Cécile L Bandet

Rana Mahfouz

Julien Véret

Athanassia Sotiropoulos

Maxime Poirier

Paola Giussani

Mélanie Campana

Erwann Philippe

Agnieszka Blachnio-Zabielska

Raphaelle Ballaire

Agnieszka Blachnio- Zabielska

Raphaëlle Ballaire

Xavier Le Liepvre

Olivier Bourron

Dušan Berkeš

Jan Górski

Pascal Ferré

Hervé Le Stunff

Fabienne Foufelle

Dr Eric Hajduch
email: eric.hajduch@crc.jussieu.fr

Ceramide Transporter CERT Is Involved in Muscle Insulin Signaling Defects Under Lipotoxic Conditions

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Introduction

A worldwide obesity and diabetes epidemic is spreading in humans for four decades now. It is concomitant with alterations of carbohydrate/lipid metabolism, particularly with dyslipidemia, and which have major consequences for cardiovascular diseases and insulin resistance (IR).

IR is a metabolic condition in which cells fail to respond to normal levels of insulin and is a key actor of type-2 diabetes (T2D). Numerous studies performed in animals and humans have demonstrated a strong relationship between IR and increased intramyocellular lipid content.

Ceramide has been described in many studies as the lipid species involved in muscle insulin resistance [START_REF] Hassan | Defect of insulin signal in peripheral tissues: Important role of ceramide[END_REF], although it is worth mentioning that other studies did not find such a relationship and rather privileged diacylglycerols [START_REF] Szendroedi | Role of diacylglycerol activation of PKCtheta in lipid-induced muscle insulin resistance in humans[END_REF].

According to various studies, skeletal muscle accounts for 30 to 70 % of insulin-stimulated glucose disposal in the postprandial state and is thus a primary target for ceramide anti-insulin action (3;4). In the context of visceral obesity, ceramides are primarily produced de novo from saturated FA (palmitate) [START_REF] Hassan | Defect of insulin signal in peripheral tissues: Important role of ceramide[END_REF]. This synthesis takes place in the ER and starts with the condensation of L-serine with palmitoyl-CoA to yield ceramides after several reactions.

Pioneering in vitro data showed an involvement of ceramides in the development of IR via the direct addition of these lipids on muscle and adipocyte cell lines [START_REF] Hajduch | Ceramide impairs the insulin-dependent membrane recruitment of protein kinase B leading to a loss in downstream signalling in L6 skeletal muscle cells[END_REF][START_REF] Summers | Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide[END_REF][START_REF] Wang | Effects of cell-permeable ceramides and tumor necrosis factor-alpha on insulin signaling and glucose uptake in 3T3-L1 adipocytes[END_REF]. Ceramides inhibit insulin-stimulated glucose uptake and glycogen synthesis by blocking insulin signaling at the level of both IRS1 and Akt [START_REF] Blouin | Plasma membrane subdomain compartmentalization contributes to distinct mechanisms of ceramide action on insulin signaling[END_REF][START_REF] Hajduch | Targeting of PKCzeta and PKB to caveolin-enriched microdomains represents a crucial step underpinning the disruption in PKB-directed signalling by ceramide[END_REF][START_REF] Mahfouz | Characterising the Inhibitory Actions of Ceramide upon Insulin Signaling in Different Skeletal Muscle Cell Models: A Mechanistic Insight[END_REF][START_REF] Hassan | Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE[END_REF]. These results indicate that saturated FAs in cells induce IR via ceramide synthesis.

Once ceramides are synthesized de novo in the ER, they are transported to the Golgi apparatus and metabolized into other sphingolipids such as sphingomyelin (SM) and glucosylceramides (GlcCer). The intracellular transport of ceramides from the ER to the Golgi involves both ATP-independent and -dependent specific carriers [START_REF] Yamaji | Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins[END_REF]. Ceramides intended to be metabolized into GlcCer at the cis side of the Golgi apparatus are transported via an ATP-independent vesicular carrier. This carrier is not well characterized, except that its activity is phosphatidylinositol-3-kinase dependent [START_REF] Yamaji | Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins[END_REF]. Whereas, to be processed into SM, ceramides are mainly transported from the ER to the Golgi via a non-vesicular ATP-dependent transporter called ceramide transporter (CERT) [START_REF] Yamaji | Sphingolipid metabolism and interorganellar transport: localization of sphingolipid enzymes and lipid transfer proteins[END_REF]. Through CERT, ceramides are extracted from the surface of the ER and transported towards the Golgi where they are metabolized into SM by SM-synthase-1 (SMS1).

Transformation of ceramide into SM may be a critical step in preventing negative actions of ceramides in cells. A metabolomic study demonstrated that reduced levels of plasma C16:1-SM species is predictive of T2D [START_REF] Floegel | Identification of serum metabolites associated with risk of type 2 diabetes using a targeted metabolomic approach[END_REF]. Inhibition of SMS in muscle cells induces a rise in ceramide content and impairs insulin signaling [START_REF] Park | A Role for Ceramides, but NOT Sphingomyelins, as antagonists of insulin signaling and mitochondrial metabolism in C2C12 myotubes[END_REF]. Obese and glucose intolerant individuals show increased muscle ceramide content and lower muscle SM compared to obese and normal glucose tolerant individuals [START_REF] Straczkowski | Increased skeletal muscle ceramide level in men at risk of developing type 2 diabetes[END_REF]. These data suggest that the biosynthesis of SM from ceramides could be protective for maintaining insulin sensitivity. Since CERT is involved in the transfer of ceramides to the Golgi for the synthesis of SM, we tested the hypothesis that modulation of CERT activity impacts muscle insulin signaling.

Research Design and Methods

Materials

Insulin, palmitate, and BSA were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Gedunin was from Tocris Bioscience (Bristol, UK). Broad caspase inhibitor (Q-VD-OPh) was from Merck Chemicals Ltd (Nottingham, UK). Antibodies against Akt, Akt Ser-473, Akt Thr-308, PKD Ser-916, GSK3α/β Ser-21/9, ERK Thr-202/Tyr-204, GAPDH, cleaved caspase-3 and -9 were from Cell Signaling (New England Biolabs, Ipswich, USA).

The antibody against CERT was from Bethyl Laboratories (Montgomery, TX, USA) and the one directed against β-actin from Sigma-Aldrich. Secondary horseradish peroxidase antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA, USA) and the chemiluminescent substrate from ThermoFisher Scientific (Waltham, MA USA).

Culture and transfection of C2C12 muscle cells

C2C12 myoblasts were grown and differentiated as myotubes as described previously [START_REF] Hassan | Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE[END_REF].

Cells were treated with palmitate or oleate conjugated to FA free BSA, as described [START_REF] Hassan | Endoplasmic reticulum stress does not mediate palmitate-induced insulin resistance in mouse and human muscle cells[END_REF].

Both HPA12 and gedunin were reconstituted in DMSO (0.4% final concentration). Control cells were incubated with the same quantity of DMSO. siRNA (25 nM) directed against CERT (Santa Cruz Biotechnology, Dallas, USA) or the same concentration of a non-specific siRNA were transfected for 96 h into C2C12 myotubes using the transfection reagent DharmaFECT (Dharmacon, Cambridge, UK). C2C12 myoblasts were seeded into 12-well plates and were transfected for 48h with a pEGFP N1/hCERT (a gift of Pr. Thierry Levade, Toulouse, France) or pCMV-GFP plasmids (1µg per well) using the Transfex transfection reagent (ATCC, Molsheim, France).

C2C12 glucose transport

Glucose transport was measured by incubating C2C12 myotubes with 10 µM [ 3 H] 2-deoxy-Dglucose (1 µCi/ml, 26.2 Ci/mmol) for 10 min as previously described [START_REF] Blair | Regulation of glucose transport and glycogen synthesis in L6 muscle cells during oxidative stress. Evidence for cross-talk between the insulin and SAPK2/p38 mitogen-activated protein kinase signaling pathways[END_REF].

Caspase activity

C2C12 myotubes were treated either with BSA (1.5%) or with palmitate (0.75 mM) complexed with BSA, in the presence or absence of the caspase inhibitor Q-VD-OPh (10µM) and the activity of caspase-3/-7 was measured 24 h later using the Apo-ONE kit Homogeneous Caspase-3/-7 Assay (Promega, Madison, USA).

CERT immunoprecipitation

C2C12 cells were lysed and CERT immunoprecipitated from 200 µg lysates using a CERT antibody. Immunocomplexes were captured by incubation with protein A-agarose beads and solubilized in Laemmli buffer prior to SDS-PAGE and immunoblotted.

[ 3 H]-sphingosine metabolism C2C12 myotubes were treated with 0.1 or 0.75 mM palmitate in the presence or absence of 10 µM N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)-dodecanamide (HPA12) [START_REF] Berkes | Chemistry and Biology of HPAs: A Family of Ceramide Trafficking Inhibitors[END_REF] for 16h at 37°C, then the cells were pulsed for 2 h with [C3-3 H]-sphingosine (0.3 µCi/ml) at 10 °C [START_REF] Giussani | Ceramide traffic in C6 glioma cells: evidence for CERT-dependent and independent transport from ER to the Golgi apparatus[END_REF].

Stock solutions of [ 3 H]Sphingosine in absolute ethanol were prepared and added to conditioned medium. The final concentration of ethanol never exceeded 0.1% (v/v). At the end of the pulse time total lipids were extracted and processed as previously described [START_REF] Giussani | Ceramide traffic in C6 glioma cells: evidence for CERT-dependent and independent transport from ER to the Golgi apparatus[END_REF].

The methanolyzed organic phase was analyzed by HPTLC using chloroform/methanol/water (55:20:3 by vol.) as solvent system. Digital autoradiography of HPTLC plates was performed with a Beta-Imager 2000 (Biospace, France) and radioactivity associated with individual lipids was determined using software provided with the instrument. The 3 H-labeled sphingolipids were recognized and identified as previously described [START_REF] Giussani | Ceramide traffic in C6 glioma cells: evidence for CERT-dependent and independent transport from ER to the Golgi apparatus[END_REF].

