocytic cells to the infection site, which may deliver increased amounts of hydroxychloroquine [START_REF] Gladue | In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection[END_REF]. But, in the absence of clear information on the influence of the inflammatory status reported in COVID-19 patients as to the accumulation of hydroxychloroquine in lungs, this point should be considered as a limit of our study. Plasma concentration is not predictive of lung concentration, as shown in Figure 1. Therefore, drug dosage determinations in studies assuming equilibrium between epithelium and plasma concentrations may lead to overly high dosages [START_REF] Group | Effect of Hydroxychloroquine in Hospitalized Patients with Covid-19[END_REF]. Furthermore, in clinical practice, low plasma concentrations should not induce an increase in the drug dosage because the lung exposure may already be high. The various in vitro studies have shown that the EC50 of hydroxychloroquine ranged from 0.72 to 4.4 µM (i.e. 0.241 to 1.4 mg/l) at 48 hours and 72 hours post-infection, respectively [START_REF] Oscanoa | A pharmacological perspective of Chloroquine in SARS-CoV-2 infection[END_REF][START_REF] Maisonnasse | Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates[END_REF][START_REF] Yao | In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)[END_REF]. However, EC50 could be determined in Vero cell lines, but not in a human epithelial cell model [START_REF] Maisonnasse | Hydroxychloroquine use against SARS-CoV-2 infection in non-human primates[END_REF]. This discrepancy may explain that hydroxychloroquine has not shown efficacy in clinical trials [START_REF] Group | Effect of Hydroxychloroquine in Hospitalized Patients with Covid-19[END_REF]. Thus, whether for a dose of 400 mg x 1 /day or 200 mg x 3 /day, the median ELF concentration of hydroxychloroquine is above the maximum EC50 value. Therefore, both regimens lead to a median lung exposure that could be sufficient to eradicate the virus. However, the heterogeneity of EC50s raises the problem of selecting the "right" threshold used to determine the dosage, in particular through modelling techniques. In conclusion, despite all its imperfections, BAL fluid provides a rough idea of lung exposure.

Figure legend Figure 1 :

 legend1 Figure legendFigure 1: Relationship between lung epithelial lining fluid (ELF) and plasma hydroxychloroquine (HCQ) concentrations. The red line is the identity line (i.e. the plasma and ELF concentrations are equal). The dots above the red line indicate that the concentrations in the ELF are higher than the plasma concentrations (ELF/plasma HCQ concentrations: 38.072[8.338 to 138.521]). This figure shows that (i) even with small plasma concentrations, the ELF concentration can be high and (ii) the plasma concentration is a poor predictor of the ELF concentration.

Table legends Table 1 :

 legends1 Socio-demographic and clinical data of 22 severe COVID-19 patients treated with hydroxychloroquine and for whom a bronchoalveolar lavage was performed (BMI: body mass index; CKD-EPI: Chronic Kidney Disease EPIdemiology; IQ: interquartile; SAPS II: simplified acute physiology score; SOFA: Sepsis-related Organ Failure Assessment).

Table 2 :

 2 Hydroxychloroquine (HCQ) plasma and epithelial lining fluid (ELF) concentrations (IQR: interquartile range).

Table 1 :

 1 Socio-demographic and clinical data of 22 severe COVID-19 patients treated with hydroxychloroquine and for whom a bronchoalveolar lavage was performed (ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BMI: body mass index; CKD-EPI: Chronic Kidney Disease EPIdemiology; D0: day 0 of the hydroxychloroquine initiation, D7: day 7 after hydroxychloroquine initiation; IQR: interquartile range; SAPS II: simplified acute physiology score; SOFA: Sepsis-related Organ Failure Assessment).

