Cecile Lebon

Francine Behar-Cohen

Alicia Torriglia
email: aliciatorriglia@inserm.fr

Cell Death Mechanisms in a Mouse Model of Retinal Degeneration in Spinocerebellar Ataxia 7

Keywords: Spinocerebellarataxia type 7, retina, caspase-independentcelldeath, polyglutaminedisorder, photoreceptors, toxicity, autophagy, unfoldedproteinresponse

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

Introduction

Spinocerebellar ataxia type 7 (SCA7) belongs to the heterogeneous family of neurodegenerative disorders. Clinically, SCA7 is characterized by a progressive ataxia and a gradual vision loss leading to blindness. It is a rare disease caused by an unstable CAG expansion in the ATXN7 gene leading to the expression of ataxin7 with an abnormal polyglutamine tract (polyQ) [START_REF] David | Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion[END_REF]. Nine polyQ neurodegenerative disorders have been described so far, including SCA1, SCA2, SCA6, SCA7, SCA 17, Huntington's disease, spinal and bulbar muscular atrophy (SBMA) and dentatorubralpallidoluysian atrophy (DRPLA). The particularity of SCA7, in addition to its great instability of the CAG-repeat length, is the fact that it affects not only brainstem and cerebellum but also leads to retinal degeneration [START_REF] Michalik | Spinocerebellar ataxia type 7 associated with pigmentary retinal dystrophy[END_REF].

The protein encoded by the ATXN7 gene is ubiquitously expressed. It is a subunit of two transcriptional repressor complexes: STAGA (SPT3/TAF9/GCN5) and TFTC (TAFcontaining complex) involved in chromatin remodeling and histone acetylation. While the normal range of CAG repeats is under 35, pathological alleles contains 36 repeats and over [START_REF] Garden | Molecular pathogenesis and cellular pathology of spinocerebellar ataxia type 7 neurodegeneration[END_REF]. Like most polyQ disorders, CAG-repeat length is directly correlated to the age of onset and the disease progression: the longer the CAG repeat, the earlier the symptoms appear and the quicker the degeneration occurs [START_REF] Michalik | Spinocerebellar ataxia type 7 associated with pigmentary retinal dystrophy[END_REF]. Moreover, SCA7 family studies demonstrate a genetic anticipation: the CAG length tends to expand through generations [START_REF] Garden | Molecular pathogenesis and cellular pathology of spinocerebellar ataxia type 7 neurodegeneration[END_REF]. SCA7 patients present with a loss of neuronal cells in the cerebellum and degeneration of photoreceptors in the retina. This degeneration affects cones first but progressesto encompass the entire retina, leading to a cone-rode dystrophyand complete blindness [START_REF] Michalik | Spinocerebellar ataxia type 7 associated with pigmentary retinal dystrophy[END_REF]. However, the mechanisms of photoreceptor cell death due to the expression of polyQ ataxin7 remain poorly understood. SCA7 R7E transgenic mice were used to study the retinal degeneration induced by polyQ toxicity. These mice express the human ataxin7 with a 90 glutamines expansion under the rhodopsin promoter [START_REF] Yvert | Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice[END_REF]. Previous studies have shown that despite the loss of visual function with aging, these mice show a slow photoreceptor degeneration without a complete loss of the retinal structure [START_REF] Helmlinger | Disease progression despite early loss of polyglutamine protein expression in SCA7 mouse model[END_REF].

Rod photoreceptors die through different cell death mechanisms including apoptotic and non-apoptotic cell death that remain to be elucidated, and can also show morphological transformation and even cell division [START_REF] Yefimova | Polyglutamine toxicity induces rod photoreceptor division, morphological transformation or death in spinocerebellar ataxia 7 mouse retina[END_REF]. In this study, we investigate the molecular pathways of cell death in the SCA7 R7E mouse.

Experimental Procedures

SCA7 mice genotyping

The SCA7 R7E mice line was kindly provided by Dr. Yvon Trottier from IGBMC, Illkirch, France. They have been engineered on a C57/Bl6 background by Jean-Louis Mandel's team.

The mice were maintained in the Ecole Vétérinaire of Maison-Alfort's animal facilities (ENVA). Identification, biopsies and genotyping were performed at PN2 weeks (postnatal). DNA was extracted according to the manufacturer's specifications with the Kapa Express Extract kit (Kapabiosystems). A PCR was then performed with the Kapa2G Robust Hotstart Ready Mix PCR kit (KapaBiosytems) and transgene specific primers: 5'-AGGCGATG ACAAAGAAGAG-3' and 3'-AGGGGATGAGGATGAAGAG-5'. Reaction conditions were: 1 cycle at 95°C for 2 min, 35 cycles as following: 94°C for 10 sec, 58°C for 10 sec and 72°C for 30 sec, and finally 1 cycle at 72°C for 7 min. PCR products were then separated on a 1 % agarose electrophoresis gel. Transgenic mice present a 470bp band while no band is detected in WT mice.

Mice sacrifice and eye removal

Most mice used in this study (140 mice) were sacrificed at PN 5 wk, according to previous studies showing that cell death is maximum between PN 5 and PN 6 wk. Procedures were performed in accordance with the animal use and care committee of the National Veterinary School of Alfort, France. Mice were sacrificed with CO 2 and eyes were immediately enucleated. Eyes were either directly mounted in Tissue-Tek O.C.T., frozen with liquid nitrogen and then cut in 10 µm frozen sections on a Leica CM3050 S freezing microtome. Alternatively, the eye was dissected, the retina removed and frozen at -20°C for protein studies.

Immunohistochemistry and TUNEL staining

Eye sections were washed in Ca 2+ /Mg 2+ supplemented PBS (phosphate-buffered saline, Life Technologies), incubated in 4% formaldehyde in PBS for 15 min for fixation, and washed with PBS. For permeabilization, they were incubated in 0.3% Triton X-100/PBS for 15 min and washed with PBS. Saturation was performed with 1 % nonfat milk in PBS for 1 hr. Sections were then incubated 1 hour with primary antibody in 0.1 % nonfat milk in PBS. Antibodies used in this study are listed in Table 1. After washing the first antibody, samples were incubated with the second antibody in PBS for 1 hr in a dark chamber (used until the end of the staining). Nuclei were stained by 5 min incubation with DAPI (4-6 diaminidino-2-henyl indoledichloride, Sigma) diluted 5000-fold in PBS. The tissue was mounted following the last wash: sections were cover slipped with fluoromount (Sigma).

Negative controls with a non immune serum were run for each experiment. Due to the total negative result they were not incuded in the figures.

The TUNEL assay was performed according to the manufacturer's instructions (Roche Diagnostics, Mannheim, Germany), after the immunohistochemistry or just alone. In most of the cases, the TUNEL was preceded by a dephosphorylation step according to the protocol described by [START_REF] Lebon | On the use of an appropriate TdT-mediated dUTP-biotin nick end labeling assay to identify apoptotic cells[END_REF]. Briefly, sections were dephosphorylated with 10 U of calf intestinal alkaline phosphatase (CIAP, Invitrogen) in the associated buffer for 30 min at 37°C and washed. Images were acquired with a fluorescent U-25ND25 Olympus microscope. Image brightness and contrast were adjusted for display purposes when necessary with Image J software.

Western Blot

Retinas were defrosted on ice and homogenized in 100µl of M-Per buffer (Mammalian Protein Extraction Reagent, Thermo Scientific) supplemented with protease inhibitor cocktail (Roche Diagnostics) and incubated on ice for 15 min. Samples were then centrifuged at 8000 rpm for 3 min at 4°C. Supernatant was collected for protein measurement with the Pierce BCA Protein assay kit (Thermo Scientific). Laemmli sample buffer was added to the extracted material. Proteins were separated in a 12% SDS-PAGE and transferred on a nitrocellulose membrane (Whatman, GE Healthcare). Saturation was performed with 5% non-fat milk in PBS for 1 hr. The membrane was then incubated with primary antibody for 1 hr. at room temperature, washed with 0.1% tween in PBS, and incubated for 1 hr. with the secondary peroxidase labeled antibody. Antibodies used in this study are listed in Table1. After final washes with PBS, Luminata Forte Western HRP substrate (Millipore) was used to reveal the signal with the Microchemi 4.2 (DNR Bio-Imaging Systems). Image analysis and quantification was performed with ImageJ .

