Quentin-Arthur Poutrel

Ianis Retailleau

Ugo Lafont

Olivier Orcid Damiano

François Tournilhac

Thermolatent heterogeneous epoxy acid formulation for manufacture of pre-impregnated sheets and composites with vitrimer properties

Keywords: CFRP composites, carbon fibers, epoxy vitrimer, prepreg, latency

The goal of this work is to develop carbon fibers reinforced polymer (CFRP) composites showing vitrimer properties and manufacturable by the prepreg technique. Since their invention in 2011 epoxy vitrimer matrices have shown appealing potential to produce CFRP with healing, adhesion and recycling abilities. While techniques such as resin infusion or resin transfer moulding have been successfully applied, production of composites by handling of pre-impregnated sheets requires a specific set of properties which is still lacking. Starting from commercially available monomers, we hereby investigate the design and preparation of a thermolatent reactive composition complient with the prepreg technique. The designed composition is heterogeneous, being comprised of a crystalline hardener dispersed in novolac-based epoxy resin. It thus offers i) latency and the required viscosity profile for impregnation of carbon fibers around 100°C; ii) chemical inertness and tackiness for storage and handling at room temperature, and iii) awakened reactivity for cure at higher temperature (>130°C). The whole CFRP composites manufacture (from resin mixing, impregnation and composite curing) was transferred from laboratory to an industrial environment. The resulting CFRP vitrimer composites are investigated in regard to mechanical properties, interfacial welding, damage healing and reprocessing through chemical depolymerization allowing to recover carbon fiber sheets with limited damage.

Introduction

Modern composites, comprised of a polymer matrix and fibers (carbon, glass, polyamide, etc.) are widely used in demanding applications such as automotive, aerospace, building, sporting goods, boats, etc. [START_REF] Mangalgiri | Composite Materials for Aerospace Applications[END_REF][START_REF] Diamanti | Structural Health Monitoring Techniques for Aircraft Composite Structures[END_REF] These composites, called fibers reinforced polymers (FRP) often incorporate an epoxy based polymer as matrix. [START_REF] Jin | Synthesis and Application of Epoxy Resins: A Review[END_REF] The assets of the epoxy chemistry are i) the wide range of commercially available resins and curing agents with virtually infinite combinations thereof, ii) a state of low viscosity before curing, permitting efficient impregnation of fibers and iii) curability without solvent, with almost no shrinkage and without by-products. [START_REF] Rebizant | Chemistry and Mechanical Properties of Epoxy-Based Thermosets Reinforced by Reactive and Nonreactive SBMX Block Copolymers[END_REF][START_REF] Vidil | Control of Reactions and Network Structures of Epoxy Thermosets[END_REF] The existence of a liquid state before curing is of utmost practical importance as it determines the suitability of the system to be implementated by any manufacturing method. Before curing, the reactants are usually combined as to form a more or less fluid mixture, giving the possibilities to be processed in various manners such as infusion, resin transfer moulding, as well as assembly of preimpregnated filaments or sheets. Epoxy resins are thus very well suited for manufacturing complex and high quality parts but they come with the inherent drawbacks of thermosets: repair is not possible, assembly of composite parts requires gluing or bolting, inducing weight increase in lightweight structures or even compromising the mechanical properties of those structures. [START_REF] Diamanti | Structural Health Monitoring Techniques for Aircraft Composite Structures[END_REF] Moreover, in current time (2023), the environmental and economic impact of such thermoset structures is questioned. The chemically crosslinked nature of such polymer matrices makes them hard to recycle, giving choice of either waste landing, incineration, pyrolysis, or using harsh chemicals to attack the polymer matrix. [START_REF] Oliveux | Current Status of Recycling of Fibre Reinforced Polymers: Review of Technologies, Reuse and Resulting Properties[END_REF] On top of being energetically intensive, those recycling techniques damage fibers making them only usable in the form of short strands, degrading the most expensive component of FRP composites.

In this context, vitrimers constitute an interesting alternative to regular thermosets. [START_REF] Montarnal | Silica-Like Malleable Materials from Permanent Organic Network[END_REF] Vitrimers are fully crosslinked chemical networks, yet able to exchange bonds upon thermal excitation. Unlike chemically adaptable networks where bonds are said reversible, being opened and closed by effect of temperature, bonds in vitrimers are said exchangeable, meaning the topology of the network can change but the number of links in the system is constant. [START_REF] Montarnal | Silica-Like Malleable Materials from Permanent Organic Network[END_REF][START_REF] Capelot | Catalytic Control of the Vitrimer Glass Transition[END_REF] Vitrimers are intrinsically insoluble, like thermosets but have the ability to flow upon elevation of temperature, unlike thermosets. The link-exchange ability leads to attractive properties such as stress relaxation, controlled creep, healing, reprocessing or even chemical depolymerization in relatively innocuous solvents (e.g., ethylene glycol) presently investigated through experiment [START_REF] Altuna | Epoxy Vitrimers: The Effect of Transesterification Reactions on the Network Structure[END_REF][START_REF] Ricarte | Linear Viscoelasticity and Flow of Self-Assembled Vitrimers: The Case of a Polyethylene/Dioxaborolane System[END_REF][START_REF] Poutrel | Graphene Functionalisations: Conserving Vitrimer Properties towards Nanoparticles Recovery Using Mild Dissolution[END_REF][START_REF] Yu | Carbon Fiber Reinforced Thermoset Composite with Near 100% Recyclability[END_REF] and simulations. [START_REF] Hamel | Reaction-Diffusion Model for Thermosetting Polymer Dissolution through Exchange Reactions Assisted by Small-Molecule Solvents[END_REF][START_REF] Perego | Microscopic Dynamics and Viscoelasticity of Vitrimers[END_REF] .

The vitrimer concept was first demonstrated on epoxy networks. Notwithstanding the existence of alternative chemistry, [START_REF] Zhang | Dynamic Polysulfide Shape Memory Networks Derived from Elemental Sulfur and Their Dual Thermo-/Photo-Induced Solid-State Plasticity[END_REF][START_REF] Liu | Tailoring Vinylogous Urethane Chemistry for the Cross-Linked Polybutadiene: Wide Freedom Design, Multiple Recycling Methods, Good Shape Memory Behavior[END_REF][START_REF] Li | Recyclable Thermoset Shape Memory Polymers with High Stress and Energy Output via Facile UV-Curing[END_REF][START_REF] Lv | Mechanical Enhancement of Amine-Functionalized TiO2 Reinforced Polyimine Composites[END_REF][START_REF] Azcune | Aromatic Disulfide Crosslinks in Polymer Systems: Self-Healing, Reprocessability, Recyclability and More[END_REF][START_REF] Huang | Reprocessable and Robust Crosslinked Elastomers via Interfacial C-N Transalkylation of Pyridinium[END_REF][START_REF] Zheng | Performance-Modified Polyimine Vitrimers: Flexibility, Thermal Stability and Easy Reprocessing[END_REF][START_REF] Demongeot | Cross-Linking of Poly(Butylene Terephthalate) by Reactive Extrusion Using Zn(II) Epoxy-Vitrimer Chemistry[END_REF] the epoxy route remains the straightforward option for transfer of the vitrimer concept in structural composite applications. Proof that epoxy vitrimers are a suitable matrix for FRP composites was made several times: [START_REF] Yu | Carbon Fiber Reinforced Thermoset Composite with Near 100% Recyclability[END_REF][START_REF] Han | Poly(Methyl Methacrylate)-Epoxy Vitrimer Composites[END_REF][START_REF] Chabert | Multiple Welding of Long Fiber Epoxy Vitrimer Composites[END_REF][START_REF] Yu | Rapid Fabrication of Malleable Fiber Reinforced Composites with Vitrimer Powder[END_REF][START_REF] Memon | An Imine-Containing Epoxy Vitrimer with Versatile Recyclability and Its Application in Fully Recyclable Carbon Fiber Reinforced Composites[END_REF] Chabert et al. produced vitrimer matrices with exchangeable ester links and a T α value > 100°C. [START_REF] Chabert | Multiple Welding of Long Fiber Epoxy Vitrimer Composites[END_REF] Composites containing more than 50 vol% of continuous glass fibers showed high bonding strength and repeated welding ability while their mechanical properties were comparable to those of non-vitrimer control samples. Odriozola and coll. investigated reprocessing, reparation and recycling of glass-fiber reinforced composites using an epoxy-amine matrix with exchangeable disulfide links. [START_REF] Ruiz De Luzuriaga | Epoxy Resin with Exchangeable Disulfide Crosslinks to Obtain Reprocessable, Repairable and Recyclable Fiber-Reinforced Thermoset Composites[END_REF] In these works, composite plates were manufactured from reactive mixtures showing an initial viscosity of no more than a few Pa.s as to be implemented by resin transfer moulding. Odriozola and coll. additionally reported the assembly of multilayers by compaction of fully cured sheets. Although nicely illustrating the original properties of vitrimers, fully cured impregnated sheets have the disadvantage of showing no tack and therefore, this process does not allow precise positioning comparable with handling of classic prepregs. Repairable and recyclable woven carbon fiber reinforced composites were also reported using malleable polyimine networks. [START_REF] Kissounko | New Material: Vitrimers Promise to Impact Composites[END_REF] These materials, can show a T g up to 135°C and are sometimes also called vitrimers. These are however networks made up of condensed small molecules (aldehydes and primary amines) as to form imine bridges.

The imination reaction is not a ring opening reaction: it produces water that has to be stripped out during the condensation. Furthermore the drawback of imine (or Schiff base) compounds, well known for their uses in the past in liquid crystals, is their inherent chemical, thermal and photophysical instability. [START_REF] De Gennes | The Physics of Liquid Crystals[END_REF][START_REF] Fritsch | An Old Dog with New Tricks: Schiff Bases for Liquid Crystals Materials Based on Isoxazolines and Isoxazoles[END_REF][START_REF] Castellano | Liquid Crystal Devices[END_REF] Aforementioned epoxy vitrimers compositions were all made from a homogeneous mixture of a so-called resin (the epoxy monomer) and the other reactant, called hardener. Desirably, the hardener is well molten and homogeneously mixed as to be poured or injected within a few minutes and reacted shortly after. This makes sense when preparing bulk samples or composites through injection, infusion or resin transfer techniques where sedimentation or separation of components may lead to inhomogeneity in the cured system. In the prepregs case, the resin has to be impregnated and stored for occasionally a very long time before lay-up. Having a heterogeneous, yet homogenizable formulation may be a route to obtain tackiness and latency at ambient temperature and awakened reactivity at higher temperature. Hence, we turned our attention to recent vitrimer formulations using a high melting point crystalline hardener. [START_REF] Tangthana-Umrung | Epoxy Homopolymerization as a Tool to Tune the Thermo-Mechanical Properties and Fracture Toughness of Vitrimers[END_REF][START_REF] Chen | Reprocessible Epoxy Networks with Tunable Physical Properties: Synthesis, Stress Relaxation and Recyclability[END_REF] Those networks proved to work with the vitrimer chemistry producing high glass transition networks, suitable to FRP composites. When attempting to transfer such formulations from laboratory to a larger scale workshop or factory, the key point is the knowledge of the operating window, including the viscosity profile, chemical conversion and exothermicity issues, as a function of temperature and time. Here, we investigate the capacity of such heterogeneous network, comprised of a viscous epoxy-novolac resin and a crystalline dicarboxylic acid, to show i) a low viscosity (< 100 Pa.s, preferably < 10 Pa.s) for impregnation of high volume fractions of fibers and latency (one or a few hours) at impregnation temperature, ii) virtually no reactivity over weeks at the so-called storage temperature (preferably room temperature, or below) and iii) fast reactivity at a temperature higher than the impregnation one, giving rise to a homogeneous polymer matrix, but desirably as low as possible. Observations combining thermalized ATR-IR spectroscopy, optical microscopy and rheology are used to determine the physical and chemical state at any step of the process. Processing conditions thus developed in the laboratory were then passed to a subcontractor to realize large preimpregnated samples (at the m 2 scale) on an industrial platform. After impregnation, layup and curing, the subsequent carbon FRP composites was mechanically tested and compared to classical thermoset CFRP. The panel was further tested to demonstrate original properties connected to the vitrimer nature of the polymer matrix such as healing, adhesion and reactive depolymerization.

