Self-mineralization and assembly of a bis-silylated Phe-Phe pseudodipeptide to a structured bioorganic-inorganic material † Saïd Jebors, a Laurine Valot, a,b Cécile Echalier, a Baptiste Legrand, a Remi Mikhaleff, a Arie Van Der Lee, c Raul Arenal, d Pascal Dumy, a Muriel Amblard, a Jean Martinez, a Ahmad Mehdi,* b and Gilles Subra * a Self-mineralization of trialkoxysilyl hybrid peptide yield in a single step a nanostructured hybrid material. A bis-silylated pseudodipeptide inspired from the Phe-Phe dipeptide was used to program the assembly by sol-gel polymerization in heterogeneous conditions, in water at pH 1.5 without any structure-directing agent. A mechanism deciphering the hybrid material assembly was proposed thanks to 1H NMR spectroscopy. First, water-insoluble hybrid building blocks were hydrolysed into their soluble silanol counterparts. Then, these transitional species, thanks to hydrogen bonding and π-π stacking, self-assembled in solution. Last, the proximity of silanol moieties favoured their polycondensation into growing siloxane oligomers, which spontaneously precipitated to produce an ordered hybrid material.

The control of shape and organization of matter at the nanoscale is of major interest for nanoscience. 1,2 This can be achieved by a bottom-up strategy involving the self-assembly of molecules. Peptides are very attractive building blocks for this approach by the functionalities afforded by the side chains of amino acids, and their three-dimensional structures offer a large diversity of potential interactions and intermolecular arrangements.

Chemists being inspired by the natural recognition systems driving the formation of nanostructures. Thus, considerable works have been done on self-assembly of synthetic peptides in the last decades. Secondary structures such as -helices, polyproline (PP) helices, and -strands, have been used to design fibrous peptide materials and hydrogels. They are obtained by self-assembly through hydrogen bonding in case of -sheets 3 and principally by side chain or N-and C-terminus interactions in the case of  helical 4 and PPII systems. 56 Peptide amphiphiles (PA) have also been used to obtain nanofibers. Their assembly is mostly driven by the lipid tail, a region devoted to -sheet formation through repetitive bonding and charged end-regions. 78 Interestingly, PA can be functionalized by bio relevant sequences such as anionic nucleation templates for biomineralization with hydroxyapatite, mimicking in some way the process of bone tissue formation. 9 Another example of biomineralization is the dynamic self-assembly of peptide silica fibres obtained from the cyclic -sheet forming peptide lanreotide.

Stacking and arrangement of dimers of the cationic lanreotide catalyse condensation of the anionic silicic acid through charge neutralization, enabling the growth of silica-peptide micrometric fibres. 10 Aside from the fundamental understanding of how nature manufactures 'hard' and highly structured macroscopic objects (bones, shells, teethes, etc.) from soluble nanometric biomolecules, mineralization of self-assembled scaffolds can be attractive to design new bioinspired nanomaterials with increased stability and resistance.

On the other hand, the sol-gel process is the route of choice to obtain silica and hybrid organic/inorganic materials. It involves the hydrolytic condensation of alkoxysilane precursors at room temperature. Noteworthy, the use of surfactants during the sol-gel process yield highly ordered mesoporous silica's (OMS). [11][12][13] Hybrid bioorganic materials are mostly obtained by post-functionalization of the silica and by non-covalent adsorption of biomolecules. On the opposite, we described that hybrid peptide units, bearing at least one triakoxysilyl group could be used as a building block to prepare peptide-silica hybrid materials through the sol-gel protocol. 1415 At the same time, we demonstrated that highly functionalized lamellar silica could be easily obtained by hydrolysis and polycondensation of a simple organosilane, in water and without any structure-directing agent (SDA). [16][17][18] In that context, we decided to use hybrid trialkoxysilyl peptides for elaboration of nano structured materials via the 'self-mineralization' of hybrid peptide building blocks displaying self-recognition properties. (Fig. 1) Fig. 1 Principle of self-mineralization of bis-silylated peptide.

On the contrary to existing methods for the synthesis of hybrid materials, some key features can be pointed out. First, our approach does not require addition of any inorganic charge such as tetraethoxysilane (TEOS), silicic acid or phosphate and calcium salts to mineralize the peptide assembly. Second, it does not require any SDA as such used to obtain ordered mesoporous silica (OMS).

To demonstrate the feasibility of this approach, we took inspiration from the dipeptide sequence Phe-Phe, perhaps the simplest self-recognition element in peptide sequences. 19 Diphenylalanine is the main Alzheimer's -amyloid recognition motif and self-assemble into ordered stiff nanotubes. 20 Since its discovery, a large number of other aromatic dipeptides 21 were used to obtain various assembled materials such as fibrils, nanowires, nanotubes, vesicles, depending on the conditions of formation. 2223 The basis of the assembly proceeds first by non-covalent assembly of a cycle formed by several dipeptides (six dipeptides in the case of the seminal H-Phe-Phe-OH sequence) brought together by carboxylic acid and amine interactions. Each non covalent cyclic peptide can seat on the top of another one, thanks to aromatic -stacking, yielding a hollow tube, which in turn packs laterally with another tube to give a supramolecular object. 24 Scheme 1 Synthesis of bis-silylated pseudodiphenylalanine building blocks 3 and 4.

Based on these mechanisms, we designed the hybrid building block 3, which satisfies four requirements: (i)-it displays the two phenylalanine side chain benzyl groups in the same orientation than the original all (L) diphenylalanine; (ii)-it presents two triethoxysilyl functionalities, which should 'freeze' covalently the structure by inorganic polymerization; (iii)-it should favour a strong H-bonding network through the urea functions replacing the carboxylateammonium interactions of the original H-Phe-Phe-OH; (iv)-the building block has to be as symmetrical as possible, presenting the silyl group in an even manner at both sides of the di aromatic template.

To address the last requirement, a gem diamino phenylalanine was synthesized to get a primary amine function substituting the Cter function of H-Phe-Phe-OH. The key step of the synthesis of H-Phe-gemPhe-H 2 (Scheme 1, and Fig. S1,ESI) is a Hoffmann rearrangement 25 to convert the C-ter amide to the transient corresponding isocyanate, which was hydrolysed into the primary amine with evolving CO2. The pseudopeptide 1 (Boc-Phe-gemPhe-H) was recovered by precipitation in diethyl ether in a 92% very good yield. Following Boc removal by TFA treatment (Figure S2), the gem-diamino peptide 2 presenting two primary amine functions was reacted with 2.2 equivalents of 3-isocyanatopropyltriethoxysilane (ICPTES) to yield the hybrid building block 3. The silylation reaction took place in dry DMF under inert argon atmosphere to avoid the premature hydrolysis and the subsequent polycondensation of alkoxysilane groups. At the end of the reaction, the hybrid pseudopeptide 3 was precipitated in diethyl ether and dried overnight under vacuum. The reaction was quantitative and compound 3 was characterized by ESI+ LC/MS and 1 H, 13 C, 29 Si NMR, (Figures S3-S6) before being stored at 4°C under argon atmosphere. Noteworthy, compound 4 containing trimethylsilyl groups was also synthesized as a stable analogue to verify the self-assembly of the building block (Fig. S7-S10, ESI). Indeed, it is unable to undergo solgel inorganic polymerization. Compound 4 was recrystallized from hot ethanol and the obtained single-crystal structure was analyzed. The structure shows three independent molecules with perfect alignment of phenyl groups and H-bonding between urea and amide parts of the pseudopeptide that confirms the lamellar arrangement (Fig. 2). The three molecules of 4 in the unit cell differ in the orientation of the trimethylsilane groups with respect to the molecule backbones (Fig. S11-S13, ESI). Molecules are connected by relatively strong classical hydrogen bonds of 2.20 Å (Fig. 2), forming a stacked sequence along the b-axis. , the distance between two aromatic cycle can be evaluated to be around 5.2 Å and this distance is largely lower than 6.5 Å, proving the presence of - stacking. Once self-assembly of the building block was evidenced, reactive precursor 3 was used for the preparation of hybrid materials by selfassembly driven sol-gel process. The sol-gel chemistry proceeds in two steps: 1) hydrolysis of the alkoxysilanes into silanols groups (SiOH), and 2) condensation of SiOH into siloxanes (Si-O-Si). In acidic aqueous solution (pH 1.5), hydrolysis of the triethoxysilane is fast. On the contrary, condensation does not proceed, and silanols do not spontaneously react together to yield siloxanes bonds. Thus, when polymerization occurs at pH 1.5, this condensation is driven by a selfassembly process, distributing the silanols in close vicinity. [25,26] Two different procedures have been assayed to obtain a hybrid material from the hybrid block 3, using homogeneous (route A) or heterogeneous (route B) conditions. In homogeneous conditions, the silylated peptide 3 was solubilized at 38.5 mM in a mixture of water (HCl, pH 1.5)/THF (1/1, v/v) solution. It is worth noting here that the organic solvent was necessary to solubilize compound 3 after sonication. Even after total hydrolysis of EtOSi groups, the solution remained limpid for several days, clearly indicating that the inorganic polymerization did not proceed. This observation could be explained by the lack of self-assembly due to the damaging effect of THF on the establishment of hydrogen bonding as a driving force of the mineralization process. 29 Failing to observe any precipitate, the solvent was then slowly removed under an extractor hood to induce the polycondensation process. In fact, after solvent evaporation, a white solid (noted M3A) was obtained in the bottom of the beaker, and was observed by SEM (Fig. S14,ESI). The worm-like solid obtained witnessed that the condensation took place during evaporation, which forced the Si-O-Si bond formation by water evolving, without any self-assembly. This result was confirmed by Xray diffraction analysis (Fig. 3). In fact, the XRD pattern for M3A exhibited only one low-angle diffraction peak (d100) at 2.69° corresponding to the d-spacing of 3.29 nm attributed to the correlation distance between the inorganic parts.