Intravitreous injections

Intravitrous injections were performed on PN 3 and 4 wk animals. Mice were anesthetized with Pentobarbital (Ceva) 50mg per kg via intraperitoneal injections and a drop of Tetracaïne (Novartis Pharma) was instilled on every eye. 1µl of solution was injected in the vitreous with the Micro4 micro-injector (World Precision Instruments). Tet-system Approved FBS, 100U/ml penicillin streptomycin, 100µg/ml G418 and 25µg/ml hygromycine). To induce transgene expression, the medium was supplemented with doxycycline (3 or 6 µg/ml) during 24 hours. For immunochemistry, cells were washed with PBS and mounted on slide with a Cytospin-4 (Thermo Scientific) for 5 min at 1000 rpm.

Immunocytochemistry was then performed according the same protocol than for immunohistochemistry as described above.

Statistical analysis

Statistics were performed using the GraphPad softweare (Prism). Due to the low number of samples non parametric tests were used. Results are shown as a median with an interquartile range. The specific test used in each experiment and the number of samples are indicated in each experiment.

Results

Ataxin7 expression induces stress in SCA7 mice retina

Previous studies reported that ataxin 790Q aggregates were detectable around and also within the nucleus in SCA7 R7E mice at PN 10 wk. [START_REF] Helmlinger | Disease progression despite early loss of polyglutamine protein expression in SCA7 mouse model[END_REF]. At the same age, transgenic mice expressing human ataxin-7 with a 10Q tract presented a cytoplasmic localization of ataxin-7-10Q. At PN 5 wk., we could detect polyQ-ataxin7 expression in rods photoreceptors (Fig. 1A). The expanded protein was found in the cytoplasm (in the outer and inner rods segment and around the nuclei) but also in the nuclei in some rods. Some polyQ accumulation was visible at this stage. Moreover, degradation of the polyQ protein was observed when studied by western blot (Fig. 1B). The specific polyQ antibody allowed the detection of small peptides (20 and 30 kDa) containing a polyQ expansion. A small amount of full sized ataxin7polyQ (approx. 110 kDa) was detected on this blot. In addition, as early as in 5 weeks old mice, rods presented a significant reduction of rhodopsin expression in outer segments (Fig. 1C andD). Interestingly, some rhodopsin positive cells were no longer in the photoreceptor layer but in the outer and inner plexiform layer. This was not observed in WT retina.Protein quantifications from western blot analysis showed the same results, with a significantly decrease of rhodopsin expression, mostly in its monomers and trimmers forms (Fig. 1D).

In order to study the retinal stress induced by the toxicity of ataxin7 polyQ expression, we used Glial Fribrillary Acidic Protein (GFAP) as a marker of glial Müller cells activation.

This intermediate filament is produced along the cell body of Müller cells during their

gliosis in response to a non-specific stress. GFAP expression is increased in PN 5 wk.transgenic mice (Fig. 2A andB) compared to their WT littermate, indicating that Müller cells were activated.

Several studies have shown that polyQ toxicity due to the expression of expanded Huntingtin induced dysfunctional ERAD (Endoplasmic Reticulum Associated Degradation) process [START_REF] Duennwald | Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity[END_REF] and UPR activation [START_REF] Leitman | Soluble forms of polyQ-expanded huntingtin rather than large aggregates cause endoplasmic reticulum stress[END_REF]. Three main pathways compose the UPR: PERK(PKR-like Endoplasmic Reticulum kinase), ATF-6 (Activating Transcription Factor 6) and IRE-1 (Inositol-requiring enzyme).We looked for UPR activation in our SCA7 mice through PERK expression.

PERK expression was increased in photoreceptor cytoplasm (inner and outer photoreceptor segment layer) in 5 wk. SCA7 mice retinas compared to their WT littermate (Fig. 3A).

However, we did not observe any increase in CHOP (C/EBP Homologous Protein) expression, a downstream marker of PERK pathway activation (not shown). Activation of IRE-1 pathway leads to an alternative splicing of XBP-1 (X-box binding protein 1) transforming it into a transcriptional factor that will modulate UPR genes expression. In SCA7 retina, we detected a small increase in XBP-1 unspliced form in both immunohistochemistry (Fig. 3B) and western blot analyzes (not shown). However, no spliced form was detected on the western blot of SCA7 retina. Interestingly, a higher expression of XBP-1 protein was also observed in HEK-293-7E cells and in HEK-293-7N cells (not shown). Recent studies have suggested a link between XBP-1 and autophagy via an increase of Forkhead box O1 (FoxO1) expression, a transcription factor regulating autophagy in neurons( 16). We studied the expression of the p62 protein (also known as sequestosome 1) because it plays a role in the clearance of protein aggregates [START_REF] Pankiv | p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF] and has been described at the intersection between autophagy and apoptosis [START_REF] Moscat | p62 at the crossroads of autophagy, apoptosis, and cancer[END_REF]. Immunohistochemistry reveals a dotted staining of this protein in ROS of SCA7 retina, which is not present in WT littermate (Fig. 3C). This specific staining suggests an accumulation of p62 in the cytoplasm and probably a decrease of the autophagic flux in SCA7 rods. However, no change in the expression level of p62 has been detected by western blot analysis (data not shown).

Taken together, these results suggest that the expression of the abnormal ataxin 7 produces retinal stress and a decrease of rhodopsin expression. The reticulum stress might be involved in this response.

SCA7 photoreceptors death did not involve the caspase pathway

It has been previously reported that SCA7 R7E mice retina displayed some photoreceptor death, most likely occurring between 5 and 6 weeks (Yefimova MG,Messaddeq N,Karam A,Jacquard C,Weber C,Jonet L,Wolfrum U,Jeanny J-C and Trottier Y, 2010). The use of the TUNEL (terminal deoxyribonucleotidyltransferase mediated dUTP-biotin nick end labeling) assay in order to detect dying rod nuclei did not allow the labeling of dying cells (Fig. 4A). However, cell death could be detected with a modified TUNEL protocol that included a prior dephosphorylation step (Lebon C,Rodriguez GV,El Zaoui I,Jaadane I,Behar-Cohen F and Torriglia A, 2015). In the retina of SCA7 mice, dying nuclei were all localized in the outer nuclei layer, suggesting that only photoreceptors were dying. No cell death was detected in the retina of WT mice with this protocol (Fig. 4B). Moreover, most cells ding are rod since the numberer of cones did not significantly change (not shown).

The modification of the cell death detection protocol could indicate the type of cell death involved in this retinal degeneration. In the case of caspase-dependent cell death, DNA degradation by Caspase Activated DNase (CAD) is systematically detected with the classical TUNEL technique. The need for a dephosphorylation step might reflect the activation of a DNase II-type endonuclease in this process. However, to further investigate if caspases were involved in this cell death,we investigated the cleavage of caspase-3 and the cleavage of ACINUS (apoptotic chromatin condensation inducer in the nucleus).In caspase-dependent cell death, ACINUS is cleaved by active caspase-3 in a 23 kDa fragment that mediate apoptotic chromatin condensation previous to DNA fragmentation [START_REF] Sahara | Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation[END_REF].Neither immunolabelling nor western blot analysis showed caspase-3 or ACINUS activation in SCA7 retinas compared to WT. Caspase-3 was only detected in the inner and outer photoreceptors segments in the retinas of WT and SCA7 mice (Fig. 5A). No cleaved ACINUS was observed in photoreceptor nuclei of control or SCA7 mice. ACINUS staining was observed only in the inner nucleus layer in both control and transgenic mice (Fig. 5C). In addition, western blot analysis did not present any cleaved form of caspase-3 or ACINUS, indicating that caspases were not involved in SCA7 rod death (Fig. 5B). Taken together, these data suggest that the photoreceptor death observed in the retinas of SCA7 mice did not involve caspase-dependent cell death.

SCA7 photoreceptors death involved caspase-independent pathways

To further investigate this cell death we focused on two proteins AIF and LEI/L-DNase II.

These cell death effectors were shown to be involved in caspase-independent cell death pathways. AIFplays an important role in mitochondrial respiration; it can be cleaved and delocalized into the nucleus upon apoptotic stimulus [START_REF] Susin | Molecular characterization of mitochondrial apoptosis-inducing factor[END_REF]. LEI belongs to the Serpin family and its native form (42 kDa) has an anti-protease activity [START_REF] Torriglia | LEI/L-DNase II: interplay between caspase-dependent and independent pathways[END_REF]. After proteolytic cleavage, it becomes an endonuclease, L-DNase II (35 and 27 kDa), which is imported into the nucleus where it degrades DNA. It was shown, moreover, that these effectors might collaborate to facilitate DNA degradation during apoptosis [START_REF] Leprêtre | Apoptosis-inducing factor (AIF) and leukocyte elastase inhibitor/L-DNase II (LEI/LDNaseII), can interact to conduct caspase-independent cell death[END_REF]. Immunostaining of these proteins combined with our adapted TUNEL staining allowed us to observe apoptotic photoreceptor nuclei in which there was a relocalization of both AIF and L-DNase II in the outer nuclear layer (Fig. 6A).