Experimental procedures

Materials

An epoxy-novolac resin, poly[(phenylglycidyl ether)-co-formaldehyde] with an average functionality of 3.6 epoxy functions per molecule, PPGEF (Epalloy 8830 from Huntsman Ltd) type was chosen for its tackiness at ambient temperature and low viscosity when heated, allowing lay-up and impregnation. Moreover, novolac resins also produce high density crosslinked networks resulting in high glass transition temperature polymers (> 120°C) usually favoured for CFRP composites. The hardener is a crystalline dicarboxylic acid (Cyclohexane-1,4-dicarboxylic acid, CHDA from Abcr) with high melting temperature to limit potential curing at ambient temperature thus offering long latency, promotor of hydroxyl-ester links (exchangeable bonds and functions) responsible for vitrimer properties (bonding and reparability). A high melting temperature catalyst (allowing to trigger the exchange upon heating) was also chosen to limit cure at ambient temperature (zinc acetylacetonate hydrate, Zn(acac) 2 from Abcr). Structure and properties of the aforementioned chemicals are given in Table 1. Octanoic acid was obtained from Alfa Aesar. All chemical were used as received without further treatment.

Table 1: Chemical structure and properties of the three compounds used in the preparation of latent reactive mixtures.

Monomers Epoxy Hardener Catalyst

Name [START_REF] Mangalgiri | Composite Materials for Aerospace Applications[END_REF] PPGEF CHDA Zn(acac) 2

Formula n ≈ 3.6 [START_REF] Jin | Synthesis and Application of Epoxy Resins: A Review[END_REF] Molar mass 171-183 g/mol [START_REF] Diamanti | Structural Health Monitoring Techniques for Aircraft Composite Structures[END_REF] 172.

Reactive mixtures

Two different compositions are hereby investigated, namely without and with the Zn(acac) 2 catalyst, they will be further on referred as PPGEF-CHDA and PPGEF-CHDA-Zn(acac) 2 respectively.

For the PPGEF-CHDA composition, PPGEF (10g) was heated to 100°C to decrease its viscosity (well below 30 Pa.s, see Table 1) whereupon CHDA (4.86g) was added via manual mixing (using a stainless steel spatula) before leaving the mixture cool down to ambient temperature. Whenever larger quantities were manipulated, the same procedure was applied using an overhead mechanical stirrer. The slurry thus obtained, contained small solid particles dispersed in a liquid.

For the PPGEF-CHDA-Zn(acac) 2 composition, Zn(acac) 2 was handled as previously reported in previous work. [START_REF] Chabert | Multiple Welding of Long Fiber Epoxy Vitrimer Composites[END_REF][START_REF] Demongeot | Coordination and Catalysis of Zn 2+ in Epoxy-Based Vitrimers[END_REF] The catalyst quantity was set to 5 mol% with respect to epoxy functional groups. At room temperature, Zn(acac) 2 is a powder solid, sometimes agglomerated; therefore, it was grinded in an agate mortar before being mixed with the CHDA powder. Then the mixture of powders was suspended in the epoxy resin (preheated to 100°C to soften it) as previously described for the non-catalyzed composition. At this point, the reactive mixture is ready for impregnation of fibers. The following only concerns the preparation of fiber-free specimens and is similar for both catalyzed and non-catalyzed compositions: a 30 min degassing at 100°C, followed by a pre-cure step at 130°C for 1h, whereupon the initially white mixture turned to transparent. The pre-cured resin temperature is then raised to 145°C over 1h and left to cure 8h.

An 8h postcure is then applied at 180°C.

Infrared spectroscopy

Attenuated total reflection infrared (ATR-IR) spectroscopy was used to monitor the variation in concentration of reactive functions and products (epoxy and carboxylic acids for the reactants, hydroxides and ester for products) throughout the curing process, and for studying the latency of reactive mixtures at ambient temperature. A Bruker Tensor 37 spectrometer equipped with a Specac Golden Gate ATR heating cell was used for the experiments. More details on the infrared analysis and spectroscopy can be found in SI and in reference. [START_REF] Poutrel | Dicarboxylic Acid-Epoxy Vitrimers: Influence of the off-Stoichiometric Acid Content on Cure Reactions and Thermo-Mechanical Properties[END_REF]

Rheological monitoring

An Anton Paar MCR 302 rheometer equipped with 25 mm disposable plates and operating at a constant 1 rad/s oscillatory shear was used to measure the viscosity of the epoxy resin at different temperatures and the evolution of the viscosity of the reactive mixture during cure and detect the occurrence of the gel point.

Thermal analysis

Differential scanning calorimetry (DSC) measurements were performed using a DSC Q1000 analyser from TA instruments, the enthalpy signal of the investigated reactive mixtures have been analysed according to the Kissinger di Benetto method. [START_REF] Ooi | DSC Studies of the Curing Mechanisms and Kinetics of DGEBA Using Imidazole Curing Agents[END_REF][START_REF] Woo | Cure Kinetics of Epoxy/Anhydride Thermosetting Matrix Systems[END_REF] More details on this method can be found in SI. Thermogravimetric analysis (TGA) was performed using a Netzsch Libra TG 209 F1

analyzer. The weight loss of the samples was measured from 25 °C to 600 °C at a constant rate of 10 °C/min under a nitrogen atmosphere. Dynamic mechanical analysis (DMA) was performed using a MCR 702 Anton Paar rheometer operating in the torsion mode at 1 rad.s -1 , using 3°C/min ramps and 1% strain. Additional experiments using the three point bending accessory are described in SI.

Optical microscopy

Polarized optical microscopy observations were made on a Leica DMRD microscope equipped with a Fluotar 5×0.12 objective, Sony XCD-100CR digital camera and Mettler FP50 heating stage. Mixtures of CHDA and PPGEF were placed between a glass slide and cover slip for temperature investigations between 80°C and 200°C.

Composites preparation

Carbon fibers impregnation of m 2 large panels was made manually in the premises of the Kalliste Composite company (Nice France). The methodology developed in the laboratory to evaluate fiber impregnation and CFRP composites was passed on to the company as follow: for initial fibers soaking, an impregnation on both sides was considered to be optimal to maximize fibers wetting. According to viscosity measurements and curing characterization, all chemicals were mixed together at 100°C, the mixture was then applied directly on either side of the carbon fiber tissue. The mixture was kept between 100°C and 120°C and spread over with the help of a smoothing iron. The impregnated carbon fiber sheet thereby obtained was then cooled down to room temperature and cut into smaller squares of ~30cm, these squares were laid up according to the following stacking sequence 0° / 60° / -60° / 0° / 90° / -30°. Once stacked, the multilayer assembly was squeezed in a vacuum bag and put under pressure in an oven with the help of iron weights. The curing conditions remained similar to those of non-reinforced polymer samples:

130°C for 1h, a 15°C/h ramp until 145°C, an isothermal cure at 145°C for 8h. Finally, the composite plate was postcured 8h at 180°C. The resulting panel was cut into 10 cm × 2 cm samples for further testing. The fiber content was evaluated on the basis of density values (detailed calculations in SI).

Compressive tests have been performed according to ASTM D3410 standard at Thalès Alenia Space facilities. Composite samples healing was performed in the hot press at 180°C for 1h at 0.5 bar pressure. This procedure also led to flatten the whole samples to equivalent thicknesses and prepare them for adhesion. Composite samples welding was achieved after sanding and cleaning 2.5 cm 2 surfaces of CFRP. Then those surfaces were pressed against each other by pairs, as to form three lap-shear joints with an overlap area of 2.5 cm 2 . The lap shear specimens were placed for 1h30 at 180°C in a clamp comprising two M5 screws tightened with a torque of 1.7 N.m, thus ensuring a force of approximately 4000 N over the total area of 7.5 cm 2 . The adhesion strength

was measured with an Instron 5900 machine with pneumatic clamps (pressure, 2 bars) at a deformation rate of 1 mm.min -1

Results & discussion

The workflow of experiments hereby reported is presented in Figure 1. First, reactive mixtures are devised with suitable latency and viscosity profiles as to be spread on a dense layer of carbon fibers and stored for long time in the form of pre-impregnated sheets. Then, individual layers are stacked, compressed and cured, as to form composite samples with a ≈ 40 % volume fraction of crossed fiber reinforcement. Then a few properties of the composite parts are evaluated in different conditions including some new properties brought by the vitrimer nature of the matrix, such as composite healing, welding and matrix depolymerization.

Formulation of a latent pre-vitrimer composition

Strategies to impart latency in epoxy formulations have been reviewed. [START_REF] Vidil | Control of Reactions and Network Structures of Epoxy Thermosets[END_REF] The most straightforward approach is to rely on the regular Arrhenius law and select an epoxy-based system showing a high activation energy, e.g. 100 kJ/mol or more. Such systems have been designed based on supramolecular chemistry. [START_REF] Vidil | Supramolecular Control of Propagation in Cationic Polymerization of Room Temperature Curable Epoxy Compositions[END_REF][START_REF] Vidil | Control of Cationic Epoxy Polymerization by Supramolecular Initiation[END_REF] High activation energy, in this case, means that the contrast between reaction rate at high temperature and reaction rate at low temperature is more pronounced. The main downside of this path is that, residual reactivity at the ambient is small but finite and this affects the viscosity of the reactive mixture over time. Other strategies use macro or microphase separation, or encapsulation. There, the idea is to prevent reaction by physically separating the reactants. Dicyandiamide (DICY) which is typically used for this purpose is crystalline. This crosslinker slowly dissolves in the epoxy resin and starts to react on heating but shows no reaction at all at room temperature. Furthermore, with this reagent, crosslinking takes place through a rather complex mechanism involving both step and chain polymerization mechanisms, eventually beneficial for latency. Curing with DICY, allows to reach a Tg of about 140°C. [START_REF] Yu | Carbon Fiber Reinforced Thermoset Composite with Near 100% Recyclability[END_REF] However, in the current state of knowledge, DICY cannot be used to produce a network with exchangeable links (Figure S1a). On the other hand, cyclic anhydrides are used for this purpose because their reaction with epoxides produces exchangeable ester links.

