Julie Dechartres

Jodi L Pawluski

Marie-Madeleine Gueguen

Amin Jablaoui

Emmanuelle Maguin

Moez Rhimi

Thierry D Charlier
email: thierry.charlier@univ-rennes1.fr

Glyphosate and glyphosate-based herbicide exposure during the peripartum period affects maternal brain plasticity, maternal behaviour and microbiome

Keywords: Neurogenesis, Neuroplasticity, Hippocampus, Cingulate gyrus, Gut microbiota Accepted Article

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

Glyphosate (N-phosphonomethyl-glycine) is the active compound found in over 750 broad-spectrum herbicides and is widely used across the world [START_REF] Myers | Concerns over use of glyphosate-based herbicides and risks associated with exposures: a consensus statement[END_REF] . In less than 20 years, Glyphosate use has significantly increased, from around 50,000 tons of bioactive compound sold in 1994 to over 825,000 tons in 2014, mostly due to the development of Glyphosateresistant genetically modified corn and soy and pre-harvest application techniques [START_REF] Myers | Concerns over use of glyphosate-based herbicides and risks associated with exposures: a consensus statement[END_REF][START_REF] Benbrook | Trends in glyphosate herbicide use in the United States and globally[END_REF] .

Glyphosate inhibits the 5-enolpyruvyl shikimate-3-phophate synthase (EPSPS), a key enzyme in the shikimate pathway [START_REF] Herrmann | The Shikimate Pathway[END_REF][START_REF] Steinrücken | The herbicide glyphosate is a potent inhibitor of 5enolpyruvylshikimic acid-3-phosphate synthase[END_REF] . This metabolic pathway is required for the biosynthesis of aromatic amino acids (tryptophan, tyrosine and phenylalanine) in plants, as well as in bacteria and fungi. Because this pathway is absent in vertebrates, Glyphosate was considered safe for animals and humans [START_REF] Herrmann | The Shikimate Pathway[END_REF][START_REF] Williams | Safety Evaluation and Risk Assessment of the Herbicide Roundup and Its Active Ingredient, Glyphosate, for Humans[END_REF] . However, a few studies link Glyphosate-based herbicide (GBH) exposure to some cancers in human [START_REF] Myers | Concerns over use of glyphosate-based herbicides and risks associated with exposures: a consensus statement[END_REF][START_REF] Guyton | Carcinogenicity of tetrachlorvinphos, parathion, malathion, diazinon, and glyphosate[END_REF] while others do not find any association [START_REF] Andreotti | Glyphosate Use and Cancer Incidence in the Agricultural Health Study[END_REF][START_REF] De Roos | Cancer incidence among glyphosate-exposed pesticide applicators in the Agricultural Health Study[END_REF][START_REF] Lee | Agricultural pesticide use and adenocarcinomas of the stomach and oesophagus[END_REF][START_REF] Mink | Epidemiologic studies of glyphosate and cancer: A review[END_REF] , making risk assessment difficult for regulatory agencies. Indeed in 2015, Glyphosate was classified as "probably carcinogenic for humans" (Group 2A) by the International Agency for Research on Cancer [START_REF]Monographs, volume 112: some organophosphate insecticides and herbicides: tetrachlorvinphos, parathion, malathion, diazinon and glyphosate. Iarc working group[END_REF] while the European Food Safety Authority [START_REF] Food | Conclusion on the peer review of the pesticide risk assessment of the active substance glyphosate[END_REF] and the European chemical agency [START_REF]Opinion proposing harmonised classification and labelling at EU level of glyphosate[END_REF] concluded that Glyphosate is not carcinogenic, mutagen or genotoxic for reproduction. The debate on the safety of Glyphosate and Glyphosate-based herbicide in term of carcinogenicity is currently open [START_REF] Portier | Glyphosate toxicity and carcinogenicity: a review of the scientific basis of the European Union assessment and its differences with IARC[END_REF][START_REF] Portier | glyphosate toxicity and carcinogenicity: a review of the scientific basis of the European Union assessment and its differences with IARC[END_REF][START_REF] Tarazona | Glyphosate toxicity and carcinogenicity: a review of the scientific basis of the European Union assessment and its differences with IARC[END_REF][START_REF] Tarazona | concerning our review 'Glyphosate toxicity and carcinogenicity: a review of the scientific basis of the European Union assessment and its differences with IARC[END_REF] .

More recently, an increasing number of studies suggest that GBH exposure might affect vertebrate development and physiology [START_REF] Dallegrave | Pre-and postnatal toxicity of the commercial glyphosate formulation in Wistar rats[END_REF][START_REF] Paganelli | Glyphosate-based herbicides produce teratogenic effects on vertebrates by impairing retinoic acid signaling[END_REF][START_REF] Zhang | Biological impacts of glyphosate on morphology, embryo biomechanics and larval behavior in zebrafish (Danio rerio)[END_REF] . More precisely, several laboratories found that Glyphosate exposure could alter brain physiology and behavior, independently of its potential carcinogenic and mutagenic properties. Indeed, GBH induced alteration in neurotransmitter dopamine, norepinephrine, serotonin and glutamate levels and their metabolites in several brain regions such as the hippocampus, substantia nigra and prefrontal cortex in adult male rats [START_REF] Cattani | Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity[END_REF][START_REF] Martínez | Neurotransmitter changes in rat brain regions following glyphosate exposure[END_REF] as well as neurotransmitter binding to their cognate receptor [START_REF] Hernández-Plata | The herbicide glyphosate causes behavioral changes and alterations in dopaminergic markers in male Sprague-Dawley rat[END_REF] .

Accepted Article

Consequently, these alterations in neurotransmission are likely to be the cause of deficits in locomotor activity and depressive-and anxiety-like symptoms observed in male mice and male and female rats following Glyphosate exposure [START_REF] Bali | Behavioral and Immunohistochemical Study of the Effects of Subchronic and Chronic Exposure to Glyphosate in Mice[END_REF][START_REF] Aitbali | Glyphosate based-herbicide exposure affects gut microbiota, anxiety and depression-like behaviors in mice[END_REF][START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF][START_REF] Gallegos | Exposure to a glyphosate-based herbicide during pregnancy and lactation induces neurobehavioral alterations in rat offspring[END_REF] . Interestingly, while several studies have investigated the effect of perinatal exposure to GBH via the exposure to gestating and lactating females, there is no information about the mother's behavior and brain plasticity and how the dam herself could be affected by the treatment. In general, the vast majority of studies focus on the effects of the chemical environment on neurodevelopmental outcomes during the perinatal period, because of the intense neuroplasticity in the developing brain.

However, the neural circuitry of adult females is also significantly remodeled during pregnancy and lactation to adapt her behavior to her offspring's needs and survival in a variable environment [START_REF] Bridges | Long-term alterations in neural and endocrine processes induced by motherhood in mammals[END_REF][START_REF] Kohl | The neurobiology of parenting: A neural circuit perspective[END_REF][START_REF] Lévy | Neuroendocrine control of maternal behavior in non-human and human mammals[END_REF][START_REF] Slattery | The maternal brain under stress: Consequences for adaptive peripartum plasticity and its potential functional implications[END_REF] . Amongst the various brain regions remodeled around the peripartum period, including the medial preoptic nucleus, the ventral tegmental area and the bed nucleus of the stria terminalis, the hippocampus has received much attention because of its persistent high level of neuroplasticity, including adult neurogenesis and because of its sensitivity to social and chemical environmental factors [START_REF] Leuner | The birth of new neurons in the maternal brain: Hormonal regulation and functional implications[END_REF][START_REF] Pawluski | Neuroplasticity in the maternal hippocampus: Relation to cognition and effects of repeated stress[END_REF][START_REF] Pereira | Structural and Functional Plasticity in the Maternal Brain Circuitry[END_REF] . It should be added that the ventral and dorsal region of the hippocampus present physiological and functional differences, with the agreed consensus that the dorsal hippocampus is involved in spatial cognition and more cognitive performance and memory in general while the ventral part is involved in the regulation of emotions, including stress and anxiety [START_REF] Fanselow | Are the Dorsal and Ventral Hippocampus Functionally Distinct Structures?[END_REF][START_REF] Moser | Functional differentiation in the hippocampus[END_REF] . Previous data suggest the GBH exposure affects hippocampal physiology and plasticity [START_REF] Martínez | Neurotransmitter changes in rat brain regions following glyphosate exposure[END_REF][START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF][START_REF] Coullery | Neuronal development and axon growth are altered by glyphosate through a WNT non-canonical signaling pathway[END_REF][START_REF] Gallegos | Perinatal Glyphosate-Based Herbicide Exposure in Rats Alters Brain Antioxidant Status, Glutamate and Acetylcholine Metabolism and Affects Recognition Memory[END_REF][START_REF] Yu | Circular RNA expression profiles in hippocampus from mice with perinatal glyphosate exposure[END_REF] . To our knowledge, there is no data on how Glyphosate could affect maternal behavior and the associated neuroplasticity.

Accepted Article

. It should also be noted that most of these effects were observed with relatively high doses of GBH (over 50 mg/kg/day Glyphosate-equivalent) and only a handful of studies investigated the impact of exposure to low dose [START_REF] Lozano | Sex-dependent impact of Roundup on the rat gut microbiome[END_REF] . Probably as important, there is little work comparing the effects of Glyphosate alone and in GBH on neural and behavioral outcomes. This is important as a few studies suggest that some of the observed adverse effects of GBH are not triggered by Glyphosate itself but by some adjuvants, or "inert" molecules, found in the pesticide formulation, such as the polyethoxylated tallowamine [START_REF] Defarge | Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels[END_REF][START_REF] Mesnage | Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity[END_REF] .