Alternatively, heterogeneous conditions were assayed. The silylated pseudopeptide 3 was poured into water (HCl, pH 1.5) with the same ratio weight/volume of solution than in the previous experiment (theoretical concentration: 38.5 mM). As already stated, compound 3 was insoluble in water and thus remained as a powder on the surface. The heterogeneous mixture was left under magnetic stirring for four days. This white sedimented solid was filtered, washed with acidic water (pH 1.5) and dried under vacuum, for 40 minutes at 50°C to give quantitatively the corresponding hybrid material called M3B. In striking contrast with the previous solid obtained by evaporation, MEB analysis showed well organized superimposed hybrid sheets of > 10µm length (Figure 4), and TEM analysis clearly showed lamellar sheets. Noteworthy, placed in the same conditions, compound 4 did not yield any precipitate and remained intact as expected since C-SiMe3 groups are very stable under these conditions. To be sure that no degradation of the pseudodipeptide occurs during the process, an NMR is done at the beginning of the reaction with the compound 4 and after 7 days, and showed its stability at this acidic pH (Fig. S15,ESI). A supposed mechanism was proposed for the formation of such organized solid (Scheme 2). First, water-insoluble hybrid building blocks 3, were hydrolysed into their soluble silanol [Si(OH)3] counterparts. Then, these transitional species, thanks to hydrogen bonding and - stacking, tended to self-assemble in solution. Last, the proximity of silanol moieties probably favours their polycondensation into siloxanes dimers and growing oligomers, which spontaneously precipitated as ordered hybrid material. Several experiments supported this mechanism. We first studied the course of events in solution using 1 H NMR spectroscopy (Scheme 2 and Fig. S16,ESI). For that, the hybrid pseudopeptide 3 (with the same ratio weight/volume of solution than in the previous experiment meaning 38.5 mM) was placed in D2O and pH was adjusted to 1.5 using DCl. We followed the hydrolysis and polycondensation steps for 78 hours, monitoring unambiguous probes, i.e. resonances of ethanol produced during the hydrolysis (CH2 = 3.6 ppm and CH3 = 1.1 ppm), aromatic protons of the peptide moiety (centred around 7.3 ppm), and methylene protons in  of the alkoxysilane groups (  CH2 = 0.5 and 0.6 ppm). At t = 15 min, we only observed very small signals corresponding to ethanol and we could not detect any trialkoxysilylated hybrid pseudopeptide 3 resonances, since it was insoluble. We observed that ethanol signals rapidly increased with time until 7 h, then moderately until 49 h, and very slowly for the last 19 hours of experiments. Concomitantly, the intensity of the hybrid pseudopeptide resonances increased, since the hydroxysilylated derivatives were soluble even at high concentrations, reaching a plateau after 49 h (Scheme 2). We could notice that phenylalanine aromatic signals came from various soluble pseudopeptide species, both hydrolysed (and partially hydrolysed) pseudopeptide 3 monomers, and partially condensed oligomers. Thus, hydrolysis of EtOSi groups occurred rapidly after the contact of 3 with the acidic aqueous solution, releasing ethanol molecules, while as expected, the hydroxysilylated hybrid pseudopeptide concentration quickly increased. Interestingly, the weak but regular increase of the ethanol concentration while the pseudopeptide concentration remained stable after 49 h, should indicate that a proportion of the either peptide precipitated or was polycondensed to form large objects that could not be detected using liquid-state NMR spectroscopy. Fortunately, the hydrolysis and condensation events could be simultaneously monitored, following the intensity and the chemical shift of the adjacent methylene groups to the silicon atoms (Scheme 2). First, the  CH2 signals at 0.5 ppm belonging to the hydroxysilylated species quickly increased like the phenylalanine aromatic protons during the early phase of the hydrolysis until 3 h. Then, it decreased regularly until 49 h and only a small residual signal remained until the end of the experiments. It is noteworthy that a  CH2 signal at 0.6 ppm probably arising from the partially condensed oligomeric species not large enough to precipitate. Being soluble, these partially condensed species could be detected after 3 h, when the drop of the  CH2 signal at 0.5 ppm occurred. Then, the intensity of the  CH2 resonances at 0.5 and 0.6 ppm evolved in an opposite way until being stable at 46 h, the 0.6 ppm signal being largely predominant. Thus, we could reasonably hypothesize that the concentration of hydroxysilylated species necessary for the condensation was reached after 3 h and the proportion of partially condensed species increased until 46 h. At the end of experiment after 78 hours, the main species in solution was the partially condensed hybrid peptide, while the organized hybrid peptide material precipitated.

The organized solid M3B was then studied. First, we verified the covalent nature of the assembly by placing the solid in usual organic solvents (alcohol, DMF, DMSO …) even in TFA. As expected, the solid did not solubilized confirming the formation of siloxane bonds. In fact, the 29 Si CP-MAS NMR spectrum of M3B (Figure S19, ESI) displayed two peaks at -57 and -66.0 ppm attributed to T 2 [C-Si(OH)(OSi)2] and T 3 [C-Si(OSi)3] substructures. The percentage of T1, T2 and T3 substructures was found to be 5.5, 46.5 and 48 respectively and the condensation rate was estimated to be around 81%. In addition, the absence of resonance near -100 ppm corresponding to Q substructures (Si(OSi)4 ) denoted also that there was no cleavage of Si-C bonds during the sol-gel process. This stability was confirmed by C/N ratio, which is in good agreement with theoretical one, obtained from elemental analysis (Table S1, ESI). Infrared spectroscopy was performed on M3B and the spectrum shows clearly the presence of an intense band centered at 1150 cm - 1 assigned to Si-O-Si bonds (Figure S20 and table S2). In addition to all bands characteristic to the peptide units, a band at 3300 cm -1 attributed to uncondensed SiOH groups. In addition, two prominent features, the amide I (~1650 cm-1) and amide II (~1560 cm-1) bands, where the former arise primarily from the C=O stretching vibration and the latter is attributed to the N-H bending. The difference (Δν) between the two vibration modes (amide I and amide II) was found to be very close to those observed in the compound 3 and 4 at solid state. Indeed, the Δν values are often used to give a good indication of the strength of the H-bonds between ureido groups, the lower the Δν values; the stronger the H-bonding interactions are (Table S3, ESI). By contrast to M3A, the XRD pattern for M3B (Figure 3) exhibited two low-angle diffraction peaks (d100 and d200) at 2.84 and 5.70° corresponding to the d-spacings of 3.11 nm and 1.55 nm respectively. The peak observed at the higher angles is attributed as the second-order harmonic of the first peak and its presence indicates the well-ordered layered structure of M3B material obtained in water. Such differences of the solid structures obtained in different conditions was already observed and discussed in the literature, [28][29][30][31] and endorse the importance of the solvent in the control of the assembly of hybrid material during the sol-gel process. There is no one-to-one correspondence between the observed dspacings in the diffractogram of M3B and the length of the monomer which can be approximately deduced from the single-crystal structure of compound 4, The repeat distance in the hydrolysed (polymeric) structure of M3B deduced from this monomer structure is about 2.10 nm, which obviously does not correspond to the observed d-spacing of 3.11 nm. This means most probably that the principal repeat vector in M3B is not aligned with the monomer long axis and/or that a centred unit cell in M3B is formed. Scheme 2 Putative mechanism of self-assembly and self-mineralization of hybrid block 3 in water, pH 1.5 and integration of 1 H NMR signals of some relevant proton signals with their time evolution.

To assess that the structure of the material is made by the selfassembly of the peptide, an unsymetric silylated PhegemPhe pseudosipeptide 5 was prepared with a non-reactive trimethylsilyl moiety and a reactive triethoxysilyl one (Scheme S1 and Fig. S23-S28, ESI). A new material M5 is prepared with the same process than M3B. TEM images showed a semi organized material (Fig. 5A), and XRD analysis confirmed this (Fig. S30, ESI).

Another possibility was to prepare a building block 6 without pistacking but sterically overloaded (and unpolar) to compare with compound 3 and their relative materials (M6 and M3B). This compound is made with a bis-silylated pseudodileucine (Scheme S2 and Fig. S31-S37, ESI) and the material is prepared with the same process than M3B. It is interesting to notice that because of lack of self-assembly, the yield is relatively low compared to other materials. When analysed by TEM (Fig. 5B), only amorphous objects are observed, proving that the peptidic sequence did not lead to organized structure. XRD analysis also confirmed this (Fig. S39, ESI).

Conclusions

Using a tailored building block, which displays self-recognition properties, it is possible to edify supramolecular assemblies, formed though the establishment of a network of non-covalent weak interactions. However, by nature, most of these architectures only transiently exist, and must undergo chemical cross-linking to acquire better stability. Alternatively, these building blocks may be used as templates for the deposition of an inorganic layer and the recovery of the hybrid material. Herein we have demonstrated it was possible to obtain in a single step bioorganic-inorganic architectures, by adding alkoxysilanes at suitable position on a building block programmed for the irreversibility of the self-assembly. Combining the wide range of peptide recognition motives and the simplicity of sol-gel 'soft' chemistry, we expect to provide a simple and predictable tool to organize matter at the nanometric scale.