No nuclear staining was observed in the retinas of WT mice. Moreover, a laser microdissection of photoreceptor nuclei followed by a slot blot analysis showed an increase of LEI-L/DNase II and AIF in SCA7 photoreceptors nuclei compared to WT, for the same dissected surface (Fig. 6B). A western blot of WT and SCA7 retina and its quantification demonstrated significant increase of cleaved AIF (57 kDa) and of both forms of L-DNase II in SCA7 retina (Fig. 6C, D andE). These results suggested the involvement of both AIF and LEI/L-DNase in SCA7 mice retinal degeneration. A cellular model for Sca7 has also been used to determine if LEI and AIF pathways activation depends on ataxin-7 polyQ expression. HEK-293 cells expressing either a 10Q ataxin7 (HEK-7N) or a 60Q ataxin7 (HEK-7E) were used. Exogene ataxin7 expression in those cells did not induced caspase-3 activation (data not shown). In case of expanded-ataxin7, its expression led to LEI-L/DNase II nuclear relocalization (Fig. 7) while no activation is observed when normal ataxin7 is expressed. Concerning AIF, its expression is increased in HEK-293-7E compared to HEK-293-7N but very few nuclear AIF was detected, suggesting that its release from mitochondria and its nuclearisation did not massively occur or occured after L-DNase II activation in this cellular model.

Inhibition of AIF and LEI expression lead to a decrease of photoreceptors death.

In order to validate these observations, we chose to inhibit AIF and LEI/L-DNase II expression in mice to see if retinal degeneration is affected. siRNA were injected intravitreously twice to 3-and 4 week-old mice and retinas were analyzed at 5 weeks as before. Dying cells were counted after injection using our adapted TUNEL staining.

Quantification of both LEI and AIF expression after siLEI and siAIF injections, respectively, were performed on western blot analysis (Fig. 8 and9). siLEI injection led to a decrease of LEI expression only in SCA7mice (Fig. 8A andB). A significant reduction of dying photoreceptor nuclei was observed in these mice (Fig. 8C). Intravitreous injection of siAIF induced a significant decrease of AIF expression in both WT and SCA7 retinas Fig 9A andB) and an important reduction of TUNEL positive cells was detected in SCA7 mice (Fig. 9 C). It is important to note that inhibition of either AIF or LEI expression did not affect WT mice retina. The counting of photoreceptors death after siCtrl injection also permitted to evaluate cell death in SCA7 retinas compared to WT littermates where almost no dying photoreceptors were observed.Moreover, evaluation of the number of nuclei remaining in the ONL (Fig. 9D) indicates a stabilization of this layer upon siRNA treatment. Taken together these results tendedto confirm the implication of AIF and LEI in SCA7 photoreceptors cell death.

Calpains but not cathepsins seem to be related to AIF and LEI/L-DNase II activation

AIF released from mitochondria during cell death can be controlled by several factors. Among them calpains can directly cleave AIF and cathepsins are involved in AIF release through Bax activation [START_REF] Delavallée | AIF-mediated caspase-independent necroptosis: A new chance for targeted therapeutics[END_REF][START_REF] Norberg | Mitochondrial regulation of cell death: processing of apoptosis-inducing factor (AIF)[END_REF]. On the other hand, LEI can be cleaved into L-DNase II by cathepsin D [START_REF] Torriglia | LEI/L-DNase II: interplay between caspase-dependent and independent pathways[END_REF]. In order to determine whether calpains or cathepsins were involved in AIF and L-DNase II activation in our model, we decided to inhibit each protease activity with intravitreous injections of their specific inhibitors. We used calpain inhibitor VI to inhibit calpains (both micro and mini calpains also called calpains 1 and 2) activities and pepstatin A to inhibit cathepsin D activity. The same protocol used for siRNA injection was applied and photoreceptor death was evaluated in the same way (Fig. 10). Calpain inhibitor VI injections in SCA7 mice induced a significant reduction of cell death (Fig. 10A). However, pepstatinA injection did not show any significant effect (Fig. 10B). Neither pepstatin A nor calpain inhibitor VI induced toxicity in WT mice retina.

Discussion

In this study we analyzed the mechanisms of photoreceptors cell death due to polyQ toxicity induced by expanded ataxin7 expression in mice retina. We showed that two caspase-independent pathways mediate this cell death: LEI/L-DNase II and AIF.

Previous studies on SCA7 R7E mice demonstrated that this is a relevant model to study mice to extend the possibility to observe and investigate dying photoreceptors, as it is a rare event.

Our first observation was that no apoptotic cells were detected with the TUNEL technique, widely used to detect dying cells. With an adjustment of the TUNEL protocol (by adding a dephosphorylation step prior the staining), we succeed in detecting apoptotic photoreceptor nuclei. At the same time, we observed a significant reduction in rhodopsin staining, which is consistent with previous studies and with the disappearance of outer and inner photoreceptor segments prior to nuclear layer reduction. This is an important result because it indicates that the DNase activity involved in this cell death exposes 3'P ends. The changes in the photoreceptors layer are accompanied by a deformation of this entire layer, forming "waves" along the retina. There is also an increase of GFAP staining, which suggests Müller cell activation. Those observations (rods loss, GFAP increase) have also been observed in model mice for human retinitis pigmentosa: the rd/rdmouse [START_REF] Strettoi | Morphological and functional abnormalities in the inner retina of the rd/rd mouse[END_REF]. However, in this retinal degeneration, the cell layer reduction is quicker than in the SCA7 mice. It is also important to note that rod loss happens while the polyQ ataxin-7 does not form detectable aggregates. According to Yvert et al., who set up the SCA7 R7E mice, aggregation (or nuclear inclusions) starts around PN 8 wk [START_REF] Yvert | Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice[END_REF]. We can therefore assume that soluble polyQ-ataxin-7 expression induces rods segment loss, as well as rod death and Müller cell activation.