The problem of this type of curing is that the produced networks lack of hydroxide groups, which are also necessary to activate the transesterification exchange reaction. To solve this problem, it was recently proposed to blend the reactive mixture with a rigid diglycol co-monomer to serves as a hydroxide source. [START_REF] Chappuis | Enriching an Exchangeable Network with One of Its Components: The Key to High-TgEpoxy Vitrimers with Accelerated Relaxation[END_REF] This strategy enabled to produce high-Tg epoxy-anhydride vitrimer networks with accelerated relaxation potentially suitable for CFRP composites. Yet, anhydrides show relatively low melting points and a moderate activation energy making them poorly suitable towards formulation of thermolatent compositions. Retaining the idea of a monomer or comonomer both rigid and protic but also crystalline, our choice fell here on the 1,4-cyclohexane dicarboxylic acid. [START_REF] Tangthana-Umrung | Epoxy Homopolymerization as a Tool to Tune the Thermo-Mechanical Properties and Fracture Toughness of Vitrimers[END_REF][START_REF] Chen | Reprocessible Epoxy Networks with Tunable Physical Properties: Synthesis, Stress Relaxation and Recyclability[END_REF] As for the chosen resin, it is a prepolymer of the novolac epoxy ether type, conventionally used in prepregs because it has both the appropriate viscosity and a high density of crosslinkable groups. The expected curing reaction is shown in Figure 1.

Figure 2A shows the IR measurements of the reactive mixture at 25°C over a period of 25 days.

There is no noticeable change in the IR spectrum of the PPGEF/CHDA mix, the characteristic signal of the epoxide (Figure 2A right panel) does not evolve over a period of 25 days indicating latency on this time scale. As for C=O stretching vibrations, neither the starting material (acids)

nor the expected product (ester) is detectable (Figure 2A right panel). To explain this, it must be reminded that in the ATR technique utilized here the region scanned by the infrared radiation is not deeper than the depth of the evanescent wave, D p , as given by equation 1.

2 2 1 2 1 sin 2         - = n n n D p θ π λ (1)
Where λ is the IR wavelength, n 1 and n 2 the indexes of refraction of the ATR crystal and sample and θ the incidence angle. Practically, D p is of the order of a few microns, or less. In case of a two-phase sample, the heterogeneous distribution of the constituents in the vicinity of the interface may cause one of them to disappear. This is what is happening here, due to the granular shape of CHDA crystals, there is poor contact of this component along the interface. Thus, CHDA molecules, blocked in the crystals did not intercept the evanescent wave (to produce an ATR-IR signal the substance must form a liquid film of 0.5 -4 µm thickness) and did not react with the epoxy either. Hence, as long as the substance is in its crystals form, it is not detectable using ATR-IR spectroscopy, nor reactive which explains why nothing is visible in the C=O stretching region. This is further confirmed by the stability of the signal in the 2500 cm -1 -4000 cm -1 region (Figure 2A left panel) corresponding to the stretching of alkyl and hydroxyl functions. The mix PPGEF/CHDA without thermal treatment therefore shows chemical stability for at least 25 days at room temperature.

Latency thus proven has significance for storage, the next step is to monitor how the reactivity of the mixture at temperatures suitable for impregnation of carbon fibers. As the novolac-based epoxy resin has a drop in viscosity above 60°C (cf Table 1), we study the reactivity at three different temperatures: 100, 110, and 130°C. To assess the impregnation parameters in this processing window, we recorded the evolution of chemical species over 24h using thermallycontrolled ATR-IR spectroscopy. In parallel, rheology was performed to record viscosity evolution and time to gel point. The gel point is estimated by the crossover of the storage modulus G' and loss modulus G" at 1Hz (For the curing of epoxy resins, this determination was found close to the frequency-independent value in ref. [START_REF] Vidil | Control of Gelation and Network Properties of Cationically Copolymerized Mono-and Diglycidyl Ethers[END_REF] ) indicating that the polymerization is started but not completed (as revealed by the remaining epoxy signal after 24h (right panel). At 130°C (Figure 2D), the characteristic signals of carboxylic acid and epoxy completely disappear within 24 h and a clear ester vibration is seen, together with the OH stretching band at 3500 cm -1 thus indicating that the polymerization reaction is completed. Further analysis of these monitoring was performed by integrating the signals defined earlier (cf.

Figures 2B-2D
experimental procedures) over this 24h period. Figure 3 shows the evolution of the characteristic species: epoxy (a), esters (b), and the hydroxyl signal (c) of the β-hydroxy ester fragment. It is clear that at 100°C, the system barely reacts over 24h, upon increasing to 110°C it comes evident that the system reacts faster but is still continuously evolving after 24h. However, at 130°C, the system reacts fast for ~500-600 min (i.e., > 10h), then the conversion slows down for the rest of the measurement. This indicates that most chemical moieties reacted through polymerization and once most of reactants are consumed (~80%), [START_REF] Stockmayer | Theory of Molecular Size Distribution and Gel Formation in Branched-Chain Polymers[END_REF] the reaction slows down. Another classical reason for slackening is the vicinity of the T cure and T ∞ (T g of fully cured network) once the monomers are exhausted. Figure 3D shows the rheology measurements made on the same mixture (PPGEF/CHDA), displaying the complex viscosity over time at these temperatures. At 100°C, the system has little to no reactivity, hence, the measurement does not give particular information except that the gel point is not reached in 24h. This confirms what was observed in ATR-IR -that no esters are produced by the PPGEF/CHDA reaction. At 110°C, conversion of epoxides and carboxylic acids happens leading to effective cross-linking. Following definition given above, the gel time at 110°C is of about 12 -13h (crossover of G' and G" in Fig S3). At 130°C, the gel time is reduced to about 3h. Before gelation, the viscosity of the reactive system is of 18-40 Pa.s at 110°C and 2-6 Pa.s at 130°C. At 110°C, the viscosity is in the required range with little to no reactivity for nearly 10h. The impregnation temperature was set at 100-110°C, which leaves enough time to manually impregnate large carbon fibers as shown later in this work.

The crystallinity of CHDA with high melting temperature gives, therefore, a satisfactory latency for storing, handling and impregnation of this resin type. Control experiments with octanoic acid and sebacic acid confirm this relationship: Sebacic acid is a solid (MP = 135°C), its mixture with epoxy shows no sign of reaction over 2 hours at 90°C while the ester typically forms in less than 20 min at 125°C. [START_REF] Poutrel | Dicarboxylic Acid-Epoxy Vitrimers: Influence of the off-Stoichiometric Acid Content on Cure Reactions and Thermo-Mechanical Properties[END_REF] Octanoic acid is a liquid. At 25°C, its mixture with PPGEF shows the carbonyl vibration in ATR. Effective dissolution at 25°C induces a drop in viscosity, detrimental to prepregs' manipulation (Figure S4); and the epoxy-acid system is known to be reactive at 100°C once dissolved. [START_REF] Bakkali-Hassani | Epoxy Vitrimer Materials by Lipase-Catalyzed Network Formation and Exchange Reactions[END_REF] However, heterogeneous formulations bring challenges to obtain homogenous samples: a fast curing at high temperature led to samples with bubbles and high quantity of unreacted CHDA crystals. Meanwhile, with a temperature too low, the slow curing unfavourably allows time for sedimentation of CHDA at the bottom of the samples. Few systems already reported examples of heterogeneous networks to prepare vitrimers from crystalline hardener, including a degassing phase at elevated temperature. [START_REF] Tangthana-Umrung | Epoxy Homopolymerization as a Tool to Tune the Thermo-Mechanical Properties and Fracture Toughness of Vitrimers[END_REF][START_REF] Poutrel | Dicarboxylic Acid-Epoxy Vitrimers: Influence of the off-Stoichiometric Acid Content on Cure Reactions and Thermo-Mechanical Properties[END_REF] Per se, the application of temperature during the degassing step has not for sole purpose to decrease the resin viscosity and allow bubbles removal. In those reports, the heterogeneous mixture was reported to pass from a white color to transparent state indicating dissolution of crystals in the resin. [START_REF] Tangthana-Umrung | Epoxy Homopolymerization as a Tool to Tune the Thermo-Mechanical Properties and Fracture Toughness of Vitrimers[END_REF][START_REF] Poutrel | Dicarboxylic Acid-Epoxy Vitrimers: Influence of the off-Stoichiometric Acid Content on Cure Reactions and Thermo-Mechanical Properties[END_REF] Hence, we used optical microscopy to observe the crystals behavior within the epoxy when temperature is increased. Inspection of the raw mix in polarized optical microscopy (Figure S5) demonstrates that the CHDA crystals initially seen at room temperature are still present until 130°C, whereupon they gradually melt. At 170°C most of the crystalline phase is molten and dissolved in the optically isotropic mixture. Yet, a minority of birefringent areas are still detected, and this even at 200°C (Figure S6). This revealed that the mixture of cis/trans CHDA was comprised of crystals with inhomogeneous melting temperature. The majority of them show a melting temperature of ~170°C and are easily dissolved in PPGEF Yet, a minority of crystals show a higher melting temperature (>200°C) and proved difficult to react.

Nonetheless, from the unreacted crystals size and remaining quantity, it was concluded that the CHDA was still suitable as hardener for the formulation of vitrimer resin aiming to impregnate carbon fibers.

Rather than combining degassing and dissolution steps as in previous reports, degassing was performed at 100°C, where crystals remained in their solid form, thus keeping the latency of the system when temperature is brought back to ambient. The homogenization of the system was then performed following IR and rheology results from our data previously shown (Figure 3). We set the reactive mixture for 1h at 130°C to allow melting of most crystals while remaining far below the gel time. This "pre-curing" step led the system to pass from a white opaque aspect to transparent (Figure S7) -indicating that most CHDA crystals were dissolved in the epoxy. The remaining opacity of the resin can be attributed to the unsolvable crystal fraction observed through microscopy (Figure S5 andS6).

The infrared spectrum of the reactive mixture before heating and after 1h and 24 h curing at 130°C are presented in Figure S8. As already observed, the signal of the carboxylic acid hardener is not detected at room temperature. At 130°C, the carboxylic acid signal (doublet at 1712 and 1740 cm -1 ) is detected: dissolved molecules can access the interface and give rise to detectable IR absorption signals. As for the epoxy signal, its position changes between room temperature and 130°C, then, it decreases but remains present after 24 h of curing, showing slow reaction at this temperature. This is all truer since the decrease in the epoxy signal is partially due to increased volume fraction of dissolved CHDA crystals.

Right after this precuring step, characteristic signals of the carboxylic acid are then evident. As already mentioned, the C=O stretching vibration is split due to different forms of H-bonding.

After long stage at 130°C, the C=O stretching vibration of the ester, has appeared at ~1725 cm -1 but the carboxylic acid is still detected, showing effective dissolution of CHDA with slow reaction at this temperature.