	All Patients (n=22)	Median [IQR]	Min -Max
	Age	59.5 [53 to 70]	30 -81
	BMI	28.3 [26 to 31.3]	20.7 -37
	SAPS II	37 [32 to 46]	8 -76
	SOFA score	6 [3 to 7]	2 -14
	Protidemia (g/L) D7	61 [59 to 68]	50 -77
	AST(UI/L) D7	65[69 to 179]	28 -135
	ALT (UI/L) D7	99 [69 to 179]	18 -257
	Bilirubin (µmol/L) D0	7.6 [5.15 to 11.2]	4 -29
	CKD-EPI D7 (mL/min/1.73 m²) 97 [60.5 to 105.8]	9 -123
	Duration of invasive ventilation	19.5 [11 to 28]	0 -22
	(day)

Table 2 :

 2 Hydroxychloroquine (HCQ) plasma and epithelial lining fluid (ELF) concentrations (IQR: interquartile range).

		All dosages	400mg x1/day	200 mgx3/day
		Median [IQR]	Min -Max	Median [IQR]	Min -Max	Median [IQR]	Min -Max
	Plasma HCQ	0.09 [0.06 to	0.03 -0.19	0.09 [0.06 to	0.03 -0.19	0.07 [0.05 to 0.08]	0.03-0.09
	concentrations (mg/L)	0.14]		0.14]
	ELF HCQ	3.03 [1.10 to	0.13 -36.75	3.74 [1.1 to	0.13 -36.75	1.807 [1.2 to 7.25]	0.34 -10.08
	concentrations (mg/L)	6.78]		7.26]
	ELF/plasma HCQ	38.07 [8.34 to	2.1 -290.4	39.96 [7.33 to	2.1 -290.4	21.22 [18.41 to	13.4 -168
	concentrations	138.52]		162.66]		109.49]

Acknowledgements

We would like to thank Dr Marion Grare and Dr Sophie Cassaing for their help in sampling bronchoalveolar fluid, the technicians of the Pharmacokinetics and Toxicology Laboratory of Toulouse University Hospital, Magali Centelles for her help in data collection, and Della Hoy for her kind help proofreading the English.

Declarations

Funding: This study was supported by internal funding.

Competing interests: None to declare

Ethical Approval: This study was entered in the Toulouse University Hospital register of retrospective studies (registration number: RnIPH 2020-33) and is covered by MR-004 (CNIL number: 2206723 v 0). This study was approved by Toulouse University Hospital and all ethical requirements were complied with.

Authors' contributions: SR, DC, JMC and PG conceptualised the research aims, designed the study, and took responsibility for the integrity of the data and the accuracy of the data analysis. PG, HV, DR, VM and BG contributed to the acquisition of data. TL, SB, ML and CM performed HCQ concentration determinations in BALs and plasma. DC and JMC performed the statistical analysis. SR, DC, JMC and PG wrote the first draft of the paper, and other authors provided comments and approved the final manuscript.


Moutel

Sophie Baumann

Sylviane Darquy
email: silviane.darquy@u-bordeaux.fr

Claire Miry

Nathalie Duchange

Grégoire Moutel

Darquy Sylviane

Termination of pregnancy for foetal indication in the French context analysis of decision-making in a Multidisciplinary Centre For Prenatal Diagnosis

Keywords: termination of pregnancy for foetal anomaly (TOPFA), prenatal diagnosis, collegiality, particular severity, decision-making

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

Introduction

Medical and technical advances in prenatal care have made prenatal diagnosis increasingly possible for a continually lengthening list of abnormalities presenting various levels of severity, with sometimes-uncertain prognosis [START_REF] Stoll | Detection of Congenital Anomalies by Fetal Ultrasonographic Examination across Europe[END_REF][START_REF] Garne | EUROCAT website data on prenatal detection rates of congenital anomalies[END_REF]. Meanwhile, therapeutic capabilities have not increased to the same degree, leading to the difficult situation of choosing between continuation of pregnancy and abortion. Most developed countries have some form of prenatal screening for Down syndrome and other major foetal anomalies [START_REF] Garne | Termination of pregnancy for fetal anomaly after 23 weeks of gestation: a European register-based study[END_REF][START_REF] Mansfield | Termination rates after prenatal diagnosis of Down syndrome, spina bifida, anencephaly and turner and Klinefelter syndromes: a systematic literature review[END_REF].