Experimental section

Details on the experimental procedures are provided in the ESI. †

26

 26

Fig. 2

 2 Fig. 2 Crystal structure of compound 4 in the unit cell.

Fig. 3

 3 Fig. 3 XRD patterns of materials M3A and M3B.

Fig. 4

 4 Fig. 4 SEM (a and b) and TEM images (c and d) of nanometric hybrid superimposed sheets M3B obtained by sol-gel polymerization of 3 in heterogeneous conditions in water at pH 1.5.

Fig. 5

 5 Fig. 5 SEM image of nanometric mateirals M5 (a, 2 µm scale bar) and M6 (b, 1 µm) obtained by sol-gel polymerization of 5 or 6 in heterogeneous conditions in water at pH 1.5.

Acknowledgements

This work was funded by the ANR (Agence Nationale de la Recherche), the French National Research Agency (ANR-16-CE18-0003).

Peptide syntheses and purifications were performed using SynBio3 platform facilities supported by GIS IBISA and ITMO Cancer.

Conflicts of interest

There are no conflicts to declare.


MD Mathurin Beve

MD, MSc Vincent Auffret
email: vincent.auffret@chu-rennes.fr

MD Reda Belhaj Soulami

MD b , Jacques Tomasi

MD PhD Amedeo Anselmi

MD Antoine Roisne

MD a , Dominique Boulmier

MD a , Marc Bedossa

MD a , Guillaume Leurent

MD PhD Erwan Donal

MD Hervé Le Breton

MD PhD Jean-Philippe Verhoye

Comparison of the Transarterial and Transthoracic Approaches in Nontransfemoral Transcatheter Aortic Valve Implantation

come

A C C E P T E D M

A N U S C R I P T 2 bleeding, stage 2 or 3 acute kidney injury, coronary obstruction, or major vascular complications.

During the study period, 104 patients underwent transthoracic TAVI (transapical: 60.6%, transaortic: 39.4%) whereas 87 patients underwent transarterial TAVI (sub-clavian: 83.9%, transcarotid: 16.1%).

Logistic EuroSCORE I tended to be higher among transthoracic-TAVI recipients. In-hospital and 30day composite endpoint rates were 25.0% and 11.5% (p = 0.025), and 26.0% and 14.9% (p=0.075) for the transthoracic and transarterial cohorts, respectively. Propensity score-adjusted logistic regression demonstrated no significant detrimental association between the 30-day composite endpoint and transthoracic access (OR: 2.12 95% CI: 0.70-6.42; p=0.18). Transarterial TAVI was associated with a shorter length of stay (median: 6 vs. 7 days, p<0.001). TAVI approach was not an independent predictor of mid-term mortality. In conclusion, non-transfemoral transarterial-approach TAVI is safe, feasible, and associated with comparable rates of major perioperative complications, and mid-term mortality compared with transthoracic-approach TAVI.

KEYWORDS: Aortic stenosis, Transcatheter aortic valve implantation, Surgical approaches

Since the publication of the randomized Placement of Aortic Transcatheter Valves (PARTNER) trials [START_REF] Leon | Transcatheter aortic-valve implantation for aortic stenosis in patients who cannot undergo surgery[END_REF][START_REF] Smith | Transcatheter versus surgical aortic-valve replacement in high-risk patients[END_REF][START_REF] Leon | Transcatheter or Surgical Aortic-Valve Replacement in Intermediate-Risk Patients[END_REF] , transcatheter aortic valve implantation (TAVI) is recognized as an efficient therapy for treatment of severe aortic stenosis in inoperable, high and intermediate-risk patients.

Transfemoral approach, as the less invasive and safest access, stands as the reference access route for TAVI. Although non-transfemoral approaches are decreasing with the miniaturization of delivery systems, in a recent national registry 17.2% of patients were still treated by non-transfemoral accesses [START_REF] Auffret | Temporal Trends in Transcatheter Aortic Valve Replacement in France: FRANCE 2 to FRANCE TAVI[END_REF] . Alternative approaches requiring a surgical contribution include transaortic, transapical, sub-clavian, and, lately, transcarotid access. Transapical access is well-described [START_REF] D'onofrio | Medium term outcomes of transapical aortic valve implantation: results from the Italian Registry of Trans-Apical Aortic Valve Implantation[END_REF] and provides acceptable results. The transaortic approach requires to expose the ascending aorta using a ministernotomy or a right thoracotomy, and is associated with similar outcomes as transapical access [START_REF] Arai | Direct Comparison of Feasibility and Safety of Transfemoral Versus Transaortic Versus Transapical Transcatheter Aortic Valve Replacement[END_REF][START_REF] Thourani | Transapical and Transaortic Transcatheter Aortic Valve Replacement in the United States[END_REF] .

Considering their invasiveness, the use of these transthoracic approaches is decreasing. Sub-clavian access is a safe method, showing comparable results to transfemoral approach [START_REF] Gleason | Subclavian/Axillary Access for Self-Expanding Transcatheter Aortic Valve Replacement Renders Equivalent Outcomes as Transfemoral[END_REF] , however with the same limitations regarding vessel anatomy, and being unsuited for patients with thoracic artery grafts. Transcarotid access tends to represent a growing proportion of alternative accesses, despite

A C C E P T E D M A N U S C R I P T 3
ongoing questions regarding the risk of stroke [START_REF] Kirker | The Carotid Artery as a Preferred Alternative Access Route for Transcatheter Aortic Valve Replacement[END_REF][START_REF] Folliguet | Transcarotid transcatheter aortic valve implantation: multicentre experience in France[END_REF] . Given the paucity of direct comparisons of alternative accesses in the current literature, the aim of this study was to compare short and mid-term outcomes between patients undergoing transthoracic (transapical or transaortic) TAVI and transarterial (transcarotid or sub-clavian) TAVI.

METHODS

All patients who underwent a non-transfemoral approach TAVI at our institution (Rennes University Hospital, Rennes, France) from May 2009 to September 2017 were included. Details regarding the pre-procedural evaluation and follow-up modalities were previously published [START_REF] Auffret | Predictors of 6-month poor clinical outcomes after transcatheter aortic valve implantation[END_REF] . Details regarding the access selection process by the Heart Team are provided in the supplementary appendix. All patients gave written informed consent for the procedures and anonymous collection of their data, which were prospectively gathered in an electronic database. The institutional review board waived specific consent for this study due to its retrospective and observational nature.

Chronic lung disease was defined as a restrictive lung disease or a chronic obstructive pulmonary disease. Cerebrovascular disease was defined as a previous carotid surgery or a stenosis ≥ 50% of carotid or vertebral arteries. Peripheral artery disease included artery stenosis ≥ 50%, claudication and previous vascular surgery. Surgical risk was estimated with the logistic EuroScore I, the logistic EuroScore II and the Society of Thoracic Surgeons Predicted Risk Of Mortality score.

Valve Academic Research Consortium 2 (VARC-2) standards were used to define hostile chest, severe liver disease and all study endpoints [START_REF] Kappetein | Updated standardized endpoint definitions for transcatheter aortic valve implantation: the Valve Academic Research Consortium-2 consensus document[END_REF] .

The primary endpoint was the 30-day rate of major perioperative complications defined as the composite of death of any cause, need for open surgery, tamponade, stroke, major or life-threatening bleeding, stage 2 or 3 acute kidney injury, coronary obstruction, or major vascular complications.

Secondary endpoints were in-hospital and 30-day rates of the components of the primary endpoint and survival at follow-up.

Patients were included, according to the access site used, in the transthoracic (transapical or transaortic access) or transarterial (sub-clavian or transcarotid access) groups. Continuous variable are

A C C E P T E D M A N U S C R I P T

presented as mean ± standard deviation or median (interquartile range) depending on their distribution, which was assessed using the Kolmogorov-Smirnov test, and were compared using t tests or the Mann-Whitney U test as appropriate. Categorical variables are summarized as numbers (percentages), and were compared using chi-square tests or the Fisher exact test. Survival rates were summarized using Kaplan-Meier estimates, and log-rank tests were used to compare groups. To evaluate the impact of access-site on the rate of the primary endpoint while adjusting for baseline differences between groups, propensity score-adjusted multivariable logistic regression was performed. Results are expressed as adjusted odds ratio (OR) with its 95% confidence interval (CI). The propensity score was calculated as the probability of undergoing transthoracic access using a non-parsimonious logistic regression model. Details regarding variables included in the propensity score can be found in the supplementary appendix. The propensity score model had adequate calibration (Hosmer-Lemeshow goodness-of-fit p=0.46) and discrimination (c-statistic=0.93, 95% CI: 0.89-0.97). Predictors of allcause mortality were analyzed using univariable and multivariable proportional hazard models (cumulative outcomes). The proportional hazard assumption was tested by plotting log-minus-log survival. Variables with p-values <0.1 in univariable analysis and the access-route were included in the multivariable analysis. Statistical analysis was performed with the use of Statistical Package for Social Sciences version 22 (SPSS, Chicago, IL, USA). All tests were 2-sided at the 0.05 significance level.