Once resin homogenization was probed, a curing cycle had to be defined to achieve most epoxide/acids conversion within a reasonable amount of time. The evolution of precured samples has been monitored by ATR-IR, for different curing temperatures (100, 110, 130, 145°C). The results are shown in Figure S8 a-c , similarities between raw mix and precured resin (homogenized) in the temperature 100-130°C range demonstrate that the precuring step has for main goal to components through formation of epoxy-CHDA adducts at early stage of the reaction (see Figure S2 for detailed explanation). Nonetheless, to obtain rapid and full curing of the epoxy resin, an increase to 145°C after precured for 8h is necessary. A postcuring step at 180°C for 8h was then applied to ensure maximum conversion of remaining monomers. To go a step further in the suitability of such heterogeneous networks for CF impregnation, the intensities of two characteristic vibrations related to the COOH group are plotted (Figure S9) as a function of cure time at different temperatures. Before experiment, all samples have been subjected to a 1h precure at 130°C. Even though O-H stretching signal has a higher level of noise, the information brought by both measurements are the same. At the initial time, the concentration of COOH increases almost linearly with temperature. Then the level remains constant at 100°C, slowly decreases at 110°C, faster at 130°C, even faster at 145°C. Interestingly, complete consumption is observed at 130 and 145°C after 24 hours but final level is almost the same as the initial one when cure is performed at 110 or 130°C.

To better visualise the behavior of the reactive mixture in temperature and understand the design of the material, Figure 4 plots the viscosity of the resin (measured by rheology), the epoxy opening reaction rates measured by the initial slope of the epoxy conversion graph (Figure 3a), and the percentage of black pixels of the images of Figures S5 andS6 (pixels with luminance under 30 in a 0-255 scale are considered black) correlated to the disappearance of CHDA crystals. On this graph appear three temperature ranges suited for the successive steps of the composite manufacturing: at low temperature (below 100°C), the reaction rate close to 0 and the high viscosity of the resin allows long latency and high tackiness for proper handling, storage and layup of prepregs sheets. At intermediate temperature (between 100 and 120°C), the reaction rate stays finite but the viscosity of the resin is low (around 200 mPa.s -1 ), allowing impregnation of carbon fiber sheets without reaching the gel point during the process. At high temperature (above 120°C), the reaction rate steeply increases whereas the melting of CHDA crystals is ascertained by POM. In Figure 4, the reaction rate data are fitted by a solid line corresponding to an Arrhenius activation energy of 159 kJ/mol. Such a value higher than 100 kJ/mol clearly corresponds to the above definition of a thermolatent system. These data demonstrate that two different factors contribute to the latency of the CHDA-epoxy system: low solubility, due to its high melting point and low reactivity between 100°C and 130°C. In this temperature range, approaching but still below the melting point of CHDA (164 -167°C), the instantaneous concentration of carboxylic acid is thus determined both by the kinetics of dissolution and that of reaction with the epoxide. Practically, this means that this composition has an excellent latency profile for impregnation of fibers but on condition that it remains in this temperature range. Otherwise, there is a risk that the system will viscosify in the event of cooling, with formation of clots as well as in the event of heating with premature hardening of the resin.

Resin with catalyst: PPGEF/CHDA/Zn(acac) 2

To produce a system with vitrimer properties, a catalyst for transesterification is required to display the capacity of the polymer to be weld and healed in a suitable time/temperature window.

Adding a catalyst to the mixture potentially leads to several changes in the resin behavior, especially latency and curing. Hence the exact same procedure was applied to the reactive mixture with catalyst to measure its latency over 25 days. Figure 5 shows the IR measurements of the raw mix at 25°C over this period of time. There are no noticeable changes in the IR signatures of the reactive mixture incorporating PPGEF, CHDA and Zn(acac) 2 , indicating that after 25 days the network has no reactivity at room temperature. This confirms latency over period of weeks for both uncatalyzed and catalyzed systems. As for stability over a longer timescale, samples of the catalyzed formulation have been stored at -18°C and re-analyzed two years later after thawing. The sample showed unchanged pasty texture at 25°C, IR analysis revealed slight solubilization of the acid and remnant epoxy groups while kinetics at 130°C demonstrates the system is still reactive (Figure S10). S1,S2). In isothermal curing at 145°C, the reaction rate is roughly the same for both systems. At higher temperatures, DSC data at variable heating rates (Kissinger method, ref [START_REF] Jourdain | Comparison of Poly(Ethylene Glycol)-Based Networks 40 Obtained by Cationic Ring Opening Polymerization of Neutral and 1,2,3-Triazolium Diepoxy Monomers[END_REF] ) give a measure of the activation energy, E a . The catalysed system shows E a = 65 kJ.mol -1 while the uncatalyzed system gives E a = 74 kJ.mol -1 . These values are by far lower than the one (159 kJ.mol -1 ) determined by fitting the data of Figure 4. In the Kissinger's method, the key for determination of the activation energy is the position of the reaction exotherm as a function of the heating rate. In the data presented in Table S1, the maximum exotherm always falls between 200 and 250°C. The activation energies thus determined are therefore relevant to that temperature range where the homogeneity of the reaction mixture is at its best and moreover the E a value is not much lowered with the catalyst. This indicates that the heterogeneity of the mixture is by far the principal factor of thermolatency, and demonstrate the interest of this design.

Polymer characterization

The glass transition temperature after cure was determined by dynamic mechanical analysis using a dual motor MCR702 rheometer. The uncatalyzed sample exhibits an α relaxation temperature of T α ≈ 100°C at 1 rad.s -1 while the catalyzed sample has T α ≈ 107°C (Figure 6A and 6B respectively). The measured T α meet the objective of a vitrimer matrix with T g > 100°C for CFRP composites. Moreover, two distinct features appear from this characterization: (i) In the vicinity of T α and above, the storage and loss modulus of the catalyzed system are both higher than that of uncatalyzed one, probably due to a higher conversion at the end of the cure stage. (ii) In presence of the catalyst, the tan δ peak deforms with shift of the maximum towards higher temperatures. This trend, (also observed in other metal-catalyzed systems) [START_REF] Tangthana-Umrung | Epoxy Homopolymerization as a Tool to Tune the Thermo-Mechanical Properties and Fracture Toughness of Vitrimers[END_REF][START_REF] Poutrel | Dicarboxylic Acid-Epoxy Vitrimers: Influence of the off-Stoichiometric Acid Content on Cure Reactions and Thermo-Mechanical Properties[END_REF] reflects a network densification, probably due to larger involvement of alcohol + epoxy additions, [START_REF] Demongeot | Cross-Linking of Poly(Butylene Terephthalate) by Reactive Extrusion Using Zn(II) Epoxy-Vitrimer Chemistry[END_REF] conducive to additional ether links. The thermal stability of the cured networks was evaluated using thermogravimetric analysis TGA under a nitrogen flow. Results are presented in Figure S15, both networks exhibit an initial weight loss in the vicinity of 100°C, probably due to the release of humidity. Then, the samples show a high temperature of degradation (> 300°C) which is classical for thermoset epoxy polymers. For the catalyzed system, the temperature of 5% weight loss is of about 325°C compared to 360°C for the uncatalyzed system. However, it can be seen that moisture evaporation is more important for the catalyzed sample. If the 5% weight loss is considered with respect to the weight at 100°C, the value is ~365°C for both samples. autoclave) during curing. This may have an impact on its compressive strength, combining with the heat sensitivity point indicated just before. S4) demonstrated that the flexural modulus of composite plates specimens decreases by about one order of magnitude between 25 and 60°C, thus confirming the effect of matrix softening.

Vitrimer-like properties

Stress relaxation properties of the cured matrix (Figure S14) were measured and show that increasing temperature (from 180 to 200°C) leads to faster relaxation, but slackening above 200°C, probably due to formation of ether links by intermolecular dehydration. [START_REF] Chappuis | Enriching an Exchangeable Network with One of Its Components: The Key to High-TgEpoxy Vitrimers with Accelerated Relaxation[END_REF] However, it is noticeable that full relaxation is not achieved at any temperature within a measurable timescale and consequently this matrix will be referred as 'vitrimer like' by analogy with materials showing the same structure and properties. [START_REF] Imbernon | Stress Relaxation and Self-Adhesion of with Exchangeable Links[END_REF][START_REF] Kaiser | Stress Relaxation and Thermally Adaptable Properties in Vitrimer-like Elastomers from HXNBR Rubber with Covalent Bonds[END_REF] This can be explained by the nature of the network. The epoxy monomer used in this work has an average functionality of 3.6 epoxy function per molecule and CHDA a functionality of 2 acid functions per molecule. Following the reasoning of ref [START_REF] Poutrel | Dicarboxylic Acid-Epoxy Vitrimers: Influence of the off-Stoichiometric Acid Content on Cure Reactions and Thermo-Mechanical Properties[END_REF] , expected conversion at the gel point for the 1:1 stoichiometry is 62%. This is the case only if transesterification reactions are not considered. If transesterification reactions are considered, conversion at gel point shifts to 56%. Thus, transesterification reactions do not influence much the gelation of the system.

However, when comparing time to gel point determined at 130°C (Figure S3) with conversion obtained in IR measurement (Figure 3B), it appears that gelation happens at much lower conversion (20%) than expected. This demonstrates that side reactions are occurring: alcohol + epoxy reaction was mentioned above; the anionic ring opening polymerization of epoxies can be also considered for off-stoichiometric compositions. [START_REF] Tangthana-Umrung | Epoxy Homopolymerization as a Tool to Tune the Thermo-Mechanical Properties and Fracture Toughness of Vitrimers[END_REF][START_REF] Poutrel | Dicarboxylic Acid-Epoxy Vitrimers: Influence of the off-Stoichiometric Acid Content on Cure Reactions and Thermo-Mechanical Properties[END_REF] This reaction is likely to take place here, due to incomplete melting of the trans-CHDA isomer. Both lead to ether bonds formation which are not exchangeable, making the network unable to reach full relaxation. The network is therefore considered as a vitrimer-like materials still able to bond or heal.

As a proper protocol is yet to be determined in order to measure relaxation of CFRP vitrimer and reshaping is rather limited by the fibers [START_REF] Chabert | Multiple Welding of Long Fiber Epoxy Vitrimer Composites[END_REF] , our focus went on showing interest of such CFRP vitrimer for sustainable applications: capacity of cracks healing, adhesion without the use of glue or bolts, and recovery of fibers at the end of the composite's life cycle. Crack healing and samples welding rely both on the system capacity to undergo transesterification upon heating (Figure 7). Firstly, we chose a damaged sample showing signs of delamination induced by the dry cutting mode. After flattening at 180°C for 30min, it can be seen that the fibers adhered to the matrix, reducing initial delamination which can be induced either during manufacturing or processing or the CFRP composite plate (Figure 7). After flattening, six samples were taken to form lap-shear specimens according to procedure explained earlier. To measure the strength of the adhesion, lap-shear tests were performed, the samples were pulled 4 times to 1 kN pulling force (≈ 4 MPa) without break, showing shear strength equivalent to classical epoxy glue. Upon 5th pulling event, one of the samples broke at the interface (Figure S18). Post mortem examination of the fracture surface showed evidence of cohesive fracture and that the effective contact was smaller than the overlap surface; meaning that the intrinsic debonding strength is between 4 and 6 MPa. This indicated that adhesion of the two CFRP pieces was achieved through effective welding of the matrix, permitted by the vitrimer bond exchanges mechanism. Finally, as one of the main issues of thermoset CFRP is the end of life, with recycling leading to potential environmental wastes and degradation of the expensive carbon fibers, we took a small sample (2 cm × 1cm) to investigate the soft chemical depolymerization of the vitrimer matrix.