Although it is hypothesized that the effects of Glyphosate in the brain might be linked to glutamate excitotoxicity and oxidative stress [START_REF] Cattani | Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity[END_REF][START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF][START_REF] Gallegos | Perinatal Glyphosate-Based Herbicide Exposure in Rats Alters Brain Antioxidant Status, Glutamate and Acetylcholine Metabolism and Affects Recognition Memory[END_REF][START_REF] Bonvallot | Metabolome disruption of pregnant rats and their offspring resulting from repeated exposure to a pesticide mixture representative of environmental contamination in Brittany[END_REF] , the molecular target(s) of Glyphosate in the brain is/are not well defined. As introduced above, Glyphosate is best known for its direct inhibitory effects on EPSPS. While this protein is mostly investigated in plants for its clear economical reason, the shikimate enzymatic pathway is also present in most bacteria. Interestingly, every vertebrate (and invertebrate) organism is the host for billions of microorganisms (mostly bacteria), collectively named microbiota. Modification of microbiota by chemical exposure (including antibiotic, diet, pre or probiotics…) is known to be associated to significant alterations of the organism's physiology, including defect in brain neurodevelopment and neuroplasticity [START_REF] Sherwin | Recent developments in understanding the role of the gut microbiota in brain health and disease[END_REF][START_REF] Tognini | Gut Microbiota: A Potential Regulator of Neurodevelopment[END_REF][START_REF] Wang | Progress in fish gastrointestinal microbiota research[END_REF] . Recently, a few studies showed that GBH exposure can affect gut microbiota, a process known as dysbiosis [START_REF] Lozano | Sex-dependent impact of Roundup on the rat gut microbiome[END_REF][START_REF] Ackermann | The Influence of Glyphosate on the Microbiota and Production of Botulinum Neurotoxin During Ruminal Fermentation[END_REF][START_REF] Motta | Glyphosate perturbs the gut microbiota of honey bees[END_REF][START_REF] Shehata | The Effect of Glyphosate on Potential Pathogens and Beneficial Members of Poultry Microbiota In Vitro[END_REF] and such an alteration could result in mental health disorders, including anxiety and depression [START_REF] Bali | Behavioral and Immunohistochemical Study of the Effects of Subchronic and Chronic Exposure to Glyphosate in Mice[END_REF][START_REF] Aitbali | Glyphosate based-herbicide exposure affects gut microbiota, anxiety and depression-like behaviors in mice[END_REF] .

Alternatively, or in addition, Glyphosate possess chelating properties and binds divalent cations (e.g. Mg 2+ , Ca 2+ , Fe 2+ , Mn 2+ , Cu 2+ , Zn 2+ ). This chelating effect in vivo is unclear and the outcome poorly investigated [START_REF] Mertens | Glyphosate, a chelating agent-relevant for ecological risk assessment?[END_REF][START_REF] Samsel | Glyphosate, pathways to modern diseases III: Manganese, neurological diseases, and associated pathologies[END_REF] . A reduction of these elements could lead to a significant alteration of most enzymatic cascades that usually require metallic cofactors but could also indirectly alter the microbiota.

Accepted Article

. Therefore, the aim of the present study was to compare the potential effects of relatively low dose (5 mg/kg/day, 1:10 th of the NOAEL, for "No Observable Adverse Effect Level") of Glyphosate and GBH on maternal behavior and maternal neuroplasticity, focusing on the hippocampus (dorsal and ventral) and cingulate gyrus. To measure changes in neurogenesis and cell structure, we focused on immunohistochemical techniques to investigate cell proliferation (Ki67-immunoreactivity(-ir)), immature neurons (doublecortinir), and pre-and post-synaptic protein densities (synaptophysin-ir and PSD95-ir) in the hippocampus. In the cingulate gyrus pre-and post-synaptic protein densities (synaptophysinir and PSD95-ir) were assessed. In addition, we also investigated signaling pathways that could be affected by Glyphosate exposure and lead to adverse neural outcomes. In parallel to brain and behavior analysis, we investigated whether Glyphosate alone or in formulation could affect the gut microbiota and/or modulate plasma concentration of several cations to affect neuroplasticity in the dam.

METHODS

Animals

Twenty-one adult pregnant (gestational day (GD) 8) Sprague-Dawley rats (239-340 g) were obtained from Janvier Laboratories, France, and singly housed upon arrival at our animal facility. They had access to rat chow and tap water ad libitum and were kept under standard laboratory conditions in a 12:12 light/dark cycle (light on at 07:00 am). All animal procedures were approved by the Ethics Committee of the French Ministry of Research (authorization number APAFlS#5294-2016050412068659 v3). The animal facility is licensed by the French Ministry of Agriculture (agreement D35-238-19). All efforts were made to minimize animal stress and pain.

Accepted Article

. Pregnant females were randomly assigned to 3 groups (n = 7/group): Control (vehicle: milliQ sterilized water), Glyphosate (5 mg/kg/day of glyphosate isopropylamine salt, Dr.

Ehrenstorfer GmbH, Germany) and Roundup® 3Plus (Roundup, 5 mg/kg/day glyphosate isopropylamine salt equivalent, S.A. Monsanto Europe L&G Dept., Belgium 170 g/L glyphosate acid, 229 g/L glyphosate isopropylamine salt) at GD9. Treatment was administered orally via biscuits [START_REF] Pawluski | Fluoxetine Dose and Administration Method Differentially Affect Hippocampal Plasticity in Adult Female Rats[END_REF] every morning between 8:30 am and 9:30 am from GD10 to the weaning of the pups at postpartum day (PD) 21. Females were fed 1/9 th of a vanilla wafer cookie (Crousti fondante, Delacre®, Belgium) injected with treatment or vehicle.

Injections were made into the vanilla portion of the wafer. Observations were made to ensure that the biscuit was completely ingested by the dam and not shared with the offspring.

Cookies were consistently eaten in their entirety but the dams. Two females had problems during parturition and were euthanized (1 Control, 1 Glyphosate). Weight was recorded on GD10, PD1 and PD22. Figure 1 provides a timeline of the study.

Maternal behavior

One day after birth (PD1), litter was weighed, sexed and culled to 10 pups per litter (balanced sex ratio). Between PD1 to PD6, maternal behavior was scored during 5 minutes twice a day, directly in the cage without disturbing the animals. Scoring took place in the morning (between 09:30 and 13:30) and in the afternoon (between 14:00 and 16:30) with at least 1 h between the two sessions. An observer blind to the treatment scored the time the dams spent licking the pups, arched-back nursing (ABN), passive nursing, blanket nursing time the dams spent off the nest and the time the dams spent building the nest (NB). Data is reported as percentage of time spent for each maternal behavior. We analyzed maternal behavior on the first day following parturition independently from the other days, because the litter was handled and characterized, causing a short (~15 minutes) maternal separation and

Accepted Article

. potentially affecting behavioral outcome. Therefore, maternal behavior was investigated separately on PD1 and aggregated across PD2-6 for additional analyses.

Microbiome

In addition to standard cage changing, the dams were transferred to new cages with clean bedding on GD9, GD19 and PD22, and feces collected with sterilized tweezer the following day and transferred to sterile 1.5 ml tubes for microbiota analysis. Analysis of the feces prior to treatment (GD9) and at the end of the study (PD22) was done. There were no differences between groups at GD9 and only differences at PD22 are reported. No samples were pooled. Samples from GD19 were not used.

16S rRNA amplicon generation for MiSeq sequencing

Accepted Article

. the run was checked internally using PhiX, and for further analysis, each pair-end sequence was assigned to its sample using the previously integrated index.

Bioinformatics analysis Sequences were trimmed for adaptors and assembled with Flash1.6.2 [START_REF] Magoč | FLASH: fast length adjustment of short reads to improve genome assemblies[END_REF] . PCR primers were removed and sequences with sequencing errors in the primers were excluded (Mothur) [START_REF] Schloss | Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities[END_REF] . 12000 reads were randomly selected for each sample.

Chimera were removed with UCHIME [START_REF] Edgar | UCHIME improves sensitivity and speed of chimera detection[END_REF] and Mothur 54 software. Reads were clustered into Operational Taxonomic Units (OTUs) at the 97% identity level using Esprit-tree [START_REF] Sun | A large-scale benchmark study of existing algorithms for taxonomy-independent microbial community analysis[END_REF] . A reference sequence was picked for each OTU and assigned it at different taxonomic levels (from phylum to species) using the Greengenes database (release 13-5) [START_REF] Desantis | Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB[END_REF] and the RDP classifier [START_REF] Wang | Naive Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy[END_REF] .

After rarefaction of all communities to even sampling depths, the abundances of all families were computed by agglomerating the OTUs assigned to those families. For each such family, Kruskall Wallis test with BH correction [START_REF] Hochberg | More powerful procedures for multiple significance testing[END_REF] were performed to detect the combinations (treatment) that were significantly different in terms of abundance. The same procedure was applied for each genus and for each OTUs. All analyses were done using R (R Core Team, 2015, R Foundation for Statistical Computing, Vienna, Austria).

Euthanasia

At PD22, one day after weaning of the offspring, dams were deeply anaesthetized with sodium pentobarbital (i.p, 150 mg/kg BW, Ceva Santé Animale) and euthanized by a rapid decapitation. Trunk blood was collected and kept on ice until processing within 8 h.

Blood samples were centrifuged at 2375 x g for 10 minutes at 4°C. Serum was stored at -80°C until use. The brains were quickly removed, immersion-fixed in 4% paraformaldehyde at 4°C for 24 h. Brains were then transferred in 30% sucrose/phosphate-buffered saline

Accepted Article

. solution for 7 days for cryoprotection before snap-freezing on dry ice. Brains were stored at -80°C until processing (see below).

Serum Analysis

Serum transaminases analysis. Serum alanine (ALT) and aspartate (AST) transaminases were measured according to the International Federation of Clinical Chemistry (IFCC) primary reference procedures using Olympus AU2700 Auto-analyser® (Olympus Optical, Tokyo, Japan). Serum transaminases levels in dams were measured to evaluate potential hepatotoxic effect [START_REF] Amacher | Serum transaminase elevations as indicators of hepatic injury following the administration of drugs[END_REF] .

Interleukin 6 (IL-6).

Serum levels of interleukin 6 (IL-6) were determined using a commercial ELISA kit with pre-coated plates (Legend Max TM , BioLegend Inc) and performed according to the provider instructions. All samples were analyzed in triplicate. The plates were read with iMark microplate reader (Bio-rad) and analyzed with MPM6 software (Bio-rad). The assay had a minimum detectable concentration of 5.3 pg/mL.

Cations. Serum levels of calcium (Ca), iron (Fe), magnesium (Mg) and manganese (Mn) were quantified by ICP/MS/MS at the LERES analytical platform. Briefly, plasma was acidified with HNO3 (1% final concentration) and diluted 1/10 in ultra-pure water before analysis on quadrupole-based ICP-MS instrument (ICP-MSMS 8800, Agilent) equipped with an extra octopole-containing cell to remove interferences by selective reaction with a reactive gas (e.g., H2, O2, NH3) or collisions of the ions with a non-reactive gas (e.g., He). Technical triplicate for each sample were measured following a calibration curve (8 points for each ions) and internal standards (Iridium, Scandium, Germanium, Indium, Rhénium and Rhodium).