RESULTS

During the study period, 191 patients (female sex: 39%) had a non-transfemoral approach TAVI, among which 87 patients (mean-age: 79.3±6.7 years) underwent a transarterial approach and 104 patients (mean-age: 78.0±9.9 years) a transthoracic approach. Sub-clavian access was performed in 73 patients (38.2%), transcarotid in 14 patients (7.3%), transaortic in 41 patients (21.5%), and transapical in 63 patients (33.0%). Supplemental Figures 1 and2 show the annual number of each alternative access, and the annual proportion of all TAVI represented by each of these accesses.

Baseline characteristics of the study population according to the approach are summarized in Table 1.

Coronary artery disease was significantly more frequent in the transthoracic group than in the

A C C E P T E D M A N U S C R I P T 5
transarterial group (68.3% vs. 48.4%; p=0.008). Patients of the transthoracic group harbored a higher burden of cerebrovascular disease (18.4% vs. 3.4%; p=0.001) and hostile chest (5.8% vs. 0.0%; p=0.03). Prevalence of peripheral artery disease was similar in both groups. Mean Logistic EuroScore I tended to be higher in patients treated with the transthoracic approach (14.9% vs. 12.0%; p=0.053).

Echocardiographic parameters were similar between groups.

Procedural characteristics are described in Table 2. General anaesthesia was used in all transthoracic procedures, and in 90.8% of the transarterial ones (p = 0.002). Balloon-expendable valves were much more frequently implanted in the transthoracic group (96.2% vs. 26.4%; p<0.001). Device success was achieved in 95.2% of transthoracic TAVI recipients compared with 85.1% of their transarterial counterparts (p=0.024). Major intra-procedural complications were comparable between groups (Table 2).

In-hospital and 30-day outcomes of the 2 groups are depicted in Table 3. Supplemental Tables 1 and2 present a comparison of subclavian vs. transcarotid approaches, and transaortic vs. transapical approaches, respectively. In univariate analysis, the in-hospital composite endpoint rate was significantly higher in the transthoracic access cohort (25.0% vs. 11.5%; p=0.025), yet this difference did not persist at 30-day (26.0 vs. 14.9%, p=0.075). Propensity score-adjusted logistic regression confirmed the absence of a significant association between the 30-day composite endpoint and transthoracic access (OR: 2.12, 95% CI: 0.70-6.42; p=0.18). Albeit numerically higher in the transthoracic cohort, there was no significant difference between groups for in-hospital (4.6% vs. 7.7%; p=0.55) and 30-days (4.6% vs. 8.7%; p=0.39) mortality. Also numerically higher in the transthoracic group, incidences of most major periprocedural complications were statistically comparable between groups. One contralateral transient ischemic attack occurred 24h post-procedure in a patient who underwent left transcarotid TAVI under general anesthesia without balloon predilation. New-onset atrial fibrillation and acute kidney injury stage 2 or 3 were more prevalent in the transthoracic cohort. In the transthoracic access group, consistently with the lower use of selfexpendable valves, TAVI resulted in a lower rate of permanent pacemaker implantation than in the transarterial cohort. Length of hospitalization was higher (7.0 days vs. 6.0 days; p<0.001) and patients

A C C E P T E D M

A N U S C R I P T 6 were less often discharged at home (42.0% vs. 68.7%; p=0.002) in the transthoracic cohort.

Echocardiographic findings at discharge are presented in Table 3.

Median follow-up was 395 days (interquartile range [IQR]: 320-974) and was significantly longer in the transthoracic cohort (676 days, IQR: 367-1182, vs. 367 days, IQR: 204-427; p<0.001).

The 1-year Kaplan-Meier survival curves are shown in Figure 1. At 1 year, overall survival was similar in both groups (89.6%; 95% CI: 80.1-94.7 and 84.5%; 95% CI: 75.9-90.2 with the transarterial and transthoracic approach, respectively; p=0.30). Figure 2 summarizes the multivariable predictors of all-cause mortality at follow-up. Atrial fibrillation (HR: 2.52; 95% CI: 1.33-4.78; p=0.005), STS-PROM score (HR: 1.18; 95% CI: 1.01-1.39; p=0.04) and periprocedural stroke (HR: 5.85; 95% CI: 1.51-22.63; p=0.011) were found as independent predictors of overall mortality. Transthoracic accessroute was not independently associated with mortality at follow-up (HR: 1.16; 95% CI: 0.55-2.45; p=0.70)

DISCUSSION

The main findings of this study are the following: despite a numerically lower incidence among transarterial TAVI patients, transthoracic approaches were not significantly associated with an increased rate of 30-days major perioperative adverse events or decreased mid-term survival (Figure 3). However, a shorter length of stay and a higher likelihood of being discharge at home were observed among transarterial TAVI recipients.

At the inception of the TAVI era, when a transfemoral approach was not feasible, a "transapical-first" policy was usually applied. Currently, strategies tend to evolve, with a priority for less invasive approaches. In a recent analysis of temporal trends in French registries [START_REF] Auffret | Temporal Trends in Transcatheter Aortic Valve Replacement in France: FRANCE 2 to FRANCE TAVI[END_REF] , transapical TAVI drastically decreased over time (from 27.9% in 2010 to 4.7% in 2015 among patients receiving a balloon-expandable valve). This evolution can be explained by an increase of transfemoral TAVI (73.4% vs. 82.8%) and the expansion of alternative access-sites of more recent emergence such as the direct aortic, and, particularly, transcarotid routes (5.5% and 3.4% of 12804 patients included in the FRANCE TAVI registry, respectively). Comparable findings were previously reported in the UK

A C C E P T E D M

A N U S C R I P T 7 registry [START_REF] Ludman | Transcatheter aortic valve implantation in the United Kingdom: temporal trends, predictors of outcome, and 6-year follow-up: a report from the UK Transcatheter Aortic Valve Implantation (TAVI) Registry, 2007 to 2012[END_REF] . However, the transcarotid approach being the latest implemented in routine practice, the above-mentioned trend will likely accentuate regarding this specific access.

Transapical and transaortic TAVI are associated with similar rates of complications and comparable outcomes [START_REF] Arai | Direct Comparison of Feasibility and Safety of Transfemoral Versus Transaortic Versus Transapical Transcatheter Aortic Valve Replacement[END_REF][START_REF] Thourani | Transapical and Transaortic Transcatheter Aortic Valve Replacement in the United States[END_REF] . Our results regarding the in-hospital and 30-days morbidity and mortality of transthoracic TAVI are consistent with these recent studies. On the contrary to transthoracic TAVI, some groups reported favourable outcomes, sometimes comparable with the transfemoral approach, with the use of transcarotid and subclavian TAVI [START_REF] Gleason | Subclavian/Axillary Access for Self-Expanding Transcatheter Aortic Valve Replacement Renders Equivalent Outcomes as Transfemoral[END_REF][START_REF] Paone | Transcatheter Aortic Valve Replacement: Comparing Transfemoral, Transcarotid and Transcaval Access[END_REF][START_REF] Petronio | 2-year results of CoreValve implantation through the subclavian access: a propensity-matched comparison with the femoral access[END_REF] . Therefore, these alternative transarterial accesses could conceivably be superior to transthoracic ones, and should represent the first option when transfemoral TAVI cannot be performed. However, the paucity of direct comparisons between non-transfemoral approaches supporting this assumption is highlighted by the observation that transthoracic access still represents the majority of alternative access TAVI in some regions, such as the US [START_REF] Damluji | Alternative access for transcatheter aortic valve replacement in older adults: A collaborative study from France and United States[END_REF] . The present study adds to a small number of publications, which investigated the potential benefits of practicing a transarterial instead of a transthoracic approach [START_REF] Kirker | The Carotid Artery as a Preferred Alternative Access Route for Transcatheter Aortic Valve Replacement[END_REF][START_REF] Fröhlich | Comparative survival after transapical, direct aortic, and subclavian transcatheter aortic valve implantation (data from the UK TAVI registry)[END_REF][START_REF] Ciuca | Trans-subclavian versus transapical access for transcatheter aortic valve implantation: A multicenter study[END_REF][START_REF] Chamandi | Transcarotid Compared With Other Alternative Access Routes for Transcatheter Aortic Valve Replacement[END_REF] . As in the present study, a 3 Italian centers retrospective study also reported a non-significant trend to increased peri-procedural events, which did not impact mid-term survival, with the use of transapical versus subclavian TAVI [START_REF] Ciuca | Trans-subclavian versus transapical access for transcatheter aortic valve implantation: A multicenter study[END_REF] .

Within the larger UK registry, Fröhlich et al demonstrated a significantly higher 2-year mortality following transapical or transaortic access than after transfemoral or subclavian TAVI [START_REF] Fröhlich | Comparative survival after transapical, direct aortic, and subclavian transcatheter aortic valve implantation (data from the UK TAVI registry)[END_REF] . Recently, using propensity score-matching, Chamandi et al published the largest comparative analysis specifically involving alternative access [START_REF] Chamandi | Transcarotid Compared With Other Alternative Access Routes for Transcatheter Aortic Valve Replacement[END_REF] . Ninety-four transcarotid TAVI recipients were matched with 163 transthoracic TAVI patients. Transcarotid access was associated with reduced 30-days rates of new-onset atrial fibrillation, major or life-threatening bleeding, stage 2 or 3 acute kidney injury, and a shorter length of stay. Mortality, stroke and device success were comparable between groups while early safety favored transcarotid access. The present study reports largely consistent findings.

