Volodymyr Malytskyi

Virginie Gadenne

Younal Ksari

Lionel Patrone
email: lionel.patrone@im2np.fr

Jean-Manuel Raimundo
email: jean-manuel.raimundo@univ-amu.fr

Synthesis and characterization of thiophene-based push

Introduction

Controlled spatial organization of chromophores, by covalent bonding, on solid surfaces has sparked a growing interest in the last decades due to its importance in the field of molecular nanotechnology for the fabrication, for instance, of innovative smart materials and surfaces, and biosensors. 1 Moreover, the control of the composition and orientation of chromophores at the nanometer scale is of crucial importance in order to optimize both their photophysical and photochemical properties. In this respect, the formation of self-assembled monolayers (SAMs) 2 appears to be a method of choice to produce robust natureinspired [START_REF] Boynton | Human Color Vision[END_REF] thin films that are highly ordered, dense, associated with a good uniformity in thickness and in composition. Among the myriad of chromophores, linear push-pull derivatives (consisting of a π-conjugated system (π) end-capped with a donor (D) on one side and an acceptor (A) on the other side (namely Dπ-A)) 4 represent one of the most widely studied structures that have been used in several molecular nanotechnology applications comprising non-linear optics (NLO), 5 organic photovoltaic cells (OPVs), 6 organic light-emitting diodes (OLEDs), 7 organic fieldeffect transistors (OFETs) 8 and so on. However, their grafting onto surface has been still scarcely studied despite their great importance in order to improve the device performances. 9 As exemplified by Facchetti et al, 10 D-π-A chromophores have been incorporated in insulating monolayers of OFETs, leading to high dielectric constant due to the perfect control of the dipole orientation at the surface (i.e. self-assembled nanodielectrics SANDs). Ashwell et al have sturdily anchored hemicyanines dyes onto a gold surface and have demonstrated a diode-type behavior exhibiting a reversible current rectification upon acidic treatment or dipole inversion. 11 More recently Meskers et al have proven that the uniform arrangement of the hemicyanine-type dipoles leads to an enhancement of the light harvesting properties associated with an efficient generation of photocurrent 12 Although these results are very promising and afford interesting properties, the presence of intrinsic charges within these molecules (pyridinium, zwitterion) may be problematic in certain cases because of the existence of coulombic interactions that could alter the self-organization process and/or properties. To circumvent this issue, uncharged linear D-π-A chromophores can be considered and used as effective alternatives. 13 Based on these considerations, we report herein on the behavior of a chromophore SAM grafted on gold surfaces using scanning probe microscopy and spectroscopic methods as well as on the synthesis of a set of new chromophores with several structural modifications that are suitable for surface modifications.

A B S T R A C T

Due to their efficient light absorption properties and their electronic dipole, "push-pull" chromophores with thiophene-based spacer between donor and acceptor groups are very promising for potential use in optoelectronics, photovoltaics, non-linear optics, or electronics. Such applications could benefit from chromophore deposition on a surface in an ordered way with oriented dipoles. For this purpose, self-assembled monolayers (SAMs) constitute an interesting approach. In this work we present a multi-step synthesis of a series of push-pull structures based on 2,2'-bithiophene or dithieno [3,2-b:2',3'-d]thiophene frameworks born on an alkyl chain terminated with an anchoring group for further use in surface modification. Obtained molecules and SAMs were analyzed by cyclic voltammetry, spectroscopy and scanning probe microscopy. We show that varying the acceptor moiety allows to tune the absorption band in a wide spectral range, and that a longer chain length improves the organization of the dipoleoriented chromophore SAM.

Keywords:

Organic synthesis Donor-acceptor systems Push-pull chromophores Self-assembled monolayers Electronic properties.