Animals

Male C57Bl6 mice (5 weeks old, Charles River Laboratories, Saint Germain Nuelle, France) were adapted to their environment for 1 week prior to the study. Mice were housed with a 12 h light/12 h dark cycle in a temperature-controlled environment and had free access to water and regular diet (energy: 65% carbohydrate, 11% fat, 24% protein) or a high fat diet (HFD) (EF D12492, energy: 21% carbohydrate, 60% fat, 19% protein, gross energy, 24.0 MJ/kg, ssniff Spezialdiäten GmbH, Soest, Germany) for 12 weeks. All procedures were approved by the Regional Ethics Committee for animal experiments no.5 of Ile-de-France (agreement no.

02852.03).

Electrogene transfer in mice

Mice were anesthetized with Aerane (Baxter) and their tibialis anterior muscles were injected with 8 U of hyaluronidase 2 h prior to the injection of 15 µg pEGFP N1/hCERT or 15 µg pCMV-GFP plasmids. Six 65 V/cm pulses of 60-ms, with a 100-ms interval, were applied [START_REF] Guerci | Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy[END_REF]. 14 days after gene delivery and before sacrifice, mice were injected or not with 0.75 UI/kg insulin (Actrapid, Novonordisk, La Défense, France) for 15 min. Then, muscles were collected under microscope. All experiments were conducted in accordance with European guidelines for the care and use of laboratory animals and were approved by the institutional animal care and use committee (agreement no. 00315.01).

Lipid extraction

Sphingomyelins and ceramide were extracted according to Bielawski et al [START_REF] Bielawski | Simultaneous quantitative analysis of bioactive sphingolipids by high-performance liquid chromatography-tandem mass spectrometry[END_REF]. Muscles were crushed in an Omni Bead Ruptor 24 apparatus (Omni International, Kennesaw, USA) with 950 µl of saline and circa twenty 1.4 mm OD zirconium oxide beads. An aliquot equivalent to 3 mg muscle (60 µl of lysate) was diluted with 1.94 ml saline and finally spiked with an internal standard mix containing 30 ng and 125 ng of d18:1/12:0 Ceramide and d18:1/12:0 SM respectively. Lipids were extracted with 2 ml of propanol2/water/ethyl acetate 30/10/60 for 30 min. After centrifugation (1100g, 5 min) the organic phase was kept and the aqueous phase further extracted. After centrifugation both organic phases were combined and evaporated to dryness under vacuum. Samples were solubilized with 200 µl of methanol and transferred to injection vials, again evaporated to dryness under vacuum and finally solubilized with 40 µl of methanol.

Quantification of ceramides and SM by LCMS

Ceramide analysis was carried out on a 1200 6460-QqQ LC-MS/MS system equipped with an ESI source (Agilent technologies, Les Ulis, France) as previously described [START_REF] Blondelle | Targeted Lipidomic Analysis of Myoblasts by GC-MS and LC-MS/MS[END_REF]. Samples were injected on a Poroshell C8 2.1x100 mm, 2.7 µm column (Agilent technologies) at a flow rate of 0,3 ml/min, 50°C, and separation was achieved with a linear gradient of (solvent A) formic acid/ammonium formate (0,2 % /1 mM final concentrations) and (solvent B) methanol containing formic acid/ammonium formate 1 mM . Acquisition was performed in positive Single Reaction Monitoring (SRM) mode. Relative quantitation of ceramide related compounds was performed by calculating the response ratio of the considered ceramide to d18:1/12:0 Ceramide, used as internal standard. Two microliter samples were used for quantitation of sphingomyelins.

Human skeletal muscle cells

Biopsies from lean healthy adult volunteers were obtained in the context of agreed preclinical and clinical experiences [START_REF] Vilquin | Normal growth and regenerating ability of myoblasts from unaffected muscles of facioscapulohumeral muscular dystrophy patients[END_REF] via the Tissue Bank for Research (Myobank) of the French Association against Myopathies (AFM) in agreement with the French bioethical law (Law No 94-654 of 29 July 1994, amended 22 January 2002). Samples of patients with type-2 diabetes were obtained from healthy tissue after leg amputation on informed consent. Ethical approval for the use of human muscle tissue was given by the Ethics Committee of Pitié-Salpêtrière hospital (CPP-Ile de France VI-Paris, France). Fresh muscle samples were sliced and dissociated in collagenase. Satellite cells were purified, cultured and differentiated into myotubes, as previously described [START_REF] Hassan | Endoplasmic reticulum stress does not mediate palmitate-induced insulin resistance in mouse and human muscle cells[END_REF].

Preparation of whole cell lysates

Cells were lysed following experimental manipulation (see figure legends) in an appropriate volume of lysis buffer and frozen at -80°C until required [START_REF] Hajduch | Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells[END_REF].

Real time quantitative RT-PCR

Total RNA was extracted from muscle cells and real-time quantitative RT-PCR analyses were performed as described previously [START_REF] Hassan | Endoplasmic reticulum stress does not mediate palmitate-induced insulin resistance in mouse and human muscle cells[END_REF]. One microgram RNA was retro-transcribed using Superscript II (Invitrogen, Carlsbad, USA). Sequences of sense and antisense primers of the gene to be amplified (CERT) are respectively 5'-TCTGCTTATCTCCTGGTCTCCC-3' and 5'-CGAATCAAGCCAGCCTTGAC-3'.

Immunoblotting

Frozen tissues or cells were homogenized following experimental manipulation in an appropriate volume of lysis buffer, and cell lysates were subjected to SDS/PAGE and immunoblotted as previously described [START_REF] Hajduch | Constitutive activation of protein kinase B alpha by membrane targeting promotes glucose and system A amino acid transport, protein synthesis, and inactivation of glycogen synthase kinase 3 in L6 muscle cells[END_REF].

Statistics

Data were analyzed with GraphPad Prism 6.07 by unpaired or paired two-tailed t-test when two groups were compared, and one way Anova followed by Bonferroni's multiple comparison tests when more than two groups were compared. p value <0.05 was considered significant.

Results

CERT expression is altered in lipotoxic conditions in muscle cells.

Palmitate treatment (0.75 mM) for 16 h induced a 50% decrease in CERT protein expression (fig. 1A), concomitantly to a 60% increase in total ceramide content (fig. 1B) and a 35% increase in total SM content (fig. 1B) in C2C12 myotubes. In gastrocnemius muscle lysates from mice fed a HFD (12 weeks) we also observed a 58% decrease in CERT protein content (fig. 1C) and a 28% increase in total ceramide content (fig. 1D) when compared to controls.

However, no difference in total SM content was observed between both groups (fig. 1D). We then studied human myotubes differentiated from human satellite cells obtained from either insulin sensitive or T2D donors [START_REF] Mahfouz | Characterising the Inhibitory Actions of Ceramide upon Insulin Signaling in Different Skeletal Muscle Cell Models: A Mechanistic Insight[END_REF]. Figure 1E shows that insulin-stimulated Akt phosphorylation in myotubes derived from muscles from T2D patients was drastically reduced compared to non-diabetic myotubes. Interestingly, a concomitant decrease in CERT expression was observed in T2D patient myotubes compared to control myotubes (fig. 1E).

Next, we tested whether the decrease in insulin-induced Akt phosphorylation usually observed after 16 h palmitate exposure (10;11;16) was concomitant to a decreased CERT expression. C2C12 cells were treated with palmitate up to 16 h and insulin-induced Akt phosphorylation and CERT expression were assessed in the same time-frame. Supplemental fig. 1A shows that palmitate needed 16 h to induce both defect in insulin signaling (decrease in Akt phosphorylation in response to insulin) and decreased CERT protein content. CERT mRNA levels were not decreased after 16 h palmitate incubation in C2C12 myotubes (supplemental fig. 1B) and in muscle of mice fed a HFD compared to control mouse muscles (supplemental fig. 1C), suggesting that the alteration of CERT observed in lipotoxic conditions was post-transcriptional.

Then we tested whether palmitate could act through ceramide production to alter CERT protein expression in muscle cells. C2C12 myotubes were treated with palmitate in the presence of myriocin (inhibitor of the first enzyme of ceramide biosynthesis) for 16h before assessing CERT expression. Decreased CERT protein content observed after palmitate treatment (fig. 1F) was concomitant to an increase in ceramide content in cells (fig. 1G).

Interestingly, both the decreased-expression of CERT and the increased-ceramide content observed in response to palmitate were completely abrogated in the presence of myriocin (fig. 1F-G), suggesting that ceramides produced from palmitate were accountable for the observed CERT alteration in muscle cells.

The type of free FA, saturated or unsaturated, is critical for the development of insulin resistance. While saturated FAs induce insulin resistance (25;26), unsaturated FAs have no deleterious effect and even protect cells from the negative action of saturated FAs [START_REF] Dimopoulos | Differential effects of palmitate and palmitoleate on insulin action and glucose utilization in rat L6 skeletal muscle cells[END_REF][START_REF] Listenberger | Triglyceride accumulation protects against fatty acid-induced lipotoxicity[END_REF][START_REF] Ryan | Diabetes and the Mediterranean diet: a beneficial effect of oleic acid on insulin sensitivity, adipocyte glucose transport and endothelium-dependent vasoreactivity[END_REF]. To determine whether unsaturated FAs exert a protective effect on the expression of CERT in the presence of palmitate, C2C12 myotubes were incubated with palmitate, oleate or linoleate. Supplemental fig. 2A shows that while palmitate altered CERT expression, the other two unsaturated FA displayed no significant effects. More interestingly, treatment of C2C12 myotubes with both oleate and palmitate together protected cells against the harmful effect of the latter on CERT expression (supplemental fig. 2B).

Overall, these data demonstrate that lipotoxic conditions negatively regulate CERT content and activity in muscle cells.

Influence of the modulation of CERT activity/expression on muscle cell insulin signaling in vitro.