We further studied the mechanisms involved in this cell death by first looking at the caspase pathway, although failing to label dying cells by regular TUNEL suggested a non-CAD (Caspaseactivated DNase) dependent cell death. As expected, neither caspase-3 nor ACINUS activation have been detected in the retina of SCA7 mice. Actually, no caspase-3 expression has been detected in the retinas of 5 wk old SCA7 and WT mice, which is consistent with previous studies on C57/bl6 retinal development showing that Apaf-1 and caspase-3 expression are strongly repressed in adult retina [START_REF] Donovan | Caspase-independent photoreceptor apoptosis in vivo and differential expression of apoptotic protease activating factor-1 and caspase-3 during retinal development[END_REF]. In addition, light-induced retinal degeneration in mice has been shown to be caspase-3 independent as well as cell death in rd mouse photoreceptors [START_REF] Arango-Gonzalez | Identification of a common non-apoptotic cell death mechanism in hereditary retinal degeneration[END_REF][START_REF] Donovan | Light-induced photoreceptor apoptosis in vivo requires neuronal nitric-oxide synthase and guanylate cyclase activity and is caspase-3-independent[END_REF]. Taken together, these results suggest that photoreceptor cell death does not depend on caspase pathways and might involve a caspase-independent mechanism. Indeed, the need of a dephosphorylation step to detect DNA degradation informs us on the type of DNA breaks. 3'OH breaks are recognized by the terminal tranferase in the TUNEL assay, while 3'P ends are not, leading to a TUNEL negative labeling. However, after a dephosphorylation step, 3'P ends become 3'OH and are now recognized by the TUNEL enzyme. This particularity has been described previously by our team and others (Lebon C,Rodriguez GV,El Zaoui I,Jaadane I,Behar-Cohen F and Torriglia A, 2015) [START_REF] Hayashi | Quantitative differentiation of both free 3'-OH and 5'-OH DNA ends between heat-induced apoptosis and necrosis[END_REF], specifically the in case of LEI/L-DNase II activation which is a 3'P producing endonuclease. This protein was described by Torriglia et al. in 1995 during lens cell differentiation (Torriglia A et al., 1995;[START_REF] Torriglia | L-DNase II, a molecule that links proteases and endonucleases in apoptosis, derives from the ubiquitous serpin leukocyte elastase inhibitor[END_REF][START_REF] Padron-Barthe | Conformational modification of serpins transforms leukocyte elastase inhibitor into an endonuclease involved in apoptosis[END_REF] and has been involved in different rats retinal paradigms: glucocorticoids-induced retinal toxicity or different types of light-induced retinal degeneration [START_REF] Chahory | The LEI/L-DNase II pathway is activated in light-induced retinal degeneration in rats[END_REF][START_REF] El Zaoui | Glucocorticoids exert direct toxicity on microvasculature: analysis of cell death mechanisms[END_REF][START_REF] Jaadane | Retinal damage induced by commercial light emitting diodes (LEDs)[END_REF]. It has also been demonstrated that LEI/L-DNase II can collaborate with AIF, another caspaseindependent pathway, to induce cell death in vitro [START_REF] Leprêtre | Apoptosis-inducing factor (AIF) and leukocyte elastase inhibitor/L-DNase II (LEI/LDNaseII), can interact to conduct caspase-independent cell death[END_REF]. AIF nuclear translocation was described in an induced retinal detachment rat model and in a LED-induced retinal degeneration in rats (Jaadane I,Boulenguez P,Chahory S,Carré S,Savoldelli M,Jonet L,Behar-Cohen F,Martinsons C and [START_REF] Lebon | On the use of an appropriate TdT-mediated dUTP-biotin nick end labeling assay to identify apoptotic cells[END_REF]. So that, we have investigated these two cell death effectors and shown here that both are translocated to the nucleus of TUNEL positive photoreceptors. In addition, LEI/L-DNase II is also translocated to the nucleus after expanded ataxin7 expression in our in vitro cellular model in a more systematic way than AIF. This observation is consistent with previous results showing that L-DNase II precedes AIF into the nucleus and with the hypothesis that L-DNase II might, in some cases, contribute to the nuclear translocation of AIF via the activation of PARP and the release of PAR [START_REF] Leprêtre | Apoptosis-inducing factor (AIF) and leukocyte elastase inhibitor/L-DNase II (LEI/LDNaseII), can interact to conduct caspase-independent cell death[END_REF]. AIF and LEI/L-DNase II activation in SCA7 photoreceptors death have been confirmed by intravitreous injections of specific siRNA. When either LEI or AIF are downregulated, the number of apoptotic photoreceptors is significantly decreased. However, there are still dying photoreceptors after two siRNA injections suggesting that either inhibition was not enough efficient or others pathways could be involved. It might be interesting to inhibit both AIF and LEI in the same time to see if there is a cumulative effect on cell death receptors. However, this is a difficult task because both LEI and AIF have anti-apoptotic functions in their cytoplasmic/ mitochondrial forms [START_REF] Daugas | Apoptosis-inducing factor (AIF): a ubiquitous mitochondrial oxidoreductase involved in apoptosis[END_REF][START_REF] Padron-Barthe | Conformational modification of serpins transforms leukocyte elastase inhibitor into an endonuclease involved in apoptosis[END_REF].

In order to explore the activation mechanism of those two caspase-independent cell death effectors, we chose to inhibit calpain activation pathway with calpain inhibitor VI and cathepsin D pathway with Pepstatin A injection, both inhibitors already used in previous studies [START_REF] Chahory | Light induced retinal degeneration activates a caspase-independent pathway involving cathepsin D[END_REF][START_REF] Rodriguez | Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2[END_REF]. Our results suggest that AIF and LEI activation depends, at least in part of calpain 1 (or micro-calpain). Indeed, calpain 1 is the protease responsible for the release of AIF from mitochondria and it plays also a role in lysosomal permeabilisation, leading to the release of cathepsin D from the lysosome among others lysosomal proteins [START_REF] Chen | Heart mitochondria and calpain 1: location, function, and targets[END_REF][START_REF] Rodriguez | Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2[END_REF]. As cathepsin D is also responsible for LEI cleavage into L-DNase II, calpain 1 inhibition activity may prevent both cell death effectors activation. It is the case in this SCA7 degeneration since injection of calpain inhibitor VI (at a dose that specifically inhibits calpain 1 activity) [START_REF] Rodriguez | Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2[END_REF] , produced a decrease in the number of dying cells. Calpain1 inhibition has also been used to reduce cardiac cell death in a myocardial infarction model [START_REF] Smith | Calpains, mitochondria, and apoptosis[END_REF]. Concerning cathepsin D activity inhibition by pepstatine A, no significant effect on photoreceptors cell death has been observed, suggesting that LEI cleavage into L-DNase II involves other proteases such elastase which is its specific substrate, AP24 (Apoptotic Protease 24 kDa) or other serine proteases (O'connell A et al., 2006). These results differ from what has been observed in light-induced retinal degeneration in rats in which pepstatin A injection prevented photoreceptors death due to LEI activation [START_REF] Chahory | Light induced retinal degeneration activates a caspase-independent pathway involving cathepsin D[END_REF]. Another possibility could be that, when LEI cleavage is inhibited by pepstatin A, AIF is still released from mitochondria and relocated into the nucleus where it can mediates DNA degradation through endonuclease G (endo G) activation. Indeed, endo G is also a type II nuclease, originally located in the mitochondria and involved in mitochondrial replication [START_REF] Li | Endonuclease G is an apoptotic DNase when released from mitochondria[END_REF].

It can be released from the mitochondria in a caspase-independent manner, such as AIF, and translocate into the nucleus where it degrades DNA [START_REF] Van Loo | Endonuclease G: a mitochondrial protein released in apoptosis and involved in caspase-independent DNA degradation[END_REF]. Endo G has been involved in neuronal cell death due to glutamate excitotoxity in mice hippocampus [START_REF] Wu | Role of endonuclease G in neuronal excitotoxicity in mice[END_REF].

It has been previously reported that polyQ proteins induce endoplasmic reticulum stress accompanied by an unfolded protein response. Studies, mostly concerning huntingtin, the protein responsible for Huntington's disease, demonstrated that the three branches of UPR are involved in the response to polyQ toxicity and ERAD inhibition (Jiang Y et al., 2016;[START_REF] Ogen-Shtern | Protein aggregation and ER stress[END_REF]. This phenomenon is shared by other misfolding protein diseases such Alzheimer's or Parkinson's [START_REF] Leitman | Soluble forms of polyQ-expanded huntingtin rather than large aggregates cause endoplasmic reticulum stress[END_REF].

Our 5 wk. SCA7 mice rods do not present ataxin7 aggregates but nuclear inclusions are detected at 10 wk. [START_REF] Helmlinger | Disease progression despite early loss of polyglutamine protein expression in SCA7 mouse model[END_REF]). Here we show that some branches of the UPR are already activated at 5 wk. such as the PERK pathway. XBP-1 expression is increased but no spliced form has been detected. However, CHOP up-regulation has not been observed.

When ERAD and UPS (Ubiquitin Proteasome System) are not sufficient to manage misfolded protein, autophagy can be activated. In our model, p62 bodies are detected suggesting that p62 binds to autophagosomes to clear misfolded proteins and aggregates. However, it also suggests an impairment in the autophagy machinery since p62 accumulates in ROS [START_REF] Giansanti | Conversation between apoptosis and autophagy:"Is it your turn or mine?[END_REF]. If all the systems involved in protein clearance are deficient or overwhelmed by expression of polyQ proteins, cells activate apoptotic pathways. The mechanisms involved in the switch between autophagy and caspase-independent apoptosis remain to be elucidated.

Studies on polyQ diseases are mainly focused on Huntington's disease and concern brain or in vitro models. SCA7 is the only polyQ disease affecting the retina and deciphering the toxicity and cell death mechanisms induced by abnormal ataxin 7 in retina may contribute to the advancement of knowledge on neuro-proteinopathies.

  siCtrl (Santa Cruz),siLEI (CCAUAAAUGUCCUCACUAU, Eurofins) and siAIF (GGAU CCUGACAUGCCAGACA, Eurofins) was diluted at 25 µM and 1 µl of this solution was incubated 5 min in 11 µl of TransIT buffer (Mirius) prior the injection. Calpain Inhibitor VI (Calbiochem) was diluted in PBS at concentration 600 nM and Pepstatin A (Sigma) was diluted at 40 µM. The control solution consisted of DMSO (Sigma) diluted 1/130 in PBS. HEK293 cells 2 lines of stably transfected Human Embryonic Kidney 293 (HEK293) cells have been used in this study, kindly provided by Dr. YvonTrottier. Each line express either the wild-type human ataxin 7 protein (293-7N) or the mutant protein containing a polyQ expansion (293-7E), both containing a HA tag. Transgene expression is under doxycycline inducible promoter. Cells were cultured in Dulbecco's DMEM (4,5% glucose) supplemented with 5%

  SCA7 human retinal degeneration since it reproduces what is seen in human pathology. SCA7 mice retinal degeneration is characterized by a very slow reduction of photoreceptors and intermediate cells layers. Outer and inner photoreceptor segments are first and soon disrupted while the amount of photoreceptors nuclei remains almost constant. However, Yefimov aet al, showed a peak of cell death between PN 5 and 6 wk (Yefimova MG,Messaddeq N,Karam A,Jacquard C,Weber C,Jonet L,Wolfrum U,Jeanny J-C and Trottier Y, 2010). The morphology of dying cells reminds apoptosis but no extensive study on the mechanisms involved in this cell death were performed. We chose to use PN 5 wk.