Accepted Article

.

Brain histology

Brains were sliced with a cryostat (Microm HM560) at 40 m, in series of 10.

Sections were stored at -16°C in ethylene glycol-based antifreeze solution until immunohistochemical processing.

Immunohistochemistry. Immunohistochemistry was performed as previously published [START_REF] Pawluski | Fluoxetine Dose and Administration Method Differentially Affect Hippocampal Plasticity in Adult Female Rats[END_REF][START_REF] Gemmel | Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: Interactions with pre-gestational maternal stress[END_REF][START_REF] Pawluski | Stress and the pregnant female: Impact on hippocampal cell proliferation, but not affective-like behaviors[END_REF][START_REF] Pawluski | Effects of stress early in gestation on hippocampal neurogenesis and glucocorticoid receptor density in pregnant rats[END_REF] . Briefly, 1 series of sections was used for each marker (Ki67, Doublecortin, Synaptophysin and PSD95; see Table 1 for details). Sections were brought to room temperature and extensively rinsed in PBS before processing. For PSD95 staining only, sections were incubated in 10 mM sodium citrate buffer (pH 6.0) for 10 min at 80°C for antigen retrieval then rinsed in PBS before further processing. For all markers, endogenous peroxidase activity was blocked in 0.6 % H 2 O 2 for 30 min at room temperature. Sections

were then incubated at room temperature in normal donkey serum (NDS, Sigma 5% in PBS containing 0.1% of triton (PBST), for Ki67 and Doublecortin) or in normal goat serum (NGS, 5%, synaptophysin or 2%, PSD95) for 30 min as described in Table 1. Tissue was then incubated at 4°C in primary antibody (Ki67, DCX, synaptophysin, PSD95, as described in Table 1). Sections were rinsed in PBST then incubated in secondary biotinylated antibody.

Signal amplification was performed using avidin-biotin-horseradish peroxydase complex (Vectastain® ABC Elite kit; 1:1000; Vector Laboratories) for 2 h at room temperature and signal was visualized using DAB (3,3-diaminobenzidine) peroxidase substrate kit (SK-4100, Vector Laboratories). Sections were mounted on SuperFrost Plus slides (Thermo Scientific), dried overnight, counterstained with Cresyl Violet acetate (only for Ki67), dehydrated and cover with Permount (Fischer Scientific).

Accepted Article

.

Plasticity measures

Hippocampal volume. Cresyl-stained sections were observed under 2X magnification using an Olympus AX70 TRF and a DP71 digital camera with Cell^F software (Olympus).

Areas of the granular cell layer and subgranular zone (GCL/SGZ) throughout the dentate gyrus rostro-caudal extent were measured using ImageJ software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF] . Areas were summed and multiplied by the sampling interval (10) to derive an estimate of volume.

Ki67 and DCX quantification. DCX-and Ki67-positive cells were counted under X40 objective (Olympus BX60 optic microscope) throughout the dentate gyrus (GCL/SGZ) by an observer blind to the experimental treatment. Ki67-positive cells were also counted in the hilus. The number of cells counted was multiplied by 10 (1 series of sections analyzed out of 10) to obtain an estimate of the total number cells in the dentate gyrus.

We also quantified and characterized DCX-positive cells based on their morphology in 2 dorsal and 3 ventral sections. A minimum of 60 DCX-positive cells were counted per brain section and categorized as either proliferative (no process or short process), intermediate (medium process with no branching) or post-mitotic neurons [START_REF] Plümpe | Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation[END_REF][START_REF] Workman | Parity modifies the effects of fluoxetine and corticosterone on behavior, stress reactivity, and hippocampal neurogenesis[END_REF]

Accepted Article

. photomicrographs in the cingulate gyrus (CG) bregma 2.76 mm to -0.12 mm 67 were taken under 40X objective using a Olympus AX70 TRF and a DP71 digital camera with Cell^F software (Olympus). For representative photomicrographs of synaptophysin density see Figure 5A.

Optical densities (OD) were quantified with ImageJ (NIH) software. The relative optical density was calculated as the difference between optical density (grey level after calibration) of the area of interest and the background of an adjacent region [START_REF] Pawluski | Fluoxetine Dose and Administration Method Differentially Affect Hippocampal Plasticity in Adult Female Rats[END_REF][START_REF] Gemmel | Perinatal fluoxetine effects on social play, the HPA system, and hippocampal plasticity in pre-adolescent male and female rats: Interactions with pre-gestational maternal stress[END_REF] .

Statistical analysis

Data were analyzed using the software Statistica 13 (Dell Inc.) and GraphPad Prism version 5.03. One-way ANOVAs were computed on separate maternal behaviors on PD1 and across PD2-6, litter characteristics, hepatic toxicity (AST/ALT), hippocampal volume, measures of synaptic plasticity (synaptophysin, PSD95), neurogenesis and serum analysis of cations and IL6. Repeated measures ANOVAs were done on maternal weight, Ki67-ir cells in the GCL/SGZ and hilus as well as for the morphology of DCX-ir cells. Post hoc comparisons were performed using Fisher LSD when necessary. Due to the high variance between groups for maternal behavior on PD1, values were normalized before statistical analysis but untransformed data are presented in the bar graph (figure 2) to feature natural individual variability. For all statistical analysis, p < 0.05 was considered significant.

Microbiome statistical analysis.

Following rarefaction of all communities to even sampling depths, the abundances of all families were computed by agglomerating the Operational Taxonomic Units (OTUs) assigned to those families. For each comparison between all groups, Kruskal-Wallis tests with BH correction [START_REF] Hochberg | More powerful procedures for multiple significance testing[END_REF] were carried out to identify the significant effect of treatment on abundance at Phylum, family, genus and OTU levels.

Probabilities were considered significant when p < 0.05.

Accepted Article

.

RESULTS

General toxicity

Several parameters were measured to define potential general toxic effects of Glyphosate or Roundup. As expected, the weight of the dams was significantly different during pregnancy and the postpartum period (F (2,32) = 13.24, p < 0.0001; 2B).

Hippocampal Plasticity

Hippocampal volume. There were no significant effects of treatment by Glyphosate or Roundup on the volume of the dentate gyrus (F (2,16) = 0.2633, p = 0.7718, Table 3).

Therefore, total number of cell counts were used throughout.

Hippocampal cell proliferation (Ki67). Using repeated-measures ANOVAs between

the GCL/SGZ and hilus, there were no significant effects of treatment and no interaction between the treatment and the area on the number of Ki-67-ir cells in the dorsal region of the GCL/SGZ and hilus (respectively F (2, 16) = 0.=65158, p = 0.5345 and F (2, 16) = 0.15107, p = 0.8610, Figure 3A). As expected there were significantly more proliferative cells in the GCL/SGZ than in the hilus (F (1, 16) = 147.65, p < 0.0001).

In the ventral region of the GCL/SGZ, there was strong tendency toward an interaction between the treatment and area (F (2, 16) = 3.6168, p = 0.05059, Figure 3B).

Similarly to the dorsal hippocampus, significantly more Ki67-ir cells were found in the ventral GCL/SGZ compare to the hilus (F (1, 16) = 383.16, p < 0.0001). There was no significant main effect of treatment on the number of proliferative cells (F (2, 16) = 2.7492, p = 0.0941).

Hippocampal neurogenesis (DCX).

There was no effect of the treatment on total number of immature DCX-ir neurons in the dorsal (F (2,15) = 1.0597, p = 0.3711, Figure 4A) or ventral GCL/SVZ (F (2,15) = 2.0139, p = 0.1680, Figure 4B).

Accepted Article

. DCX cell morphology. There were no significant differences between groups in the number of DCX-ir cells quantified in terms of morphology in the dorsal or ventral hippocampus (p's > 0.16). Repeated measures ANOVAs on the morphology of DCX-ir cells in the dorsal GCL/SGZ revealed a significant interaction between the treatment and the maturation of the cells (F (4,30) = 3.0955, p = 0.0302, Figure 4C) with a significantly higher proportion in post-mitotic neurons in Roundup-treated dams compared to the Control dams (p = 0.0016). There was also a significant difference in the maturation stages in the dorsal GCL/SGZ (F (2,30) = 16.7734, p < 0.0001, Figure 4C) with significant differences between the number of cells at each stage of development ( p's < 0.04). There was no significant main effect of the treatment on the morphology of the new neurons in the dorsal GCL (F (2,15) = 1.7504, p = 0.2074).

For the ventral hippocampus, there was also no significant main effect of the treatment on DCX cell morphologies (F (2,15) = 2.014, p = 0.1680) nor an interaction effect (F (4,30) = 1.567, p = 0.2086, Figure 4D). Similarly to the dorsal GCL, there was a significant difference in the distribution of the morphologies (F (2,30) = 1515.907, p < 0.0001, Figure 4D) with significant differences between the number of cells at each stage of development ( p's < 0.001).

Synaptophysin. There was a significant main effect of the treatment on synaptophysin OD in the dorsal region of the dentate gyrus (F (2,16) = 9.807, p = 0.0017, Figure 5A) with a significantly greater density of synaptophysin in Glyphosate and Roundup-treated dams compared to the Controls (p < 0.02). There was also a significant main effect of treatment on synaptophysin density in the ventral dentate gyrus (F (2,16) = 7.064, p = 0.0063, Figure 5B)

with Glyphosate-treated dams having significantly greater synaptophysin density compared to both Control and Roundup treated dams (respectively p = 0.0025 and 0.0121). In the CA3

Accepted Article

. region, there was also a significant main effect of treatment on synaptophysin density (F (2,16) = 5.817, p = 0.0126, Figure 5C) with Glyphosate dams having a significantly greater synaptophysin density compared to Control and Roundup groups (p's < 0.02).

When looking at synaptophysin density in the cingulate gyrus there was also a significant main effect of treatment (F (2,15) = 4.908, p = 0.0229, Figure 5D) with synaptophysin density being reduced in both Glyphosate and Roundup groups compared to Controls (p's < 0.03).

PSD95. There was no significant effects of the treatment on PSD95 density in the dorsal (F (2,16) = 0.4293, p = 0.6582, Table 4) or ventral (F (2,16) = 0.9545, p = 0.4059, Table 4) dentate gyrus or the CA3 region (F (2,16) = 1.701, p = 0.2140, Table 4).

Microbiome

We also evaluated the impact of the Glyphosate and Roundup on the gut microbiota.