We next determined whether an artificial reduction of CERT function could potentiate palmitate-induced defects in insulin signaling in myotubes. We used a concentration of palmitate (0.1 mM) that had a minimal impact on total ceramide content (supplemental fig. 3) and CERT expression (fig. 2B). We inhibited the activity of CERT in muscle cells using a CERT inhibitor called N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)-dodecanamide (HPA12) [START_REF] Berkes | Chemistry and Biology of HPAs: A Family of Ceramide Trafficking Inhibitors[END_REF]. HPA12 inhibited CERT activity through its interaction with the START domain of CERT that usually binds ceramides [START_REF] Kudo | Crystal structures of the CERT START domain with inhibitors provide insights into the mechanism of ceramide transfer[END_REF]. HPA12 treatment enhanced the ceramide concentration induced by a low concentration of palmitate (supplemental fig. 3), suggesting that the inhibition of CERT activity prevented the ceramide produced in the ER to be metabolized into SM in the Golgi. To evaluate the effect of both palmitate and HPA12 on ceramide utilization for the biosynthesis of SM, we studied ceramide metabolism using [ 3 H]sphingosine as a metabolic precursor, as it is rapidly internalized into cells and N-acylated to ceramide and then metabolized to form SM and GlcCer [START_REF] Gjoni | Glucolipotoxicity impairs ceramide flow from the endoplasmic reticulum to the Golgi apparatus in INS-1 beta-cells[END_REF]. The experiment was performed at 10 °C, a non-permissive temperature for the ER-to-Golgi vesicle flow, allowing to assess essentially a CERT dependent transport. At that temperature and after a short time pulse, we found comparable levels of radioactivity incorporated in control and palmitate-treated cells (data not shown) and most of the radioactivity remained associated with ceramides (fig. 2A).

A 0.1 mM or 0.75 mM palmitate treatment inhibited synthesized [ 3 H]-SM levels by 31% and 70% respectively (fig. 2A), but did not affect GlcCer biosynthesis (fig. 2A). HPA12 treatment mimicked high concentration levels of palmitate and blocked the conversion of ceramide into SM. Addition of 0.75 mM palmitate did not enhance the negative action of HPA12 (fig. 2A).

Taken together, the data demonstrate that both high palmitate concentration and HPA12 inhibit SM biosynthesis in the Golgi apparatus. We then assessed insulin signaling. At 0.1 mM, palmitate did not affect CERT expression (fig. 2B), but only partially blocked SM biosynthesis (fig. 2A) and did not inhibit insulin-induced Akt phosphorylation (fig. 2B).

However, at the same concentration of palmitate, but in the presence of HPA12, we observed a complete inhibition of ceramide transport from the ER to the Golgi (fig. 2A) and an accentuated inhibitory action of the lipid on insulin signaling (fig. 2B). Interestingly, at 0.75mM, the negative effect of palmitate on CERT expression and SM biosynthesis was maximal, and HPA12 did not potentiate any further the deleterious action of the lipid on Akt phosphorylation (fig. 2B).

Similar results were obtained by using another CERT inhibitor, gedunin which inhibited CERT-mediated extraction of ceramides from the ER membranes [START_REF] Hullin-Matsuda | Limonoid compounds inhibit sphingomyelin biosynthesis by preventing CERT proteindependent extraction of ceramides from the endoplasmic reticulum[END_REF]. Figure 2C shows that, like HPA12, gedunin unmasked the inhibitory action of 0.1 mM palmitate on insulin signaling.

To confirm the importance of the negative effect of a decreased-CERT activity on insulin signaling in muscle cells, we used a small interfering-RNA directed against CERT (siCERT).

Figure 3A-B shows that siCERT decreased CERT mRNA and protein content in C2C12 myotubes. Figure 3B demonstrates that 0.1mM palmitate induced only a slight reduction in CERT protein content and had no significant effect on Akt phosphorylation. However, in cells transfected with siCERT, CERT protein was strongly reduced (fig. 3B), along with an increased action of palmitate on insulin-induced Akt phosphorylation, confirming that the absence of CERT expression unmasks the action of a low palmitate concentration on insulin signaling (fig. 3B).

To demonstrate that a correct ceramide transport from the ER to the Golgi apparatus is essential in preventing the inhibitory effect of ceramides on insulin signaling, we overexpressed CERT in C2C12 myoblasts before treating them with palmitate and insulin.

For this experiment, we used myoblasts instead of myotubes because of their higher transfection efficiency. Endogenous CERT expression was identical before and after C2C12 myoblast differentiation into myotubes (data not shown). A 16 h palmitate exposure induced a 7-fold increase in total ceramide content in C2C12 myoblasts (fig. 4A) (a much higher increase than in myotubes, e.g. fig. 1B). CERT overexpression, however, reduced the total ceramide increase in response to palmitate to 4.1-fold (fig. 4A). Overexpression of an exogenous CERT prevented endogenous CERT downregulation in response to a high palmitate concentration (fig. 4B). In addition, CERT overexpression also counteracted palmitate's deleterious action on insulin signaling. Indeed, palmitate induced an inhibition of the insulin-induced phosphorylation of Akt, GSK3 and ERK. CERT re-expression, however, induced a major improvement of insulin signaling (fig. 4B).

Mechanism of alteration of CERT action in lipotoxic conditions: a PKD and caspasedependent mechanism.

CERT function is downregulated by phosphorylation on its serine-132 residue by protein kinase D (PKD). PKD is activated by phosphorylation of its serine-960 residue in response to various stresses [START_REF] Franz-Wachtel | Global detection of protein kinase D-dependent phosphorylation events in nocodazole-treated human cells[END_REF], and CERT phosphorylation on serine-132 by PKD decreases CERT affinity to phosphatidylinositol-4-phosphate in the Golgi, thus reducing ceramide transfer activity [START_REF] Fugmann | Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein[END_REF]. Palmitate induced both phosphorylation of PKD on its serine-960 residue (Supplemental fig. 4A) and CERT on its serine-132 residue (Supplemental fig. 4B). Treatment of cells with a PKD inhibitor (kb-NB-142-70), however, reduced palmitate-induced CERT phosphorylation (Supplemental fig. 4B).

A recent study showed that CERT can be cleaved by caspases during pro-apoptotic stress in HeLa cells, resulting in a loss of function of CERT and a decrease in SM de novo synthesis in the Golgi [START_REF] Chandran | Inactivation of ceramide transfer protein during pro-apoptotic stress by Golgi disassembly and caspase cleavage[END_REF]. Since palmitate activates both caspase-3 and -9 in C2C12 myotubes [START_REF] Peterson | Bax signaling regulates palmitate-mediated apoptosis in C(2)C(12) myotubes[END_REF], we wondered whether a similar mechanism could occur in our muscle cell model. To test this hypothesis, we treated C2C12 myotubes with palmitate for 16h in the presence or absence of a broad caspase inhibitor (Q-VD-OPh) [START_REF] Caserta | Q-VD-OPh, a broad spectrum caspase inhibitor with potent antiapoptotic properties[END_REF]. Palmitate induced the cleavage of both caspases-3/-9 (fig. 5A) and activity (fig. 5B) in C2C12 myotubes, together with a loss of insulin response (fig. 5C). Interestingly, Q-VD-OPh, which blocks caspase activity in the presence of palmitate (Fig. 5B), prevented the alteration of CERT expression in response to palmitate (Fig. 5C), and improved insulin signaling (fig. 5C).

We next evaluated whether changes in CERT expression have consequences on glucose metabolism downstream of insulin signaling. Insulin induced a 40% increase in glucose transport in C2C12 myotubes (fig. 6). If at 0.1mM, palmitate had no significant effect on insulin-stimulated glucose transport, in the presence of HPA12, palmitate inhibited the insulin-induced stimulation of glucose transport (fig. 6). This result confirms the importance of an active ceramide transport to counteract the action of palmitate on insulin signaling. The stimulation of insulin was completely lost when cells were pre-treated with high palmitate concentrations (fig. 6). The caspase inhibitor, Q-VD-OPh, however, prevented the inhibition by palmitate of insulin-stimulated glucose transport (fig. 6).

Influence of CERT overexpression on muscle insulin sensitivity and ceramide content in vivo.

We fed mice a HFD for 10 weeks and 2 weeks before the end of the diet, we overexpressed a CERT-GFP construct through electrogene-transfer [START_REF] Guerci | Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy[END_REF] in the tibialis anterior muscle of the left leg of the animals (fig. 7A). In the right leg, a GFP construct was transferred. Two weeks later we sacrificed the mice and isolated tibialis anterior muscles that were overexpressing CERT (GFP-CERT visible under fluorescence microscope, fig. 7A). Ceramide species contents (except for C18 and C20) were decreased up to 30% in CERT-overexpressing muscle fibers (fig. 7B) demonstrating that CERT overexpression counteracts ceramide accumulation in muscles. We also observed a 10% decrease in total ceramide content (although at p<0.0533). In addition, both caspases-3/-9 were cleaved in HFD muscle mice, and CERT overexpression completely abrogated caspase cleavages (fig. 7C), indicating a decrease in lipotoxicity-induced caspase activation in vivo. Next, we assessed insulin signaling in these muscle fibers. CERT overexpression in the tibialis anterior muscle prevented endogenous CERT degradation observed in response to lipotoxicity and improved significantly the poor in vivo stimulation by insulin of Akt, GSK3 and ERK observed in HFD treated mice (fig. 7D).

Discussion

In the present study, we demonstrate that CERT plays a pivotal role for the control of ceramide content and insulin response in muscle cells. In lipotoxic conditions, i.e. in the presence of saturated FA, a decrease in CERT content induces ceramide accumulation in cells through a defective ceramide transport from the ER to the Golgi. This leads to an inhibition of SM synthesis and a concomitant loss in insulin response. Importantly, a direct inhibition of CERT expression/activity has an effect on ceramide content and insulin signaling similar to lipotoxic conditions. Conversely, an increased CERT expression in vitro and in vivo counteracts the deleterious effects of lipotoxic conditions on muscle insulin signaling. As a reflect of insulin action we show here mainly AKT, GSK3 and ERK phosphorylation. Defects in insulin mediated phosphorylation of these targets are usually concomitant with a loss of tissue insulin sensitivity [START_REF] Manning | AKT/PKB Signaling: Navigating the Network[END_REF], although a number of studies show a dissociation between Akt phosphorylation and insulin sensitivity [START_REF] Fazakerley | Distinct patterns of tissue-specific lipid accumulation during the induction of insulin resistance in mice by high-fat feeding[END_REF](40)[START_REF] Hoehn | IRS1-independent defects define major nodes of insulin resistance[END_REF]. Thus, it remains to directly demonstrate that CERT activity/expression modulation translates in vivo into functional changes of muscle glucose metabolism.