Legislations and practices vary from one country to another, but prenatal diagnosis and termination of pregnancy for foetal anomaly (TOPFA) raise important ethical dilemmas everywhere in the world [START_REF] Garel | Ethical decision-making in prenatal diagnosis and termination of pregnancy: a qualitative survey among physicians and midwives[END_REF][START_REF]Registry Prenatal Screening Policies in Europe[END_REF].

In France, termination of pregnancy (TOP) has been legal since 1975 [7] and has been allowed without medical indication up to 14 weeks since 2001: "After this time period, a medical reason is necessary and abortion can be performed at any time […] if there is a strong probability that the child to be born has a particularly severe disorder, recognised as incurable at the time of diagnosis" [8].

TOP for medical reasons is performed at the mother's request after examination by a Multidisciplinary Centre for Prenatal Diagnosis (MCPD). These Centres were established by the French bioethics law of 1994 and have been operating since 1999. Their mission is to assist medical teams and couples in the analysis, decision-making and follow-up of pregnancy when foetal anomaly is detected or suspected [9]. After collegial discussion, they issue a TOP authorisation on medical grounds by certifying the seriousness of the situation.

However, a decree of June 2015 states: "The autonomy of pregnant women is a fundamental principle in prenatal diagnosis. Her will and choices are at the core of the MCPD process and functioning" [START_REF] Law | [END_REF].

The cross expertise of different specialists and their plurality of values are at the heart of the modus operandi of MCPDs. Their members take into account the nature and degree of the pathology, the possible treatment modalities as well as the specific situation and familial environment. The process ensures the transparency of reasoned decisions as well as traceability. A MCPD comprises gynaecologists and obstetricians, paediatricians, geneticists, sonographers, radiologists, paediatric surgeons, midwives, psychiatrists or psychologists and genetic counsellors. It must also ensure the collaboration of other specialists such as foetal pathologists or biologists specialised in foetal abnormalities. It meets regularly, on a weekly basis, and functions through a collegial decision-making procedure. If the MCPD agrees, two physicians who are members of this MCPD must still ratify the decision, and the cost of the TOPFA is borne by the national health insurance. To date, there are 49 MCPDs throughout the French territory.

The rate of acceptance of TOPFA by MCPDs has remained stable over the last 5 years in relation to applications. In 2010, 6949 particular severity statements for TOPFA acceptance and 119 refusals were recorded, and in 2015, 7084 applications were accepted and 132 refused; the acceptance percentage remains around 98.5% [START_REF]Le rapport médical et scientifique de l'Agence de la biomédecine[END_REF].

The objective of this study is to consider the decision-making process within MCPDs. First, we analysed the elements supporting the professionals' decision to accept or refuse a TOP.

Second, we investigate MCPD members' vision about their role, and how they interpret the concept of particular severity according to the law. Finally, we explored their view on the evolution of diagnostic tools.

Methods

This study (file analysis and interviews) was carried out in 2015.

Files Analysis

This study consisted of a review of all TOPFA applications submitted to one MCPD selected for its high level of activity and number of files studied each year. Files for the two consecutive years of 2013 and 2014 were studied. The following elements were gathered: indications of TOP; number of presentations to MCPD; time between application and performance of TOP.

Semi-structured interviews with MCPD members

All MCPD professionals regularly present at MCPD meetings were contacted and eight accepted to be interviewed: three obstetricians, three midwives, one psychologist and one geneticist (five women and three men). No paediatrician accepted to participate.

Each interview lasted between 20 and 30 minutes and was recorded with the agreement of the professional. The main themes explored were TOPFA for Down syndrome; the notion of "particular gravity, serious disease or condition" in situations of uncertainty; the principle of women's autonomy; and collegiality. A qualitative analysis was performed from the transcription of the interviews.

Ethical background: Data collection was strictly anonymous in accordance with medical secrecy; a person from the healthcare team carried out anonymisation.

Results

Medical records analysis

We reviewed 280 files submitted to the MCPD for the period 2013-14. Fifteen files were not included because they concerned maternal indications of TOP. Thus, 265 cases fell within our objectives of foetal indications. All but one (i.e. 264) resulted in a TOP acceptance. The refusal concerned a case of mosaic Klinefelter syndrome.