Results and discussion

Synthesis and SAM characteristics of push-pull chromophores

Classical linear D-π-A chromophores can be easily customized in order to improve their intrinsic photophysical properties by selecting the appropriate structural elements. The typical structure of the push-pull chromophores studied in this paper is presented in Scheme 1. In this archetypical molecular structures, electrical and optical properties are determined by the energy of frontier orbitals: the HOMO level is mostly depended on the nature of the donor group while the LUMO level is associated with the acceptor group. Moreover, electron is transferred from the donor (D) to the acceptor (A) through the pi-conjugated spacer acting as an electron relay. Thus tailoring the nature and structure of the spacer for a specific couple of D and A makes it possible to tune the dipole formation 14 . Molecular engineering on π-conjugated spacers has revealed that planar and rigid systems are highly powerful relays compared to their non-rigid analogues. Hence, design of effective flat rigid electron relay is needed either to modulate the dipole moment and/or to help the spatial organization in order to afford more densely packed layers. Other determinant factors governing the compactness of the monolayers include the length of the linker between the chromophore and the surface, the intermolecular π-π interactions, the nature of the anchoring group, the surface structure and so on. From a selected family of chromophores we compared at early stage the influence of the chain length on the self-assembling properties by synthetizing two identical chromophores (6-7) possessing the same anchoring group but different linkers. The synthesis is depicted in Scheme 2. The bithiophene derivative 1 was synthesized, in 81% yield, according to a synthetic procedure developed in our group in three steps 13 from commercially available bithiophene (BT). Then the latter was subjected to a Wittig reaction with the corresponding phosphonium salts (prepared extemporaneously from N-methyl aniline and known procedures) 13,15 affording the key intermediates 2 and 3 in 94% and 93% yield, respectively. These intermediates were successfully saponified and have been reacted with malonodinitrile (Knoevenagel-type condensation) yielding the chromophores 4 and 5 in 77% and 95% respectively. The last step consists to convert the hydroxyl group into a more active surface-anchoring group such as a thiol or thioacetate group. This was accomplished in two consecutive steps by converting first the OH group into a more efficient leaving group such as Br in presence of PBr 3 followed by nucleophilic substitution reaction with NaSH or KSAc leading to the compounds 6 and 7, after purification, in 7% and 31%, respectively (Scheme 2). A successful direct grafting, on pre-cleaned substrates, has been achieved with the chromophores 6 and 7, without any deprotection step in the case of the thioacetate group 13,16 at room temperature using a CH 2 Cl 2 solution soak at 1 mM during 24h. In both cases, complete adsorptions were achieved but slower kinetics was observed for the thioacetate functionalized chromophore 7 as previously speculated. 17 After grafting, surfaces were abundantly rinsed, dried, and annealed at 85°C over few hours to remove solvent traces and to achieve the dense molecular packing.

SAM of 6 appears rougher than SAM of 7 with calculated values of RMS surface roughness of ~0.23 nm vs. ~0.07 nm respectively. Indeed, SAM of 6 is covered by disordered aggregates appearing as white spots, whereas flat SAM surface of 7 enables to see etch pits (black holes), typical of sulfur reaction with gold surface, as well as gold steps (e.g. right side of the image) thus indicating the presence of a grafted single monolayer. This is further supported by a water contact angle value about closer to that of ~69-75° reported for single cyano surface group (CN) 18 for 7 (~79°) than for 6 (~88°) for which side groups of disordered tilted molecules should contribute (as a blank, we measured 57° for cleaned bare gold surface immersed in the solvent). Therefore, although the free thiol-anchoring moiety of compound 6 is more reactive, a two-carbon linker appears not long enough to provide the required flexibility for the organization of the SAM. Hence, in the following we will focus on SAMs of compound 7. The surface enhanced Raman spectroscopy (SERS) spectrum of 7 chromophore SAM (Fig. 2) shows a band splitting of the symmetric C=C stretching mode of thiophene moieties at 1420 and 1440 cm -1 in respect with the SERS spectra of unsubstituted bithiophene. 19 Collado et al. 20 demonstrated that these findings are correlated with a zwitterion form due to the introduction of electron donor and electron withdrawing substituents on the bithiophene core. Thus, donor groups reinforce the π-conjugated system leading to a vibration mode of the thiophene at higher frequency (1440 cm -1 ) whereas electron withdrawing groups conduct to a C=C vibration at a lower frequency (1420 cm -1 ). In addition, stretching bands associated with the cyano (CN) groups (at 2000-2300 cm -1 ) are not observed in the spectrum indicating that these groups are not directly in interaction with the gold surface, which is in favor of the speculated chromophores orientation within the SAM, i.e., cyano moieties on top. Spectroscopic analyses were performed in order to determine the electronic properties of push-pull 7 SAMs on Au. Filled and empty states were studied using Ultraviolet Photoelectron Spectroscopy (UPS) and Inverse Photo-Emission Spectroscopy (IPES) respectively in Fig. 3 (for details see reference 21 and references therein). In the UPS spectrum the highest occupied molecular orbital (HOMO) appears as a bump just below the Fermi level. The zoom in the inset shows the HOMO position at ~0.20 eV below the Fermi level. IPES spectrum exhibits clearly a broad band above the Fermi level, the onset of which should correspond to the position of the lowest unoccupied molecular orbital (LUMO), measured at ~1.60 eV above the Fermi level. This indicates the whole push-pull chromophore behaves as a donor with a charge transfer from the molecule to the substrate surface.