We demonstrate here the direct involvement of CERT in the transport of ceramides from the ER to the Golgi and in their synthesis into SM. However, the fact that the GlcCer content did not change with either palmitate or CERT inhibitor confirms that GlcCers are coming from ceramide transported to the Golgi through the vesicular transport, not through CERT [START_REF] Ishibashi | New insights on glucosylated lipids: metabolism and functions[END_REF]. It is important to stress that we do not observe a decrease in total SM content in cells in lipotoxic condition (fig. 1A-B) since the total SM content exceeded the total ceramide content by more than 20-fold in muscle cells (unpublished data).

It appears that a targeted decrease in some ceramide species (C16, C22, C24:1 and C24) without a statistically significant change in total ceramide content was sufficient to modulate the insulin response of the cells in lipotoxic conditions. C16-ceramides have been demonstrated to attenuate the hepatic insulin response (43;44), thus similar ceramide species could also mediate lipotoxicity in muscle cells. However, and in opposite of what was suggested in some studies (40;45), C18-ceramides did not seem to play a role in the inhibition of insulin response in our experimental models. At present we have no explanation for this discrepancy. It is likely that, depending on the relative abundance of specific FA in the HFD, ceramide species are affected differently.

Processing of newly synthetized ceramides to give more complex sphingolipid derivatives such as SM, GlcCer and complex glycosphingolipids (e.g. gangliosides) [START_REF] Perry | Molecular mechanisms and regulation of ceramide transport[END_REF], occurs in the Golgi apparatus. Therefore, an efficient, rapid and regulated transport is required since the t 1/2 for spontaneous interbilayer movement of ceramide is in the order of days [START_REF] Simon | AR: Exchange of C(16)-ceramide between phospholipid vesicles[END_REF]. In mammalian cells, transport of ceramides from the ER to the Golgi occurs through two different mechanisms; vesicular and non-vesicular. In yeast, it has been shown that the nonvesicular CERT-dependent transport contributes to 50% of transport ceramide from the ER to the Golgi [START_REF] Funato | Vesicular and nonvesicular transport of ceramide from ER to the Golgi apparatus in yeast[END_REF]. Our data demonstrate that a default in CERT content in response to lipotoxicity is enough to stop the conversion of ceramide to SM in the Golgi and thus to increase ceramide concentration in cells and to trigger their negative action on insulin signaling. This suggests that the loss in CERT-dependent transport of ceramides from the ER to the Golgi is not compensated by the vesicular transport. However, lack of knowledge about the mechanism by which this ceramide vesicular transport is regulated precludes any conclusion concerning an effect of lipotoxicity on its function.

A key and original result is that, in lipotoxic conditions, CERT function is modulated through two mechanisms. Firstly, palmitate inhibits CERT activity through phosphorylation on its serine-132 residue by PKD in muscle cells. The importance of this mechanism for the control of CERT activity has already been demonstrated in rat islet β-cells where high dose palmitate treatment increased PKD-induced phosphorylation of CERT and its dysfunction, and deleterious effects on islet β-cells [START_REF] Guo | Palmitate-induced inhibition of insulin gene expression in rat islet beta-cells involves the ceramide transport protein[END_REF]. Secondly, CERT protein content is strongly reduced in lipotoxic conditions, and the decreased protein content is secondary to an activation of caspases. The underlying mechanism is not completely resolved but is likely to be mediated by ceramides. Indeed, decreased CERT expression did not occur when the de novo ceramide biosynthesis pathway was inhibited (fig. 1F). In addition, when CERT was overexpressed in diabetic mouse muscle, ceramide content was decreased (fig. 7B) and lipotoxicity-induced caspase cleavage was abrogated (fig. 7C). It is likely that the caspase-dependent inhibition of CERT activity prevailed over the PKD-dependent one. Indeed, inhibition of caspase activity restored on its own insulin signaling (fig. 5C).

In lipotoxic conditions, GFP-CERT-overexpressing muscle cells displayed higher endogenous CERT concentrations compared to control muscle cells (fig. 4B and7D). This difference in expression could be explained by an enhanced ceramide transport from the ER to the Golgi in CERT-overexpressing cells, resulting in less ceramide accumulated and a decrease in ceramide-induced caspase activation. Thus CERT plays a crucial role in the regulation of sphingolipid metabolism in lipotoxic conditions. Changes in CERT expression observed in our study are not unprecedented. Indeed, another study has shown that a pro-apoptotic stress (induced by TNFα) can also result in an inactivation of CERT via its cleavage by caspases in HeLa cells [START_REF] Chandran | Inactivation of ceramide transfer protein during pro-apoptotic stress by Golgi disassembly and caspase cleavage[END_REF] and results in a decreased biosynthesis of SM [START_REF] Chandran | Inactivation of ceramide transfer protein during pro-apoptotic stress by Golgi disassembly and caspase cleavage[END_REF]. Interestingly, another study demonstrated that the inhibition of the de novo ceramide biosynthesis prevents caspase-3 activation in response to palmitate in L6 myotubes [START_REF] Turpin | Apoptosis in skeletal muscle myotubes is induced by ceramides and is positively related to insulin resistance[END_REF]. As we have observed (fig. 5), the authors of the study also showed that inhibition of caspase-3 partially improves insulin-stimulated glucose uptake in palmitate-treated L6 myotubes [START_REF] Turpin | Apoptosis in skeletal muscle myotubes is induced by ceramides and is positively related to insulin resistance[END_REF]. They did not explain, however, how ceramide-activated caspases could affect insulin signaling but they showed that it is not through proteolysis of insulin signaling proteins [START_REF] Turpin | Apoptosis in skeletal muscle myotubes is induced by ceramides and is positively related to insulin resistance[END_REF]. Very recently a study conducted in yeast also demonstrated that a non-vesicular ceramide transfer out of the ER prevents the buildup of ceramide content [START_REF] Liu | An inducible ER-Golgi tether facilitates ceramide transport to alleviate lipotoxicity[END_REF]. Although S. cerevisiae do not express a CERT homologue, under certain conditions, the protein Nvj2p can play a similar role by facilitating lipid exchange between the ER and the Golgi. During ER stress or ceramide overproduction, Nvj2p relocalizes to and increases ERmedial Golgi contacts, facilitating ceramide exit from the ER and preventing ceramide toxic accumulation [START_REF] Liu | An inducible ER-Golgi tether facilitates ceramide transport to alleviate lipotoxicity[END_REF]. All of these data strengthen our results in mammalian cells by demonstrating the importance of a functional ceramide transport from the ER to the Golgi to prevent lipotoxicity. Nevertheless, it will be particularly important to completely elucidate CERT regulation in response to an excess of FA.

We previously showed that the mechanisms inhibiting insulin action in the presence of ceramides take place at the plasma membrane (PM) (8;10). A crucial question thus remains:

during lipotoxicity, what is the mechanism linking ER-accumulated ceramides to their effects at the PM? One hypothesis would be that ceramides could directly transfer from the ER to the PM at specialized membrane contact sites (MCS). MCS are regions of close apposition between two cellular membranes, and the ER can form MCS with virtually any other organelle within cells. Based on electron microscopy, the ER also has contacts with the PM [START_REF] Perry | Molecular mechanisms and regulation of ceramide transport[END_REF]. ER-PM MCS exist in striated muscles between the T-tubule (a specialized plasma membrane network) and the sarcoplasmic reticulum [START_REF] Fernandez-Busnadiego | Supramolecular architecture of endoplasmic reticulum-plasma membrane contact sites[END_REF]. Interestingly, most of the insulin-regulated glucose transporter GLUT4, gets translocated into T-tubules in response to the hormone [START_REF] Ploug | Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions[END_REF]. As such, ER-T-tubule MCS could form important hubs for lipid metabolism and exchange between membranes. To date, however, no information on sphingolipid transfer from the ER to the PM by such a mechanism is available.

In summary, our study shows that ceramide transport from the ER to the Golgi is altered in lipotoxic conditions and that its artificial recovery prevents ceramide to accumulate and to act negatively on insulin signaling. These results could open avenues to identify new therapeutic targets allowing the amelioration of insulin sensitivity. access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

  [ 3 H]-2deoxy-D-glucose (26.2 Ci/mmol) and D-erythro-[3-3 H]-sphingosine (Sph) (18.6 Ci/mmol) were from PerkinElmer Life Science (Boston, MA, USA). High performance thin layer chromatography (HPTLC) silica gel plates were from Merck (Darmstadt, Germany). Lipid internal standards (d18:1-12:0 Ceramide, d18:1-12:0 sphingomyelin and d17:1-Sphingosine-1-Phosphate) were obtained from Avanti Polar Lipids (Coger SAS, Paris, France). LCMSMS quality grade solvents were purchased from Fischer Scientific (Illkirch, France).