Stroke was numerically higher in the transthoracic cohort, without significant difference between groups (3.8% vs. 1.1%; p = 0.38), which may be related to the small sample size. Beyond their heavier atherosclerotic burden, new-onset atrial fibrillation, a condition considerably more prevalent after transthoracic TAVR and associated with a higher risk of 30-days cerebrovascular events [START_REF] Auffret | Predictors of Early Cerebrovascular Events in Patients With Aortic Stenosis Undergoing Transcatheter Aortic Valve Replacement[END_REF] , may significantly contribute to an increased risk among recipients of this approach. This

A C C E P T E D M A N U S C R I P T 8
finding may be of paramount importance, as consistently with our results, periprocedural stroke was an independent predictor of mortality in previous studies [START_REF] Nombela-Franco | Timing, predictive factors, and prognostic value of cerebrovascular events in a large cohort of patients undergoing transcatheter aortic valve implantation[END_REF] . Moreover, although local anesthesia was not consistently associated with better outcomes [START_REF] Maas | General or Local Anesthesia for TAVI? A Systematic Review of the Literature and Meta-Analysis[END_REF] , a higher rate of periprocedural stroke has been suggested with general anesthesia following transcarotid TAVI [START_REF] Debry | Transcarotid Transcatheter Aortic Valve Replacement: General or Local Anesthesia[END_REF] . Only 50% of transcarotid approach patients were treated under local anesthesia with sedation in the present study. Whether a broader use of local anesthesia and sedation with growing experience with this approach improves neurological outcomes should be the focus of future studies.

On the contrary, permanent pacemaker implantation was more frequent in the transarterialaccess cohort. As reported in previous studies [START_REF] Auffret | Conduction Disturbances After Transcatheter Aortic Valve Replacement: Current Status and Future Perspectives[END_REF] , this finding is related to the higher implantation rate of self-expandable valves in the transarterial group. With the expansion of TAVI to lower-surgical risk and younger patients, this result raises the major issue of potentially negative effects of long-term pacing, even if the impact of permanent pacemaker implantation after TAVI remains debated [START_REF] Auffret | Conduction Disturbances After Transcatheter Aortic Valve Replacement: Current Status and Future Perspectives[END_REF][START_REF] Chamandi | Long-Term Outcomes in Patients With New Permanent Pacemaker Implantation Following Transcatheter Aortic Valve Replacement[END_REF] .

However, during the early experience of TAVI, subclavian approach was almost exclusively performed with self-expandable valve. Yet, with growing experience, balloon-expandable valves are increasingly implanted through this access, which should mitigate its detrimental association with post-procedural pacemaker implantation.

Several limitations need to be acknowledged. First, there were significant differences in baseline characteristics between the two groups, which we attempted to adjust for by propensity scoreadjustment. Nonetheless, no statistical method can provide the degree of bias reduction obtained with randomization. Therefore, residual confounding, related to the higher burden of comorbidities observed in the transthoracic group, cannot be ruled out. Second, this is a retrospective analysis, based on a single center population with a limited number of patients in both cohorts, which implies a significant risk of type II error. Furthermore, because of the relatively recent development of this strategy in our center, the transcarotid approach was less represented than the subclavian access-route in the transarterial cohort. Considering the well-known learning-curve effect with other approaches [START_REF] Minha | Learning curves for transfemoral transcatheter aortic valve replacement in the PARTNER-I trial: Success and safety[END_REF][START_REF] Suri | Learning curves for transapical transcatheter aortic valve replacement in the PARTNER-I trial: Technical performance, success, and safety[END_REF] , we can hypothesized that our lower experience regarding transcarotid procedures in comparison with others approaches potentially influenced our results. Conversely, transcarotid TAVI was likely

Figure 1 -

 1 Figure 1 -Rates of all-cause mortality

Figure 2 -Figure 3 -

 23 Figure 2 -Predictors of all-cause mortality at follow-up

Table 1 -

 1 Baseline characteristics of the study population according to TAVI approach Coronary artery disease defined as previous myocardial infarction, or previous percutaneous coronary intervention, or previous coronary artery bypass grafting, or presence of at least one lesion ≥ 50% on the preoperative coronary angiogram. †Systolic pulmonary artery pressure was measurable by echocardiography in 145 patients.

	Transarterial	Transthoracic	p-
	TAVI group	TAVI group	value
	(n=87)	(n=104)

*

No extra-mural funding supported this work CONFLICT OF INTEREST STATEMENT Dr Vincent Auffret received research grants from Abbott, Edwards Lifescience, Medtronic, Biosensors, Terumo and Boston Scientific. Dr Le Breton received lecture fees from Edwards Lifesciences.

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A N U S C R I P T 9 performed using more advanced valve technologies among patients with a lower surgical risk, which may have balanced our limited experience.

CONCLUSION

Non-transfemoral transarterial-approach TAVI is safe, feasible, and associated with comparable rates of major perioperative complications, and mid-term mortality compared with transthoracic-approach TAVI. Nonetheless, transarterial access may be associated with lower rates of new-onset atrial fibrillation, acute kidney injury, and shorter hospitalization.


K Nyangoh Timoh

J Deffon

D Moszkowicz

C Lebacle

M.D M Creze

J Martinovic

M Zaitouna

D Diallo

V Lavoue

A Fautrel

G Benoit

T Bessede

Dr Nyangoh Krystel

MD. Timoh

Smooth muscle of the male pelvic floor: an anatomic study

Keywords: levator ani-neuro-anatomysurgerypelvic floorsmooth musclerectumproctectomyprostatectomyanal canalmaleurethra calcitonin gene related protein CRL, crown rump length IHP, Inferior hypogastric plexus LAM, Levator ani Muscle

Introduction: Knowledge of the anatomy of the male pelvic floor is important to avoid damaging the pelvic floor muscles during surgery. We set out to explore the structure and innervation of the smooth muscle of the whole pelvic floor using male fetuses.

Material and Methods:

We removed en-bloc the entire pelvis of three male fetuses. The specimens were serially sectioned before being stained with Masson's trichrome and hematoxylin and eosin, and immunostained for smooth muscles, and somatic, adrenergic, sensory and nitrergic nerve fibers. Slides were digitized for 3D reconstruction.

Introduction

Genito-urinary dysfunctions, such as erectile dysfunction and urinary incontinence, occur respectively in 10 to 46% and 16% of patients after radical prostatectomy (Ficarra et al., 2012a;Ficarra et al., 2012b). They may be induced by lesions of the cavernous nerves or of the nerves that supply the urethra after passing through the rectourethral muscle [START_REF] Costello | Anatomical studies of the neurovascular bundle and cavernosal nerves[END_REF][START_REF] Strasser | Anatomic and functional studies of the male and female urethral sphincter[END_REF][START_REF] Takenaka | Variation in course of cavernous nerve with special reference to details of topographic relationships near prostatic apex: histologic study using male cadavers[END_REF]. The relationship between nerve injury and muscle injury is unclear but it is anatomically relevant to study the nerve supply and the targeted muscles together.

Knowledge of the anatomy of the male pelvic floor is thus important to avoid damaging the pelvic floor muscles during surgery. Recently, two anatomic studies described the presence of smooth muscle (SM) within the male pelvic floor [START_REF] Muro | Spatial distribution of smooth muscle tissue in the male pelvic floor with special reference to the lateral extent of the rectourethralis muscle: Application to prostatectomy and proctectomy[END_REF][START_REF] Wu | Architecture of structures in the urogenital triangle of young adult males; comparison with females[END_REF]. Wu introduced the concept of "the smooth muscle of the middle compartment" (SMMC) in the urogenital triangle, i.e., between the urethra and the rectum [START_REF] Muro | Spatial distribution of smooth muscle tissue in the male pelvic floor with special reference to the lateral extent of the rectourethralis muscle: Application to prostatectomy and proctectomy[END_REF][START_REF] Wu | Architecture of structures in the urogenital triangle of young adult males; comparison with females[END_REF]. However, complete studies of the whole male pelvic floor (i.e., laterally and posteriorly to the rectum) are lacking. Moreover, to date no study has detailed the innervation of the SMMC with a view to clarifying its function.

Classically, autonomic nerves are reported to run above the levator ani muscle (LAM) and somatic nerves below [START_REF] Benoit | Supra and infralevator neurovascular pathways to the penile corpora cavernosa[END_REF]. We have previously described communications between the pudendal nerve and the inferior hypogastric plexus (IHP) [START_REF] Alsaid | Autonomic-somatic communications in the human pelvis: computer-assisted anatomic dissection in male and female fetuses[END_REF], and dual autonomic (IHP) and somatic control (pudendal nerve) of pelvic viscera with a communicating loop within the LAM [START_REF] Alsaid | Autonomic-somatic communications in the human pelvis: computer-assisted anatomic dissection in male and female fetuses[END_REF]. Subsequently, in two previous studies based on female human fetuses with 3D reconstruction, our team reported that the medial and posterior part of the LAM was smooth and under autonomic nerve control coming from the IHP (Nyangoh [START_REF] Timoh | Levator ani muscle innervation: Anatomical study in human fetus[END_REF][START_REF] Timoh | Detailed muscular structure and neural control anatomy of the levator ani muscle: a study based on female human fetuses[END_REF].

Therefore, we hypothesized that the SMMC within the male pelvic floor muscle is extensive and controlled by the autonomic system. Thus, the aim of the present study was to describe the SMMC

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and detail its innervation using specific muscular and neuronal antibody markers with 3D reconstruction.