Fig. 3. UPS (left) and IPES (right) spectra of 7 SAMs on gold. Estimated values of HOMO and LUMO were determined by the intersection of the tangent to the band (red dashed line) with the spectrum horizontal baseline (black dotted line). 21 To bring out LUMO position, IPES spectrum with subtracted background signal is plotted in SI (see Fig. S1).

Based on these results it was demonstrated in this series that hexyl chains, used as linker, are the best candidates for the formation of self-assembled monolayers. These features led us to develop chromophores with different acceptors and spacers in order to possibly control both the electro-optical properties and the density of the monolayers. To this end we focused on changing the bithiophene spacer used for the synthesis of chromophores 6 and 7 with a more rigid and planar one, namely the dithieno[3,2-b:2',3'-d]thiophene (DTT) 8. The DTT conjugated spacer was synthesized in four steps from tetrabromothiophene accordingly to reported procedures. 22 Key derivative 11 was synthesized in 52% overall yield from 8 in 3 steps following the same procedure as described for compound 1 (Scheme 3). 13,23 Similarly to the synthesis of compound 3 (Scheme 2) the analogous derivative 12 was obtained in 83% after purification (Scheme 4). This procedure has the main advantage to afford the desired compounds in significantly higher yields compared to reported procedures using unprotected bisaldehyde spacers (20-30%). 24 In addition, the 1 H NMR analysis of the 3 J-coupling constants for the vinylic protons of compounds In the last step the OH group was converted into thioacetate instead of the thiol group in order to avoid its oxidation and to promote stability under storage conditions. The conversion proceeds smoothly but with low yields, probably due to the harsh conditions used during the first step of the reaction (Scheme 5).