Table 1 presents the indications of TOP for all 264 applications authorised by the MCPD. The majority correspond to malformations without chromosomal abnormalities that are classified according to the body regions affected. Chromosomal abnormalities also represent a high number of cases among which trisomy 21, which is responsible for Down syndrome, accounts for more than half of cases. Less frequently, indications correspond to genetic disorders, infections or other unclassified situations.

Table 2 presents the number of times files were presented to MCPD meetings. Depending on the indications, the cases required one or more meetings to reach the decision.

In 203 cases out of the 264, the decision was reached in one meeting. Among them, 107 corresponded to chromosomal indications detected during the first half of the pregnancy: trisomy 21 for the majority (n=64); trisomy 18 (n=19); trisomy 13 (n=10); Turner's syndromes (n=8); and others (n=6). Decisions reached in one meeting were found for 75 of the 123 indications of malformations, 11 of the 13 indications of genetic diseases, 2 of the 3 indications of foetal infections, and 8 of the 10 other unclassified indications.

In 61 cases, consecutive meetings two to three weeks apart were required to reach the decision. They corresponded to 48 malformations, 8 chromosomal abnormalities, 2 genetic disorders, 1 infection and 2 other indications.

When files were presented several times, the opinion of an organ specialist or new imaging exploration was required. The mean gestational age at TOPFA was delayed from 16 to 24 weeks of gestation (wg) according to the number of presentations in MCPD meetings (Table 2). An analysis of the records shows that all women received at least one ultrasound and chromosomal analysis and a discussion with their obstetrician.

The term for carrying out TOPFA varies greatly: it extends throughout the pregnancy (Table 3) and depends on the number of MCPD presentations and on abnormalities. The majority are performed before 21 wg, but 5.7% are carried out after 30 wg. The average time elapsed between the decision at the MCPD and TOP is 6.6 days and this period is often reduced at the request of the parental couple, especially in cases of trisomy 21 and 13 (5.4

days).

Semi-structured interviews with professionals (Table 4)

Consensus concerning Down syndrome

All the professionals interviewed confirm the decision-making consensus regarding Down syndrome and the lack of debate around this indication. Most of them explain that the fact of carrying out a systematic prenatal screening is a choice of society. By setting up this screening, the French legislator initiates a list of foetal TOPs' indications leading to consensual and "automatic" acceptance of TOP requests for Down syndrome. For this pathology, our study shows that the period of implementation of TOP is the shortest (under one week).

In addition, seven of the eight professionals question the lack of research, care and acceptance of children with Down syndrome, unlike in other countries, especially in Europe where these children and adults are better accepted and integrated into the school and professional communities.

Situations of uncertainty and notion of particular severity

All professionals reported that some indications are still debated within the MCPD due to prognostic uncertainty. The most exemplary case is corpus callosum agenesis. Regarding this disorder, some point out an evolution in diagnosis leading to a decrease in TOP over the last 15 years. They emphasised that more powerful diagnostic tools would reduce the degree of uncertainty.

All agreed that the concept of "high probability" is difficult to determine. In their opinion, face to the probability of a potential severe clinical picture, couples have full legitimacy to decide.

As regards the concept of a "particularly severe" disorder, most professionals do not believe that a list of foetal anomalies creating a right to TOP should be drawn up because new therapies, particularly in genetics, will change the vision of some disorders in the future.

Role of MCPDs in relation to women's autonomy

The professionals recalled that the legal framework is based on women's autonomy. They are also concerned by their role in protecting the foetus, especially in the event of a disability that is viable or accessible for treatment. Professionals all focused on the ability of MCPD to refuse TOP if necessary, especially in the event of a disability that is viable or accessible for treatment. However, their responses show that they are aware of the fragility of the legitimacy of the MCPD. Several stated that they are not opposed to those couples asking for a second opinion from another MCPD in case of a first refusal. They all recall situations in which women were forced to continue their pregnancy, leading to severe postpartum depression, with all the harmful and possibly irreversible consequences on the mother-child bond.