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Methods

Material and Methods

Human fetuses

The fetal specimens were obtained from late miscarriages. All parents gave written consent and authorization for the scientific use of the cadaver. Only specimens without maceration and without morphologic or neurologic macroscopic abnormalities on pathology examination were used. No infectious conditions were reported and no abnormalities of the central nervous system were observed during the autopsy of the fetuses.

The French Biomedicine Agency approved the study (PFS15-011). The work was compliant with the provisions of the 2013 revised version of the Declaration of Helsinki.

Three male fetuses were selected for study. Fetal age was determined from the crown rump length (CRL) and fetal heel-to-toe length, corrected for the first-trimester ultrasound CRL measurement and confirmed by postmortem examination estimating organ maturation. We analyzed the whole pelvis of three male human fetuses with a CRL of 150-185 mm. Fetuses A and B were at 23 weeks of gestation, and fetus C at 40 weeks of gestation.

The external body aspect of each fetus was carefully restored after the autopsy out of respect and in case the parents expressed a wish to see their fetus once more.

Macroscopic dissection

The entire pelvis was removed en-bloc with the pelvic organ and pelvic bone, and was fixed in formalin (10% formaldehyde) for 48 h. The tissues were then cut into transverse or sagittal slices at 4-mm intervals. The tissue slices were placed in baskets, processed, and embedded in cardboard molds filled with paraffin. To avoid alteration of the visceral topographic relationship, the slices were kept warm in water at 37°C and then mounted whole on Superfrost glass slides. They were then dried at 37°C overnight. Series of 5 µm-thick sections

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were then created without intervals. We obtained a total of 150-320 sections from each fetus, depending on the age and size of the specimen.

Staining and immunolabeling

The first section of each level was taken as the reference and was stained with Masson's trichrome or hematoxylin and eosin (H&E) to provide information about the topography and localization of anatomic structures. Smooth muscles were detected with polyclonal antibodies against alpha-Smooth Muscle Actin (SMA) [START_REF] Kobayashi | A simple method of isolating mouse aortic endothelial cells[END_REF] (Table 1).

Neuronal markers were detected with polyclonal antibodies against protein S100 (PS100) for all nerves and peripheral myelin protein (PMP 22) for the somatic peripheral nervous system [START_REF] Bremer | Axonal prion protein is required for peripheral myelin maintenance[END_REF]; tyrosine hydroxylase (TH) for adrenergic nerves [START_REF] Butler-Manuel | Pelvic nerve plexus trauma at radical and simple hysterectomy: a quantitative study of nerve types in the uterine supporting ligaments[END_REF]; the neural isoform of nitric oxide synthase (nNOS) for the pro-erectile nerve bundles; and the calcitonin gene-related peptide (CGRP) for primary sensory neuropeptide immunolabelling [START_REF] Moszkowicz | Neural supply to the clitoris: immunohistochemical study with three-dimensional reconstruction of cavernous nerve, spongious nerve, and dorsal clitoris nerve in human fetus[END_REF] (Table 1).

Every tenth section was treated according to the following sequences:

(1) The first section was stained with Masson's trichrome or H&E (annex 1).

(2) The second was used to study smooth muscle with smooth anti-actin polyclonal antibody.

(3) The third was used to study nerves by immunolabeling with anti-protein S100.

(4) The fourth was used to study nerves by immunolabeling with anti-peripheral myelin protein (PMP 22).

(5) The fifth was used to study nerves by immunolabeling with anti-tyrosine hydroxylase antibody (TH).

(6) The sixth was used to study nerves by immunolabeling with an anti-neural isoform of nitric oxide synthase (anti-nNos).

The remaining non-immunolabeled sections were stored for further additional investigations.

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The avidin-biotin-peroxidase detection procedure was carried out with a Vectastain ABC kit (ref. PK6100; Vector Laboratories, Burlingame, CA, USA). Chromogenic detection was performed with a DAB detection kit (DAB, ref. SK-4100; Vector Laboratories, Burlingame, CA, USA). Non-immune serum or IgG at an equivalent dilution was used as a control for all immunohistochemical analysis.

Histologic examination

Serial stained and immunolabeled two-dimensional (2D) sections were used for threedimensional (3D) reconstruction. The various anatomic structures (organs, bones, and fascia) were visible under high magnification (4× to 40×) analysis of the Masson's trichrome and H&Estained sections. Subsequent sections, treated with an antibody against S100, were used to identify the pelvic-perineal nerves and communicating branches. By comparing the Masson's trichrome-stained sections with sections stained with specific antibodies against SMA, S100, TH, PMP22, nNOS, and CGRP it was possible to determine the structure of the muscle (smooth (SMA) or striated (myogenin)) and the nature of the nerve fibers identified: i.e., somatic (PMP22), autonomic (TH, VAChT), or nitrergic (nNOS).

The sections were taken at almost the same level with a negligible interval between sections (5 µm). The computer system comprised a personal laptop computer (Windows XP) equipped with an Epson Perfection V750 digitization system, Silverfast AI digitization software (Ref B11B178071), Adobe Photoshop image-processing software, and Surfdriver software for Windows (Winsurf image reconstruction software, version 4.3). All sections were digitized at a resolution of 4800 dots per inch, and the images were then stacked and aligned. The brightness and contrast of the histologic tissue images were adjusted using Adobe Photoshop™. The pelvic anatomic structures and nerve fibers were outlined manually on all histologic sections. A 3D analysis of the location, course, and distribution of the nerve fibers and muscle structure was

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then carried out. To realize the reconstruction, sections stained with Masson's trichrome and/or H&E, and by anti-PS100 and anti-SMA were used.

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Results

Global anatomy of the male pelvic floor muscles

The pubovisceral and puborectal part of the LAM are attached anteriorly to the pubis. The puborectal part of the LAM forms a muscle loop behind the rectum. A bulge of the medial aspect of each pubovisceral muscle between the urethra and rectum is medially linked to the contralateral bulge by a central SM mass that is located between the urethra and the rectum (Fig. 1 and Fig. 2).

The iliococcygeal part of the LAM is attached to the internal obturator muscle by the conjunctive tissue of the tendinous arch of the LAM (Fig. 1A, 1B, and1C).

The deep transverse perineal muscle lies below the LAM. The superficial transverse perineal muscle is anterior to the deep transverse perineal muscle and lateral to the bulbospongiosus muscle.

Spatial distribution of smooth muscle areas within the male pelvic floor

We individualized a middle compartment that contains SM cells. This compartment is in close relation with the LAM, rectum, and urethra (Fig. 1A, 1B, 1C, and 1D). We will describe it posteriorly and anteriorly to the anterior rectal wall:

The posterior part of the middle compartment:

From the sacrum to the rectum, an SM layer upholsters the internal aspect of the LAM with SM digitations into the rectal wall. Laterally, this layer is continuous with the anterior part of the middle compartment, and more specifically with the endo-levator area (Fig. 1 and Fig. 2).

The anterior part of the middle compartment:

Using the LAM as an anatomic landmark, three areas can be described:

1. The centro-levator area. A "core" of SM cells is localized between the right and left LAM in the transversal plane, and between the rectum and urethra in the sagittal plane. At its cranial pole, the core is in continuity with the external longitudinal layer of the rectum (Fig. 1 and Fig. 2).

2. The endo-levator area. An SM layer upholsters the internal aspect of the LAM. On both sides of the SM layer, digitations anchor the layer within the rectum internally and within

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the LAM externally. The endo-levator area corresponds to the medial part of the LAM and to the external anal sphincter (Fig. 1 and Fig. 2).

3. The infra-levator area. SM cells lie in a fan shape below the LAM and infero-posteriorly to the endo-levator area (Fig. 1).

Ventrally to the "SM-core" (sagittal and transversal sections), these three areas merge in the extension of the bulbospongiosus muscle and of the internal urethral sphincter. (Fig. 2 and Fig. 3) Laterally to the "SM-core" (coronal reconstruction and transversal sections) the endo-and infralevator areas cover the LAM (Fig. 1 and Fig. 2). These SM expansions can be described to fins of the SM-core (Fig. 1).

All of the above-described SM structures of the anterior and posterior parts are in continuity with each other and can be defined as a "smooth muscle middle compartment" (SMMC) of the male pelvic floor.

Innervation of the smooth muscle area

The SM areas are innervated by autonomic nerves which arise from the IHP. The IHP is comprised of hypogastric and pelvic splanchnic nerves. It is possible to individualize retro-rectal, latero-rectal, inter-prostato-rectal, latero-prostatic, and antero-prostatic fibers (Fig. 4A et Fig. 5D).

The distal part of the IHP is localized in a triangular zone between the LAM laterally, the urethra anteriorly and the rectum posteriorly. The nerves (positive S100 antibody labeling) are: sympathetic (positive TH antibody), sensory (positive CGRP antibody), and nitrergic (positive NOS antibody).

There are no somatic nerves (negative PMP22 antibody) (Fig. 5).

The posterior part of the SMMC

The nerves of the posterior part of the SMMC are retro-rectal and come from the IHP. Some fibers continue to the rectum and others terminate in the posterior part (Fig. 5 and Fig. 6).

The anterior part of the SMMC

Nerve fibers come from the IHP complex.