Figure 1 :

 1 Figure 1: degeneration induced by polyQ expression. (A) polyQ (in red) staining in 5 wk. WT and SCA7 retinas. Black arrows show ONL enlargement. Nuclei are stained in blue with Dapi. (B) Western Blot analysis of WT and SCA7 retinas with anti-polyQ antibodies. Actin was used as a charge control. (C) Rhodopsin immunostaining (in green) of WT and SCA7 retinas. Nuclei are stained in blue with Dapi. ONL indicates the outer nuclear layer, INL the inner nuclear layer. Scale bar represents 30 µm. (D) Representative western blot of rhodospin expression in WT and SCA7 retinas, and its densitometric analysis (quantification was normalized to actin level).T, D and M represent the different forms of rhodopsin namely trimers, dimers and monomers. Medians are represented by lines. Statistical calculations were performed using Mann Whitney test, U(n3,n3)=0.0, p ≤ 0.05 .

Figure 2 :

 2 Figure 2: Müller cells are activated in SCA7 retina. (A) anti-Glial Fibrillary Acid Protein antibody (in red) was used to observe Müller cells activation in WT and SCA7 retinas. Nuclei are stained in blue with Dapi. ONL indicates the outer nuclear layer, INL the inner nuclear layer. Scale bar represents 30 µm. (B) Western blot and its densitometric analysis of WT and SCA7 retinas with anti-GFAP antibodies. Actin and ponceau red were used as charge controls. Medians are represented by lines Statistical calculations were performed using Mann Whitney test U(n3,n3)=0.0, p ≤ 0.05.

Figure 3 :

 3 Figure 3: The ER stress is activated in SCA7 retina.(A) PERK immunostaining (in red) of 5 wk. WT and SCA7 retinas. (B) XBP-1 staining (in red) in WT and SCA7 5 wk. retinas.(C) p62 staining (in red) in SCA7 retina compared to WT littermate. Black arrows indicate enlargement of the outer nuclear and receptor layers. Nuclei are stained in blue with Dapi. ONL indicates the outer nuclear layer, INL the inner nuclear layer. Scale bar represents 30µm.

Figure 4 :

 4 Figure 4: Detection of photoreceptors death in SCA7 retina. (A) No cell death (in green) is detected in WT and SCA7 retinas using classic TUNEL protocol. Dying photoreceptors are only detected with a dephosphorylation prior to the TUNEL assay in

Figure 5 :

 5 Figure 5: Caspase 3 and ACINUS are not activated in photoreceptors death. (A) Staining of activated caspase3 (in green) in WT and SCA7 retinas. Nuclei are stained in blue with Dapi. (B) Western blot analysis of WT and SCA7 retinas with anti-activated-caspase3 and anti-ACINUS antibodies. Actin and red ponceau are used as a charge control. Etoposide-treated HeLa cells are used as a positive control for caspase3 activation and acinus cleavage.(C) Staining of ACINUS (in red) in WT and SCA7 retinas. Nuclei are stained in blue with Dapi. ONL indicates the outer nuclear layer, INL the inner nuclear layer. Scale bar represents 30µm.

Figure 6 :

 6 Figure 6: LEI/L-DNase II and AIF are activated in SCA7 retina. (A) Co-staining of LEI/L-DNase II (in blue), AIF (in red) and modified TUNEL assay (in green). White arrows indicate TUNEL positive nuclei in which both LEI/L-DNase II and AIF are translocated in SCA7 retina. Nuclei are stained in blue with Dapi. ONL indicates the outer nuclear layer, INL the inner nuclear layer. Scale bar represents 30µm. (B) Quantification of the expression of LEI/L-DNase II and AIF in the ONL. The samples were obtained by laser microdissection of the ONL. The amount of the proteins present in this layer was quantified by dot blot, Medians are represented by lines Statistical calculations were performed using Mann Whitney test. LEI: U(n8,n8)= 2.0, , p ≤ 0.005. AIF: U(n6,n6)= 3.0, , p ≤ 0.05 . (C) Western blot analysis of WT and SCA7 retinas with anti-AIF and anti-LEI/L-DNase II antibodies (representative image). Actin and ponceau red were used as a charge control (D) A densitometric analysis of the represented western blot for AIF and activated AIF (AIFt) Statistical calculations were performed using Mann Whitney test. Medians are represented by lines Statistical calculations were performed using Mann Whitney test. AIF: U(n4,n4)=

Acknowledgments: We are grateful to Julia Pardo and Laurent Jonet for technical assistance and to Cathy Claramonte and Dr. Sabine Chahory for their assistance in the animal care. We are thankful to Alejandro Arboleda for his English editing. This work was supported by INSERM and Cecile Lebon had a fellowship from Paris Descartes University and from the CRO association. CL made the experiments and wrote de paper, FBC gave scientific advice, AT conducted the work designed the experiments and wrote the paper.

. (E) densitometric analysis for and its two activated forms. Quantification was normalized to actin level. Medians are represented by lines. Statistical calculations were performed using Mann Whitney test. LEI: U(n4,n4)= 0.0, p = , p ≤ 0.05. p35: U(n4,n4)= 11.0, p =, p ≤ 0.05


A Rosefort

MD C Huchon

MD S Estrade

MD A Paternostre

MD J.-P Bernard

Prof Arnaud Fauconnier
email: afauconnier@chi-poissy-st-germain.fr

Is training sufficient for ultrasound operators to diagnose deep infiltrating endometriosis and bowel involvement by transvaginal ultrasound?

Keywords:

Objectives To assess and compare the diagnostic accuracy of transvaginal ultrasonography (TVUS) by trained or untrained ultrasound operators in deep infiltrating endometriosis (DIE) imaging, for diagnosing DIE and bowel involvement.

Methods

This was an observational study of patients with clinically suspected DIE operated in a reference center. TVUS was performed pre-operatively by a trained or/and untrained ultrasound operator to search for DIE and rectal involvement. During surgery, DIE was diagnosed according to macroscopic and histological criteria. Sensitivity (Se), specificity (Sp) and c-index were calculated with 95% confidence intervals for trained and untrained operators, if TVUS results were significantly predictive of DIE and rectal involvement at p<0.05.

Results 115 patients were included: 100 (87%) had DIE and 34 (29.6%) had bowel involvement. TVUS was performed by a trained ultrasound operator for 70 patients and by an untrained one for 56 patients. When performed by a trained operator, TVUS significantly predicted DIE with a Se of 58% (95% CI, 46-70), a Sp of 87.5% (95% CI, 63-100) and a cindex of 0.73 (95% CI, 0.59-0.87). TVUS performed by an untrained operator was not significantly predictive of DIE (p=0.58).

Rectal involvement was significantly predicted by TVUS performed by a trained operator with a Se of 40% (95% CI, 23-59), a Sp of 93% (95% CI, 86-100) and a c-index of 0.67 (95% CI, 0.56-0.77). None of the untrained ultrasound operators diagnosed a bowel involvement.

Conclusion TVUS is not sufficient to diagnose DIE and bowel involvement, in particular when performed by untrained ultrasound operators.

Introduction

Endometriosis is defined as the growth of functional and hormone-dependent endometrial tissue outside the uterine cavity. Deep infiltrating endometriosis (DIE) involves lesions infiltrating the retroperitoneal space by 5 mm or more [START_REF] Koninckx | Deeply infiltrating endometriosis is a disease whereas mild endometriosis could be considered a non-disease[END_REF] . It can affect different regions of the pelvis, including the digestive organs. Laparoscopic resection of all DIE lesions significantly reduces pain and improves the patient's quality of life. Thus, the thorough preoperative evaluation of DIE lesions is essential to define the best therapeutic strategy [START_REF] Abbott | Laparoscopic excision of endometriosis: a randomized, placebo-controlled trial[END_REF][START_REF] Chopin | Operative management of deeply infiltrating endometriosis: results on pelvic pain symptoms according to a surgical classification[END_REF][START_REF] Thomassin | Symptoms before and after surgical removal of colorectal endometriosis that are assessed by magnetic resonance imaging and rectal endoscopic sonography[END_REF] .