Figure 1 :

 1 Figure 1: Effect of lipotoxicity on CERT expression in muscle cells.

Figure 2 :

 2 Figure 2: Effect of palmitate and CERT inhibitors on SM synthesis and insulin sensitivity in

Figure 3 :

 3 Figure 3: Effect of CERT siRNA on palmitate-induced IR in C2C12 myotubes.

Figure 4 :

 4 Figure 4: Effect of CERT overexpression on palmitate-induced IR in C2C12 myoblasts.

Figure 5 :

 5 Figure 5: Involvement of caspases in the regulation of CERT expression and insulin

Figure 6 :

 6 Figure 6: Effects of modulating CERT activity on insulin-induced glucose uptake in C2C12

Figure 7 :

 7 Figure 7: Consequences of CERT overexpression on insulin signaling and ceramide content in

Figure 1 180x238mm

 1 Figure 1 180x238mm (300 x 300 DPI)

Figure 2 180x230mm

 2 Figure 2 180x230mm (300 x 300 DPI)

Figure 3 90x123mm

 3 Figure 3 90x123mm (300 x 300 DPI)

Figure 4 180x241mm

 4 Figure 4 180x241mm (300 x 300 DPI)

Figure 5 180x224mm

 5 Figure 5 180x224mm (300 x 300 DPI)

Figure 6 90x107mm

 6 Figure 6 90x107mm (300 x 300 DPI)

Figure 7 180x236mm 1 :

 71 Figure 7 180x236mm (300 x 300 DPI)

(

  A) C2C12 myotubes were treated with 0.75 mM palmitate (Pal) from 8h to 16 h, then with insulin during the last 10 min. Lysates were immunoblotted with the indicated antibodies. Blots were quantified and are shown as means +/-SEM (n=6). *Significant change (*p<0.05) relative to insulin alone. Quantification of CERT mRNA levels was performed by qRT-PCR in C2C12 myotubes treated with 0.75 mM palmitate for 16 h (n=6) (B) or in muscles of high fat diet (HFD) and chow diet (control) mice (n=4 to 6) (C). Results are mean +/-SEM. Supplemental figure 2: Effects of saturated and unsaturated fatty acids on CERT protein content in C2C12 myotubes. (A) C2C12 myotubes were treated for 16 h with 0.3 mM oleate, 0.75 mM palmitate (Pal) or 0.3 mM linoleate (Lino) before being lysed. Histograms show densitometric quantification of CERT. Results are means +/-SEM (n=5). *Significant change (* p<0.05) relative to untreated cells. (B) C2C12 myotubes were treated with 0.3 µM oleate in the presence or absence of 0.75 mM palmitate (Pal) before being lysed. All cell lysates were immunoblotted with the indicated antibodies. Histograms show densitometric quantification of CERT. Results are means +/-SEM (n=4). *Significant change (* p<0.05) relative to control and a ( a <0.05) relative to palmitate treated myotubes.

Acknowledgments:

We are obliged to N. Venteclef (INSERM U1138) for his constructive comments and to F. Hajduch for professional editing. We would like to thank J.-T. Vilquin (UPMC UM76, Groupe Hospitalier Pitié-Salpêtrière, Paris, France), F. Koskas, J. Gaudric, C. Goulfier, C. Jouhannet and T. Khalife (Service de Chirurgie Vasculaire, AP-HP, Hôpital Pitié-Salpêtrière , Paris, France) for providing the human muscle samples. We are grateful for the technical assistance of the staff of the Centre d'Exploration Fonctionnelle (Cordeliers Research Centre and Faculté de médecine -Sorbonne Université, Paris, France). We thank J.-P. Pais de Barros (INSERM UMR866, Université de Bourgogne, Dijon, France) for the assessment of sphingolipids. We are grateful to the tissue bank of the Association Française des Myopathies for control human biopsy samples. This work was supported by INSERM, the Société Francophone du Diabète, the Agence Nationale de la Recherche (ANR) project (ANR 11 BSV1 03101-Crisalis) and the Fondation pour la Recherche Médicale (équipe FRM DEQ20140329504). This study was supported by Piano di sostegno alla ricerca BIOMETRA -Linea B (grant 15-6-3003005-9) to P. G. C. B. who is the recipient of a doctoral fellowship from Sorbonne Université. Eric Hajduch is the guarantor of this work and, as such, had full

Abbreviations

(B) C2C12 myotubes were incubated with 0.75 mM palmitate for 16 h, with or without 10 µM PKD inhibitor (PKDi, kd-NB-142-70). Then CERT was immunoprecipitated and immunoblotted against antibodies directed against phospho-serine, CERT and β-actin.


Charlene Herbemont

Solmaz Sarandi

Jeremy Boujenah

Isabelle Cedrin-Durnerin

Nathalie Sermondade

Alexandre Vivot

Christophe Poncelet

Michael Grynberg

Christophe Sifer
email: christophe.sifer@aphp.fr

Should we consider day-2 and day-3 embryo morphology before day-5 transfer when blastocysts reach a similar good quality?

Keywords:

Should we consider day-2 and day-3 embryo morphology before day-5 transfer when blastocysts reach a similar good quality?

C Herbemont, S Sarandi, J Boujenah, I Cedrin-Durnerin, N Sermondade, A

quality embryo groups. No signficiant differences in morphology of transferred blastocysts at day 3 were found. Multivariable analysis highlighted that poor or good embryo quality at day 2 and day 3 were not predictive of the implantation of good-quality blastocysts (at day 2: adjusted odds ratio = 0.82 CI 95% 0.49 to 1.38; at day 3: adjusted odds ratio = 1.39; CI 95% 0.77 to 2.52).

Good-quality blastocyst transfer should, therefore, be carried out irrespective of embryo quality at cleavage stage, as it may not compromise success rates in a good-prognosis population. KEYWORDS: extended culture, good-quality blastocyst, cleavage stage embryo quality, implantation rate, live birth rate. Introduction Over the past decade, improvements in culture conditions have allowed the possibility of extended embryo culture until day 5, which has been increasingly preferred to a transfer at day 2 or day 3. In fact, blastocyst transfer is considered more physiological compared with transfers at cleavage stage, as it theoretically enables a better synchronization between the embryonic stage and the endometrial receptivity [START_REF] Valbuena | Increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo[END_REF]. Moreover, it is well known that not all embryos obtained at the early cleavage stage develop into blastocysts. Indeed, the morphological criteria commonly used to select embryos at cleavage stage are insufficient to predict their developmental potential, notably because, in many cases, they fail to discard chromosomally abnormal embryos [START_REF] Fragouli | Morphological and cytogenetic assessment of cleavage and blastocyst stage embryos[END_REF].

Embryo culture generally improves the selection of good-quality embryos available for fresh transfer, cryopreservation, or both, and displaying a good implantation prognosis. Blastocyst morphology is usually evaluated according to three criteria defined in Gardner and Schoolcraft's classification [START_REF] Gardner | Culture and transfer of human blastocysts[END_REF]: the degree of blastocoele expansion, graded from B1 to B6; the inner cell mass (ICM); and the trophectoderm morphology, both graded from A to C (A corresponding to the best quality score). A high blastocyst quality has undeniably been related to the success rates after IVF or intracytoplasmic sperm injection (ICSI) attempts [START_REF] Rehman | Late stages of embryo progression are a much better predictor of clinical pregnancy than early cleavage in intracytoplasmic sperm injection and in vitro fertilization cycles with blastocyst-stage transfer[END_REF][START_REF] Guerif | Does early morphology provide additional selection power to blastocyst selection for transfer?[END_REF][START_REF] Van Den Abbeel | Association between blastocyst morphology and outcome of singleblastocyst transfer[END_REF]. A longer period of culture, however, requires optimal culture conditions and an efficient blastocyst freezing programme to give patients all the benefit of embryo culture strategy. When these conditions are fulfilled, vitrified-warmed blastocyst transfers might provide equivalent live birth rates (LBR) as fresh transfers [START_REF] Roy | Single-embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers[END_REF].

The efficiency of embryo culture strategy has already been demonstrated, especially in goodprognosis populations, when numerous good-quality embryos are available at cleavage stage [START_REF] Guerif | Top quality embryos at day 2: a prerequisite for single blastocyst transfer? An observational cohort study in women under 36[END_REF][START_REF] Glujovsky | Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology[END_REF]. The overall [START_REF] Guerif | Top quality embryos at day 2: a prerequisite for single blastocyst transfer? An observational cohort study in women under 36[END_REF] and individual embryo qualities [START_REF] Rehman | Late stages of embryo progression are a much better predictor of clinical pregnancy than early cleavage in intracytoplasmic sperm injection and in vitro fertilization cycles with blastocyst-stage transfer[END_REF] at cleavage stage, however, do not seem to influence the clinical outcomes after the transfer of a good-quality blastocyst, but data on this particular topic are still too scarce.

Therefore, the aim of the present study was to assess, in a good-prognosis population, the value of considering the individual embryo quality at day 2, day 3, or both, when equivalent goodquality blastocysts were available for a transfer at day 5.

Study groups

Two groups were retrospectively created depending on whether the transferred blastocysts originated from a good-quality embryo at day 2 or day 3 (three to five adequately-sized blastomeres at day 2; six to 10 at day 3, <20% fragmentation, no multinucleation) or from a poorquality embryo (embryos that did not meet the criteria mentioned above). Sample size A difference of 30% was previously reported by [START_REF] Silber | Blastocysts that came from better quality day 3 embryos give higher pregnancy rates than blastocysts that derive from poor quality day 3 embryos[END_REF] between the implantation rate of good-quality blastocysts deriving from day-3 good-quality embryos (implantation rate = 70%) and day-3 poor-quality embryo (implantation rate = 40%). Therefore, considering an alpha risk less than 0.05 and a power greater than 0.95, we assumed that the sample size required to conclude was at least 69 observations in each group.

IVF procedures <C>Semen preparation

Semen was prepared, using a two-layer density technique (45% and 90%) of PureSperm (Nidacon International, Göteborg, Sweden) diluted in Ferticult HEPES culture media (FertiPro NV, Beernem, Belgium). After a 20-min centrifugation at 300 g, semen pellet was washed using Ferticult HEPES media (FertiPro N.V) and then centrifuged for 5 min at 600 g. <C>Ovarian stimulation and oocyte retrieval Ovarian stimulation was conducted as previously described with standard agonist or antagonist protocols [START_REF] Huirne | Cetrorelix in an oral contraceptivepretreated stimulation cycle compared with buserelin in IVF/ICSI patients treated with r-hFSH: a randomized, multicentre, phase IIIb study[END_REF][START_REF] Sifer | A prospective randomized to assess the benefit of partial zona pellucida digestion before frozen-thawed embryo transfers[END_REF]. In brief, within the 3 months preceding ovarian stimulation, antral follicle counting and serum oestradiol, FSH, and anti-Müllerian (AMH) levels were measured on cycle day 3. During a subsequent cycle, recombinant FSH therapy (Gonal-F, Serono Pharmaceuticals, Boulogne, France) was started at a dosage of 150-300 IU/day for at least 5 days, and continued until the day of HCG (Gonadotrophine Chorionique "Endo", Organon Pharmaceuticals, Saint-Denis, France, 10,000 IU, IM) administration. From the sixth day of exogenous FSH therapy onwards, daily FSH doses were adjusted according to oestradiol levels, the number of growing follicles, or both. During the last days of ovarian stimulation, patients had daily visits at our Institution for ultrasonographic and hormonal examinations to define the proper timing for HCG administration. Administration of HCG (day of HCG administration) was carried out as soon as three or more preovulatory follicles (16-22 mm in diameter) were observed and oestradiol levels per preovulatory follicle were greater than 200 pg/ml. Oocytes were retrieved 36 h after HCG administration by transvaginal ultrasound-guided aspiration.