Composition of the gut microbiota did not display significant differences between groups at GD9, i.e before treatment (data not shown). At PND22, we found several significant differences at phylum, family and genus level between groups (Figure 6). We found a significant effect of the treatment on Bacteroidetes (p = 0.009) and Firmicutes (p = 0.006),

with respectively an increase and a decrease in Roundup group compared to the Control and Glyphosate (p's < 0.04; Figure 6B). We further analyzed the gut microbiota variation at the family level and showed a significant effect of the treatment on Lachnospiraceae (Phylum:

Firmicutes, p = 0.033) which were less abundant in Roundup compared to Glyphosate dams (p = 0.013), with a trend in Roundup group compared to Control (p = 0.065; Figure 6C andD). We also found a significant effect of the treatment on Erysipelotrichaceae (Phylum:

Firmicutes, p = 0.039), which were conversely more abundant in Roundup-treated dams

Accepted Article

. compared to Glyphosate (p = 0.017), and with a strong tendency in Roundup dams compared to Controls (p = 0.056; Figure 6C andD). At the genus level, there was a significant main effect of the treatment on Alloprevotella (Family: Prevotellaceae, Phylum: Bacteroidetes), Butyricicoccus (Family: Clostridiaceae, Phylum: Firmicutes), Ruminococcaceae UCG-013 (Family: Ruminococcaceae, Phylum: Firmicutes) and Turicibacter (Family:

Erysipelotrichaceae, Phylum: Firmicutes) abundancy (respectively p = 0.03, p = 0.021, p = 0.04 and p = 0.039). Alloprevotella were more represented in the Roundup compared to Control and Glyphosate groups (p's < 0.05; Figure 6E). Turicibacter were more abundant in Roundup-treated dams compared to Glyphosate (p = 0.017), and there was strong tendency in Roundup compared to Control group (p = 0.056). In contrast Butyricicoccus genus decreased in Glyphosate group comparatively to Control and Roundup groups (p's < 0.04; Figure 6E).

Moreover, Ruminococcaceae UCG-013 genus were significantly decreased in both Glyphosate and Roundup groups compared to Control (p's < 0.04; Figure 6E).

DISCUSSION

Main findings of the present study show that peripartum exposure to a low dose of Glyphosate or Roundup affect maternal behavior, neuroplasticity and induce dysbiosis in dam rats. More specifically, we showed that perinatal exposure to Glyphosate or Roundup significantly altered maternal licking behavior without affecting other characteristics of the maternal behavior. In addition to the behavior, we also showed that Glyphosate alone or in formulation affected aspects of neurogenesis in the maternal brain postpartum. Indeed, there were more post-mitotic immature neurons in the dorsal dentate gyrus of Roundup dams, while cell proliferation and the total number of immature neurons were not affected.

Moreover, synaptophysin was modified following Glyphosate and Roundup perinatal exposure in dorsal and ventral dentate gyrus and CA3 of the hippocampus, as well as in the

Accepted Article

. cingulate gyrus, albeit with different profiles depending on the region of interest. It is important to highlight here that the behavioral and physiological outcomes following exposure to the active compound alone (Glyphosate) or to the formulation (Roundup) were also significantly different, confirming previous observations that the effects of formulations could be linked to adjuvants within these formulations [START_REF] Defarge | Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels[END_REF][START_REF] Mesnage | Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity[END_REF] .

Absence of general toxicity.

In the present study, Glyphosate and Roundup had no significant effect on dam weight throughout pregnancy and the postpartum period. Similarly, the litter characteristics such as length, weight, sex ratio were not altered by the treatment. This observation is in agreement with recent studies that investigated dams weight during pregnancy [START_REF] Dallegrave | Pre-and postnatal toxicity of the commercial glyphosate formulation in Wistar rats[END_REF][START_REF] Gallegos | Exposure to a glyphosate-based herbicide during pregnancy and lactation induces neurobehavioral alterations in rat offspring[END_REF] and litter characteristics [START_REF] Dallegrave | Pre-and postnatal toxicity of the commercial glyphosate formulation in Wistar rats[END_REF][START_REF] Gallegos | Perinatal Glyphosate-Based Herbicide Exposure in Rats Alters Brain Antioxidant Status, Glutamate and Acetylcholine Metabolism and Affects Recognition Memory[END_REF][START_REF] Sof A Gimenez | Effect of the Herbicide Glyphosate on Enzymatic Activity in Pregnant Rats and Their Fetuses[END_REF] following exposure to relatively low dose of GBH. Only higher doses were found to reduce weight in juvenile or in pregnant rat (500 and 1000 mg/kg BW/d) peripartum exposure in dams [START_REF] Sof A Gimenez | Effect of the Herbicide Glyphosate on Enzymatic Activity in Pregnant Rats and Their Fetuses[END_REF] . We also confirm the absence of visible toxicity at the biochemical level, reflected by the low plasma levels of AST and ALT, two hepatic transaminases released in the vascular system upon liver aggression [START_REF] Amacher | Serum transaminase elevations as indicators of hepatic injury following the administration of drugs[END_REF] . These results are in line with those obtained in adult male rats exposed for 5 weeks to 5, 50 and 500 mg/kg BW/day of Glyphosate, where only the highest dose led to a significant increase in AST and ALT circulating levels [START_REF] Tang | Ion Imbalance Is Involved in the Mechanisms of Liver Oxidative Damage in Rats Exposed to Glyphosate[END_REF] .

Glyphosate and maternal behaviors.

In the present study, Control dams tended to spend more time licking their pups on the day following parturition, compared to Glyphosate or Roundup-treated animals. Additional maternal behaviors were not different between groups and time spent licking in these untreated Control dams was significantly reduced the following days. It is important to

Accepted Article

. highlight here that on the day following parturition the offspring were handled and separated from their mother for up to 10 minutes to record offspring characteristics and cull the litter.

Previous studies show that pup handling or isolation after parturition disrupts basal maternal behaviors, with most changes being associated with an increase in licking and grooming behaviors following the reintroduction of the pups with their mother [START_REF] Barnett | Early Stimulation and Maternal Behaviour[END_REF][START_REF] Kosten | Immediate and enduring effects of neonatal isolation on maternal behavior in rats[END_REF][START_REF] Lee | Changes in licking behaviour of rat mother following handling of young[END_REF][START_REF] Liu | Maternal care, hippocampal glucocorticoid receptors, and hypothalamic-pituitary-adrenal responses to stress[END_REF][START_REF] Marmendal | Maternal separation alters maternal care, but has minor effects on behavior and brain opioid peptides in adult offspring[END_REF][START_REF] Pryce | Comparison of the effects of early handling and early deprivation on maternal care in the rat[END_REF] . Our observations, therefore, suggest that Glyphosate-and Roundup-treated dams do not present this normal increase in licking behavior upon reuniting with the pups. While a large number of studies have focused on the short-and long-term consequences of pup isolation on the offspring [START_REF] Giachino | Maternal deprivation and early handling affect density of calcium binding protein-containing neurons in selected brain regions and emotional behavior in periadolescent rats[END_REF][START_REF] Veenema | Early life stress, the development of aggression and neuroendocrine and neurobiological correlates: What can we learn from animal models?[END_REF] , little is known about the outcome on the mother and the underlying physiology. It is, however, likely that separation causes some form of anxiety, not only for the pup, but also for the mother. Interestingly, previous work suggests that early or adult exposure to Glyphosatebased herbicide increases anxiety and depression-like behavior in rodents [START_REF] Bali | Behavioral and Immunohistochemical Study of the Effects of Subchronic and Chronic Exposure to Glyphosate in Mice[END_REF][START_REF] Gallegos | Exposure to a glyphosate-based herbicide during pregnancy and lactation induces neurobehavioral alterations in rat offspring[END_REF][START_REF] Baier | Behavioral impairments following repeated intranasal glyphosate-based herbicide administration in mice[END_REF] . Therefore, we can speculate that the altered interpretation of the social environment associated with anxiety or depressive-like behavior will result in modification in maternal care.

Rather surprisingly, the maternal behavior was later affected in the Roundup-treated animal between PD2 to 6, with an increase in licking behavior compared to dams from Control and Glyphosate groups. It could be that one of the adjuvant, such as polyethoxylated tallowamine, present in the Roundup formulation might be responsible for the effect as suggested by Mesnage and colleagues [START_REF] Defarge | Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels[END_REF][START_REF] Mesnage | Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity[END_REF][START_REF] Mesnage | Ignoring Adjuvant Toxicity Falsifies the Safety Profile of Commercial Pesticides[END_REF] , but this hypothesis remains to be tested.

It is also interesting to note that the offspring influences the amount of parental care given by the mother through direct tactile contact, olfactory cues, and ultrasonic vocalizations. For example in rodents, the male is more likely to initiate contact with the mother and in turn, licking and grooming from the dam toward their male offspring is usually more important [START_REF] Seta | Bisphenol-A exposure during pregnancy and lactation affects maternal behavior in rats[END_REF][START_REF] Moore | Maternal contributions to the development of masculine sexual behavior in laboratory rats[END_REF][START_REF] Porrini | Early exposure to a low dose of bisphenol A affects sociosexual behavior of juvenile female rats[END_REF] . A misinterpretation of these cues by the mother exposed to Glyphosate or Roundup could result in different amounts of licking as observed in the present study. It

Accepted Article

. should be kept in mind that in utero exposure of the offspring could have affected the sensory cues emitted by the offspring [START_REF] Zimmerberg | Neonatal social isolation alters both maternal and pup behaviors in rats[END_REF] . For example, perinatal exposure to organochloride pesticides such as chlorpyryfos or lindane significantly affected the ultrasonic vocalization emitted by pups [START_REF] Rivera | Increase in rat pup ultrasonic isolation calls induced by lindane[END_REF][START_REF] Venerosi | Prenatal chlorpyrifos exposure alters motor behavior and ultrasonic vocalization in cd-1 mouse pups[END_REF] . The modification of the pup behavior following in utero exposure to Glyphosate would alter the care provided by the mother but this hypothesis remains to be investigated.

While most studies, including the present one, investigated only one part of the mother/offspring dyad (the mother), the behavior results from the interaction and constant feedback between the mother and the pups. Indeed, the polychlorinated biphenyl congener PCB 77 affected maternal behavior when both the mother and offspring were exposed, as confirmed by cross-fostering experiments [START_REF] Cummings | A cross-fostering analysis of the effects of PCB 77 on the maternal behavior of rats[END_REF] . This is an important reminder that the effects of many substances on offspring outcomes may be due, in part, to their effects on maternal caregiving behaviors. Future research needs to acknowledge the importance of maternal caregiving on offspring outcomes and investigate both parts of the mother/offspring dyad.