According to the literature, there is currently no consensus strategy for the pre-operative evaluation of DIE and several imaging techniques may be used. Magnetic resonance imaging (MRI) can detect both uterosacral ligaments (USL) and rectal involvement with good sensitivity (Se) (80 to 85% and 53 to 96%, respectively) [START_REF] Dumontier | Comparison of endoscopic ultrasound and magnetic resonance imaging in severe pelvic endometriosis]. Gastroenterologie clinique et biologique[END_REF][START_REF] Camagna | Surgical management of rectovaginal septum endometriosis from a continuous series of 50 cases[END_REF][START_REF] Bazot | Accuracy of magnetic resonance imaging and rectal endoscopic sonography for the prediction of location of deep pelvic endometriosis[END_REF][START_REF] Bazot | Diagnostic accuracy of physical examination, transvaginal sonography, rectal endoscopic sonography, and magnetic resonance imaging to diagnose deep infiltrating endometriosis[END_REF] . Rectal endoscopic sonography (RES) is considered the gold standard for diagnosing rectal involvement of DIE lesions, and has a Se of 82 to 100%. However, the use of these techniques is limited due to their availability and cost.

Transvaginal ultrasound (TVUS) is a non-invasive, cost-effective, well-accepted and accessible test for diagnosing benign gynecological diseases. Signs of DIE on ultrasound, including abnormal hypoechoic linear thickening, nodules or masses with or without regular contours that are visible in particular regions such as the retrocervical area, have been successfully used for diagnosis [START_REF] Bazot | Sonography and MR imaging for the assessment of deep pelvic endometriosis[END_REF] . Furthermore, several teams have described specific signs on TVUS indicative of bowel involvement associated with DIE lesions, and have reported that such an examination can effectively diagnose bowel involvement [START_REF] Goncalves | Transvaginal ultrasonography with bowel preparation is able to predict the number of lesions and rectosigmoid layers affected in cases of deep endometriosis, defining surgical strategy[END_REF][START_REF] Bazot | Diagnostic accuracy of transvaginal sonography for deep pelvic endometriosis[END_REF][START_REF] Bazot | Accuracy of transvaginal sonography and rectal endoscopic sonography in the diagnosis of deep infiltrating endometriosis[END_REF][START_REF] Valenzano Menada | Does transvaginal ultrasonography combined with water-contrast in the rectum aid in the diagnosis of rectovaginal endometriosis infiltrating the bowel?[END_REF][START_REF] Hudelist | Can transvaginal sonography predict infiltration depth in patients with deep infiltrating endometriosis of the rectum?[END_REF][START_REF] Abrao | Comparison between clinical examination, transvaginal sonography and magnetic resonance imaging for the diagnosis of deep endometriosis[END_REF] . However, the present role of TVUS in the diagnosis of DIE is disputed. Some authors [START_REF] Bazot | Diagnostic accuracy of physical examination, transvaginal sonography, rectal endoscopic sonography, and magnetic resonance imaging to diagnose deep infiltrating endometriosis[END_REF][START_REF] Bazot | Diagnostic accuracy of transvaginal sonography for deep pelvic endometriosis[END_REF][START_REF] Bazot | Accuracy of transvaginal sonography and rectal endoscopic sonography in the diagnosis of deep infiltrating endometriosis[END_REF][START_REF] Hudelist | Can transvaginal sonography predict infiltration depth in patients with deep infiltrating endometriosis of the rectum?[END_REF][START_REF] Piketty | Preoperative work-up for patients with deeply infiltrating endometriosis: transvaginal ultrasonography must definitely be the first-line imaging examination[END_REF] consider that TVUS should be used as the first-line test to diagnose DIE and rectal involvement, whereas others found that this technique has low sensitivity and specificity [START_REF] Valenzano Menada | Does transvaginal ultrasonography combined with water-contrast in the rectum aid in the diagnosis of rectovaginal endometriosis infiltrating the bowel?[END_REF][START_REF] Dessole | Sonovaginography is a new technique for assessing rectovaginal endometriosis[END_REF][START_REF] Delpy | Value of endorectal ultrasonography for diagnosing rectovaginal septal endometriosis infiltrating the rectum[END_REF] . However, few teams have published their results and such studies have been conducted in departments that are highly specialized in DIE employing practitioners who are very skilled in ultrasound imaging [START_REF] Bazot | Accuracy of transvaginal sonography and rectal endoscopic sonography in the diagnosis of deep infiltrating endometriosis[END_REF][START_REF] Abrao | Comparison between clinical examination, transvaginal sonography and magnetic resonance imaging for the diagnosis of deep endometriosis[END_REF] .

The aim of the study was to assess the accuracy of TVUS for diagnosing DIE and bowel involvement, and to compare performance between ultrasound operators who were trained or untrained in DIE imaging.

Methods

This was an observational, single-center study conducted at the gynecologic surgery unit of the Poissy-Saint-Germain-en-Laye Hospital. All patients who underwent surgery for clinical suspicion of endometriosis between October 1, 2004 and April 30, 2011 were collected in a prospective database. Patients were included prospectively when they had symptoms advocating endometriosis at pre-operative examination with no obvious pathology.

Painful symptoms were chronic pelvic pain of more than six months duration, including severe dysmenorrhea, deep dyspareunia, cyclic pelvic pain and painful defecation, with or without infertility. Patients with a myoma or an ovarian cyst (including endometrioma) of more than 40 mm and those who had previously undergone surgery for the resection of a DIE nodule, or those without an available complete preoperative ultrasound report, were excluded from the study. Epidemiological, ultrasound, surgical and histological data were collected with a standardized method. The practitioner (A.R.) who collected ultrasound results was blind to surgical findings collected during surgery. The recorded ultrasound data included the number, size, depth of infiltration and localization of DIE nodules, and the number and size of ovarian cysts. Ultrasound operators were blind to the results of other imaging tests at the time of the TVUS. Surgical reports were based on ASRM classification [START_REF]Revised American Society for Reproductive Medicine classification of endometriosis[END_REF] and immediately completed by the surgeon after the intervention in a standardized datasheet. The report described the surgical approach used (laparoscopy or laparotomy), the number, size, depth of infiltration, localization and treatment of DIE nodules, and the number and size of ovarian cysts. Surgeons were trained in endometriosis surgery and aware of the results of imaging tests performed before surgery. DIE was diagnosed according to surgical and histological criteria [START_REF] Kennedy | ESHRE guideline for the diagnosis and treatment of endometriosis[END_REF] . A positive surgical diagnosis was based on obvious retroperitoneal infiltration of more than 5 mm, visible nodule or infiltration associated with palpable induration, or visible dark blue nodule of the posterior vaginal fornix [START_REF] Chapron | Presurgical diagnosis of posterior deep infiltrating endometriosis based on a standardized questionnaire[END_REF] . A positive histological diagnosis was based on the presence of endometrial glands and stroma on resected specimens. Digestive DIE was defined as the involvement of the muscularis propria of the digestive wall. Histological tests were performed on the resected specimen if the nodule was resected, and on biopsies if the nodule was not resected. DIE was considered absent if the surgical and/or histological diagnosis was negative as follows: (i) a negative surgical and histological diagnosis; (ii) a negative surgical diagnosis and a positive histological diagnosis, corresponding to peritoneal endometriosis with retroperitoneal infiltration of less than 5 mm; or (iii) a positive surgical diagnosis and a negative histological diagnosis corresponding to retroperitoneal fibrosis without endometriotic lesions (scar tissue from previous intervention, infections, malignant lesions).

All ultrasound operators (N=47) had a national degree in gynecological ultrasound and regularly practiced gynecological ultrasound scans. Forty-six ultrasound operators were classified as untrained in endometriosis imaging based on their replies to a questionnaire about their experience of ultrasound; 28 were radiologists (60.9%), 15 were obstetrician gynecologists (32.6%), two were general practitioners (4.3%), and one was a medical gynecologist (2.2%). One ultrasound operator (J-P.B.) was an obstetrician gynecologist who had practiced gynecologic and obstetrical ultrasound for over 15 Ordinal variables were compared with the Chi-squared test when the expected numbers were ≥ 5, and Fisher's exact test when they were < 5. The Student's t-test was used to compare continuous variables if they were normally distributed. Association between ultrasound and the diagnosis of DIE was studied by the Chi-squared test or Fisher's exact test for ordinal variables for both types of ultrasound operator. The diagnosis of DIE and the diagnosis of digestive involvement were examined separately. If there was a significant association, sensitivity, specificity and the c-index were calculated with 95% confidence intervals (95% CI). The c-index measures the overall predictive information of each of a diagnostic test prediction and is defined as the proportion of all usable patient pairs in which the predictions and outcomes are concordant, which is, for a binary outcome prediction mathematically equivalent to the area under the receiver operating characteristic curve.