About collegiality

The decision of the MCPD relies on the principle of collegiality, defined in this case as multiprofessional expertise. All the interviewees stated that they are convinced of the importance of this collegiality for themselves, but also for women and couples: also, each professional has his own position on what he considers important. This system provides support for professionals and helps reassure them because a mistake is always possible. Professionals also think that it is important and reassuring for couples because of the guilt experienced with termination of pregnancy. Finally, a midwife pointed out that the presence of an ethicist or philosopher would sometimes be a plus to take a step back from some complex cases.

Discussion

Since the introduction of MCPDs, few studies have investigated their functioning and the contribution of these centres to antenatal diagnosis and support for couples. Our results

show that unfavourable opinions are exceptional. They represent one refusal out of 265 requests (0.37%). This tallies with the national percentage, with ranges from 0.24% to 0.31% of the files for the period 2011 to 2015, as reported by the French biomedicine agency [START_REF]Le rapport médical et scientifique de l'Agence de la biomédecine[END_REF].

This underlines the fact that files submitted to MCPDs following couples' requests are the result of high-quality evaluation by clinicians and constructive discussion with couples. This leads to a better targeting of files presented to MCPDs. Malformations considered as not severe and able to benefit from surgery are referred directly to an adapted care pathway. In contrast, severe malformations are examined by the MCPD and usually lead to an acceptance of interruption.

In our study, the majority of TOP indications are the result of pathologies of particular severity, recognised as incurable at the time of diagnosis. The main indications for TOP found in the medical records were malformations without chromosomal abnormality and chromosomal abnormalities with or without associated ultrasound anomaly. These represent the main indications at the national level, as shown in a 2010 study by Dommergues et al. [START_REF] Dommergues | ICI Group-Club de médecine foetale. Termination of pregnancy following prenatal diagnosis in France: how severe are the foetal anomalies?[END_REF] where the indications for TOP from 48 MCPDs where reported.

A number of chromosomal abnormalities such as trisomy 21 or trisomy 18 are paradigmatic examples of indications where the decisions have become consensual between MCDP members and other specialists. Since the prenatal screening model and management for trisomy 21 became the norm, the consequence has implicitly been an automatic acceptance of the principle of TOPFA. The legislation governing prenatal diagnosis requires that every pregnant woman has to be informed of the existence of trisomy 21 screening. It also requires information to be given on the possibility of revealing other potentially identifiable conditions. This obligation to inform can be seen as a strong incentive for screening and termination of pregnancy in the event of abnormalities [START_REF] Ukuhor | A Framework for Describing the Influence of Service Organisation and Delivery on Participation in Fetal Anomaly Screening in England[END_REF][START_REF] Gruchy | Pregnancy outcomes in prenatally diagnosed 47, XXX and 47, XYY syndromes: a 30-year French, retrospective, multicentre study[END_REF]. For these indications, decisions are made within one meeting, generally.

For some malformations and some chromosomal abnormalities, diagnostic uncertainties or late detection require several meetings, usually one to three weeks apart, to better explore the situation. Decisions are particularly critical when the viability threshold is reached.

Indeed, the number of pregnancies with a foetal pathology that would previously have given rise to a TOP and that have been continued increased between 2011 (762) and 2015 (1296).

For some well-defined pathologies, treatment protocols, most often surgical, are established. For others for which there is not necessarily a surgical indication, it is mainly a question of ensuring medical care from birth, deciding on the conditions of birth and preventing certain complications by organising paediatric follow-up.

Following TOP authorisation, the discussion continues between the woman and the clinician, to allow a period of reflection and maturation of the decision. The medical teams are in favour of a reflection period of not less 6 to 7 days, as recommended by law. Couples usually want things to go fast. For professionals, the importance not to rush is essential to give to couples the time to accept the end of the pregnancy and the loss of the future child. However, our results show that for some pathologies such as trisomy 21 and trisomy 13, the period is reduced according to the decisions of women and couples (see comments in Table 4).

What about a list of pathologies?