<C>Oocytes and embryo culture Conventional IVF and ICSI procedures were carried out 3-6 h after oocyte retrieval. All cumulus oocyte complexes (COCs) were denuded before ICSI in a ready to use 80 IU/ml hyaluronidase medium (Fertipro NV) in accordance with the manufacturer's instructions. For an IVF attempt, COCs were inseminated with a concentration of 150,000 progressive motile sperm cells/ml. The embryos were cultured in a humidified 5% CO 2 atmosphere maintained at 37°C until day 6. The media used for embryo culture was Continuous Single Culture® (Irvine Scientific, Santa Ana, USA), from day 0 to the day of embryo transfer, and up to day 5 or day 6 if necessary. To cryopreserve potential supernumerary embryos, assessment of fertilization was carried out under an inverted microscope (x 200 magnification) 18 h after insemination (conventional IVF) or sperm injection (ICSI), by the presence of two pronuclei and two polar bodies.

Embryos were evaluated according to the conventional classification system currently used in our IVF laboratory: the number of blastomeres; the degree of cytoplasmic fragmentation; and the equality of blastomeres. Embryo morphology was scored as grade A, no cytoplasmic fragmentation; grade B, less than 20% fragmentation; grade C, 20-50% fragmentation; grade D, greater than 50% fragmentation. The number of cells per embryo was also recorded. Grade A and B embryos with three to five cells at day 2 or six to 10 at day 3, and no multinucleation were considered as good-quality embryo, respectively. Embryo quality was systematically recorded by the embryologist 44 h after insemination (day 2). Embryo culture strategy was applied to goodprognosis cases, mainly depending on the number of good-quality embryos available at cleavage stage (day 2, day 3, or both). Hence, assessment at day 3 remained optional when embryo culture strategy was already decided on day 2, thus avoiding unnecessary removals of culture dishes from optimal incubation conditions. At day 5, blastocyst morphology was evaluated according to Gardner and Schoolcraft's classification [START_REF] Gardner | Culture and transfer of human blastocysts[END_REF]. The blastocysts were scored according to size: B1, early blastocyst with a blastocoel less than one-half volume of that of the embryo; B2, with a blastocoel one-half volume of that of the embryo or more; B3, full blastocyst with a blastocoel completely filling the embryo; B4, expanded blastocyst with a blastocoel volume larger than that of the blastocyst, and a thinning zona; B5, hatching blastocyst with a trophectoderm starting to herniate through the zona; and B6, hatched blastocyst, having completely escaped the zona. For blastocysts graded as B3 to B6, the development of the inner cell mass and trophectoderm was assessed. The inner cell mass was graded as A, tightly packed, many cells; B, loosely grouped, several cells; C, very few cells. The trophectoderm was graded as: A, many cells forming a tightly knit epithelium; B, few cells; C, very few cells forming a loose epithelium. Good-quality blastocysts were defined as follows: blastocoel B3 or higher, inner cell mass A/B, trophectoderm A/B.

<C>Blastocyst cryopreservation and frozen embryo cycles

In this study, only frozen embryo transfer of day-5 good-quality blastocysts were included (n = 166). Vitrification and warming procedures were carried out according to Kuwayama et al.'s method (2005), using embryo vitrification; freeze; and Thaw kits (Irvine Scientific, Santa Ana, USA) and high security vitrification straws HSV™ (CryoBioSystem, L'aigle, France).

<C>Embryo transfer

Transfer of the best graded fresh or day-5 cryopreserved blastocyst(s), according to the criteria mentioned above and irrespective of their morphology at cleavage-stage, was carried out 5 days after oocyte retrieval. When several and equivalent good-quality blastocysts were available, day 2, day 3, or both, qualities were considered, explaining the higher number of cases included in the good-quality embryo groups. Luteal phase was supported with micronized progesterone (Utrogestan, Besins Iscovesco, Paris, France), 600 mg/day, administered continuously by vaginal route, starting on the evening of oocyte retrieval. Cryopreserved blastocyst transfers were scheduled during the course of either a natural, stimulated or an artificial cycle using hormone replacement treatment. In the two first cases, blastocysts were transferred on the seventh day after LH surge, and in the latter case on the fifth day after introduction of progesterone supplementation.

Clinical pregnancy was defined as presence of a gestational sac observed at ultrasound scan at around 7 weeks of amenorrhoea. All pregnancies were followed up until delivery. Miscarriages were defined as the loss of a biochemical or clinical pregnancy before 20 completed weeks of gestational age.

<C>Statistical analysis

The primary end-point of the present investigation was implantation rate. The secondary endpoints were clinical pregnancy rates (CPR) per embryo transfer, miscarriage rates and LBR. Analysis according to quality at day-3 of the transferred blastocyst A similar analysis was conducted for the same cycles, when embryo quality was assessed at day 3 (day-3 good-quality embryos group, n = 147 transfers, 174 transferred blastocysts; day-3 poorquality embryo group, n = 74 transfers, 86 transferred blastocysts). As reported in Table 3, patient characteristics and clinical parameters of the cycles were comparable in each group, except body mass index, which was higher in day-3 poor-quality embryo group (24.2 [22.2-27.6] kg/m² versus 22.9 [20.4-26.4] kg/m², respectively; P = 0.02).

Embryologic and IVF-ICSI outcomes were similar whatever the quality at day 3 of the transferred blastocyst(s) (Table 4). As shown, implantation rate (40.2% versus 31.4%, respectively), HCG positive rates per transfer (48.3% versus 43.2%), CPR (42.2% versus 35.1%), miscarriage rate (23.9% versus 18.8%) and LBR (36.7% versus 35.1%) were not significantly different in day-3 good-quality embryo and poor-quality embryo groups.

Multivariable analysis

Finally, the results of multivariable analysis using implantation, HCG and live birth as clinical outcomes are presented in Table 5 andTable 6. When adjusting for potential confounding factors, both embryo qualities at day 2 (Table 5) andday 3 (Table 6)

Discussion

The efficiency of embryo culture strategy has already been evaluated in a meta-analysis [START_REF] Glujovsky | Cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology[END_REF]) that compared the outcomes of transfers at day 2 and day 3 versus day 5 and day 6 reported in several prospective studies. In the case of fresh blastocyst transfer, LBR were significantly higher (12 randomized control trials, 1510 women; 31.2% at day 2 and 3 versus 38.9% at day 5 and 6; P = 0.02; OR 1.40 1.13 to 1.74), and especially when numerous good-quality embryos were available at day 2, day 3, or both, justifying the decision of embryo culture strategy (two RCTs, 364 women; 31.5% versus 50.0%; P = 0.0004; OR 2.17 1.42 to 3.33). In an observational cohort study conducted in a good-prognosis population, [START_REF] Guerif | Top quality embryos at day 2: a prerequisite for single blastocyst transfer? An observational cohort study in women under 36[END_REF] confirmed the relevance of opting for an embryo culture on the basis of embryo quality observed at day 2. They actually reported that the number of top-quality embryos at day 2 (four even cells, <20% fragmentation rate) was predictive of the blastocyst rate (68% when more than two top-quality embryos versus 50% when no top-quality embryos was obtained at day 2; P < 0.05). They reported similar LBR per blastocyst transfer; however, whatever the number of topquality embryos obtained at day 2, suggesting that once blastocysts are available, the overall embryo quality at day 2 has no affect on the subsequent clinical outcomes. Another study, published by [START_REF] Rehman | Late stages of embryo progression are a much better predictor of clinical pregnancy than early cleavage in intracytoplasmic sperm injection and in vitro fertilization cycles with blastocyst-stage transfer[END_REF], evaluated three markers of the quality and progression of 2000 embryos until blastocyst stage: the blastocyst quality score, a numerical blastocyst morphology-grading system calculated according to Gardner's criteria; the embryo progression index, calculated from the number of observed or estimated cells in the developing embryo, between day 1 and day 3; and the embryo progression index between day 1 and day 6). A receiver operator characteristic curve analysis showed that blastocyst quality score and embryo progression index day 1-6 seemed to be good predictors of clinical pregnancy, whereas embryo progression index day 1-3 seemed to be less powerful. These conclusions reinforce the fact that the better the quality of the transferred blastocyst, the higher its implantation potential.

To the best of our knowledge, no study has previously evaluated the effect of the individual embryo quality at cleavage stage on the outcome of good-quality blastocyst transfers. In fact, the present study has shown that, in a good-prognosis population, and using an appropriate statistical methodology, clinical outcomes are not influenced by good or poor quality at day 2 or day 3 when good-quality blastocysts are transferred at day 5. Even if some parameters were significantly different between good-quality embryos and poor-quality embryo at day 2 and day 3, the observed difference did not seem to negatively affect IVF-ICSI clinical outcomes. Furthermore, using multivariable logistic regression models, clinical outcomes after good-quality blastocyst transfers were not related to day 2 and day 3 embryo quality. Recently, [START_REF] Kaartinen | Can repeated IVF-ICSIcycles be avoided by using blastocysts developing from poor-quality cleavage stage embryos?[END_REF] analysed the outcomes of 134 vitrified-warmed blastocyst transfers, using good-quality blastocysts arising from supernumerary poor-quality embryos at day 2 or day 3. A CPR of 24.6% was reported in the cryopreserved blastocyst group, whereas CPR in the group of good-quality embryos cryopreserved at day 2 or day 3 reached 29.4%. Although these results were not compared with the outcome of blastocyst transfers derived from good-quality embryos, they still suggest that poor-quality embryos at cleavage stage may develop into good-quality blastocysts, displaying good implantation potential. Moreover, [START_REF] Poulain | Is it acceptable to destroy or include human embryos before day 5 in research programmes?[END_REF] reported an implantation rate of 29.4% and a CPR of 30.3% after the transfer of frozen-thawed high-quality blastocysts arising from day-3 poor-quality embryos. These findings are in line with previous results that suggest that only the quality of the transferred blastocyst may be predictive of the subsequent clinical outcome, whereas morphological aspects at day 2 or day 3 have limited interest.