Differences between trained and untrained ultrasound operators were considered significant if the confidence interval of the c-index of one group did not include that of the other.

Results

A total of 175 women underwent surgery at our institution for suspected endometriosis between October 2004 and April 2011. Sixty patients were excluded and 115 patients were included in the study (Figure 1). There were 110 (95.6%) successful laparoscopic procedures, three laparotomies and two laparoscopies that were converted into a laparotomy. There was no significant difference between patients with DIE and patients without DIE regarding age, body mass index (BMI), desire for pregnancy, and mean delay between ultrasound and surgery. A total of 126 TVUS were performed: 70 (60.9%) by the trained ultrasound operator and 56 (48.7%) by an untrained ultrasound operator. There were no significant differences regarding the general characteristics of the population between these two groups (Table 1).

Patients received a single ultrasound by the trained (59/115) or an untrained ultrasound operator (45/115), or received an ultrasound from both successively (11/115).

Fifteen patients did not have deep infiltrating endometriosis: nine had both a negative surgical and histological diagnosis; three had a negative surgical diagnosis and a positive histological diagnosis, corresponding to peritoneal endometriosis with retroperitoneal infiltration of less than 5 mm; and three showed macroscopic aspects of endometriosis but histological diagnosis after complete resection of the lesion was negative, and revealed retroperitoneal fibrosis without endometriotic lesions. Thus, 100 patients (86.9%) had DIE.

Thirty-four patients (29.6%) had anterior DIE, including eight patients (7.0%) with detrusor infiltration. All women with anterior DIE lesions also had posterior DIE. Thirty-four patients (29.6%) had digestive involvement, including rectal involvement in 26 cases, involvement of the recto-sigmoid junction in seven cases and sigmoid involvement in two cases. Surgical data including the location, size and number of nodules are described in Table 1. Overall, 186 DIE nodules were observed during surgery. Nodules were removed or left intact in 71.5% and hysterectomy (6.5%) or a proctectomy (2.7%).

Overall TVUS (performed by either the trained or an untrained ultrasound operator) significantly predicted the presence of DIE (Fisher's exact test, p=0.005) with a sensitivity of performed by untrained ultrasound operators did not significantly predict the presence of DIE (Fisher's exact test, p=0.58), sensitivity and specificity were not calculated, and the c-index was 0.56 [0.52-0.61]. The c-index of the untrained ultrasound operators was not within the range of the confidence interval of the trained ultrasound operator; thus, the diagnostic value of TVUS performed by the trained ultrasound operator was significantly higher than that of TVUS performed by an untrained ultrasound operator (Figure 2). TVUS performed by the trained ultrasound operator significantly predicted the presence of rectal involvement (Fisher's exact test, p=0.001) with a sensitivity of 40% [95% CI, 23-59], a specificity of 93% [95% CI, 86-100], and a c-index of 0.67 [95% CI, 0.56-0.77].

Untrained ultrasound operators did not diagnose any cases of rectal involvement, although 14 women (25% of their patients) had DIE involving the digestive wall.

Discussion:

Our study shows that routine TVUS has an insufficient diagnostic accuracy to detect DIE and to diagnose rectal involvement, even when performed by a trained ultrasound operator, experienced in the field of endometriosis. We also demonstrate that the expertise in the field of endometriosis of the ultrasound operator has an important impact on the diagnostic performance of the examination.

Our study is the first one to examine how the level of training of ultrasound operators affects the accuracy of TVUS for the diagnosis of DIE, according to well-defined criteria for training in endometriosis imaging. The gold standard method used here to confirm diagnosis was very strength: all patients had surgery and histological analysis in accordance with CNGOF [START_REF] Roman | Guidelines for the management of painful endometriosis[END_REF] and ESHRE [START_REF] Kennedy | ESHRE guideline for the diagnosis and treatment of endometriosis[END_REF] criteria. Furthermore, all DIE implants were characterized with precision. Indeed, if an implant was not resected, positive surgical diagnosis was based on the appearance of the nodule [START_REF] Chapron | Presurgical diagnosis of posterior deep infiltrating endometriosis based on a standardized questionnaire[END_REF] and biopsies were performed for histological diagnosis. In addition, our exclusion criteria limited potential confounders that might decrease the sensitivity of TVUS for the diagnosis of DIE lesions, such as endometriomas and myomas > 40 mm [START_REF] Bazot | Diagnostic accuracy of transvaginal sonography for deep pelvic endometriosis[END_REF] .

There are nonetheless several limitations to our study. At first, the prevalence of DIE was very high in our population suggesting the possibility of referral bias [START_REF] Miller | Effects of adjustment for referral bias on the sensitivity and specificity of single photon emission computed tomography for the diagnosis of coronary artery disease[END_REF] . Indeed, 86.9% of patients had posterior DIE and the prevalence of bowel involvement in DIE patients was particularly high (34%) compared to other sample of women operated for DIE. Chapron et al. [START_REF] Chapron | Anatomical distribution of deeply infiltrating endometriosis: surgical implications and proposition for a classification[END_REF] reported that only 9.9% of DIE lesions infiltrated digestive organs. These figures can at first be explained by the fact that, in our study, the patient inclusion criteria were rather strong and surgical procedures for diagnosis were highly standardized. Nonetheless, it is obvious than those patients who had an abnormal TVUS will have surgery to confirm the diagnosis more frequently than those who had a normal TVUS. Therefore, our population contained a higher proportion of abnormal TVUS than in the population of women having TVUS for chronic pelvic pain. Thus, the 58% sensitivity rate which we have calculated for TVUS might in turn have been artificially increased and the actual diagnostic accuracy of TVUS may be lower than that reported in our study.

Secondly, untrained ultrasound operators performed "first-line" ultrasound scans whereas the trained ultrasound operator performed first or second line scans on those patients who have been referred specifically to them: the indications for TVUS in these patients may be better defined and they probably have more severe endometriosis than patients sent for first-line scans. This may affect the difference of predictive value of TVUS between trained and untrained operators, but we were not able to take this into account in our statistical analysis.

Finally, we assessed the diagnostic accuracy of TVUS by only one trained ultrasound operator, compared with many untrained ultrasound operators. This could have an impact in our result, taking into account inter-operator variability. Untrained ultrasound operators may have various experience of endometriosis imaging. The diagnostic accuracy of TVUS performed by untrained ultrasound operators might have been different if there were fewer operators and with the same experience. Conversely, there are probably different levels of experience for endometriosis imaging. Our results might have been more representative if the diagnostic accuracy had been assessed by several trained operators.