Optical and electrochemical properties of push-pull chromophores

The optical properties of the new chromophores 3, 12, 7 and 16-18 have been investigated by UV-Visible spectroscopy in CH 2 Cl 2 (Fig. 4, Table 1). In both series, identical behaviors were found and all spectra are characterized by a predominant absorbance band at low energy which is red-shifted when the strength of the electron acceptor is increased successively from the aldehyde (3 or 12) to the dicyanovinyl (7 or 17) and then to the N,N-diethyl thiobarbituric group (16 or 18). This low energy band corresponds to the intramolecular charge transfer (ICT) band, related to the intrinsic push-pull effect of these structures. Thus, the highest red shift is observed for the derivatives 16 and 18 (603 and 604 nm respectively). Moreover, it is clearly evidenced that the replacement of the BT -conjugated spacer by the DTT does not lead to a significant modulation of the absorption properties. Consequently, similar optical bandgaps (E opt ) were obtained for the compounds in each series possessing the same acceptor (Table 1). Note that the optical HOMO-LUMO gap of compound 7 (~1.9 eV) is similar to that obtained from UPS/IPES experiments in Fig. 3 (1.8 eV). Fluorescence measurements have been performed on these series after excitation at the maximum of absorbance of the ICT band. All chromophores show a high Stokes shift (from 163 to 221 nm) after excitation of the emission band associated with low quantum yields that did not exceed 0.10% (Table 1). Nevertheless, DTT based-chromophores present the highest quantum yields that could be explained by the rigidity and planarity of this -spacer leading to a decrease of the energy losses by the internal vibrations. Additionally, as exemplified in Fig. 5, the emission shows a nontrivial band structure having a shoulder. This shoulder could be attributed to the excited internal charge transfer state while the main band is ascribed to the locally excited state. Furthermore, vibrational spectra give additional insights about the structure and properties of the above chromophores. For instance, compounds 7 and 17 display a stretching vibration for the CN bond ((CN)) centered at 2220 cm -1 and 2213 cm -1 , respectively, which is lowered compared to the expected theoretical value (2280 cm -1 ). 26 Thus, these values reflect a strong push-pull effect along the -conjugated backbone, which is the most effective in the DTT-bridged chromophores.

Stretching vibrations for the C=C bonds (specifically those connected to the donor (D; (C D =C)) and to the acceptor (A; (C=C A )) parts) have also been analyzed. Interestingly, the stretching vibrations for the C D =C bonds appear to be the same for all studied chromophores whereas important differences are noticed for the C=C A stretching. These differences have been related to the acceptor strength; indeed a stronger acceptor will lower the frequency of molecular vibration. At the same time, DTT-based chromophores possess lower frequency of C=C A vibration than corresponding BT analogs. This can be explained by less aromatic and more quinoidal nature of DTT-relay (Table 2). 27,28 Analysis of the cyclic voltammograms of compounds 3, 12, 7, 16-18 (Fig. S2 in the Supporting Information) has revealed similar characteristics. All displayed two irreversible oneelectron oxidation redox systems ascribed to formation of the radical cation and dication species and one irreversible oneelectron reduction redox system (Table 3). The first oxidation has been attributed to the dialkylaniline donor part (0.660-0.685 V) while the second (0.97-1.04 V) to the -conjugated spacer. The reduction potentials exhibit the main differences, which have been related to the acceptor strengths. However, similar values are obtained when the comparison is made on the spacer changes corroborating the observed optical behaviors.

Table 3. Oxidation and reduction potentials for compounds 3, 12, 7, 16-18 10 -3 M in CH3CN/CH2Cl2 (1:1), 0.1 M Bu4NPF6, Pt (WE) Ag/AgCl (RE), v= 100 mV/s, V vs Fc/Fc + (see Fig. S2 in the Supporting Information for the voltammograms). Molecular structure of the compounds 7, 16-18 were obtained thanks to DFT calculations using the 6-31G (d,p) basis set (Table 4). [START_REF] Frisch | Revision D.01[END_REF] All HOMO levels are located along the π-conjugated system whereas the LUMO levels are mainly centralized on the BT or DTT core and the lateral acceptor groups. The energy values of these frontier orbitals can be correlated with the cyclic voltammetry experiments for the HOMO levels while calculated LUMO levels appear at higher energies than expected. The main differences could be explained by the fact that in the excited state the molecules exhibit a higher polarization due to the intramolecular charge transfer which is more stabilized by solvation effects in solution leading to lowered LUMO energy levels.

Epa(1) (V) Epa(2)(V) Epc(V) HOMO (eV) LUMO (eV) E elec (eV) 3 0.

Conclusion

Previously published works on donor-acceptor chromophores for surface modification [10][11][12] deal with charged compounds for which coulombic interactions could hinder the self-organization process and/or alter the properties of the functionalized surface, and surface modification has been scarcely studied. Conversely, in this study we have reported on an innovative approach with the synthesis of uncharged novel D--A linear chromophores that are suitable for surface modifications, and a global engineering of the electrical and optical properties using different structural modifications namely the -conjugated spacer, the acceptor, and the donor. Moreover their SAMs have also been analyzed.