Glyphosate and plasticity in the maternal brain

Neurogenesis in the dentate gyrus of the hippocampus.

Two brain regions, the sub-ventricular zone of the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus are known to produce a large number of new neurons throughout adulthood [START_REF] Lim | Adult neural stem cells stake their ground[END_REF][START_REF] Migaud | Emerging new sites for adult neurogenesis in the mammalian brain: a comparative study between the hypothalamus and the classical neurogenic zones[END_REF][START_REF] Ming | Adult Neurogenesis in the Mammalian Brain: Significant Answers and Significant Questions[END_REF][START_REF] Zhao | Mechanisms and Functional Implications of Adult Neurogenesis[END_REF] . Importantly, hippocampal neurogenesis is strongly modulated during pregnancy and the postpartum period in rodents [START_REF] Pawluski | Neuroplasticity in the maternal hippocampus: Relation to cognition and effects of repeated stress[END_REF][START_REF] Darnaudéry | Early motherhood in rats is associated with a modification of hippocampal function[END_REF][START_REF] Galea | Hippocampal Plasticity during the Peripartum Period: Influence of Sex Steroids, Stress and Ageing[END_REF][START_REF] Hillerer | Lactation-induced reduction in hippocampal neurogenesis is reversed by repeated stress exposure[END_REF][START_REF] Leuner | Maternal experience inhibits the production of immature neurons in the hippocampus during the postpartum period through elevations in adrenal steroids[END_REF][START_REF] Pawluski | Reproductive experience alters hippocampal neurogenesis during the postpartum period in the dam[END_REF] . Research has also shown that neuroplasticity and neurogenesis in the hippocampus is strongly affected by the chemical environment [START_REF] Hossain | Hippocampal ER Stress and Learning Deficits Following Repeated Pyrethroid Exposure[END_REF][START_REF] Mishra | Prenatal Carbofuran Exposure Inhibits Hippocampal Neurogenesis and Causes Learning and Memory Deficits in Offspring[END_REF][START_REF] Pizzurro | Diazinon and diazoxon impair the ability of astrocytes to foster neurite outgrowth in primary hippocampal neurons[END_REF][START_REF] Wang | Reversible effect of developmental exposure to chlorpyrifos on late-stage neurogenesis in the hippocampal dentate gyrus in mouse offspring[END_REF] including medication [START_REF] Workman | Parity modifies the effects of fluoxetine and corticosterone on behavior, stress reactivity, and hippocampal neurogenesis[END_REF]100,101 . In the present work we found a significant increase in the number of post-mitotic (more mature) doublecortinpositive neurons in the dorsal granule cell layer of Roundup-treated dams compared to Controls. The effect of Glyphosate alone on this parameter was not statistically significant

Accepted Article

. but a trend toward an increase was also observed. No treatment effects were evident in the ventral hippocampus or in measures of cell proliferation. The upregulation of the number of post-mitotic cells in the dorsal hippocampus with Roundup treatment is likely a result of a combination of several mechanisms related to a faster maturation of these neurons or a longer period cells of this stage survive. The consequences of this increase are not known but it is suggested that adult neurogenesis is not needed for learning per se but rather for the modulation and plasticity of spatiotemporal contextualization of new information. Indeed, the new neurons in the dentate gyrus of the hippocampus contribute to synaptic plasticity to the dentate gyrus 102-104 while other surrounding neurons are usually inhibited by local GABAergic interneurons. The present increase in postmitotic doublecortin neurons might therefore leads to stronger plasticity within this region and the behavioral consequences remain to be investigated.

Synaptic plasticity in the hippocampus

Findings of the present study also show that peripartum exposure to Glyphosate and Roundup increased synaptophysin expression in the dorsal region of the dentate gyrus and interestingly, decreased synaptophysin expression in the cingulate gyrus, an area of the medial prefrontal cortex. Glyphosate alone increased synaptophysin expression in both ventral dentate gyrus and CA3 regions of the hippocampus. This suggests region specific effects of Glyphosate and Roundup on synaptic proteins throughout the brain.

Synaptophysin is an membrane protein expressed at the surface of synaptic vesicles and can be found in most neurons within the central nervous system 105,106 . To our knowledge, the precise function of this protein is still not clear but studies suggest that synaptophysin is involved in calcium binding 107 , chanel formation 108,109 , exocytosis 110,111 , as well as synaptic vesicle recycling 112 . The change in synaptophysin expression following

Accepted Article

. peripartum exposure to Glyphosate is perhaps not surprising as several studies have demonstrated a change in neurotransmitter release and metabolism in various regions of the rat brain after exposure to Glyphosate or Glyphosate-based herbicides [START_REF] Cattani | Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity[END_REF][START_REF] Martínez | Neurotransmitter changes in rat brain regions following glyphosate exposure[END_REF][START_REF] Hernández-Plata | The herbicide glyphosate causes behavioral changes and alterations in dopaminergic markers in male Sprague-Dawley rat[END_REF][START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF][START_REF] Gallegos | Exposure to a glyphosate-based herbicide during pregnancy and lactation induces neurobehavioral alterations in rat offspring[END_REF] . For example, high doses of Glyphosate lead to an increase in serotonin release and turnover in the striatum and hypothalamus and in dopamine turnover in prefrontal cortex and hippocampus [START_REF] Martínez | Neurotransmitter changes in rat brain regions following glyphosate exposure[END_REF] . The change in dopamine is likely to be related to the reduction of tyrosine hydroxylase expression [START_REF] Bali | Behavioral and Immunohistochemical Study of the Effects of Subchronic and Chronic Exposure to Glyphosate in Mice[END_REF] , the key enzyme responsible for dopamine synthesis. The modulation of dopamine levels can also be associated to the reduction in D1-dopamine specific receptor binding in the nucleus accumbens in adult male Sprague-Dawley rats exposed to Glyphosate [START_REF] Hernández-Plata | The herbicide glyphosate causes behavioral changes and alterations in dopaminergic markers in male Sprague-Dawley rat[END_REF] . It should be noted that the nucleus accumbens is a fundamental brain region involved in the motivation aspect of maternal behavior and modulation of activity within this brain region only can alter proper maternal care toward the offspring [113][114][115] . In addition to monoamines, acetylcholine esterase, the enzyme responsible for the breakdown of acetylcholine in the synaptic cleft to terminate synaptic transmission, is inhibited in the prefrontal cortex, striatum and hippocampus of male and female adult offspring after a perinatal exposure to Glyphosate [START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF][START_REF] Gallegos | Perinatal Glyphosate-Based Herbicide Exposure in Rats Alters Brain Antioxidant Status, Glutamate and Acetylcholine Metabolism and Affects Recognition Memory[END_REF] . Extracellular glutamate concentration and the reuptake of amino acids are also hindered in the hippocampus following exposure to Glyphosate-based herbicide [START_REF] Cattani | Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity[END_REF][START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF] .

Whether or not the effects of glyphosate on the maternal brain reported in this study are unique to motherhood remain to be determined. Previous research shows that pregnancy and the postpartum period both affect plasticity in the hippocampus [START_REF] Pawluski | Neuroplasticity in the maternal hippocampus: Relation to cognition and effects of repeated stress[END_REF] . How exposure to this herbicide interacts with the normative changes in the female brain during pregnancy and the postpartum period is needed to understand the complexities of these relationships and how they may affect behavioral outcomes.

Accepted Article

.

Molecular target of Glyphosate

As introduced earlier, the molecular target of Glyphosate is the protein EPSPS, an enzyme found in plants and bacteria. This mode of action suggests that vertebrates are relatively safe if exposed to Glyphosate-based herbicides. However, the increased number of reports pointing to effects of Glyphosate on animal physiology open the rarely asked question about the molecular target of the Glyphosate in vertebrates. Only a handful of studies have suggested direct molecular target for Glyphosate.

First, as mentioned in the introduction, the inhibition of the enzyme EPSPS not only impacts the growth and survival of plants, but also that of bacteria. Multicellular organisms are colonized with hundreds of species of bacteria, collectively known as microbiota and they exert a profound influence on key physiological processes. More precisely, microbiota present in the digestive system is known to exert major influence on neuroinflammation, activation of the stress axes, neurotransmission, and neurogenesis to name a few, resulting in the modulation complex behaviors, such as sociability and anxiety [116][117][118][119][120] . Gut microbiota influences the central nervous system through its ability to modulate the immune system, to provide bioactive metabolites such as fatty acids and synthesize neurotransmitters including GABA, noradrenaline, and dopamine 121,122 . Any dysregulation of the gut microbiome, or dysbiosis, is known to significantly affect the central nervous system and the resulting behavior, leading to increased susceptibility to psychiatric disorders 120,123 . Our results clearly demonstrate that gut microbiota is affected by glyphosate and glyphosate-based herbicide, albeit in formulation-specific manner. Indeed, we showed an increase in the phylum Bacteroidetes and a reduction of Firmicutes following Roundup exposure, the Glyphosate alone having no impact on the 2 phyla. The effect of glyphosate or glyphosate based herbicide on bacterial population is not a complete surprise. A few studies, mostly on soil bacteria 124 , but also in animal species, from insects [START_REF] Motta | Glyphosate perturbs the gut microbiota of honey bees[END_REF]125 to reptiles 126 and mammals 127,128 ,

Accepted Article

. investigated the effect of the pesticide on microorganism ecology. While the precise role of each of these bacteria phyla for brain physiology is, to our knowledge, far from understood, a few studies have suggested that a dysregulation of Bacteroidetes or Firmicutes populations is associated to mental health disorders, including autism 129-131 and Alzheimer's disease 132,133 .