The sample was plunged into an ethanol/acetone solution (50:50), and heated for 4h at 180°C in a pressurized reactor. After removal and cooling, the fiber sheets were separated with the help of a spatula and recovered. Figure 1 shows the full story from monomers to CFRP with vitrimer properties and eventually chemical depolymerization allowing fibers recovery. Despite small damage induced by the separation process, it is clear that the depolymerization allows to separates laminates in the size of the initial sample without the use of high temperature or harsh chemical usually used to remove classical thermoset epoxy resin.

Product of the depolymerization was analyzed with GPC (Figure S19) and compared to initial monomers, the trace overlay shows a larger proportion of higher oligomers in the depolymerization mixture, consistent with the occurrence of etherification side reactions above discussed.

Conclusions and outcomes

The use of a heterogeneous reactive mixture (liquid epoxy and crystalline hardener) shows potential to produce long latency prepreg sheets and CFRP composites with vitrimer properties and demonstrated to be feasible on an industrial platform. When the hardener is chosen to be insoluble at ambient temperature, tackiness and viscosity are governed by the epoxy resin properties, allowing impregnation, layup and storage. Insured by the low reactivity of components and without need of cold storage, up to 25 days of latency are allowed by the chosen composition. If the mix remains below its homogenization temperature, the layup can also be undone and remade in another configuration due to the high latency of the system. Simple combination of thermalized IR spectroscopy and optical microscopy was used to determine impregnation and homogenization temperatures which allowed to prepare and handle the prepreg vitrimer without risk of premature curing. The impregnation, made in an industrial environment, demonstrates an ease of transferring the vitrimer technology where it is still lacking applications:

high performance CFRP composites.

As for the PPGEF/CHDA system, the current preliminary results on such CFRP composites demonstrates that vitrimer properties, such as crack healing, adhesion without bolts or glues (for weight reduction) or reactive depolymerization can ameliorate both life cycle and end of life of the materials. Nonetheless, outgassing and mechanical results (at high temperatures) also emphasis remaining marge of progress in the development of vitrimer resins for space applications. The broad glass transition and pretransitional softening of vitrimers when approaching T g are currently limiting factors for their use in high performance CFRP.

Formulation of high glass transition vitrimer, would need to carefully consider up to which temperature the system exhibits high strength. Increasing these properties means reducing chains mobility and getting closer to the degradation temperature of the polymers (either through unwanted secondary reaction or proper thermal degradation). This reduces the temperature windows to obtain healing or adhesion. Recent progress in high glass transition temperature vitrimer suggest this issue is solvable through careful design of the formulation. Hence, there is no doubt that heterogeneous systems with high latency can be used for prepregs FRP combining both requirement of thermoset resins and the dynamic properties of vitrimers.

Figure 1 :

 1 Figure 1: Thermolatent epoxy formulation with vitrimer-like properties: workflow from

  Figures 2B-2Dshow the IR spectra of the PPGEF/CHDA mix at t = 0 h and t = 24h for each

Figure 2 :

 2 Figure 2: Infrared spectra of the PPGEF/CHDA mix at (A) 25°C; over a span of 25 days, no noticeable change is detected; absence of bands in the 1700 cm -1 region demonstrate insolubility

Figure 3 :

 3 Figure 3: Time dependence of characteristic IR signals and complex viscosity of the

Figure 4 :

 4 Figure 4: Diagram showing the correlation between epoxy resin viscosity (▲), effective melting

Figure 5 :

 5 Figure 5: Infrared spectra of PPGEF/CHDA/Zn(acac) 2 mixed manually at 100°C and left to cool

Figure 6 :

 6 Figure 6: Dynamic mechanical measurement of the PPGEF CHDA system. A: without catalyst,

Figure 7 :

 7 Figure 7: Cracks healing and adhesion through chemical exchange leading to bonding strength

Table 2 :

 2 Calculated and measured Yong's modulus and compressive strength for CFRP vitrimer composite following ASTM D3410 standard. *Due to pretransitional softening on approaching the T g .

	Environment Young's modulus (Gpa)	Mechanical strength
		Expected Measured	Expected Measured Agreement
	-70 to -40°C	38	38-39	360	344	Good
	+25°C	38	35-72	360	95-225	Fair
	+60 to +90°C 38	N/A	360	N/A	Poor*
	At high temperature, compression tests failed to produce reliable values. Measurements in the
	three point bending mode (Table

Table of

 of Contents (TOC) graphic

Acknowledgements

ESPCI, CNRS, the Thales Group and the European Space Agency (ESA Contract 4000128430/19/NL/KML/afm) are thanked for financial support. Anton Paar France SAS is gratefully acknowledged for loan of the MCR702 rheometer. Paolo Edera is thanked for help in three-point bending measurements. Mickaël Pomès Hadda (CNRS), Maelys Lahoré (TAS) and Renaud Viala Artigues (Kalliste Composite) are thanked for technical support in resin, textile and composite handling.

CFRP composites

The preparation of large size preimpregnated sheets is illustrated in Figure S16: After fibers impregnation, layup and curing, the resulting panel was then cut in small samples of 10 × 2 cm with the help of a dry diamond saw, to proceed of CFRP characterization.

The outgassing properties of the neat matrix and its CFRP composites have been measured based upon the micro-VCM test (ECSS-Q-70-02). Data are given in Table S3, the CFRP composite presents a CVCM (Collected Volatile Condensable Material) of 0.17 % and a recovered mass loss (RML) of 0.98 %. The European Space agency recommends values of CVMV < 0.1% and RML < 1% for aerospace applications. This CFRP composite already satisfy the requirement of RML, and improvement of the formulation or manufacture are envisioned to reach the CVMV requirement making those long latency of vitrimer formulation suitable for use in the aerospace industry.

Compression tests were also performed and compared to the expected value of a reference thermoset CFRP composite cured with DICY. Those tests, made at different temperature (-70°C, 25°C, 60°C/90°C) allow to strongly engage the resin and are thus a relevant test for assessing the health and quality of composite laminates, the results are collected in Table 2. For Young's modulus, the measured values bracket the expected one. As for the compressive strength, three samples were tested giving an average of about 180 ± 72 MPa at ambient temperature. This result is at least 40% lower than expected, considering the strength at low temperatures (see Table 2, -70°C value), matching the prediction, this low strength at room temperature is likely linked to early softening of the matrix, visible on the DMA plot (Figure 6). Another cause, reflected on the large dispersion of results may be the non-optimal pressure cycle (i.e., with weight rather than in Supporting information. Strategy to impart vitrimer properties in a latent epoxy formulation; IR band assignment; fiber content; activation energies; IR and rheology monitoring, optical microscopy of curable compositions; latency in presence of catalyst; stress relaxation and thermal degradation of cured compositions; preparation of large size preimpregnated sheets; composites' outgassing, three point bending, facies of fracture after bonding and analysis of the depolymerization products.


Sarah Leclaire

Mandar Bandekar

Melissah Rowe

Jarmo Ritari

Annalaura Jokiniemi

Jukka Partanen

Pia Allinen

Liisa Kuusipalo

Jukka Kekäläinen

Women reproductive microbiota varies with HLA

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

Introduction

In mammals, follicular fluid is the liquid filling the follicular antrum and thus forms the microenvironment of the oocyte during its development and maturation. It is composed of several biologically active molecules that acts as major oocyte nutrients or important mediators of the interaction between male and female gametes (e.g., [START_REF] Fitzpatrick | Chemical signals from eggs facilitate cryptic female choice in humans[END_REF][START_REF] Jokiniemi | Postcopulatory genetic matchmaking: HLA-dependent effects of cervical mucus on human sperm function[END_REF]. Follicular fluid composition is thus a key element to the success of fertilization and the development of viable embryos [START_REF] Kim | Proteomic analysis of recurrent spontaneous abortion: identification of an inadequately expressed set of proteins in human follicular fluid[END_REF][START_REF] Dumesic | Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health[END_REF][START_REF] Ciepiela | Oocyte matched follicular fluid anti-Müllerian hormone is an excellent predictor of live birth after fresh single embryo transfer[END_REF].

Recently, a few studies have shown that many bacteria colonize the follicular fluid, even in the absence of symptomatic infection [START_REF] Einenkel | Microorganisms in the healthy upper reproductive tract: from denial to beneficial assignments for reproductive biology[END_REF][START_REF] Salary | Detection of bacteria in bovine ovarian follicular fluid[END_REF][START_REF] Usman | The presence of microorganisms in follicular fluid and its effect on the outcome of in vitro fertilization-embryo transfer (IVF-ET) treatment cycles[END_REF][START_REF] Wu | Microbiological composition of follicular fluid in patients undergoing IVF and its association with infertility[END_REF]. One of the plausible way of follicular fluid colonization is the ascension of bacteria along the women reproductive tract. Therefore, in humans, bacteria commonly detected in follicular fluid include for instance Lactobacillus, Propionibacterium and Gardnerella (Pelzer, Allan, et al. 2013;[START_REF] Wu | Microbiological composition of follicular fluid in patients undergoing IVF and its association with infertility[END_REF], which are normal bacteria of the women's lower reproductive tract [START_REF] Chen | The microbiota continuum along the female reproductive tract and its relation to uterine-related diseases[END_REF][START_REF] Pelzer | The fallopian tube microbiome: implications for reproductive health[END_REF]. Follicular fluid microbiota also contain bacteria commonly detected in the gastrointestinal tract, skin or oral mucosa (Pelzer, Allan, et al. 2013), which may spread via hematogenous dissemination.

Several studies have shown that the microbiota of follicular fluid may be a fundamental modulator of host reproduction [START_REF] Pelzer | Microbial colonization of follicular fluid: alterations in cytokine expression and adverse assisted reproduction technology outcomes[END_REF]Pelzer, Allan, et al. 2013;[START_REF] Dong | Urogenital tract and rectal microbiota composition and its influence on reproductive outcomes in infertile patients[END_REF][START_REF] Wu | Microbiological composition of follicular fluid in patients undergoing IVF and its association with infertility[END_REF]. For instance, in women, the presence of Lactobacillus in follicular fluids has been associated with higher pregnancy rates (Pelzer, Allan, et al. 2013;[START_REF] Wu | Microbiological composition of follicular fluid in patients undergoing IVF and its association with infertility[END_REF]) and, in mouse and pigs, the presence of several microorganisms compromise oocyte quality or maturation [START_REF] Sluss | Presence of Bacteria in Porcine Follicular Fluid and Their Ability to Generate an Inhibitor of Follicle-Stimulating Hormone Binding to Receptor[END_REF][START_REF] Pelzer | TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes: The effect of microorganisms within human follicular fluid collected during IVF cycles[END_REF]. Mechanisms include deleterious effects of bacteria on DNA fragmentation within oocytes [START_REF] Pelzer | TUNEL analysis of DNA fragmentation in mouse unfertilized oocytes: The effect of microorganisms within human follicular fluid collected during IVF cycles[END_REF][START_REF] Usman | The presence of microorganisms in follicular fluid and its effect on the outcome of in vitro fertilization-embryo transfer (IVF-ET) treatment cycles[END_REF]. Therefore, unraveling the factors that governs follicular fluid microbiota may be crucial for the ethiology of infertility.