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1. The centro-levator area: Inter-prostato-rectal and latero-prostatic nerves pass through the endo-levator area of the anterior part of the SMMC (Fig. 5C, 6A, 6B, and 6D). The area is characterized by dense innervation: several nerves pass through this region to reach the posterior face of the internal urethral sphincter or continue towards the erectile bodies. (Fig. 4C, 4D, 6A, 6B and 6D) 2. The endo-levator area: This area is innervated by nerve fibers from the lateral wall of the rectum. Some provide innervation to the rectum following digitations of the endo-levator area within the rectum. Others innervate the prostate. (Fig. 5G and5H).

3. The infra-levator area: Nerve fibers are latero-rectal, inter-prostato-rectal and lateroprostatic. Some provide innervation for the rectum. Some nerves pass through this region to reach the posterior face of the internal urethral sphincter or continue towards the erectile bodies. Others innervate the infra-levator area. (Fig. 4D and6C).

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Innervation of striated muscles

The striated muscle is innervated by the pudendal nerve and the levator ani nerve.

Contribution of the pudendal nerve:

The pudendal nerve passes through the pudendal canal. Proximally, the pudendal nerves are somatic (positive PMP22 antibody) and sympathetic (positive TH antibody), but not CGRP or NoS positive.

The perineal branches of the pudendal nerve innervate the inferior part of the puborectal, pubovisceral and iliococcygeal muscles, the ischiocavernosus muscle and the bulbospongiosus muscle, as well as the superficial transverse perineal muscle (Fig. 5). Terminal perineal branches of the pudendal nerve are sympathetic (positive TH antibody), and somatic (positive PMP22 antibody). They are also nitrergic (positive NOS antibody) and sensory (positive CGRP antibody) (Fig. 7).

Contribution of the levator ani nerve:

The fibers run horizontally in the pelvis and then vertically to take a pre-rectal and latero-prostatic pathway. They innervate the superior part of the LAM. (Fig. 7) The fibers are somatic (positive PMP22 antibody), sympathetic (positive TH antibody), sensory (positive CGRP antibody), and nitrergic (positive NOS antibody) (Fig. 7).

Summary

The SMMC of the male pelvic floor is a continuous area surrounding the pelvic organs. It is anchored laterally into the striated muscular structures and medially into the pelvic and perineal viscera. The SMMC is under autonomic influence contrary to the lateral striated muscles that are under somatic control. We provide a video of the male pelvic floor of the fetus B (supporting information)

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Discussion:

The findings of the current study support the existence of the SMMC: a middle muscle compartment formed by smooth muscle behind (posterior part) and in front of (anterior part) the rectum. These smooth muscles are continuous and connected to the LAM, the rectum and the internal urethral sphincter. The present study also suggests that the SMMC contains a high density of autonomic nerves coming from the IHP.

The human fetal model has previously been used to study pelvic muscle and neuroanatomy. This model offers many advantages over surgical dissection. First, the small size of the fetus pelvis enables en-bloc removal and en-bloc processing with mega-cassette embedding, and data loss and structure displacement is limited. Consequently, the whole neuro-anatomy and muscular pelvic floor (i.e., anterior and posterior to the rectum) can be studied as muscular insertions are preserved.

Moreover, 3D reconstruction is easier and faster and there is less reconstruction bias. Second, the nerve diameter/body ratio is higher in the fetus versus the adult thus simplifying neuroanatomy observations. Third, the use of very fresh tissue means that multiple specific muscular and neuronal markers can be used to provide functional results.

In an effort to systematize our description, we considered the SMMC as a whole and named its different parts according to their relationship with the LAM. This topographic denomination illustrates and highlights the common histology of these structures. However, when referring to anatomic literature, which includes several descriptions of the muscular architecture of the male urogenital region, one has to make the connection with other terminologies [START_REF] Aigner | The rectogenital septum: morphology, function, and clinical relevance[END_REF][START_REF] Brooks | Anatomy of the rectourethralis muscle[END_REF][START_REF] Fritsch | Topography of the pelvic autonomic nerves in human fetuses between 21-29 weeks of gestation[END_REF][START_REF] Muro | Spatial distribution of smooth muscle tissue in the male pelvic floor with special reference to the lateral extent of the rectourethralis muscle: Application to prostatectomy and proctectomy[END_REF][START_REF] Nakajima | Macroscopic and histotopographic study of the deep transverse perineal muscle (musculus transversus perinei profundus) in elderly Japanese[END_REF][START_REF] Porzionato | Histotopographic study of the rectourethralis muscle[END_REF][START_REF] Smith | Studies in the Anatomy of the Pelvis, with Special Reference to the Fasciae and Visceral Supports: Part II[END_REF][START_REF] Wu | Architecture of structures in the urogenital triangle of young adult males; comparison with females[END_REF][START_REF] Zhai | The male rectourethralis and deep transverse perineal muscles and their relationship to adjacent structures examined with successive slices of celloidinembedded pelvic viscera[END_REF] Similarly to previous authors, we observed a central mass coming from the anterior longitudinal layer of the rectum. We chose to describe this mass as a "centro-levator area" because of its very central position in the pelvic floor between the two sides of the LAM or a "core" in relation to its Accepted Article compact aspect. Due to the presence of a loose smooth muscle cell connection between this "centrolevator area" and the external and internal urethral sphincter, bulbourethral gland and bulbospongiosus muscle, the centro-levator area may correspond a structure that has successively been called: the rectourethral [START_REF] Brooks | Anatomy of the rectourethralis muscle[END_REF][START_REF] Porzionato | Histotopographic study of the rectourethralis muscle[END_REF]Uchimoto et al., 2007) muscle, the recto-perineal [START_REF] Aigner | The rectogenital septum: morphology, function, and clinical relevance[END_REF][START_REF] Sebe | An embryological study of fetal development of the rectourethralis muscle--does it really exist?[END_REF] muscle, or the deep transverse perineal muscle [START_REF] Oelrich | The urethral sphincter muscle in the male[END_REF].

We also observed SMs running antero-inferiorly to the LAM which we describe as an "infra-levator area". They are linked to "the centro-levator area" and connected to the external wings of the urethral sphincter [START_REF] Nakajima | Macroscopic and histotopographic study of the deep transverse perineal muscle (musculus transversus perinei profundus) in elderly Japanese[END_REF], the bulbourethral gland, and the bulbospongiosus muscle. Muro et al. described a lateral extension of the rectourethral muscle located anteroinferiorly to the LAM as the deep transverse perineal muscle [START_REF] Muro | Spatial distribution of smooth muscle tissue in the male pelvic floor with special reference to the lateral extent of the rectourethralis muscle: Application to prostatectomy and proctectomy[END_REF]. Our results illustrate that the "infra-levator area" does indeed correspond to the deep transverse perineal muscle. Although many discrepancies remain about the smooth or skeletal nature of the muscle and about the existence of the deep transverse perineal muscle [START_REF] Kokoua | Maturation of the external urinary sphincter: a comparative histotopographic study in humans[END_REF][START_REF] Murakami | Individual variations in aging of the male urethral rhabdosphincter in Japanese[END_REF][START_REF] Nakajima | Macroscopic and histotopographic study of the deep transverse perineal muscle (musculus transversus perinei profundus) in elderly Japanese[END_REF]Uchimoto et al., 2007;[START_REF] Zhai | The male rectourethralis and deep transverse perineal muscles and their relationship to adjacent structures examined with successive slices of celloidinembedded pelvic viscera[END_REF], our description is consistent with Henle, Muro et al., and Zhai et al. (Henle;[START_REF] Muro | Spatial distribution of smooth muscle tissue in the male pelvic floor with special reference to the lateral extent of the rectourethralis muscle: Application to prostatectomy and proctectomy[END_REF][START_REF] Zhai | The male rectourethralis and deep transverse perineal muscles and their relationship to adjacent structures examined with successive slices of celloidinembedded pelvic viscera[END_REF].

We describe the SM located medially to the LAM and to the external anal sphincter as an "endolevator area". We have already referred to this structure in the female as the medial part of the LAM [START_REF] Timoh | Detailed muscular structure and neural control anatomy of the levator ani muscle: a study based on female human fetuses[END_REF]. This area directly connects the rectum and the LAM. Shafik called it the hiatal ligament [START_REF] Shafik | Levator ani muscle: new physioanatomical aspects and role in the micturition mechanism[END_REF], and Arakawa et al. the smooth muscle-mediated interface [START_REF] Arakawa | Development of the external anal sphincter with special reference to intergender difference: observations of mid-term fetuses (15-30 weeks of gestation)[END_REF].

The part posterior to the rectum, the posterior part, also contained smooth muscle cells and forms the posterior part of the endo-levator area. It corresponds to the anococcygeal ligament that goes from the coccyx to the anus between the slings of the LAM [START_REF] Kinugasa | Anococcygeal raphe revisited: a histological study using mid-term human fetuses and elderly cadavers[END_REF][START_REF] Kinugasa | Anatomical reevaluation of the anococcygeal ligament and its surgical relevance[END_REF][START_REF] Muro | Dynamic intersection of the longitudinal muscle and external anal sphincter in the layered structure of the anal canal posterior wall[END_REF][START_REF] Shafik | New concept of the anatomy of the anal sphincter mechanism and the physiology of defecation. II. Anatomy of the levator ani muscle with special reference to puborectalis[END_REF]. Muro et al. already reported smooth muscle cells behind the rectum [START_REF] Muro | Dynamic intersection of the longitudinal muscle and external anal sphincter in the layered structure of the anal canal posterior wall[END_REF].