Nevertheless, [START_REF] Silber | Blastocysts that came from better quality day 3 embryos give higher pregnancy rates than blastocysts that derive from poor quality day 3 embryos[END_REF], during the Ovarian Club 2014 meeting, reported the results of a study conducted in a similar manner to our study. They compared implantation rate and CPR after good-quality blastocysts transfers in patients younger than 35 years, according to embryo quality at day 3 only. Finally, this study yielded conflicting results, as blastocysts arising from poor-quality embryos displayed lower implantation rate and CPR compared with good-quality embryos (39% versus 73%; P < 0,01; and 34% versus 54%; P = 0.02, respectively) [START_REF] Silber | Blastocysts that came from better quality day 3 embryos give higher pregnancy rates than blastocysts that derive from poor quality day 3 embryos[END_REF]. These discrepancies between our results and those reported by [START_REF] Silber | Blastocysts that came from better quality day 3 embryos give higher pregnancy rates than blastocysts that derive from poor quality day 3 embryos[END_REF] could partly be explained by a different definition of good-quality embryos. Indeed, in the study by [START_REF] Silber | Blastocysts that came from better quality day 3 embryos give higher pregnancy rates than blastocysts that derive from poor quality day 3 embryos[END_REF], only embryos having six to eight cells of equal size and less than 20% fragmentation were considered as good-quality embryos, whereas in our IVF unit, as defined in the 'Materials and methods' section, embryos with six to 10 blastomeres are usually quoted as good-quality embryos. Moreover, the standard microscopic scoring of embryo morphology remains necessarily a subjective method, often subject to a wide inter and intra-observer variability [START_REF] Baxter Bendus | Interobserver and intraobserver variation in day 3 embryo grading[END_REF][START_REF] Paternot | Intra-and interobserver analysis in the morphological assessment of early stage embryos during an IVF procedure: a multicentre study[END_REF], and represents one of the main weaknesses of this study.

In order to minimize these variations, and above all to exploit innovative markers of embryo development, new technologies such as time lapse imaging have been developed. Morphokinetic variables are increasingly evaluated, from fertilization to cleavage and blastocyst stages, improving embryo scoring and selection, and at the same time enhancing the subsequent IVF-ICSI outcome [START_REF] Wong | Non-invasive imaging of human embryos before embryonic genome activation predicts development to the blastocyst stage[END_REF][START_REF] Conaghan | Improving embryo selection using a computer-automated time-lapse image analysis test plus day 3 morphology: results from a prospective multicenter trial[END_REF][START_REF] Herrero | A time to look back: analysis of morphokinetic characteristics of human embryo development[END_REF][START_REF] Adamson | Improved implantation rates of day 3 embryo transfers with the use of an automated timelapse-enabled test to aid in embryo selection[END_REF]Motato et al., 2015). Reliability, however, of such morphokinetic markers for predicting the implantation potential of blastocysts remains a matter of debate. Some authors have analysed the timing of each early event during the development of good-quality blastocysts, depending on whether they implanted or not [START_REF] Chamayou | The use of morphokinetic parameters to select all embryos with full capacity to implant[END_REF][START_REF] Desai | Analysis embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles[END_REF]. Finally, few parameters, such as time to syngamy (tPNf), times to 2 (t2), 3 (t3), 5 (t5) and eight (t8) cells, t2-tPNF, t5-t2 [START_REF] Desai | Analysis embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles[END_REF], or cc3 (t5-t3) [START_REF] Chamayou | The use of morphokinetic parameters to select all embryos with full capacity to implant[END_REF], were significantly different between implanted and non-implanted blastocysts. Furthermore, [START_REF] Motato | Morphokinetic analysis and embryonic prediction for blastocyst formation through an integrated time-lapse system[END_REF] have recently built a hierarchical classification of blastocysts according to the following morphokinetic parameters: (i) the time of morulation (tM), and (ii) timing of transition from 5blastomere embryo to until eight-blastomere embryo (t8-t5). In this study, they demonstrated the capacity of these variables to predict the ability of obtained blastocysts to implant. Clearly, either a wide variation between these implantation-associated markers or the lack of validation on an external independent series can be pointed out, probably because of the limitations of these studies, in particular the retrospective design and the limited number of enrolled blastocyst transfers. Yet, the value of morphokinetic patterns, helping to select good-quality blastocysts, needs to be further evaluated in a prospective way.

The present study, however, presents several limitations deserving discussion. First, the retrospective nature and the number of included cycles necessarily affect the statistical power of the present investigation, and no formal conclusion should be drawn from these results before proceeding to a prospective randomized study that would confirm them. Over a 4-year period, only 291 good-quality blastocyst transfers have been conducted in our IVF unit. In fact, embryo vitrification, authorized by the French legislation since the end of 2010, was concomitant and required a preliminary evaluation before its general implementation for embryos both at cleavage and blastocyst stages. As a consequence, the improvement of embryo culture strategy in our centre has been delayed. Moreover, only blastocyst transfers with known implantation data could have been included, excluding heterogeneous transfers of two blastocysts (one good-quality and one poor-quality blastocyst), or double blastocyst transfers displaying a 50% implantation rate, thus reducing the number of useable cycles. Therefore, fresh and cryopreserved blastocyst transfers were deliberately included, enabling in this way a consequent increase in the number of analysed transfers. In fact, the freeze-all policy was used, notably in case of elevated serum progesterone level on the day of ovulation triggering or in patients at high risk of ovarian hyperstimulation syndrome, so that the cycle outcomes for such patients were not compromised.

Yet, a recent study reported similar clinical outcomes after fresh or vitrified-warmed blastocyst transfers [START_REF] Roy | Single-embryo transfer of vitrified-warmed blastocysts yields equivalent live-birth rates and improved neonatal outcomes compared with fresh transfers[END_REF], suggesting that the inclusion of vitrified-warmed blastocyst transfers might not affect the outcomes in either of our study groups. Finally, male factor infertilities were not excluded from this analysis, as the euploidic status of day 5 blastocysts, when available, and consequently their potential to implant, might not be impaired by semen alterations, as suggested by several authors during the ASRM Congress in 2016 (unpublished data).

In the light of the present findings, the embryologist may be encouraged to no longer assess embryo quality at day 2 or day 3 in case of embryo culture strategy. In addition, avoiding unnecessary retrievals of culture dishes from the incubator could reduce deleterious stresses on embryos without jeopardizing blastulation or implantation rate [START_REF] Zhang | Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate[END_REF].

In conclusion, good-quality blastocyst transfer should be conducted irrespective of embryo quality at cleavage stage, as it may not compromise success rates in a good-prognosis population.

A prospective randomized study, when at least two similar good-quality blastocysts arising from both a good-quality embryo and a poor-quality embryo at day 2 or day 3 are available at day 5, would be the only way to accurately answer the question raised. Furthermore, other non-invasive methods, such as the use of morphokinetic parameters, could represent valuable tools to predict the blastocyst implantation potential, and need further evaluation. range] and as number; proportion (%). There were no statistically significant differences between the two groups. 1.00 e 0.58 to 1.72 1.00 e 0.58 to 1.72 a Multivariable logistic regression models: OR adjusted for variables when P < 0.20 (initial) or P < 0.05 (final) in the univariable analysis. b Adjusted for number of blastocysts transferred, primary/secondary infertility, duration of infertility and number of days of ovarian stimulation. c Adjusted for primary or secondary infertility and duration of infertility. d Adjusted for number of blastocysts transferred and indication. e Adjusted for number of blastocysts transferred, primary and secondary infertility and duration of infertility.

f All P-values were not statistically significant. b Adjusted for number of blastocysts transferred and number of oocytes retrieved and primary and secondary infertility. c Adjusted for number of blastocysts transferred, primary and secondary infertility, duration of infertility and IVF and intracytoplasmic sperm injectino rank. d Adjusted for number of blastocysts transferred, primary and secondary infertility and duration of infertility. e All P-values were not statistically significant.

  of prospectively collected data was conducted in the assisted reproduction technique unit of Jean Verdier University Hospital, Bondy, France, after being approved by the Local Ethics Committee on 18 November 2014. Two hundred and ninety-one blastocyst transfers (125 fresh blastocyst transfers, 166 cryopreserved blastocyst transfers) carried out between January 2012 and March 2016 were retrospectively analysed. All patients met the following inclusion criteria: female age less than 37 years; first or second IVF-ICSI cycle; quality of the transferred blastocyst: blastocoele B3 or higher, inner cell mass A/B, trophectoderm A/B (according to Gardner and Schoolcraft's criteria) (Gardner and Schoolcraft 1999); known implantation outcome for each transferred blastocyst (including double blastocyst transfers when implantation rate was 0 or 100%). Patients may have been included twice.