The microbiota is shaped by multiple environmental and host factors, including diet, habitat, sex, age and host genetics [START_REF] Grice | The skin microbiome[END_REF][START_REF] Kers | Host and Environmental Factors Affecting the Intestinal Microbiota in Chickens[END_REF][START_REF] Rothschild | Environment dominates over host genetics in shaping human gut microbiota[END_REF]). Among the host genetic factors, several immune genes may play a critical role, because they encode for proteins involved with detecting specific bacteria products [START_REF] Marietta | Immunogenetic control of the intestinal microbiota[END_REF]. The major histocompatibility complex (MHC; called HLA in humans), one of the most polymorphic group of genes in vertebrates, is crucial for the recognition of self and non-self protein antigens by T-cells. Consequently, MHC genes are also involved in defence against bacterial pathogens and tolerance towards symbiotic bacteria [START_REF] Roland | How MHCII signaling promotes benign hostmicrobiota interactions[END_REF]. As shown in several vertebrates including humans, inter-individual variation in MHC genotype may thus drive inter-individual difference in microbiota [START_REF] Toivanen | Influence of major histocompatibility complex on bacterial composition of fecal flora[END_REF]Bolnick et al. 2014a;[START_REF] Marietta | Immunogenetic control of the intestinal microbiota[END_REF][START_REF] Khan | Does MHC heterozygosity influence microbiota form and function?[END_REF]Andeweg et al. 2021a). However, although this association has been studied for the human gut microbiota (Andeweg et al. 2021a), the association between the MHC and the human reproductive tract microbiota remains unknown.

Here we investigated the association between HLA profiles and follicular fluid microbiota in women. First, because individuals with higher MHC diversity are expected to recognize a broader range of antigens [START_REF] Pierini | Divergent Allele Advantage at Human MHC Genes: Signatures of Past and Ongoing Selection[END_REF], we expected microbiota diversity to decrease with MHC diversity, as shown in human gut, fish gut and bird feathers (Bolnick et al. 2014b;[START_REF] Leclaire | Plumage microbiota covaries with the major histocompatibility complex in blue petrels[END_REF]Andeweg et al. 2021a). Second, because two individuals with the same HLA genotypes are hypothesized to have similar immune control of their microbiota, we expected similarity in microbiota composition between individuals to covary with HLA similarity.

Material and methods

Sample collection

The participants are 27 women, with mean age 31.7 ± 2.7 s.e. years, who underwent transvaginal follicular aspiration for in vitro fertilization. Among these women, 3 were sampled twice at several months interval. Women were recruited via three fertility clinics: North Karelia Central Hospital (Joensuu,Finland,n = 20), ISLAB (Kuopio, Finland, n = 4) and InOva (Kuopio, Finland, n = 10) between April 2017 and December 2020. All the participants were Caucasian except one who was Asian.

However, since the inclusion of this Asian woman did not affect our results, we included this sample in our final dataset. All results remained similar if we exclude the Asian woman from the analysis. Thus, we report results including her. Transvaginal follicular punctures were performed under local anesthesia, using ultrasound guidance. Prior to follicular fluid collection, follicle maturation was hyperstimulated with follicle-stimulating hormone, and premature ovulation was prevented using a gonadotrophin-releasing hormone antagonist. When the diameter of the largest follicle reached 18-20 mm, human corion gonadotrophin was administered. After collection, the follicular fluid was centrifuged at 500 × g for 10 min. The supernatant was aliquoted and stored in liquid nitrogen for later use. Nine of the women had been diagnosed with female factor infertility (i.e., anovulation, polycystic ovarian syndrome, or endometriosis; n = 9 women), whereas 18 women were not diagnosed with female factor infertility problem (e.g., unexplained couple infertility or pair infertility due to the man; n = 18 women). All the women donated a blood sample for genetic analyses.

Because the follicular fluid is obtained transvaginally, and it is thus prone to bacterial contamination from the lower genital tract, we collected additional follicular fluid samples and vaginal swabs from four women. Before follicular fluid collection, a sterile cotton swab (FLOQSwabs, COPAN, California) was inserted in the vagina to collect mucus from the area of the transvaginal puncture.

Swabs were stored in sterile 5 ml cryotubes.

Microbiota analyses

To characterize the bacterial community present in each sample, we performed 16S rRNA amplicon high-throughput sequencing. PCR amplification, Miseq library preparation and amplicon sequencing were performed at Genome Quebec, Montréal, Canada. Staggered primers 515F [START_REF] Caporaso | Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample[END_REF] and 806R [START_REF] Caporaso | Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample[END_REF] were used to amplify the variable region V4 of 16S rRNA gene from bacteria. We included four DNA extraction blank controls and three PCR blank controls to detect and withdraw potential contaminants. Follicular fluid DNA was extracted in five different batches. To control for batch effect, one sample was repeatedly extracted in each batch. In addition, to control for repeatability in extraction, PCR and sequencing, DNA extraction was repeated twice for six follicular fluid samples.

We first analysed the sequence reads using the OBITools package (Boyer et al., 2016). Briefly, after assembly of paired-end reads, we assigned reads to their respective samples and excluded reads with low assembly scores or reads that contained ambiguous bases (i.e., "N"). We dereplicated strictly identical reads using the "obiuniq" algorithm (Boyer et al., 2016). We then analysed the sequences using the FROGS 4.1 pipeline (Escudié et al., 2018). Briefly, ASVs were formed using the swarm denoising method with the fastidious method with d = 1 [START_REF] Mahé | Swarm v3: towards tera-scale amplicon clustering[END_REF]. Chimeras were removed using VSEARCH (Rognes et al., 2016). Taxonomic assignment was then performed using blast affiliation and the SILVA v138 database (Quast et al., 2012). ASVs sharing same taxonomy with at least 99% identity and 99% alignment coverage were aggregated together. We removed ASVs that were taxonomically classified as mitochondria, chloroplasta or Archaea. We also removed ASVs that had fewer than 10 reads (n = 50 ASVs) or that were detected in a single sample (n = 22 ASVs). Finally, we used the isContaminant function with the prevalence option (package decontam in R) to remove potential contaminants from the data set [START_REF] Davis | Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data[END_REF]. 14 ASVs were found to be contaminants.

They belonged to the genus Escherichia-Shigella or the family Burkholderiaceae. We found that the number of reads of the remaining OTUs belonging to Escherichia-Shigella were similar in follicular fluid samples and extraction controls (t-test: t13 = -1.5, P = 0.10). It has been suggested that reads attributed to Escherichia-Shigella may represent remnant DNA from the Escherichia coli used to produce Taq DNA polymerase [START_REF] Stinson | Identification and removal of contaminating microbial DNA from PCR reagents: impact on low-biomass microbiome analyses[END_REF]. Thus, we decided to exclude all ASVs belonging to Escherichia-Shigella (n = 5 additional ASVs). In addition, using the one sample that was extracted at least once in each extraction batch, we observed high variation in the relative abundance of Acinetobacter between replicates (Fig. S1). When considering all follicular fluid samples, the relative abundance of Acinetobacter was found to vary with the DNA extraction batch (χ 2 4 = 29, P < 0.00001; Fig. S1).

Acinetobacter are frequent cleanroom contaminants because they resist sterilization procedures and disinfectants [START_REF] Ridgway | Chlorine resistance patterns of bacteria from two drinking water distribution systems[END_REF][START_REF] Hendrickson | Clean room microbiome complexity impacts planetary protection bioburden[END_REF]. In a conservative way, we decided therefore to exclude all OTUs belonging to Acinetobacter from the analyses (n = 53 ASVs). We obtained mean ± SE per follicular fluid sample: 51 518 ± 11 512 sequences (median: 20 388 sequences; range:

1 480 -255 176 sequences). DNA extraction replicates were repeatable (Permanova: F6,10 = 5.35, r² = 0.76, P < 0.001; Fig. S2) and were therefore averaged.

To analyse α-diversity of follicular fluid microbiota, we used the weighed phylogenetic diversity (Faith's PD) based on the nonrooted phylogenetic tree (function "pd" in package picante in R). Faith's PD is the sum of total phylogenetic length of OTUs in each sample. Prior calculating Faith's diversity, we rarefied the dataset to the smallest library size (i.e., 1480 sequences). While there have been considerable debates concerning rarefaction in microbiome datasets (McMurdie and Holmes 2014; [START_REF] Weiss | Normalization and microbial differential abundance strategies depend upon data characteristics[END_REF][START_REF] Cameron | Enhancing diversity analysis by repeatedly rarefying next generation sequencing data describing microbial communities[END_REF], the wide variability in our library sizes, ranging from 1 480 to 255 176 sequences in follicular fluid samples, justifies our decision to rarefy. However, because rarefaction can result in a substantial loss of reads (McMurdie and Holmes 2014) and can introduce a stochastic component to the analysis because the rarefied taxa count table depends on the specific rarefaction replicate, we used multiple rarefactions, which has been proposed to recover some of the information lost through a single rarefaction [START_REF] Cameron | Enhancing diversity analysis by repeatedly rarefying next generation sequencing data describing microbial communities[END_REF]).

To analyse β-diversity, we used the phylogenetic isometric log-ratio transform (PhiLR) which allows analysis of compositional data where the parts can be related through a phylogenetic tree [START_REF] Silverman | A phylogenetic transform enhances analysis of compositional microbiota data[END_REF]. PhiLR has been suggested to be a replacement for the weighted UniFrac distances when accounting for the compositional nature of the data is needed (Gloor et al., 2017). OTU sequences were aligned using Decipher (Wright, 2015). Then a maximum-likelihood tree was constructed using the package phangorn (Schliep, 2011). This tree was used as a reference from which the Euclidean distances were calculated on phylogenetic isometric log-ratio transformed OTU abundance (i.e., the PhILR transform) between samples (Egozcue et al., 2003;Gloor et al., 2017) using the package PhiLR (Silverman et al.,2 017). In the PhiLR transform, we added a pseudocount of 0.1 reads. Rarefaction is unnecessary when using PhILR transformation [START_REF] Silverman | A phylogenetic transform enhances analysis of compositional microbiota data[END_REF].