To support our findings, other studies, in rats but also in bees, report similar changes in Bacteroidetes and/or Firmicutes following Glyphosate or Glyphosate-based herbicide exposure [START_REF] Lozano | Sex-dependent impact of Roundup on the rat gut microbiome[END_REF][START_REF] Motta | Glyphosate perturbs the gut microbiota of honey bees[END_REF]127,128 . Similarly, the population of the genera Alloprevotella is increased in our study, while studies have found an association between Parkinson and Alzheimer's disease and reduced population of the family of Prevotellaceae [134][135][136][137] . It is obviously important to note that these studies are only correlational and the precise consequences of the impact of Glyphosate or Glyphosate-based herbicide on various species of bacteria, as well as on the complex microbiota ecosystem, deserve to be investigated in more detail to understand how Glyphosate, alone or in combination with adjuvants, might impact the organisms. Indeed, it is interesting to note that the most pronounced effects on the microbiome in the present study are linked to Roundup treatment. One hypothesis is the presence of multiple, yet mostly undefined, adjuvants added to the formulation to increase the penetration of glyphosate within the target organisms 138,139 . A low penetration of glyphosate in the absence of adjuvant could result in less consequences for the potential targeted organisms. Alternatively, these adjuvants by themselves could be affecting the microbiota population, as suggested Mesnage and collaborators for vertebrate toxicity [START_REF] Defarge | Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels[END_REF][START_REF] Mesnage | Ethoxylated adjuvants of glyphosate-based herbicides are active principles of human cell toxicity[END_REF][START_REF] Mesnage | Ignoring Adjuvant Toxicity Falsifies the Safety Profile of Commercial Pesticides[END_REF] . A very few studies, on soil bacteria suggest that this might be the case 124 . This differential effect should be investigated.

A second molecular target of Glyphosate is the direct activation of glutamatergic NMDA receptor, suggested by Cattani and Colleagues [START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF] . Indeed, their molecular dynamic simulation demonstrates that Glyphosate may bind to glutamate and glycine cavities of NMDA receptors, with lower stability of Glyphosate binding at glycine binding pocked

Accepted Article

. compared with glutamate one. This interaction would likely increase the neuronal Ca 2+ entry, triggering mitochondrial dysfunction and oxidative damage [START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF] . This direct interaction remains to be tested experimentally. It should be pointed out that oxidative stress, independently of NMDA receptor activation, was also observed in other tissues, such as the liver and the kidney following exposure to Glyphosate or various Glyphosate-based herbicides [140][141][142][143] .

A third hypothesis for molecular target is the chelating role of Glyphosate spleen and kidney in a dose dependent-way in adult male rat [START_REF] Tang | Ion Imbalance Is Involved in the Mechanisms of Liver Oxidative Damage in Rats Exposed to Glyphosate[END_REF] . In addition, they found a significant increase of Mg 2+ in the brain following exposure for 35 days to 50 and 500 mg/kg

Glyphosate. There was no information about the plasma levels. It is clear that additional studies should investigate this potential effect of Glyphosate on divalent cations and define whether the observed changes could lead to alter physiology in the organism.

Future Directions

This is the first study to investigate how glyphosate may affect the maternal brain and behavior. However, we acknowledge the much research is needed to determine the extent of these effects on the mother and offspring. Of particular importance in the present study would be to investigate how changes in the maternal brain are causally related to changes in the Funding sources had no involvement in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication. There was a significant interaction between the cell morphology and the treatment in the dorsal GCL. * denotes significance (p < 0.05). Scale bar is 20 µm.

Figure 1 .

 1 Figure 1. Timeline of the present study. Dams were treated from GD10-PD22. Maternal

Figure 2 .

 2 Figure 2. Mean (+SEM) percentage of total time spent in maternal behavior (%) A: during

Figure 3 .

 3 Figure 3. Representative photomicrographs of A: Ki67-ir cells in the granule cell layer and

Figure 4 .

 4 Figure 4. Representative photomicrographs of A: doublecortin-ir cells in the GCL with B:

Figure 5 .

 5 Figure 5. Representative photomicrographs of A: synaptophysin-ir in the GCL/SGZ. Mean

Figure 6 .

 6 Figure 6. Analysis of the effects on gut microbiota by 16S rRNA gene high-throughput

Table 2

 2

	). But there

= 0.2770, p = 0.7616; Table

2

). A trend toward a change in sex ratio was observed (F (2,16) = 3.559, p = 0.0526; Table

2

), with Roundup treatment showing a slight increase in the number of male offspring compared to female offspring. There were also no significant effects of treatment on AST (F (2,16) = 1.857, p = 0.1883) or ALT (F (2,16) = 1.347, p = 0.2881) or serum levels of cations and IL-6 serum (Table

2

).

Maternal behavior

On PD1, treatment affected pup licking behavior in dams but this did not reach significance (F (2,16) = 2.9103, p = 0.08357, Figure

2A

). No other maternal behaviors were significantly affected on PD1 (ABN F (2,16) = 0.68192, p = 0.51975; blanket F (2,16) = 0.42441, p = 0.66131; passive F (2,16) = 0.58698, p = 0.56754; off nest F (2,16) = 0.77431, p = 0.47755; nest building F (2,16) = 0.18884, p = 0.82974, Figure

2A

). Glyphosate-treated dams (p's < 0.04). There were no other significant main effects on additional maternal behaviors (ABN F (2,16) = 1.5988, p = 0.23281; blanket F (2,16) = 0.62086, p

  [START_REF] Mertens | Glyphosate, a chelating agent-relevant for ecological risk assessment?[END_REF][144][145][146] , particularly with divalent cations. The presence of divalent cations, including Ca 2+ , Fe 2+ or Mg 2+ is of fundamental importance for the organisms as they participate in various physiological functions, including bone formation and cellular communication (Ca 2+ ), oxygen transport (Fe 2+ ) and enzyme cofactors (Ca 2+ , Mg 2+ ) to name a few. An alteration of these elements would therefore lead to major adverse effects for the organism. In the present study plasma levels of Ca 2+ , Fe 2+ , Mn 2+ and Mg 2+ were quantified but not affected by treatments. To our knowledge, only one study investigated the effect of Glyphosate on ion concentrations in vertebrate. Tang and colleagues revealed that low (5 mg/kg), medium (50 mg/kg) or high doses (500 mg/kg) of Glyphosate altered levels of Zn 2+ and Fe 2+ in liver,

  In the present study we focused on hippocampal plasticity at the time of weaning, but future work would benefit from investigating brain changes in areas playing a key role in maternal care-giving behaviors (i. e. MPOA) at the time the care-giving is at its peak, early in the postpartum period.The present study used a relatively low dose compared to most other studies (usually ≥ 50 mg/kg/day) but high compared to authorized daily intake (ADI) in the USA (1.75 mg/kg/bw/day or in the European Union (0.5 mg/kg/bw/day). Further research investigating a range of doses on both the mother and offspring would be valuable to determine the extent of this exposure on neurobehavioral outcomes.It remains to be determined whetherGlyphosate or Roundup alters peripheral and central actions of gonadal and adrenal hormones in the mother. Previous research on human cell lines, human breast cancer cells and rodents have shown that glyphosate-based herbicides in formulation can disrupt steroid secretion 147-149 , estrogen receptors 150 and aromatase activity 151 albeit the exact mechanisms remains to determined. The physiological and behavioral effects of glyphosate observed in the present could be linked to endocrine disruption, and future work will investigate this possibility.

	Accepted Article paradigms have been developed to investigate toxicity of various compounds on offspring development and, rather surprisingly, potential effects on the dam's brain and behavior are maternal care-giving behaviors. Conclusion Our work adds to a growing body of research highlighting that low doses of Glyphosate and Roundup can impact vertebrate physiology. In the present study we show that Glyphosate alone or in formulation (Roundup), at a tenth of the currently accepted NOAEL, alters maternal behavior, neuronal plasticity and induces gut dysbiosis in the mother. Importantly, we also show that exposure to the active compound of Glyphosate or to the formulation can have different outcomes, potentially due to a direct action of adjuvants or to almost never investigated. Further research in this area is needed so that regulatory agencies can better evaluate the outcomes following exposure to Glyphosate and Glyphosate-based herbicides. FUNDING TDC was supported by La Région Bretagne (SAD), Rennes Métropole and the University of Rennes 1 (Défis Emergents 2018). JD is funded from the University of Rennes 1 (Bourse Accepted Article fléchée UR1).
	. .

the interaction of the adjuvants and Glyphosate. Taken together, this work points to the important role that environmental chemicals can have on mother. Most experimental

Table 1 .

 1 Summary of immunohistochemistry processing. NDS = Normal Donkey Serum, NGS = Normal Goat Serum, CG = cingulate gyrus.

	ccepted Article .

Table 2 . Mean (±SEM) of measures of toxicity (AST, ALT), weight and litter characteristics and serum analysis.

 2 GD= gestation day, PD = postpartum day, AST = alanine transaminase, AST= aspartate transaminase, Ca 2+ = calcium, Fe 2+ = iron, Mg 2+ = magnesium and Mn 2+ = manganese.

	. Hippocampal volume (mm 3 ) PSD95 Dorsal DG PSD95 Ventral DG PSD95 CA3 ccepted Article Table3. Mean (	Control 11.38 ± 0.61 0.01028 ± 0.0046 0.0095 ± 0.0030 0.0142 ± 0.0040 Glyphosate Roundup 11.10 ± 0.54 11.60 ± 0.36 0.0244 ± 0.0035 0.0309 ± 0.0052 0.0335 ± 0.0053 0.00922 ± 0.0036 0.0105 ± 0.0055 0.0206 ± 0.0052	F statistics F (2,16) = 0.263 F (2,16) = 0.429 F (2,16) = 0.954 F (2,16) = 1.701	p value 0.7718 0.6582 0.4059 0.2140

±SEM) of hippocampal volume and PSD95 optical density (OD) in the dentate gyrus (DG) and CA3 hippocampal regions.

ACKNOWLEDGEMENTS

We thank the Department of Biochimie du CHU Rennes and Michel Samson and the LERES for their assistance for analysis. We also thank Charlène Renoult, Niece Nmezu, Claire Montmasson and Florence Pontais for their technical contribution to this work. We also would like to thank the 2 anonymous reviewers for their extremely useful comments on the previous version of this article.


Hélène Andre

Céline Pimentel

Ségolène Veau

Mathilde Domin-Bernhard

Hélène Letur-Konirsch

Gérard Priou

Florence Eustache

Solène Vorilhon

Béatrice Delepine-Panisset

Patricia Fauque

Florence Scheffler

Annie Benhaim

Oxana Blagosklonov

Isabelle Koscinski

Célia Ravel

Pregnancies and obstetrical prognosis after oocyte donation in Turner Syndrome : A multicentric study

Introduction : Turner syndrome is one of the most frequent chromosomal abnormalities in women, with a prevalence estimated to be 1 of 2500 live birth. Pregnancy in women with Turner syndrome is known to be at high risk, whether it is spontaneous or after oocyte donation, because of miscarriages and potential cardio-vascular complications which can be life-threatening. All of these patients should therefore be screened with a comprehensive cardio-vascular assessment before pregnancy, and have a close follow-up during and after pregnancy.