Although the donor part remains the same for all synthesized chromophores (aniline analogues) preliminary slight changes on the length of the alkyl chain (2 or 6 carbons) have been tested revealing a suitable chain length of 2  n  6 carbon atoms for successful self-assembled monolayer formation, as evidenced by both spectroscopic and microscopy studies. SAMs of molecule 7 were studied by UPS and IPES spectroscopies demonstrating a good conformity of modified surface electronic properties with molecular characteristics of the adsorbed chromophore (e.g. HOMO-LUMO bandgap from optical and electrochemical analyses). Based on these results a set of chromophores associated with various optical properties has been built around this optimized donor in combination with different acceptors and electron relays (BT or DTT). It has been shown that solely increasing the acceptor strength in these push-pull chromophores enables to tune the absorption in a wide visible spectral range from the blue (~450 nm) to the red (~600 nm). To our knowledge it is the first time such an effect has been shown which makes these molecules very promising for applications such as organic photovoltaic devices. 6 For such applications where a broad spectral absorption range is mandatory, it should be benefit to mix chromophores with complemental absorption in the SAM. We are currently working on a smart approach for that with organized SAMs of bi-chromophoric moieties borne on the same anchoring group. Interestingly and as expected, the replacement of the BT -conjugated spacer by its more planar and rigid analogue DTT has been shown not to improve the optical properties. Nevertheless, the insertion of such rigid spacer might be useful to form more dense monolayers. On this subject also, works are currently under progress. In addition, future analyses could interestingly concern optical properties of the SAMs using reflection absorption spectroscopy and spectroscopic ellipsometry. [START_REF] Toccafondi | [END_REF]