HLA genotyping

DNA of all the women was extracted from EDTA blood using automatized Qiasymphony SP instrument (Qiagen) or PureLink® Genomic DNA Kit (Invitrogen). Extracted DNA samples were genotyped on Illumina GlobalScreeningArray-24v2-0 + Multi-Disease beadchip at the Institute for Molecular Medicine Finland (FIMM). All genotypes were named with GenomeStudio 2.0.3 software, and a subset was checked manually based on pre-determined selection criteria such as low call rates, bad cluster separation, low signal intensity, quality scores, and heterozygote excess. The parameters used in the quality control of X chromosomal and autosomal markers were: call rate < 97%; cluster separation < 0.3; AB R Mean (low intensity SNPs) < 0.3; AB T Mean (SNPs where two clusters are close to each other) < 0.2 or >0.8; heterozygote excess < -0.3 (not X chromosome) or >0.2; AA or BB T Dev > 0.05; and AB T Dev (too many or CNV like clusters) > 0.07. PLINK v1.90b6.6 [START_REF] Chang | Second-generation PLINK: rising to the challenge of larger and richer datasets[END_REF] (www.cog-genomics.org/plink/1.9/) was used to manage and filter the genotyping data. For genotyping quality control, the following settings were applied: missing genotype frequency (geno) < 0.05, P-value for deviation from Hardy-Weinberg equilibrium > 1e- 5; minor allele frequency > 0.01; individual missing genotype rate (mind) < 0.05. Imputation of seven classical HLA genes (HLA-A, -B, -C, -DRB1, -DQA1, -DQB1, and -DPB1) at four-digit (i.e., protein level) resolution was carried out in R v3.4.4 (R Core Team 2019), using the package HIBAG v1.18.1 [START_REF] Zheng | HIBAG-HLA genotype imputation with attribute bagging[END_REF] with default settings and the Finnish reference panel [START_REF] Ritari | Increasing accuracy of HLA imputation by a population-specific reference panel in a FinnGen biobank cohort[END_REF]) (https://github.com/FRCBS/HLA-imputation) for the human genome build GRCh37. The one Asian individual was imputed as described above except using the HIBAG Asian refence panel (https://hibag.s3.amazonaws.com/download/hlares_param/Asian-HLA4.html) instead of the Finnish panel.

HLA diversity of the women was calculated as (1) the number of heterozygous sites and (2) the mean Sandberg divergence over the seven imputed HLA genes per women. The Sandberg divergence is the pairwise Euclidean distance between the two protein sequences represented by the five physicochemical z-descriptors [START_REF] Sandberg | New chemical descriptors relevant for the design of biologically active peptides. A multivariate characterization of 87 amino acids[END_REF]. For HLA genes, the Sandberg distance has been found to correlate with the total number of peptides bound by the alleles [START_REF] Pierini | Divergent Allele Advantage at Human MHC Genes: Signatures of Past and Ongoing Selection[END_REF]. The Sandberg divergence at each HLA gene was calculated using the DistCalc function in the MHCTools package in R [START_REF] Roved | MHCtools-an R package for MHC high-throughput sequencing data: Genotyping, haplotype and supertype inference, and downstream genetic analyses in non-model organisms[END_REF]. Number of heterozygous sites and Sandberg divergence were correlated (Spearman correlation test: S = 837, r = 0.74, P < 0.0001). Dissimilarity between each women pair was estimated (1) on a pure shared/non-shared allele basis and (2) by mean the Sandberg distance between HLA alleles. HLA dissimilarity based on a pure shared/non-shared allele basis (hereafter called allelic dissimilarity) was calculated as 100*(1-(the number of shared alleles / the total number of alleles)). For each HLA gene, Sandberg distance was calculated by constructing a UPGMA dendrogram of alleles (function hclust in R), which represents clusters of functionally similar HLA sequences. Then HLA distance between two women was estimated using the tree distance (function treedist in package vegan), which is found by combining species in a common dendrogram and seeing how much of the dendrogram height is shared and how much is unique. The allelic dissimilarity was correlated to the Sandberg dissimilarity (Mantel test: r = 0.91, P = 0.001).

Whole genome diversity and similarity analysis

The whole-genome similarity measure between each woman-woman combination was calculated as the total sum of the number of shared genotypes for all genotyped and quality-filtered bi-allelic single nucleotide polymorphisms. In other words, genotyped variants were averaged in a single value, which prevented any single variants from dominating the genome-wide similarity estimate, which in turn can be expected to increase the accuracy of the overall estimate. Genome wide heterozygosity was not correlated with HLA diversity (Spearman rank correlation test: S = 2514, r = 0.23, P = 0.24 for the number of heterozygous HLA sites, and S = 2194, r = 0.33, P = 0.09 for the Sandberg divergence, n = 27 women). In contrast, whole genome dissimilarity was positively associated with HLA dissimilarity (Mantel test: r = 0.30, P = 0.002 for allelic dissimilarity and r = 0.24, P = 0.024 for Sandberg distance).

Statistical analyses

To determine whether HLA diversity was associated with microbiome α-diversity, we used linear mixed models. The DNA extraction batch was included as a random variable. For each analysis, 500 tests were run. Each test corresponded to a new rarefaction round [START_REF] Cameron | Enhancing diversity analysis by repeatedly rarefying next generation sequencing data describing microbial communities[END_REF]). In addition, for each of the three women who were sampled twice, only one of their two samples was randomly sampled in each rarefaction round to avoid pseudoreplication. In the statistical section, we reported the mean of the statistic parameters over the 500 tests. The DNA extraction batch was not included as a covariate, because it had no effect on the microbiome diversity (F3,23 = 0.91, P = 0.46). We characterized the bacterial species preferentially associated with variations in HLA diversity using ANCOM-BC on the nonrarefied dataset (ancombc2 function in the ANCOM package) (Lin and Peddada 2020).

To determine whether women who were more similar at HLA sites had more similar microbiome composition, we used partial Mantel test. A binomial matrix of DNA extraction batch similarity (0: the two samples were extracted in the same batch and 1: the two samples were not extracted in the same batch) was included as a covariate, because women whose DNA was extracted in the same extraction batch had more similar microbiome composition (mantel test: r = 0.14, P = 0.016). To avoid pseudoreplication, for each analysis, we ran 8 different tests, each test including a unique combination of one sample of the females sampled twice. In the statistical section, we reported the mean of the statistic parameters over the 8 tests.

We performed all statistical analyses using the R statistical software (R Core Team, 2020).

Results

Characterization of the follicular fluid microbiome

Considering the 34 follicular fluid samples, the most abundant species belonged to the genus Lactobacillus and included Lactobacillus iners, Lactobacillus jensenii, Lactobacillus acidophilus / L. crispatus, Lactobacillus gasseri / L. johnsonii. Other abundant species were Gardnerella vaginalis, Sneathia amnii, Prevotella sp, Atopobium vaginae, Kocuria sp. and Alishewanella sp. (Fig. S3). ANCOM-BC analyses at the species level showed that follicular fluid microbiomes with higher Faith's diversity were characterized by higher abundance of Gardnerella vaginalis, Atopobium vaginae, Sneathia amnii, Prevotella sp, Megasphaera_sp and lower abundance of Lactobacillus jensenii, Lactobacillus acidophilus / L. crispatus and Lactobacillus gasseri / L. johnsonii (Fig. S3). The composition and αdiversity of follicular fluid microbiome were not affected by the female factor infertility diagnoses (permanova: F1,22 = 0.58, r² = 0.02, P = 0.87 and Wilcoxon rank sum test: W = 61, P = 0.33, respectively).

When focusing on the four women for whose we had both follicular fluid and vaginal swab samples, we found no differences in the composition or diversity of microbiome between sample types (PERMANOVA: F1,7 = 613, r² = 0.09 P = 0.77 and paired t-test: t3 = 0.58, P = 0.60, respectively ; Fig. S4).

When investigating the OTUs that were present in follicular fluid samples but absent in vaginal swabs, we found however that OTUs belonging to the genera Alishewanella, Lawsonella and Kocuria were absent from all vaginal swab samples but were present in all (100%), 3 (75%) or 1 (25%) follicular fluid samples respectively, albeit in low relative abundance (0.26 ± 0.19 %, 0.34 ± 0.07 % and 0.54 % respectively, when considering the samples where the OTUs were present). When considering the 30 follicular fluid samples for which we have HLA genotype, we found that Alishewanella and Lawsonella were present in all samples, and Kocuria in all except one sample, and represented (5.01 ± 1.4 %, 1.33 ± 0.39 % and 1.99 ± 0.95 %, respectively).

Microbiome α-diversity and HLA diversity

Women who had a higher heterozygosity or Sandberg divergence at HLA genes had a higher α-diversity in follicular fluid microbiome (χ²24 = 8.9, P = 0.007 and χ²24 = 9.93, P = 0.005; Fig. S5). This relationship was also significant when considering HLA class I genes only (χ²24 = 11.53, P = 0.003 and χ²24 = 4.97, P = 0.038; Fig. 2). In contrast, when considering HLA class II genes only, the association between HLA diversity and microbiome diversity was significant only for Sandberg divergence (heterozygosity: χ²24 = 3.73, P = 0.072 and Sandberg divergence: χ²24 = 7.95, P = 0.011; Fig. S5). ANCOM-BC analyses carried out at the bacterial species level showed that women who were heterozygous at the three class I genes had lower relative abundance of Lactobacillus jensenii and Lactobacillus acidophilus / L. crispatus, but higher relative abundance of Sneathia amnii, Atopobium vaginae, Megasphaera sp and Prevotella bivia than women who had at least one homozygous class I genes (Fig. 3). There was no association between follicular fluid microbiome α-diversity and genome-wide heterozygosity (χ²24 = 0.01, P = 0.94; Fig. S6).

Microbiome similarity and HLA similarity

Women who were more distant at HLA genes did not have more dissimilar follicular fluid microbiome (allelic distance: r = 0.08, P = 0.22 and Sandberg distance: r = 0.07, P = 0.22; Fig. S7). However, when we focused on the women with no female factor infertility (n = 18 women; 9 women excluded), we found that women with more distant HLA genes had more dissimilar follicular fluid microbiome (allelic distance: r = 0.34, P = 0.0058 and Sandberg distance: r = 0.33, P = 0.0079; Fig. 4). This effect was significant for both HLA Class I genes (r = 0.30, P = 0.022 and r = 0.25, P = 0.043 for allelic and Sandberg distance respectively) and HLA class II genes (r = 0.27, P = 0.012 and r = 0.30, P = 0.0081 for allelic and Sandberg distance respectively). Women with a higher genome-wide similarity did not have more similar follicular fluid microbiome (r = 0.09, P = 0.36 when considering all women, and r = -0.02, P = 0.61 when focusing on the women with no female factor infertility).