The advisability of drawing up an official list of pathologies that would lead to TOP in the framework of the law has been strongly debated. Such a list would give the practitioner the security of knowing they were acting within a framework accepted by peers. It could on the other hand put pressure on doctors to adhere to that list [START_REF] Statham | Late termination of pregnancy: law, policy and decision making in four English fetal medicine units[END_REF].

The spirit of the French law is to leave discussion on a case-by-case basis, and there are a number of arguments against the establishment of such a list. First, the particular severity of a situation has to be estimated on an individual basis, relying on medical expertise and on the familial context. Second, the risk of drawing up a list of pathologies would be able to make TOPFA systematic. Then, It would be then transition from possibility to compulsory, thus contributing to an eugenic trend [START_REF] Hemminki | Views of Finnish doctors on fetal screening[END_REF][START_REF] Savulescu | Is current practice around late termination of pregnancy eugenic and discriminatory? Maternal interests and abortion[END_REF]. The risk would be a norm of decisions and an absence of discussion leading to an erosion of informed decision-making [START_REF] De Vigan | Mothers' knowledge of screening for trisomy 21 in 1999: a survey in Paris maternity units[END_REF]. Furthermore, for the same pathology, the prognosis may differ from one individual to another; as diaphragmatic hernia.

Finally, updating this list with the evolution of knowledge and treatment seems difficult [START_REF] Statham | Late termination of pregnancy: law, policy and decision making in four English fetal medicine units[END_REF].

To date, genome research technologies allow to refine the prognosis of some malformations, such as agenesis of corpus callosum for example. At present the possibility of carrying out prenatal diagnoses of genetic diseases with late onset (hereditary cancer) raises ethical issues [START_REF] Aksoy | Antenatal screening and its possible meaning from unborn baby's perspective[END_REF][START_REF] Clancy | A clinical perspective on ethical arguments around prenatal diagnosis and preimplantation genetic diagnosis for later onset inherited cancer predispositions[END_REF].

In France, the evaluation of conditions of particular severity is collegial within MCPDs. Our results underline the importance that professionals attach to this structure and to the collegiality of decisions. Through the MCPD process, professionals agree that they do not have the task of imposing their own opinion and that the decision is based on collective and shared validation. In France, the biomedicine agency clearly specifies the methods for recording decisions and their traceability [START_REF]Recommandations professionnelles de l'agence de la biomédecine pour le fonctionnement des centres pluridisciplinaires de diagnostic prénatal[END_REF].

Considering the emergence of medical practices, such as voluntary termination of pregnancy and assisted reproductive technologies, the legislator has established In article 16 of the French Civil Code, the principle of "respect for the human being from the beginning of his or her life [START_REF] Leborne | L'embryon et le foetus, entre personne et chose, entre science et droit : des protections d'intérêts[END_REF]. (Thus, the conceived child is not a "legal person", it is a "human person". It is this distinction which makes it possible to put in place abortion regimes based on women's freedom. It is clear that the desire of the woman and the couple is largely taken into account since the majority of requests are accepted. The professionals recalled that the legal framework is based on women's autonomy and among the amendments to the law on bioethics, TOP was introduced for "psychosocial distress", an act that can be carried out almost up to the end of the pregnancy [23].

What evolution and place for MCPDs to come? Will it still be up to the medical profession in the future to decide what handicap or risk is acceptable or not for couples? This question is all the more topical as professionals perceive possible shifts in the years to come, particularly because of the advent of non-invasive technologies and the potential to analyse the full genome. Today, non-invasive prenatal diagnosis (NIPD) allows the detection of genetic abnormalities targeted to trisomy 21 and other genomic anomalies at an early stage [START_REF] Lewis | Non-invasive prenatal testing for Down's syndrome: pregnant women's views and likely uptake[END_REF][START_REF] Hill | Preferences for prenatal tests for Down syndrome: an international comparison of the views of pregnant women and health professionalswomen and health professionals[END_REF][START_REF] Metcalfe | Beyond Trisomy 21: Additional Chromosomal Anomalies Detected through RoutineAneuploidy Screening[END_REF]. This test will be covered by the health insurance in France. This may lead to the emergence of interruption requests for a number of pathological predispositions.