Accepted Article

As Muro et al., we preferred not to use the term "perineal body" in the results section, as we considered that it probably refers to the region between the urethra and rectum rather than a structure per se [START_REF] Larson | Perineal body anatomy in living women: 3-dimensional analysis using thin-slice magnetic resonance imaging[END_REF][START_REF] Muro | Spatial distribution of smooth muscle tissue in the male pelvic floor with special reference to the lateral extent of the rectourethralis muscle: Application to prostatectomy and proctectomy[END_REF].

Most studies to date describe the pelvic floor smooth muscle area separately, sometimes using different terms for the same structure. Few report smooth muscle cells behind the rectum. Here, we provide evidence for continuous abundant smooth muscle fibers within the whole pelvic floor (in front of and behind the rectum).

A few authors have published data about the innervation of the muscles of the pelvic floor [START_REF] Costello | Anatomical studies of the neurovascular bundle and cavernosal nerves[END_REF][START_REF] Strasser | Anatomic and functional studies of the male and female urethral sphincter[END_REF][START_REF] Takenaka | Variation in course of cavernous nerve with special reference to details of topographic relationships near prostatic apex: histologic study using male cadavers[END_REF]. Classically, the somatic nerves are reported to run below the LAM and the autonomic nerves above [START_REF] Benoit | Supra and infralevator neurovascular pathways to the penile corpora cavernosa[END_REF]. However, our team have already provided data about autonomic-somatic communications within the LAM [START_REF] Alsaid | Autonomic-somatic communications in the human pelvis: computer-assisted anatomic dissection in male and female fetuses[END_REF]. Moreover, we recently published data about autonomic innervation of the medial and posterior part of the female LAM [START_REF] Timoh | Levator ani muscle innervation: Anatomical study in human fetus[END_REF]. Here, we provide evidence for autonomic innervation coming from the IHP of the SMMC in the male. The posterior part is innervated by retro-rectal nerves and the endo-levator part by latero-rectal nerves. More interestingly, the centro-levator and infra-levator areas (i.e., rectourethral muscle and deep transverse perineal muscle) are important nerve pathways supplying the internal urethral sphincter and erectile bodies (cavernous nerves) as suggested by [START_REF] Strasser | Anatomic and functional studies of the male and female urethral sphincter[END_REF]. Our results are consistent with the study of Takenaka et al. [START_REF] Takenaka | Variation in course of cavernous nerve with special reference to details of topographic relationships near prostatic apex: histologic study using male cadavers[END_REF]. However, unlike the study we present here, none of these previous authors used specific neuronal markers. We were able to distinguish autonomic sensory and highly nitrergic nerves supplying the SMMC.

From a physiopathologic point of view, the presence of abundant SM linking the external striated muscles of the pelvic viscera and the pelvic viscera itself may not be circumstantial. The close connection to the LAM suggests that it may be part of a mechanism playing a major role in pelvic floor stability by providing lifting power [START_REF] Delancey | Anatomy and biomechanics of genital prolapse[END_REF]. Furthermore, as the SMMC comes from the anterior longitudinal layer of the rectum in the sagittal direction, we can hypothesize that it Accepted Article participates in the anorectal flexure [START_REF] Wesson | AN ANATOMICAL AND EMBRYOLOGICAL STUDY OF THE PERINEUM[END_REF] and consequently to fecal continence.

Moreover, the SMMC is innervated by autonomic nerves from the IHP. These data point towards the possibility that the SMMC plays a dynamic role with simultaneous contraction under autonomic nerve impulse leading to urinary and fecal continence. Finally, we cannot exclude the possibility that the SMMC plays a protective or regenerative role [START_REF] Arakawa | Morphologies of the interfaces between the levator ani muscle and pelvic viscera, with special reference to muscle insertion into the anorectum in elderly Japanese[END_REF].

From a clinical point of view, we believe that a better understanding of the path of the cavernous nerve and the nerve supplying the internal urethral sphincter is of importance for pelvic surgeons. In the field of urology, protecting erectile function and urinary continence during radical prostatectomy is a challenge. A recent study highlighted that cavernous nerve preservation improves both erectile function and urinary continence [START_REF] Reeves | Preservation of the neurovascular bundles is associated with improved time to continence after radical prostatectomy but not long-term continence rates: results of a systematic review and metaanalysis[END_REF]. Because the centroand infra-levator areas (i.e., the rectourethral muscle and deep transverse perineal muscle) are crossed by both the cavernous nerve and the nerve supplying the urethra, these areas are veritable minefields during radical prostatectomy. In order to spare these nerves, Takenaka et al. recommend incising the rectourethral muscle close to the rectum during perineal prostatectomy, and near the prostatic apex during retropubic prostatectomy [START_REF] Takenaka | Variation in course of cavernous nerve with special reference to details of topographic relationships near prostatic apex: histologic study using male cadavers[END_REF]. However, surgeons must be aware that the rectouretralis muscle comes from the anterior extension of the rectal wall. Indeed up to 11% of rectal injuries occur during radical perineal prostatectomy [START_REF] Lassen | Rectal injuries during radical perineal prostatectomy[END_REF].

Nerve sparing is also a challenge in the field of abdominal surgery. In their study, Uchimoto et al.

observed that Denonvilliers' fascia ended in the rectouretralis muscle, and thus proposes taking the surgical plane behind Denonvilliers' fascia when possible (Uchimoto et al., 2007).

Knowing that both the rectourethral and deep transverse perineal muscles are SM that are connected with the urethra is also of major importance: damage to these muscles may result in postoperative urethral incontinence.

Despite these results, this study has some limitations which are mainly linked to the use of the fetal model. For example, one could argue that the pelvic structure is not fully formed in the fetus.

However, previous studies have reported that the pelvic muscular and neuroanatomy of the fetus is Accepted Article mature at 8 weeks of gestation [START_REF] Arango-Toro | Development of the pelvic plexus in human embryos and fetuses and its relationship with the pelvic viscera[END_REF][START_REF] Fritsch | Clinical anatomy of the pelvic floor[END_REF].

Second, the very small number of specimens could lead to bias. However, we used three fetuses with different axes of section (two sagittal and one axial) to add different observation angles.

Moreover, the results were consistent in all three fetuses.

Accepted Article

Conclusion

We provide evidence that the rectourethral muscle, deep transverse perineal muscle and anococcygeal raphe have common features: they are of smooth muscle nature and autonomic innervation, and they are closely linked to each other and with the pelvic viscera and striated muscle thus forming an anatomic and functional entity which can be called the SMMC. Nevertheless, the precise function of the SMMC remains to be defined.

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Figure 1 :

 1 Figure 1: Serial sagittal sections of a 23-week-old male fetus and serial transverse sections of another 23-week-old male fetus. It was scanned at an optical resolution of 4800 dpi. (A) Serial sagittal section stained with Eosin Haematoxylin. (B) , (C) Serial sagittal section stained with anti-smooth muscle actin antibody. (C) is the same section as (B) with legends. (D), (E), (F), (G) Serial transverse sections stained with anti-smooth muscle actin antibody. Sequential sections from cranial (D) to distal (F) poles showing the emergence of the centro-levator area, "the core" (light green arrows). (G) is the same section as (F) with legends. Code: Endo-levator area (ENDO): purple; Centro-levator area (CENTRO): light green; Infralevator (INFRA): light blue; Piriformis muscle (PM), Internal obturator muscle (IOM) : dark brown; Bladder: yellow; Urethra (ur) : yellow, Levator ani muscle (LAM) : orange; External anal sphincter (EAS): dark blue; Prostate : dark red; Rectum: beige; Bulbourethral gland: grey; Bulbospongiosus muscle: pink.

Figure 2 :

 2 Figure 2: Computer-assisted anatomic dissection (CAAD) of the morphologic pelvic structure in a 23-week-old male fetus. (A) (B) Reconstruction of the SMMC (C) Reconstruction of the SMMC and levator ani muscle. (D) Reconstruction of the SMMC, the rectum, the bulbospongiosus muscle (BSM), the external anal sphincter (EAS), the levator ani muscle, the internal urethral sphincter and the prostate. (E) Right lateral view of the three-dimensional reconstruction. (F) Superior view of the three-dimensional reconstruction. Code: Endo-levator area (ENDO) and the posterior part: purple; Centro-levator area (CENTRO): light green; Infra-levator area (INFRA): light blue; Levator ani muscle (LAM): orange; External anal sphincter (EAS): dark blue; Prostate: dark red; Rectum: beige; Bulbospongiosus muscle (BSM): pink; External urethral sphincter (US): light purple.

Fig 3 :

 3 Fig 3: Serial sagittal sections of a 23-week-old male fetus

Fig 4 :

 4 Fig 4: Serial transverse sections of a 23-week-old male fetus (A), (A bis) Transverse sections stained with anti-S100 antibody.

Fig 5 :

 5 Fig 5: Serial sagittal sections of a 23-week-old male fetus

Acknowledgements

We thank Felicity Neilson, a native English speaker with experience in scientific editing, for reviewing the paper.

The authors declare no conflict of interest.

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(F) (F1) Sagittal section stained with anti-TH antibody.

(G) (G1) Sagittal section stained with anti-NOS antibody.

(H) (H1) Sagittal section stained with anti-CGRP antibody.

Black arrowheads show somatic nerves (E) (E1), autonomic sympathetic nerves (F) (F1), nitrergic nerves (G), (G1) and sensitive nerves (H), (H1).