  Continuous variables are presented as median and interquartile range and qualitative variables as number and proportion. Differences between good-quality embryos and poor-qualtiy embryos were investigated by Wilcoxon-Mann-Whitney and Fisher's exact tests.Logistic regression models were used to search for a relationship between quality of the embryo at cleavage stage (evaluated at day 2 and day 3) and other potential prognostic factors; and transfer outcomes (implantation, miscarriage and live birth). To take into account the non-independency of data that are clustered on the individual level (as a women may have several transfers), we computed clustered standard errors. Continuous variables were first entered into a univariable model using restricted cubic splines with five knots. Then, an omnibus test for the three non-linear parameters was carried out. If this test yielded a P-value less than 0.10 then the variable was entered into subsequent multivariable models using restricted cubic splines, otherwise the variable was entered as a linear term. All variables associated with transfer outcomes in univariable models (P < 0.20) were entered into the initial multivariable logistic regression model. A backward stepwise selection procedure with a P-value cut-off at 0.05 was then applied to identify a set of factors significantly associated with transfer outcomes (final model). Day-2 and day-3 embryo quality was forced into all multivariable models as this was the variable of interest of this study. All tests were two-sided, and P < 0.05 were considered statistically significant. Analyses were carried out using R statistical software, version 3.3 (R Foundation for Statistical Computing, Vienna, Austria). Results Analysis according to the quality at day 2 of the transferred blastocyst(s) Outcomes of 291 cycles were collected and compared according to the quality at day 2 of the transferred blastocyst(s) (D2-GQE group, n = 206 transfers, 238 transferred blastocysts; day 2 poor-quality embryos group, n = 85 transfers, 99 transferred blastocysts). The median age of patient was 31.0 [(28.0-34.0) and 32.0 (29.0-34.0 years in day-2 good-quality embro and day-2 poor-quality embryo groups, respectively. The overall characteristics of patients, including type of infertility, ovarian stimulation and assisted reproduction techniques were similar between both groups, except for cycle rank (proportion of first IVF-ICSI cycle: 85.4% versus 72.9%; P = 0.02), and total dose of FSH administered (1562 [1241-2250] IU versus 1800 [1425-2550] IU, P = 0.03) (Table 1). The cycle outcomes are presented in Table 2. Despite comparable number of retrieved oocytes (16.0 [12.0-21.0] versus 16.0 [12.0-19.0]), more embryos were obtained in day-2 good-quality embryo group than in day-2 poor-quality embryo group (11.0 [8.0-14.0] versus 9.0 [7.0-13.0], respectively; P = 0.02). The proportion of single blastocyst transfers was equivalent in both groups (84.5% versus 83.5%). Interestingly, no difference in the degree of expansion (B3/4/5), quality of the inner cell mass and the trophectoderm (A/B), was noticed among blastocysts derived from day-2 good-quality embryo or poor-quality embryo. Finally, implantation rate (38.7% versys 41.4%, respectively), HCG positive rates per transfer (48.1% versus 52.9%), CPR (40.3% versus 45.9%), miscarriage rate (22.2% versus 26.7) and LBR (37.4% versus 38.8%) did no differ significantly between day-2 good-quality embryos and poor-quality embryo groups.

  were not associated with the clinical outcomes of good-quality blastocysts transfers (final model for quality at day 2 and (i) implantation: adjusted odds ratio (AOR) = 0.82 (CI 95% 0.49 to 1.38); (ii) HCG: adjusted OR = 0.82 (CI 95% 0.50 to 1.36; (iii) live birth: adjusted OR = 1.00 (CI 95% 0.58 to 1.72); final model for quality at day 3 and (i) implantation: adjusted OR = 1.39; CI 95% 0.77 to 2.52); (ii) HCG: adjusted OR = 1.23; CI 95% [0.70 to 2.15); (iii) live birth: adjusted OR = 1.16; CI 95% 0.63 to 2.15).

Table 1 .

 1 Patient characteristics and clinical parameters of the cycles according to the quality at day 2 of the transferred blastocyst. a

		Good-quality embryos:	Poor-quality embryos:
		206 transfers;	85 transfers;
		238 transferred blastocysts	99 transferred blastocysts
	Female age (years)	31.0 [28.0-34.0]	32.0 [29.0-34.0]
	Female body mass index	24.2 [21.8-27.7]	23.5 [21.2-25.8]
	(kg/m²)
	Smoking, n/n (%)	34/196 (17.3)	17/82 (20.7)
	Duration of infertility (years)	4.0 [3.0-5.0]	3.0 [2.0-5.0]
	Primary infertility, n (%)	148 (71.8)	53 (62.4)
	Indication
	Female	93 (45.1)	41 (48.2)
	Male	67 (32.5)	31 (36.5)
	Combined	17 (8.3)	8 (9.4)
	Unexplained	29 (14.1)	5 (5.9)
	IVF-ICSI first rank, n (%) b	176 (85.4)	62 (72.9)
	Days of ovarian stimulation, n	10.0 [9.0-11.0]	10.0 [9.0-12.0]
	Total FSH administered (IU) c	1562 [1241-2250]	1800 [1425-2550]
	Progesterone day HCG (ng/ml)	0.8 [0.6-1.0]	0.8 [0.6-1.0]
	Endometrium thickness (mm)	9.5 [8.1-11.4]	9.1 [7.6-11.8]
	P = 0.03.
	ICSI, intracytoplasmic sperm injection; IU, international unit; NS, not statistically significant.

a Data are presented as median (interquartile range) and as number (proportion [%]). b P = 0.02. c

Table 2 .

 2 Biological and clinical outcomes of the cycles according to the quality at day 2 of the transferred blastocyst. a

		Good-quality embryo: 206 transfers; 238 transferred blastocysts	Poor-quality embryo 85 transfers; 99 transferred blastocysts
	Oocytes retrieved, n	16.0 [12.0-21.0]	16.0 [12.0-19.0]
	Embryos obtained, n b	11.0 [8.0-14.0]	9.0 [7.0-13.0]
	Single blastocyst transfers, n	174 (84.5)	71 (83.5)
	(%)
	Blastocyst expansion degree, n
	(%)
	B3	34 (14.3)	17 (17.2)
	B4	172 (72.3)	69 (69.7)
	B5	32 (13.4)	13 (13.1)
	Inner cell mass morphology, n
	(%)
	A	44 (18.5)	17 (17.2)
	B	194 (81.5)	82 (82.8)
	Trophectoderm morphology, n
	(%)
	A	39 (16.4)	17 (17.2)
	B	199 (83.6)	82 (82.8)
	Top blastocyst, n (%)	45 (18.9)	16 (16.2)
	Implantation rate, %	38.7	41.4
	HCG positive, n (%)	99 (48.1)	45 (52.9)
	Clinical pregnancy rate, n (%)	83 (40.3)	39 (45.9)
	Miscarriage rate, n (%)	22 (22.2)	12 (26.7)
	Live birth rate, n (%)	77(37.4)	33 (38.8)

a Data are presented as median [interquartile range] and as number; proportion (%).

Table 3 .

 3 Patient characteristics and clinical parameters of the cycles according to the quality at day 3 of the transferred blastocyst. a

		Good-quality embryo:	Poor-quality embryo :
		147 transfers;	74 transfers ;
		174 transferred blastocysts	86 transferred blastocysts
	Female age (years)	31.0 [28.0-34.0]	31.0 [29.0-34.0]
	Female body mass index	22.9 [20.4-26.4]	24.2 [22.2-27.6]
	(kg/m²) a
	Smoking, n/n (%)	26/139 (18.7)	11/71 (15.5)
	Duration of infertility (years)	3.0 [2.0-5.0]	4.0 [2.0-5.0]
	Primary infertility, n (%)	106 (72.1)	50 (67.6)
	Indication
	Female	65 (44.2)	30 (40.5)
	Male	53 (36.1)	28 (37.8)
	Combined	15 (10.2)	4 (5.4)
	Unexplained	14 (9.5)	12 (16.2)
	IVF-ICSI first rank, n (%)	125 (85.0)	55 (74.3)
	Says of ovarian stimulation, n	9.0 [9.0-11.0]	9.5 [9.0-11.0]
	Total FSH administered (IU)	1600.0 [1243.8-2250.0]	1543.8 [1250.0-2168.8]
	Progesterone day HCG (ng/ml)	0.8 [0.7-1.0]	0.8 [0.6-1.0]
	Endometrium thickness (mm)	9.5 [8.1-11.5]	9.0 [7.4-11.5]

a Data are presented as median [interquartile range] and as number; proportion (%). a P = 0.02.

Table 4 .

 4 Biological and clinical outcomes of the cycles according to the quality at day 3 of the transferred blastocyst.

		Good-quality embryos: 147 transfers; 174 transferred blastocysts	Poor-quality embryos: 74 transfers; 86 transferred blastocysts
	Oocytes retrieved, n	16.0 [12.0-19.5]	14.0 [10.2-19.0]
	Embryos obtained, n	10.0 [8.0-12.0]	9.0 [7.0-12.0]
	Single blastocyst transfers, n	120 (81.6)	62 (83.8)
	(%)
	Blastocyst expansion degree, n
	(%)
	B3	27 (15.5)	9 (10.5)
	B4	124 (71.3)	64 (74.4)
	B5	23 (13.2)	13 (15.1)
	Inner cell mass morphology, n
	(%)
	A	36 (20.7)	16 (18.6)
	B	138 (79.3)	70 (81.4)
	Trophectoderm morphology, n
	(%)
	A	34 (19.5)	13 (15.1)
	B	140 (80.5)	73 (84.9)
	Top blastocyst, n (%)	37 (21.3)	19 (22.1)
	Implantation rate, %	40.2	31.4
	HCG positive, n (%)	71 (48.3)	32 (43.2)
	Clinical pregnancy rate, n (%)	62 (42.2)	26 (35.1)
	Miscarriage rate, n (%)	17 (23.9)	6 (18.8)
	Live birth rate, n (%)	54 (36.7)	26 (35.1)
	Data are presented as median [interquartile

Table 5 .

 5 Multivariable analysis for clinical outcomes and embryo quality at day 2. a,b

	Initial model		Final model
	Adjusted	CI 95%	Adjusted	CI 95%
	OR		OR
	Implantation 0.86 b	0.51 to 1.45 0.82 c	0.49 to
				1.38
	HCG positive 0.87 d	0.52 to 1.48 0.82	0.50 to
				1.36
	Live birth

Table 6 .

 6 Multivariable analysis for clinical outcomes and embryo quality at day 3. e Adjusted for number of blastocysts transferred and primary and secondary infertility.

		Initial model	Final model
		Adjusted	CI 95%	Adjusted	CI 95%
		OR		OR
	Implantation 1.39 a	0.77 to 2.52 1.39 a	0.77 to
					2.52
	HCG positive 1.19 b	0.67 to 2.12 1.23	0.70 to
					2.15
	Live birth	1.19 c	0.64 to 2.22 1.16 d	0.63 to
					2.15

a

Declaration

The authors report no financial or commercial conflicts of interest.