There are currently debates about the conditions under which this test can be used, in particular for predictive risk markers of disorders such as cancer [START_REF] Clancy | A clinical perspective on ethical arguments around prenatal diagnosis and preimplantation genetic diagnosis for later onset inherited cancer predispositions[END_REF][START_REF] Munthe | A new ethical landscape of prenatal testing: individualizing choice to serve and promote public health: a radical proposal[END_REF]. It is anticipated that, in the event of a result with potential risk, parents' and health professionals' preferences may be different. The latter may be "subjected" to pressure further to women's requests [START_REF] Asplin | Pregnancy termination due to fetal anomaly: Women's reactions, satisfaction and experiences of care[END_REF]. These ongoing developments [START_REF] Martin | Introduction of non-invasive prenatal testing as a first-tier aneuploidy screening test: A survey among Dutch midwives about their role as counsellors[END_REF] will lead to questions surrounding the role and missions of the MCPDs in coming years. As NIPD can be performed at an early stage, ultrasound is completed, this will allow for the opportunity of abortion within the legal timeframe of pregnancy interruption without evaluation by a MCPD.

Under French law, this timeframe is 14 weeks of amenorrhea, but an extension of the legal delay to 16 weeks of amenorrhea is currently debated. Before this deadline, some couples may opt for abortion, even when doctors consider the risk of illness low or the potential "anomaly" of low severity. NIPD may also open up the possibility of sex selection of the future child or access to genetic testing for other markers of pathologies as well as determining risk factors, broadening the debate [START_REF] Rebouché | Non-Invasive Testing, Non-Invasive Counseling[END_REF][START_REF] Richardson | Ethical considerations in prenatal testing: Genomic testing and medical uncertainty[END_REF][START_REF] Jong | Prenatal screening: current practice, new developments, ethical challenges[END_REF]. While respecting women's and couples' autonomy, it will be important to evaluate and monitor the quality of the information delivered to women and their understanding of the issues at stake. In this context, the advisory role of the MCPD would be enhanced, based on collegial expertise to guide women in their decision-making. Limits of the study. The present work was performed five years ago in one MCPD. It is important to emphasise that within the legal framework, MCPDs may have different organisational modalities. However, the data collected form the medical files in this study reflect those collected at the national level by the French agency of biomedicine, justifying the interest of this study. The interest of our study also lies in the qualitative approach to the experience of professionals.

Conclusion

The decision-making process in the MCPD is based on discussion and multi-professional collegiality faced to complex choices. The analysis of the files and the approach of the professionals by interview showed how the latter take into account, on the one hand, the interest of the foetus and, on the other hand, the expectations of women and couples. How couples envision future possibilities for their unborn child remains a challenge. This study provides information about the place and the role of MCPDs within the field of prenatal diagnosis. These structures are essential in supporting women and couples whilst respecting their autonomy. "The organization of screening for Down syndrome in France is a kind of machine for professionals and pregnant women." (Obstetrician)

"The antenatal screening is focused on Down syndrome. When a woman asks for an abortion in this context, her request is accepted. Nothing to discuss!" (Geneticist) "Actually, there is no question, there is a consensus at the MCPD level." (Obstetrician)

"We are one of the only countries to be focused on trisomy 21. In England, for example, screening is not organised. There is a greater acceptance of disability." (Obstetrician)

"Most of the time, women and couples take their decision early, when the exams are prescribed. Their choice is already made at the time of the results. So, the MCPD would not feel legitimate in refusing their request, and this leads to automatic acceptance." (Midwife)

Situations of uncertainty and notion of particular severity

"So, we have to consider life this child could have after birth." (Obstetrician, Geneticist)

"After parents have met the different specialists, pediatrician, psychologist and others, at the end we will follow them in their request." (Obstetrician) "Couples are very anxious about the uncertainty of the prognosis. In doubt, they don't want to take any risks. Then, -the professionals reach a collegial agreement to terminate pregnancy taking into account the suffering of the couple". (Psychologue) "In such cases, we can speak of precaution: the "absence of prognosis" makes it impossible to refuse the application request for a termination of pregnancy for medical reason". (Midwife)

About collegiality

"It works well"; "it's like a firewall" (Midwife, Geneticist, and Obstetrician).