Discussion

Using high-throughput sequencing of 16S rRNA genes, we characterized the follicular fluid microbiome of 27 Finnish women visiting infertility clinics. The most abundant OTUs detected in follicular fluid belonged to the genera Lactobacillus, Gardnerella, Prevotella, Atopobium and Sneathia. This result contrasts with the few other studies that characterized the follicular fluid microbiota of women. However, these studies showed no consensus on the core microbial composition. For instance, a study using 16S sequencing found that the follicular fluid microbiome of infertile women was dominated by Achromobacter, and that Lactobacillus was not the dominant genus [START_REF] Wu | Microbiological composition of follicular fluid in patients undergoing IVF and its association with infertility[END_REF]. Achromobacter is an environmental contaminant widely distributed in soils and waters and commonly detected in hospitals [START_REF] Amoureux | Detection of Achromobacter xylosoxidans in hospital, domestic, and outdoor environmental samples and comparison with human clinical isolates[END_REF]. Other studies using cultivation methods detected Lactobacillus, but also Actinomyces, Propionobacterium, Bifidobacterium, Staphylococcus, Streptococcus and Enterococcus (Pelzer, Allan, et al. 2013;[START_REF] Usman | The presence of microorganisms in follicular fluid and its effect on the outcome of in vitro fertilization-embryo transfer (IVF-ET) treatment cycles[END_REF][START_REF] Kim | Microbiology of Human Follicular Fluid and the Vagina and Its Impact on in Vitro Fertilization Outcomes[END_REF], which are normal bacteria of the skin, the oral mucosa and the gastrointestinal tract [START_REF] Stackebrandt | Family propionibacteriaceae: the genus Propionibacterium[END_REF]. Recently, several studies have suggested that low-biomass microbial niches, such as the human fetus, the placenta or the endometrium, are sensitive to contamination and data misinterpretation [START_REF] Olomu | Elimination of "kitome" and "splashome" contamination results in lack of detection of a unique placental microbiome[END_REF][START_REF] Molina | Analysing endometrial microbiome: methodological considerations and recommendations for good practice[END_REF][START_REF] Kennedy | Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies[END_REF]). The follicular fluid, like other sites of the upper reproductive tract, have low microbial biomass, and clearly, the original source of the bacteria detected in our study and others can be questioned. The major bacteria that we detected in follicular fluid (e.g., Lactobacillus, Gardnerella, Prevotella, Atopobium and Sneathia) are common inhabitants of the woman vagina [START_REF] Gajer | Temporal Dynamics of the Human Vaginal Microbiota[END_REF][START_REF] Tabatabaei | Vaginal microbiome in early pregnancy and subsequent risk of spontaneous preterm birth: a case-control study[END_REF][START_REF] France | Towards a deeper understanding of the vaginal microbiota[END_REF]), and we did not detect differences between the composition of follicular fluid microbiome and vaginal microbiome in the four women who were sampled at both sites. We cannot exclude therefore that the follicular fluid has been contaminated with vaginal bacterial strains at the time of puncture for transvaginal oocyte aspiration.

For instance, Lactobacillus abundance is known to be increased in samples collected transcervically or transvaginally compared to samples collected using hysterectomy, laparoscopy or cesarean section [START_REF] Winters | Does the endometrial cavity have a molecular microbial signature?[END_REF][START_REF] Canha-Gouveia | The female upper reproductive tract harbors endogenous microbial profiles[END_REF]). However, we observed that Alishewanella, Lawsonella and Kocuria were present in follicular fluid samples, but in none of the vaginal swab samples. These genera are thus not contaminants from the vagina, and might be true inhabitants of follicular fluid. Similarly, a previous culture-based study detected Kocuria in follicular fluid, but not in vagina [START_REF] Kim | Microbiology of Human Follicular Fluid and the Vagina and Its Impact on in Vitro Fertilization Outcomes[END_REF], and another study found that Kocuria were increased in endometrium compared to endocervix (Pelzer, Willner, Buttini, and Huygens 2018). Alishewanella and Lawsonella have previously been detected in the women reproductive tract, including the vagina and the endometrium [START_REF] Hong | Analysis of the vaginal microbiome by next-generation sequencing and evaluation of its performance as a clinical diagnostic tool in vaginitis[END_REF][START_REF] Hernandes | Microbiome Profile of Deep Endometriosis Patients: Comparison of Vaginal Fluid, Endometrium and Lesion[END_REF][START_REF] Kuznetsova | VAGINAL MICROBIOME IN PREGNANT WOMEN WITH PRETERM PRELABOR RUPTURE OF MEMBRANES AT 22-28 WEEKS' GESTATION[END_REF][START_REF] Liu | Genital microbiota of women from six ethnic groups with and without human papillomavirus infection in shangri-la, China[END_REF]).

However, clearly, bacterial contamination is a big challenge in the studies of follicular fluid microbiome.

Whatever the true origin of the microbiome studied (vaginal vs. follicular fluid), we report the first evidence that the women reproductive microbiome varied with HLA diversity (heterozygosity and divergence). We found that women with higher HLA diversity had a higher α-diversity in follicular fluid microbiome. This result contrasts with the widespread hypothesis that a higher diversity of MHC molecules should lead to a larger number of foreign peptides recognized and thus to a larger number of bacterial species eliminated. Accordingly, individuals with higher HLA class II genes diversity have a broader antibody repertoire against common microbial pathogens ( Khan et al. 2022a), and microbiome diversity is negatively associated with MHC diversity in the human gut (Andeweg et al. 2021b), fish gut (Bolnick et al. 2014a) and bird feathers [START_REF] Leclaire | Plumage microbiota covaries with the major histocompatibility complex in blue petrels[END_REF]. However, MHC molecules may also promote benign host-bacteria interactions [START_REF] Roland | How MHCII signaling promotes benign hostmicrobiota interactions[END_REF]. For instance, in the gut of mice, the presence of MHC-II on B-cells promote the proliferation of beneficial bacteria and increased microbiota diversity [START_REF] Alexeev | B-Cell-Intrinsic MHCII Signaling Shapes Microbiota Composition[END_REF]. Therefore, a positive association between MHC and microbiome diversity may also arise if a higher diversity of MHC molecules facilitate the proliferation of a more diverse range of symbiotic microbes [START_REF] Schubert | How can the MHC mediate social odor via the microbiota community? A deep dive into mechanisms[END_REF]. Similar to our results, microbiota diversity increased with MHC diversity in the skin of two amphibians (Hernández- [START_REF] Hernández-Gómez | Influence of immunogenetics, sex and body condition on the cutaneous microbial communities of two giant salamanders[END_REF][START_REF] Belasen | Geography, Host Genetics, and Cross-Domain Microbial Networks Structure the Skin Microbiota of Fragmented Brazilian Atlantic Forest Frog Populations[END_REF].

We found that women with higher HLA diversity have higher abundance of Sneathia Prevotella bivia, Atopobium vaginae, and Megasphaera and lower abundance of Lactobacillus jensenii and Lactobacillus acidophilus / L. crispatus. In the vagina, this highly diverse microbial composition, described as "CST-IV", is commonly associated with bacterial vaginosis (reviewed in Smith and Ravel 2017), a disorder often causing vaginal discharge and foul-smelling odor. Bacterial vaginosis is associated with sexually transmitted infections and adverse reproductive outcome, including pre-term birth, low birthweight and miscarriage (reviewed in [START_REF] Chen | The Female Vaginal Microbiome in Health and Bacterial Vaginosis[END_REF]. Bacterial vaginosis plays also a role in the acquisition of human papillomavirus and the development of associated cervical neoplasia [START_REF] Gillet | Association between bacterial vaginosis and cervical intraepithelial neoplasia: systematic review and meta-analysis[END_REF]. Interestingly, the positive association between HLA diversity and a CST-IV microbiome, might therefore explain the surprising positive association between HLA diversity and increased risk of cervical cancer [START_REF] Wang | Association of HLA diversity with the risk of 25 cancers in the UK Biobank[END_REF].

Although high reproductive microbiome diversity is considered deleterious for fitness, high HLA diversity I conversely expected to be beneficial for fitness, because it confers enhanced resistance to several pathogens [START_REF] Planelles | HLA class II polymorphisms in Spanish melanoma patients: homozygosity for HLA-DQA1 locus can be a potential melanoma risk factor[END_REF]Khan et al. 2022b) and reduces risks of several cancers [START_REF] Wang | Association of HLA diversity with the risk of 25 cancers in the UK Biobank[END_REF]. The positive association between HLA diversity and follicular fluid microbiome diversity in women raises therefore a potential conflict between the fitness benefits of a highly diverse HLA and of a poorly diverse reproductive microbiome. However, the effect of the CST-IV microbial state on reproduction is mixed and, in some women, this microbial state is asymptomatic [START_REF] David A Eschenbach | Diagnosis and clinical manifestations of bacterial vaginosis[END_REF]. Some studies suggest that CST-IV state increases the risk of adverse outcome only in interaction with a specific genotype (i.e., gene-environment interaction). For instance, a study showed that, bacterial vaginosis per se is not associated with an increased risk of spontaneous preterm delivery, but its association with susceptible genotypes at several immune genes (i.e., interleukin 6, protein kinase C alpha and fms-like tyrosine kinase 1) increases the risk [START_REF] Gómez | Evidence of a gene-environment interaction that predisposes to spontaneous preterm birth: a role for asymptomatic bacterial vaginosis and DNA variants in genes that control the inflammatory response[END_REF]). We suggest that further studies should investigate whether the negative outcome associated with a CST-IV microbial communities varies with HLA diversity. For instance, HLA-B heterozygotes have higher tolerance to HIV, with high viral load but minimal disease progression [START_REF] Regoes | Disentangling human tolerance and resistance against HIV[END_REF]. The potential evolutionary conflict between HLA diversity and reproductive microbiota diversity might be solved if HLA diversity confers higher tolerance to bacterial vaginosis.

We also found that women who are more similar at HLA genes host more similar follicular fluid microbiome. Similarly, humans with similar HLA genes have similar gut microbiota (Andeweg et al. 2021a). However, the association between similarity at HLA gene and similarity in microbiome composition was significant only for women who had no female factor infertility. Although we found no differences in the microbiome composition of women with female factor infertility and that of healthy women, previous studies have detected that polycystic ovary syndrome or endometriosis alters the reproductive microbiota [START_REF] Hong | Association between polycystic ovary syndrome and the vaginal microbiome: A case-control study[END_REF][START_REF] Jiang | Intricate Connections between the Microbiota and Endometriosis[END_REF]. Our result may suggest that, compared to HLA genes, other factors might be stronger determinants of the reproductive microbiome of women with female factor infertility.

In conclusion, our study reveals that the HLA is an important genetic factor driving differences in reproductive microbiotas between women. Unraveling whether the HLA effects on microbiota drive variations in reproductive outcome would be an important next step.

Figure 1 :

 1 Figure 1: Relative abundance of the most abundant bacterial species in follicular fluid. Each bar corresponds to one of the 27 women sampled.

Figure 2 :

 2 Figure 2: Microbiota diversity (Faith's PD) in follicular fluid according to (a) the number of heterozygous HLA Class I sites and (b) the Sandberg divergence in HLA Class I sites. The red, green and blue dots correspond to the three women sampled twice. Only one of their two samples was considered in the statistical analyses during each of the 5000 rarefaction rounds. For (a), similar results were found when pooling all the women with at least one homozygous site. The black line and grey shadow in (b) represent the linear trend and confidence interval.

Figure 3 :

 3 Figure 3: Results of ANCOMBC analyses, as shown by the log fold change of the 6 bacterial species thatwere differently abundant between women who were heterozygous at the three HLA class I genes and women who were heterozygous at < 3 HLA class I genes.

Figure 4 :

 4 Figure 4 : Dissimilarity in microbiome composition in women pairs with no infertility diagnoses according to their dissimilarity in HLA class I and II genes. Dissimilarity in HLA genes was estimated (a) on a pure shared-non shared allele basis and (b) using Sandberg dissimilarity. The black line and grey shadow represent the linear trend and confidence interval. The dot at 0 HLA dissimilarity corresponds to two women who had the same HLA genotype.

Acknowledgements

We thank Ulrica Candolin for hosting SL during her stay at the University of Helsinki. This study was funded by grants from the Fondation Fyssen (to SL) and Academy of Finland (grant 308485 to JK).