Experimental section

All chemicals were purchased from Sigma-Aldrich, TCI-Europe or Alfa Aesar and used as received from their commercial sources, unless otherwise stated, without further purification. Infrared spectra were recorded on an Agilent Cary 630 FTIR spectrometer. UV-visible absorption spectra were obtained on a Varian Cary 1E spectrophotometer. The electronic absorption maxima (λ max ) are directly extracted from absorption spectra of the chromophore-based solution. Fluorescence emission spectra were recorded on a Varian Cary Eclipse spectrophotometer, relative fluorescence quantum yields were determined by measuring fluorescence of anthracene as a reference. 1 H and 13 C NMR spectra were recorded on Bruker AC 250 at 250 MHz and 62.5 MHz, respectively. 1 H NMR data are reported as follows: chemical shifts in parts per million relative to CHCl 3 (7.26 ppm), multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broadened), coupling constant (Hz), and integration. High-resolution mass spectroscopy was made with a Qstar Elite spectrometer (Applied Biosystems SCIEX) with ionization electrospray source (ESI) through the Spectropole facility of Aix-Marseille University. [START_REF]For more information[END_REF] All reactions were magnetically stirred and monitored by thin-layer chromatography (TLC) using E. Merck silica gel 60 F254 precoated plates (0.25 mm), and flash chromatography was performed on silica gel 60 (230-400 mesh) from Macherey-Nagel. Compounds 1 13 , 3 13 , 7 13 , 8 16 , 9 22,[START_REF] Zhang | [END_REF] , 10 16 have been prepared according to previously described procedures and their spectroscopic characterizations gave satisfactory analyses. Cyclic voltammetry (CV) data were acquired using a BAS 100 Potentiostat (Bioanalytical Systems) and a PC computer containing BAS100W software (v2.3). A three-electrode system based on a platinum (Pt) working electrode (diameter 1.6 mm), a Pt counter electrode and an Ag/AgCl (with 3 M NaCl filling solution) reference electrode was used. Tetrabutylammonium hexafluorophosphate (n-Bu 4 NPF 6 ) (Fluka) was used as received and served as supporting electrolyte (0.1 M). All experiments were carried out in CH 3 CN/CH 2 Cl 2 (1:1) (electronic grade purity) at 20°C. Ferrocene was used as internal standard. Electrochemical reduction/oxidation potential values were determined from the cyclic voltammogram at a concentration of 1.10 -3 M with a scan rate of 100 mV•s -1 . Calculations were carried out with the Gaussian 09 program. STM (Multimode 8 & Nanoscope V, from Bruker) was operated using Pt/Ir cut tips with tunneling resistance R th ≥ 100 G (typically I ~2 pA, V~1V) in order to be able to resolve the molecular structures without entering the SAM. Voltage was applied to the sample with respect to the grounded tip. For such analyses, SAMs of the different thiol-or thioacetate-terminated chromophores were prepared on atomically flat Au(111) substrates on mica either purchased from Scientec or prepared at IM2NP. These SAMs on flat gold represent a model system to study the arrangement of molecules and the grafting process thanks to the presence of typical etch pits in the gold surface resulting from the sulfur to gold interaction. 33 Surface enhanced Raman spectroscopy (SERS) measurements were conducted on the SAM of the chemisorbed chromophore 7 onto a nanostructured gold thin film. The SERS active surface is obtained by thermal evaporation under high vacuum (10 -6 mbar) of the gold film (7 nm thick) on a glass substrate. SERS spectrum was recorded at room temperature with a Raman spectrometer (Labram HR800-Jobin Yvon) using the 632.4 nm excitation line of a He-Ne laser in retrodiffusion geometry. The laser beam was focused through a x50 objective (50 x) to a spot around 1 m 2 . Spectral calibration was performed on silicon samples at 520 cm - 1 . Ultraviolet Photoelectron Spectroscopy (UPS) was operated with an Omicron equipment (EA125 electron analyzer) and a UV source using the HeI line (21 eV) at normal incidence. Inverse Photo-Emission Spectroscopy (IPES) experiments were performed at normal incidence in the isochromat mode, detecting photons of around 9.7 eV. The absorbed current was kept low enough (~1 µA range) to prevent any degradation of the molecules as monitored by a constant signal level for each of the ten spectra that were accumulated.

2

 2

Scheme 1 .

 1 Scheme 1. Structure of the push-pull chromophores studied. The anchoring head is connected to the donor group via a linker (alkyl chain of 2N carbon atoms).

Fig. 1 .

 1 Fig. 1. STM micrographs of obtained SAMs of 6 (a) and 7 (b) after air annealing at 85°C during 4 hours and 2 hours respectively. Image sizes are 70 nm × 70 nm², height scales are indicated on the right.

Fig. 2 .

 2 Fig. 2. SERS spectrum of chromophore 7 as SAM on nanostructured gold surface

Scheme 3 .

 3 Scheme 3. i) BuLi, -78°C in THF, 1h then N-formylpiperidine -78°C to r.t. (9, 80%); ii) HOCH2CH2OH, PTSA, C6H6 reflux 2d (10, 97%); iii) BuLi, -78°C in THF, 1h then N-formylpiperidine -78°C to r.t. (11, 67%).

3 and 12 (

 12 15.75 and 16.0 Hz respectively) evidences the only presence of the trans-isomers after I 2 /CHCl 3 treatment. Finally compounds 3 and 12 were reacted with malonodinitrile and/or N,N-diethyl thiobarbituric acid (DETB) affording derivatives 5, 13, 14 and 15, respectively (Scheme 4).

Scheme 4 .

 4 Scheme 4. i) a) [4-(N-methyl-N-6-acetoxyhexylamino)benzyl]triphenylphosphonium bromide t BuOK, 0°C CH3CN/THF (1:1) 2.5h; b) HCl 1M r.t.1h then I2 cat. CHCl3 reflux 1h (3: 93%; 12: 83%); ii) NaOH, MeOH/CH2Cl2 TBABr r.t. 3 h then CH2(CN)2, Et3N EtOH r.t. 3 h (5: 95%; 14: 40%); iii) Ac2O at 80°C, 30 min. then NaOH, MeOH/CH2Cl2 TBABr r.t. 3 h (13: 74%; 15: 72%).