The diagnostic accuracy of TVUS for the diagnosis of DIE, and in particular digestive involvement, has been reported in only a few studies [START_REF] Bazot | Diagnostic accuracy of physical examination, transvaginal sonography, rectal endoscopic sonography, and magnetic resonance imaging to diagnose deep infiltrating endometriosis[END_REF][START_REF] Bazot | Diagnostic accuracy of transvaginal sonography for deep pelvic endometriosis[END_REF][START_REF] Bazot | Accuracy of transvaginal sonography and rectal endoscopic sonography in the diagnosis of deep infiltrating endometriosis[END_REF][START_REF] Hudelist | Can transvaginal sonography predict infiltration depth in patients with deep infiltrating endometriosis of the rectum?[END_REF][START_REF] Abrao | Comparison between clinical examination, transvaginal sonography and magnetic resonance imaging for the diagnosis of deep endometriosis[END_REF][START_REF] Piketty | Preoperative work-up for patients with deeply infiltrating endometriosis: transvaginal ultrasonography must definitely be the first-line imaging examination[END_REF][START_REF] Bazot | Transvaginal sonography and rectal endoscopic sonography for the assessment of pelvic endometriosis: a preliminary comparison[END_REF] , and the results are heterogeneous (Table 2). Most publications were performed by the same team, Bazot et al. [START_REF] Bazot | Diagnostic accuracy of physical examination, transvaginal sonography, rectal endoscopic sonography, and magnetic resonance imaging to diagnose deep infiltrating endometriosis[END_REF][START_REF] Bazot | Diagnostic accuracy of transvaginal sonography for deep pelvic endometriosis[END_REF][START_REF] Bazot | Accuracy of transvaginal sonography and rectal endoscopic sonography in the diagnosis of deep infiltrating endometriosis[END_REF][START_REF] Bazot | Transvaginal sonography and rectal endoscopic sonography for the assessment of pelvic endometriosis: a preliminary comparison[END_REF] who have a great experience with TVUS and bowel endometriosis. Not surprisingly, the diagnostic accuracy of TVUS, even performed by a trained ultrasound operator, we have found is far lower than that found by Bazot et al. [START_REF] Bazot | Diagnostic accuracy of physical examination, transvaginal sonography, rectal endoscopic sonography, and magnetic resonance imaging to diagnose deep infiltrating endometriosis[END_REF][START_REF] Bazot | Diagnostic accuracy of transvaginal sonography for deep pelvic endometriosis[END_REF][START_REF] Bazot | Accuracy of transvaginal sonography and rectal endoscopic sonography in the diagnosis of deep infiltrating endometriosis[END_REF] . Peculiarly, the sensitivity of TVUS for predicting bowel involvement we have found is less than half than those reported by this author. This may be explained by the 70.0% rate of rectal infiltration among DIE patients reported by this author [START_REF] Bazot | Diagnostic accuracy of physical examination, transvaginal sonography, rectal endoscopic sonography, and magnetic resonance imaging to diagnose deep infiltrating endometriosis[END_REF][START_REF] Bazot | Accuracy of transvaginal sonography and rectal endoscopic sonography in the diagnosis of deep infiltrating endometriosis[END_REF] , versus 34% in our study. It may also relate to differences between the severity of the disease among patients referred to this center, which is well-known for colorectal endometriosis, compared to our center which takes care of various form of the disease. Fortunately, the sensitivity of TVUS reported by Dessole et al. is equivalent to ours [START_REF] Dessole | Sonovaginography is a new technique for assessing rectovaginal endometriosis[END_REF] , even if this study took place in tertiary referral center in which the prevalence of endometriosis is high. Those studies used transvaginal probes of different frequencies, that could modify the diagnostic accuracy of TVUS in diagnosing DIE.

Unlike ours, the previous published studies (Table 2) did not involve procedures carried out by several ultrasound operators and thus measures of inter-observer variability are lacking. Pooling the results of several ultrasound operators would provide a more accurate estimate of diagnostic value of TVUS representative of the population of ultrasound operators available in the everyday-life practice. TVUS, although frequently used as a first-line test for various gynecologic diseases, may thus be inadequate to screen for DIE in routine practice.

The place of TVUS should be discussed alongside the other pelvic imaging examinations to assess the extent of the disease and rectal involvement at pre-operative evaluation. MRI is expensive and less accessible but can map pelvic lesions and extension into deep organs with high sensitivity and specificity [START_REF] Dumontier | Comparison of endoscopic ultrasound and magnetic resonance imaging in severe pelvic endometriosis]. Gastroenterologie clinique et biologique[END_REF][START_REF] Camagna | Surgical management of rectovaginal septum endometriosis from a continuous series of 50 cases[END_REF][START_REF] Bazot | Accuracy of magnetic resonance imaging and rectal endoscopic sonography for the prediction of location of deep pelvic endometriosis[END_REF][START_REF] Bazot | Diagnostic accuracy of physical examination, transvaginal sonography, rectal endoscopic sonography, and magnetic resonance imaging to diagnose deep infiltrating endometriosis[END_REF] . Rectal endoscopic sonography (RES) is a good technique to assess rectal infiltration and has a great sensitivity of 82 to 100 % 4- 7,18,36-42 . However, it is an invasive test and it only examines the rectum and lower sigmoid [START_REF] Bazot | Accuracy of magnetic resonance imaging and rectal endoscopic sonography for the prediction of location of deep pelvic endometriosis[END_REF] .

TVUS is frequently used due to its accessibility but its sensitivity is poor, even with a trained ultrasound operator. Our opinion is that TVUS alone is not sufficient to rule out rectal involvement, but it may help to the diagnosis. Its association with several other tests is thus mandatory for adequate pre-operative evaluation. cannot all be performed easily [START_REF] Goncalves | Transvaginal ultrasonography with bowel preparation is able to predict the number of lesions and rectosigmoid layers affected in cases of deep endometriosis, defining surgical strategy[END_REF][START_REF] Valenzano Menada | Does transvaginal ultrasonography combined with water-contrast in the rectum aid in the diagnosis of rectovaginal endometriosis infiltrating the bowel?[END_REF][START_REF] Abrao | Comparison between clinical examination, transvaginal sonography and magnetic resonance imaging for the diagnosis of deep endometriosis[END_REF][START_REF] Dessole | Sonovaginography is a new technique for assessing rectovaginal endometriosis[END_REF][START_REF] Guerriero | Tendernessguided" transvaginal ultrasonography: a new method for the detection of deep endometriosis in patients with chronic pelvic pain[END_REF][START_REF] Guerriero | Diagnostic value of transvaginal 'tenderness-guided' ultrasonography for the prediction of location of deep endometriosis[END_REF][START_REF] Menada | Transvaginal ultrasonography combined with water-contrast in the rectum in the diagnosis of rectovaginal endometriosis infiltrating the bowel[END_REF] . Nevertheless, preparing the digestive tract before TVUS is a simple approach that could improve the sensitivity of TVUS for the diagnosis of rectal infiltration. Prospective studies should be conducted to assess the diagnostic value of these technical modifications.

According to good professional practice it is recommended that "all imaging tests should be performed by operators trained to detect DIE". Our study demonstrates clearly the impact of specialized training to identify endometriosis on ultrasound. The ultrasound operator should thus be aware of the symptoms and clinical signs that prompted the use of an ultrasound scan, should carry out the exam according to a precise protocol and should know the signs on ultrasound to look for [START_REF] Martin | Modern imaging of pelvic endometriosis. Ultrasonography, scanography and magnetic resonance imaging[END_REF] .

  years. He was considered trained in endometriosis imaging after having completed these successive steps of training: (i) theoretical training in endometriosis imaging involving courses and conferences given by experts in endometriosis; (ii) surgical training involving practical training with surgeons carrying out surgical interventions for endometriosis; and (iii) self-assessment of skills in real practice. Patients were referred primarily to this trained ultrasound operator for ultrasound when patients did not have TVUS or in cases of missing or non-conform ultrasound procedures. This trained ultrasound operator used a Voluson 730 Expert machine (GE Healthcare Europe, Buc, France) with a 7-MHz transvaginal probe. Institutional Review Board approval was given for this study. The CEROG (ethical board of research in gynecology and obstetrics) approval number was CEROG-2011-GYN-06-01.

  42.7% [95% CI, 33-52], a specificity of 93.7% [95% CI, 70-100], and a c-index of 0.68 [95% CI, 0.61-0.76]. TVUS results were also significantly predictive of rectal involvement, (Fisher's exact test, p=0.004) with a sensitivity of 22.5% [95% CI, 11-38], a specificity of 95.3% [95% CI, 88-99], and a c-index of 0.59 [95% CI, 0.52-0.66]. TVUS performed by the trained ultrasound operator significantly predicted the presence of DIE (Fisher's exact test, p=0.02) with a sensitivity of 58% [95% CI, 46-70], a specificity of 87.5% [95% CI, 63-100], and a c-index of 0.73 [95% CI, 0.59-0.87]. However, TVUS

Figure 1 :Figure 2 :

 12 Figure 1:Flow diagram of the study.

Table 1 :

 1 Characteristics of the patients of the study

	Group of the	Group of
	trained	untrained	Total
	operator	operators	N=126
	N=70	N=56

* include patients with anterior DIE

** all patients with anterior DIE also had posterior DIE. So the total of USL, vagina and rectum involvement patients was equal to the number of patients with DIE.

Table 2 :

 2 Sensitivities for the diagnosis of any DIE and of rectosigmoid DIE by TVUS in previous published studies

	Authors	N	Any DIE	Rectosigmoid DIE
	Dessole 2003 17	46	43.7	33.3
	Bazot 2004 11	142	78.5	87.2
	Bazot 2007 12	81	87.3	92.6
	Valenzano Menada 2008 13	90	92.8	56.5
	Piketty 2009 16	134	NA	90.7
	Bazot 2009 8	92	87.8	93.6
	Hudelist 2009 14	200	NA	98
	Present study	115	42.7	22.5
	N	Effective of the study
	NA	Non Available
	DIE Deep Infiltrating Endometriosis
	TVUS Transvaginal Ultrasonography
	USL Uterosacral Ligament