Patients and methods : It is a retrospective study, conducted in 10 of the 27 French oocyte donation centers between 2012 and 2016, on all the patients presenting with Turner syndrome included in an oocyte donation program.

Results : 151 embryo transfers were realized in 73 patients, resulting in 39 pregnancies. Among these pregnancies, 24 children were born healthy, 11 spontaneous miscarriages, 3 voluntary abortions, 1 extra-uterine pregnancy and 1 maternal death from non-cardio-vascular origin occurred. Pregnancies were complicated by gravid arterial hypertension in 28.2 % of cases, preeclampsia in 10.3% of cases, and gestational diabetes in 7.7 % of cases.

Conclusion : This study bring out obstetrical complications of the same magnitude than the ones described in the literature. Lead over a period of 4 years, in 10 French oocyte donation centers, it doesn't reveal any cardio-vascular complications, conversely to other studies published before French and American recommendations. This study reinforces the usefulness of specific recommendations for the care of these particular patients.

INTRODUCTION

Turner syndrome was first described in 1938 by Henry Turner, an American endocrinologist [START_REF] Turner | Classic pages in obstetrics and gynecology by. A syndrome of infantilism, congenital webbed neck, and cubitus valgus[END_REF]. This congenital disorder is the main cause of hypergonadotropic hypogonadism in women. The most common features are a short stature and a premature ovarian failure from A C C E P T E D M A N U S C R I P T gonadic dysgenesis. Various morbidities can be associated, especially cardio-vascular and immune diseases. Apart from morbidity, mortality is also increased, as shown by a British and a Danish studies [START_REF] Swerdlow | Mortality and cancer incidence in persons with numerical sex chromosome abnormalities: a cohort study[END_REF][START_REF] Stochholm | Prevalence, Incidence, Diagnostic Delay, and Mortality in Turner Syndrome[END_REF].

Turner syndrome is one of the most frequent chromosomal abnormalities, with a prevalence of 1 live born girl in 2500 [START_REF] Cabrol | Le syndrome de Turner[END_REF]. It is due to the absence or structural abnormality of one of the X chromosome, with various degrees of mosaicism. Mosaicism attenuates the phenotype of Turner syndrome, especially the anthropometric data and the cardio-vascular morbidities [START_REF] Bucerzan | Clinical and genetic characteristics in a group of 45 patients with Turner syndrome (monocentric study)[END_REF][START_REF] Rosa | The Turner Syndrome Life Course Project: Karyotype-phenotype analyses across the lifespan[END_REF]. Diagnosis of mosaic forms is established 8 years after the monosomic ones [START_REF] El-Mansoury | Chromosomal mosaicism mitigates stigmata and cardiovascular risk factors in Turner syndrome[END_REF].

In Turner syndrome, migration and differentiation of germ cells seem to be done normally at the beginning of gestation, but their number significantly decreases in the middle of the pregnancy, probably by an apoptotic mechanism [START_REF] Modi | Accelerated germ cell apoptosis in sex chromosome aneuploid fetal human gonads[END_REF]. The decrease in the number of follicles could be due to an haplo-insufficiency of genes of the X chromosome which play a role in the ovarian maintenance [START_REF] Carrel | X-inactivation profile reveals extensive variability in X-linked gene expression in females[END_REF].

Spontaneous puberty seems to appear in 10 to 20 % of the patients with Turner syndrome, but less than 5 % of these patients have spontaneous periods [START_REF] Delbaere | Syndrome de Turner et don d'ovocytes[END_REF][START_REF] Hovatta | Fertility and pregnancy aspects in Turner syndrome[END_REF]. Spontaneous pregnancy can occur in 2 to 5 % of cases, especially when the syndrome is associated with a high degree of mosaicism [START_REF] Ayuso | Two fertile Turner women in a family[END_REF][START_REF] Birkebaek | Fertility and pregnancy outcome in Danish women with Turner syndrome[END_REF][START_REF] Bernard | Spontaneous fertility and pregnancy outcomes amongst 480 women with Turner syndrome[END_REF].

The ovarian reserve of these patients being low, it would be illusive to hope for good results after In Vitro Fertilization with their own oocytes. Therefore, patients who don't have spontaneous pregnancies can turn towards oocyte donation, which has permitted sensible improvement of their fertility. Oocyte donation is practiced in France since 1984. In 2007, 65% of women who benefited from this technique had a premature ovarian failure. Among them, 18,6 % had a Turner syndrome [START_REF] Letur | État des pratiques du don d'ovocytes en France et en Europe[END_REF]. However, procreation in a context of Turner syndrome is at high risk. Obstetrical prognosis is less good, with more miscarriages, hypertensive disorders, gestational diabetes and premature delivery. Moreover, the most worrying complications are cardio-vascular, because potentially life-threatening. Indeed, Turner syndrome is associated with congenital heart defects in 30 to 50 % of cases. The most common defects are bicuspid aortic valve, coarctation of the aorta and aortic root dilatation, which can lead to aortic dissection [START_REF] Karnis | Risk of death in pregnancy achieved through oocyte donation in patients with Turner syndrome: a national survey[END_REF][START_REF] Matura | Aortic Dilatation and Dissection in Turner Syndrome[END_REF][START_REF] Bondy | Congenital Cardiovascular Disease in Turner Syndrome[END_REF]. This is why French and American scientific societies edited in 2009 and 2012 specific recommendations for the screening of these patients, before, during and after pregnancy [START_REF]Collège national des gynécologues et obstetriciens francais -Recommandations pour la pratique clinique -Syndrome de Turner et grossesse[END_REF][START_REF]Practice Committee of the American Society for Reproductive Medicine. Increased maternal cardiovascular mortality associated with pregnancy in women with Turner syndrome[END_REF]. These recommendations were following the death of two patients with Turner syndrome who had been pregnant thanks to oocyte donation. European recommendations

A C C E P T E D M A N U S C R I P T
were also updated in 2017 [START_REF]Collège national des gynécologues et obstetriciens francais -Recommandations pour la pratique clinique -Syndrome de Turner et grossesse[END_REF]. We aimed to evaluate the impact of these recommendations on the management of Turner Syndrome patients in oocyte donation.

PATIENTS AND METHODS

Study : It was a multicenter, observational, retrospective study, driven in 10 of the 26 French centers practicing oocyte donation between 2012 and 2016. These centers were retrospectively asked for the results of embryo transfers after oocyte donation done for Turner patients. The aims of the study were to evaluate the success rates of oocyte donation in patients with Turner syndrome (pregnancy and live birth rates) and the obstetrical complications, since the publication of recommendations.

Patients : Patients included were 18 to 43 years old, presented a Turner syndrome, and had benefited from at least one embryo transfer after oocyte donation. The diagnosis of Turner syndrome was based on a cytogenetic analysis.

Oocyte donation : The oocytes of the donor were collected by a trans-vaginal puncture after ovarian stimulation and triggering of ovulation. The mature ones (whether they were fresh or frozen) were injected with sperm of the of the male partner of the infertile couple. Recipients received an endometrial preparation with oral or transdermal estradiol and micronized vaginal progesterone. After controlling the endometrial thickness, the embryos obtained were transferred one by one in the uterus of the recipient. The supernumerary embryos were frozen for a subsequent transfer. Recipients all benefited from a complete screening, following the recommendations.

Obstetrical outcomes : Absence of pregnancy was defined as a negative hCG level 11 days after embryo transfer. Biochemical pregnancy was defined as a positive hCG level without gestational bag during ultrasonography examination. Clinical pregnancy was defined as the presence of a gestational bag. Miscarriage was defined as the expulsion of an intrauterine pregnancy. Extra uterine pregnancy was defined as a pregnancy implanted outside the uterine cavity. Fetal death was defined as a spontaneous stopping of the cardiac activity after 14 weeks of pregnancy Pregnancy-induced hypertension was defined as new onset of hypertension of more than 140 mm Hg systolic and/or more than 90 mm Hg diastolic, after 20 weeks of pregnancy.

Preeclampsia was defined as pregnancy induced hypertension associated with proteinuria > 300 mg/24 h. Gestational diabetes was diagnosed by measuring fasting glucose in the first trimester of pregnancy, or after an OMS test (75g of glucose) between 24 and 28 weeks of pregnancy.

A C C E P T E D

M A N U S C R I P T
Statistical analysis : All data were analyzed using Excel® and XlStat®. Data are expressed as mean and percentages. Comparisons between groups were performed using Mann-Whitney and χ2 tests. A P value < 0.05 was considered statistically significant.

RESULTS

Studied population

73 patients were included. These patients benefited from 151 embryo transfers.

Characteristics of the patients are described in table 1.

The mean age during the first embryo transfer was 32.5 ± 4 years old. Among them, 67.1 % had a normal BMI, 25.5 % were overweight and 7.1 % were obese. The mean BMI was 23.69 ± 3.78 kg/m 2 .

Oocyte donation

Among the 151 embryo transferred, 112 did not result in pregnancy (74.2 %), 39 resulted in a clinical pregnancy (25.8 % of transfers and 53.4 % of patients).

The flow chart of the studied population is described in figure 1.

Among these 39 clinical pregnancies :

-11 resulted in a miscarriage (28.2 % of pregnancies) -1 was an extra uterine pregnancy (2.6 % of pregnancies) -23 resulted in a live birth (59 % of pregnancies and 15.2 % of transfers) of 25 newborns. 2 of these pregnancies were twin ones Obstetrical issues Among the live births, the mean term of delivery was 38 weeks and 4 days of amenorrhea.

One maternal death happened at 21 weeks of amenorrhea, after hospitalization for status epilepticus. This patient had a heart ultrasonography before pregnancy which found out a moderate aortic root dilatation which was not a contraindication of pregnancy.

Another pregnancy ended with a premature delivery at 23 weeks and 2 days of amenorrhea. It was complicated by a severe pregnancy induced hypertension, with thrombotic microangiopathy and the aggravation of a preexisting renal failure.

One of the twin pregnancies was complicated with a severe preeclampsia which required a pregnancy interruption at 18 weeks of amenorrhea. Another pregnancy interruption was performed at 18 weeks of amenorrhea because of the presence of an hygroma coli at the ultrasonography examination. Finally, a third pregnancy interruption was performed on a male fetus, with a karyotype 46,XY who presented a malformative association of the caudal pole (OEIS syndrome : omphalocele-exstrophy-imperforate anus-spinal defects).