"Each person provides a little of his reality." (Psychologist).

"You think you are less likely to make a mistake when there are many of you who think the same thing." (Obstetrician, Midwife).

"The couples understand that they have been heard and understood by a collective and feel more accompanied" (Psychologist).

"The presence of a neutral person, such as a philosopher or ethicist, would be very relevant and might lead prenatal medical professionals to think a little more or differently." (Midwife)

Time between decision and termination of pregnancy for medical reason / Importance of the medical support "Couples want to move fast. The concern is to alleviate the anxiety of couples as fast as possible." (Midwife) "Consulting the specialist is not a time loss whatever the decision on the outcome of the pregnancy." (Obstetrician)

"Termination of pregnancy for medical reason is never urgent. However, compassion makes us inclined to go faster to relieve couples." (Obstetrician) "For chromosomal abnormalities, the discussion does not take place at the MCPD but rather upstream when informing parents of the screening. So, it seems logical to me that the termination of pregnancy for medical reason be carried out as soon as possible after the diagnosis" (Midwife)

Table 1 :

 1 Foetal indications for medical termination of pregnancy

	MALFORMATIONS (WITHOUT CHROMOSOMAL ABNORMALITIES)	123 (46,6%)
	BRAIN
	Anencephaly	16	Septal agenesis
	Severe degrees of abnormality	5	Holoprosencephaly
	Agenesis of the corpus callosum	3	Hydrocephalus
	Hemimegalencephaly	2	Thymic hypoplasia + vermis agenesis
	Agenesis of the corpus callosum + arachnoidian cysts	2	Arachnoidian cysts + asymmetry of two cerebral hemispheres
	Craniosynostosis	1	Ventriculomegaly
	Exencephalia Ischemo-haemorrhagic lesions	1 3	Cerebellar vermis hypoplasia
	POLYMALFORMATIVE SYNDROME			20
	RACHIS			20
	Spina bifida	17	Severe abnormality (not described)
	Caudal regression	2
	HEART			16
	Complex Heart Disorders (not described)	8	Atrioventicular Communication	1
	Tetralogy of Fallot	4	Double inlet left ventricle	1
	Aortic atresia	2
	RENAL SYSTEM			13
	Megacystis	5	Renal dysplasia
	Bilateral renal agenesis	4	Severe abnormality (not described)
	HYGROMA
	ABDOMEN (DIAPHRAGMATIC HERNIA)
	MEMBER
	Femoral Agenesis	1	Hand reduction abnormality
	CHROMOSOMAL ABNORMALITIES (WITH OR WITHOUT AN ULTRASOUND ANOMALY)	115
				(43.6%)
	Trisomy 21	65	Trisomy 22
	Trisomy 18	22	Unbalanced Translocation 18.9
	Trisomy 13	11	Chromosome 2 deletion
	Turner	9	Chromosome 4 deletion
	Triploïdy	2	Mosaic (undisclosed)
	Trisomy 9	1
	GENETIC DISORDERS	13 (5%)
	Spinal muscular atrophy	3	Steinert's myotonia
	Cystic fibrosis	2	Osteogenesis imperfecta
	Tuberous sclerosis	2	Sickle cell anaemia
	Achondroplasia	1	DiGeorge syndrome
	Duchenne muscular dystrophy	1
		INFECTIONS	3 (1.1%)
	Cytomegalovirus	1	Rubella
	Parvovirus B19	1
		OTHERS	10 (3,7%)
	Proviable premature rupture of membranes 4	Cord's syndrome
	Severe Intrauterine growth retardation	3	Foetal immobility syndrome
			Anamnios
		AMOUNT		264

Table 4 : Professional comments from semi-directed interviews Consensus concerning Down syndrome "

 4 MCPDs don't work in isolation; they work within society." (Midwife)

Acknowledgments 272

The authors thank all the MCPD staff for their help in the study of medical files and MCPD members for agreeing to take part in the interviews. 274

Conflict of interest statement

The authors have no conflict of interest to declare.