A C C E P T E D

M A N U S C R I P T
Furthermore, 11 patients suffered from pregnancy-induced hypertension (28.2 % of pregnancies). Among them, 4 suffered from preeclampsia (10.3 %). 3 pregnancies were complicated by gestational diabetes (7.7 %). There was no case of aortic dissection, HELLP syndrome or eclampsia.

Characteristics of the patients according pregnancy

The Mann Whitney test did not found a significant statistical difference between the group who succeeded in having a pregnancy and the group who did not, according to the age (p = 0.74) and to the BMI (p = 0.73). A khi2 test was performed to study the relationship between karyotype and the chances of obtaining a pregnancy. There was no significant difference between the two groups. These results are described in table 2.

DISCUSSION

Here we described a cohort of 73 patients with Turner syndrome, in whom we retrospectively studied oocyte donation outcomes. The aim of this study was to evaluate the pregnancy rates and the evolution of obstetrical complications, after the publication of national recommendations. The clinical pregnancy rate was 53.4% of patients.

The prevalence of pregnancies in this multicenter French cohort was 25.8 % of embryo transfers. However, because of an important number of miscarriages, the rate of ongoing pregnancy falls to 15.2 % of embryo transfers.

Bodri et al., in a study driven in Spain and published in 2005, reported an ongoing pregnancy rate of 30 % of transfers. This elevated rate can be explained by a median number of transferred embryos of two [START_REF] Bodri | Oocyte donation in patients with Turner's syndrome: a successful technique but with an accompanying high risk of hypertensive disorders during pregnancy[END_REF]. Khatsgir et al., found an ongoing pregnancy rate of 19.1 % without any influence of age or chromosomal abnormalities [START_REF] Khastgir | Oocyte donation in Turner's syndrome: an analysis of the factors affecting the outcome[END_REF]. [START_REF] Karnis | Risk of death in pregnancy achieved through oocyte donation in patients with Turner syndrome: a national survey[END_REF], reported an ongoing pregnancy rate of 64.1 % and suggested that there could be a bias due to the answers to their survey [START_REF] Karnis | Risk of death in pregnancy achieved through oocyte donation in patients with Turner syndrome: a national survey[END_REF].

Regarding the pregnancy loss, the miscarriages rate in our study was 28.2 % of pregnancies (7.3 % of transfers). Khatsgir et al. reported in 1997 a miscarriage rate of 50 % of embryo transfers [START_REF] Khastgir | Oocyte donation in Turner's syndrome: an analysis of the factors affecting the outcome[END_REF], whereas Karnis et al obtained low levels (6.4 %), still with the hypothesis of a response bias [START_REF] Karnis | Risk of death in pregnancy achieved through oocyte donation in patients with Turner syndrome: a national survey[END_REF]. Bryman et al., in a study on 482 patients, concluded that the miscarriage rate was significantly lower in patients with Turner syndrome who had obtained a pregnancy with donor oocytes (26 %) than patients who had obtained a pregnancy with their own oocytes (45 %). This brings out the hypothesis of lower oocyte quality in Turner syndrome [START_REF] Bryman | Pregnancy rate and outcome in Swedish women with Turner syndrome[END_REF]. Moreover, whether pregnancies are spontaneous or obtained by Assisted Reproductive Technologies, the miscarriage rates remain higher than in the French general population

A C C E P T E D M A N U S C R I P T (15%)
. Some studies raised the hypothesis of abnormalities of endometrial receptivity inherent to the absence of some X-linked genes [START_REF] Yaron | Patients with Turner's syndrome may have an inherent endometrial abnormality affecting receptivity in oocyte donation[END_REF], associated to a small uterine volume and a reduced thickness of the endometrium [START_REF] Donnell | Physiological sex steroid replacement in premature ovarian failure: randomized crossover trial of effect on uterine volume, endometrial thickness and blood flow, compared with a standard regimen[END_REF]. Another explanation proposed by Abir et al.

would be a high prevalence of auto-immune diseases in these patients [START_REF] Abir | Turner's syndrome and fertility: current status and possible putative prospects[END_REF]. Furthermore, pregnancies in patients with Turner's syndrome are at high risk for complications, especially cardiovascular ones. In our study, we found a rate of pregnancyinduced hypertension of 28.2 % of pregnancies, and a preeclampsia rate of 10.3%, without HELLP syndrome. Hagman et al. studied 106 patients with Turner syndrome and pregnant after egg donation. Their rates of hypertension were 35 %, and 20.5 % of pregnancies were complicated by preeclampsia [START_REF] Hagman | Obstetric and neonatal outcome after oocyte donation in 106 women with Turner syndrome: a Nordic cohort study[END_REF]. Alvaro Mercadal et al. had found a preeclampsia rate of 40% in 10 pregnancies obtained after oocytes donation [START_REF] Mercadal | Pregnancy outcome after oocyte donation in patients with Turner's syndrome and partial X monosomy[END_REF]. Chevallier et al. studied 82 pregnancies after egg donation. The pregnancy rate was 17.1 % and the preeclampsia rate 22 % [START_REF] Chevalier | Materno-Fetal Cardiovascular Complications in Turner Syndrome after Oocyte Donation: Insufficient Prepregnancy Screening and Pregnancy Follow-Up Are Associated with Poor Outcome[END_REF]. Bernard et al., in a study of 480 patients with Turner's syndrome and spontaneously pregnant, found rates of hypertension and preeclampsia of 13.3 % and 6.7 %, respectively [START_REF] Bernard | Spontaneous fertility and pregnancy outcomes amongst 480 women with Turner syndrome[END_REF]. Thus, it appears that post-donation pregnancies are statistically more complicated than spontaneous pregnancies in patients with Turner syndrome. To explain this difference, several hypotheses can be mentioned. First of all, the patients who succeed in obtaining a spontaneous pregnancy are those who have a less severe phenotype, and therefore less cardiovascular pathologies. In addition, these women are probably younger than those who use egg donation, because of the difficulty of this journey. In our study, the average age at first embryo transfer was 32.5 years. It was 32 years old in the Hagman's study [START_REF] Hagman | Obstetric and neonatal outcome after oocyte donation in 106 women with Turner syndrome: a Nordic cohort study[END_REF] and 30.5 years old in Bernard's study [START_REF] Bernard | Spontaneous fertility and pregnancy outcomes amongst 480 women with Turner syndrome[END_REF]. Moreover, although pregnancies are spontaneous, women with Turner syndrome are more at risk of pregnancy-induced hypertensive diseases than the general population. This suggests that there is a risk associated with the syndrome itself. We can mention cardio-vascular or kidney diseases that predispose to hypertension. It has also been shown that patients with premature ovarian failure may present anti-zona pellucida and anti-granulosa cell autoantibodies. This could lead to abnormalities of dialogue between the trophoblastic cells and the endometrium, generating hypertensive pathologies [START_REF] Kelkar | Circulating autoantibodies against the zona pellucida and thyroid microsomal antigen in women with premature ovarian failure[END_REF]. Finally, independently of Turner syndrome, there is a risk of hypertensive pathologies inherent in oocyte donation. In an article published in 2017, Tarlatzi et al. compared 144 pregnancies obtained after oocyte donation to 144 pregnancies obtained after IVF or IVF-ICSI, in patients who did not have Turner syndrome. The former were associated with a significantly higher risk of pregnancy-induced hypertension and preeclampsia [START_REF] Tarlatzi | Does oocyte donation compared with autologous oocyte IVF pregnancies have a higher risk of preeclampsia?[END_REF]. Indeed, the donor is immunologically foreign to the mother, which could lead to an inadequate immune response

A C C E P T E D M A N U S C R I P T
between the mother and the fetus and lead to hypertensive pathologies related to a reduced trophoblastic invasion [START_REF] Salha | The influence of donated gametes on the incidence of hypertensive disorders of pregnancy[END_REF][START_REF] Ohl | Pathologies des grossesses issues de don d'ovocytes[END_REF][START_REF] Savasi | Maternal and fetal outcomes in oocyte donation pregnancies[END_REF]. There is also an endothelial dysfunction resulting from an imbalance between pro and anti-angiogenic factors [START_REF] Levine | Circulating Angiogenic Factors and the Risk of Preeclampsia[END_REF].

In addition to cardiovascular complications, the risk of liver disease or gestational diabetes is high. However, we did not identify any liver dysfunction. Among the 39 cases studied, 3 pregnancies were complicated by gestational diabetes (7.7 % of pregnancies). This result seems to be in agreement with the literature. In the Bryman et al. study, 5.3 % of patients had gestational diabetes [START_REF] Bryman | Pregnancy rate and outcome in Swedish women with Turner syndrome[END_REF], whereas Hagman et al. reported a gestational diabetes rate of 9.4 % [START_REF] Hagman | Obstetric and neonatal outcome after oocyte donation in 106 women with Turner syndrome: a Nordic cohort study[END_REF]. Finally, the maternal mortality rate in our study is 2.6%, with one patient dying after status epilepticus. This death did not follow cardiovascular complications, such as those described in the literature.

The limitations of our study come from a limited number of patients, Turner syndrome being a rare pathology, which do not lead systematically leading to egg donation. Furthermore, oocyte donation in France is often a long process, with an expectation that can last several years. Thus, it is possible that a significant number of couples whose wife is suffering from Turner syndrome give up the start of the procedures in France and benefit from egg donations abroad. In addition, only 10 of the 27 centers participating at that time in oocyte donation responded to our questionnaire. It is therefore a non-exhaustive investigation, although multicentric. There may be a response bias. If centers that did not respond are those with lower success rates or higher cardiovascular complications, this could lead to an underestimation of those complications or even mortality.

CONCLUSION

Pregnancies in women with Turner's syndrome are at high risk, whether obtained with their own oocytes or with those from a donor. These complications can be cardiovascular, and potentially life-threatening. The purpose of this study was to re-evaluate the pregnancy rates and obstetric complications in patients with Turner syndrome who achieved pregnancy after oocyte donation. This reassessment follows the publication of French and American recommendations in 2009 and 2012, after the demonstration of a mortality of cardiovascular cause increased in these patients during pregnancy. We found rates of pregnancy, miscarriage and obstetric complications similar to those described so far in the literature, and did not demonstrate cardio-vascular death. Our study reinforces the usefulness of specific recommendations for the care of these particular patients.

Figure 1 -

 1 Figure 1 -Flow chart of the study

Table 1 -

 1 Characteristics of the patients (n = 73)

	n = 73