Scheme 5 .

 5 Scheme 5. i) PBr3 CH2Cl2 r.t. 2 d. then KSAc, MeOH/CH2Cl2 r.t. 18h (7: 31%; 16: 15%; 17: 16%; 18: 14%).

Fig. 4 .

 4 Fig. 4. Normalized UV-Vis absorption spectra of 3, 12, 7, 16-18 in CH2Cl2

Fig. 5 .

 5 Fig. 5. Absorption (solid line) and emission (dotted line) spectra of 7 (blue) and 17 (dark cyan) in CH2Cl2. The emission was excited at ICT λmax (550 nm for 7 and 553 nm for 17). Fluorescence intensity was normalized by the concentration (0.75•10 -5 M and 1.07•10 -5 M for 7 and 17 respectively).

  25

Table 1 .

 1 Optical properties of compounds 3

, 12, 7, 16-18

		Absorption				Emission
		max	max	0-0	E opt	max	SS	
		(nm)	(L.mol -1 .cm -1 )	(nm)	(eV)	(nm)	(nm)
	3	459	3.3.10 -4	540	2.30	640	181	ND
	12	456	3.5.10 -4	526	2.36	619	163	ND
	7	550	7.5.10 -4	664	1.87	712	162	0.042
	17	552	5.8.10 -4	651	1.90	736	184	0.093
	16	603	3.7.10 -4	723	1.71	824	221	0.004
	18	604	1.1.10 -4	715	1.73	825	221	0.015
	E

opt : optical bandgap; SS: Stokes shift; ND: not determined; : quantum yield.

Table 2 .

 2 Evolution of specific stretching vibrations for compounds 7

, 16-18.

		(CN) (cm	-1 )	(CD=C) (cm	-1 )	(C=CA) (cm	-1 )
	7	2220	1597	1571
	17	2213	1598	1558
	16	-		1597	1552
	18	-		1598	1550

Table 4 .

 4 HOMO/LUMO calculated values in eV for compounds 7, 16-18 (B3LYP/ 6-31G(d,p))

	HOMO (eV)	LUMO (eV)
	7
	-5.02	-2.88
	17
	-5.07	-2.86
	16
	-4.95	-2.84
	18
	-5.02	-2.83

Acknowledgements

The authors thank gratefully Prof. J.-J. Simon for valuable discussions on this work and Prof. M. Abel for UPS analyses. This work was supported by the Centre National de la Recherche Scientifique (CNRS) and the Ministère de l'Enseignement Supérieur et de la Recherche. Financial support from ANR program (SAGe III-V project ANR-11-BS10-012) and "Solutions Communicantes Sécurisées" (SCS) competitive cluster are also acknowledged. V.M. also thanks the Ministère de l'Enseignement Supérieur et de la Recherche for his doctoral Financial support.


Alexandra Ohannessian

Anderson Loundou

Audrey Gnisci

Odile Paulmyer-Lacroix

Jeanne Perrin

Blandine Courbiere
email: blandine.courbiere@univ-amu.fr

Unexplained infertility: live-birth's prognostic factors to determine the ART management

Keywords:

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

P R O O F M I N E R V A M E D I C A 2
MiNerVa GiNecoloGica ???? 2017

Infertility was defined by the lack of pregnancy after one year of regular unprotected intercourse. To define the unexplained infertility, no etiology must be found after infertility investigations: for the man, at least two normal semen analyses, according to World Health organization criteria. [START_REF] Cooper | World Health organization reference values for human semen characteristics[END_REF] if one semen parameter (sperm concentration, motility, morphology) was out of the reference range, the couple was not included in the study. For the woman, the assessment of the tubal patency and uterine cavity was made by hysterosalpingogram (HSG) and pelvic ultrasound. The ovarian reserve was assessed by antral follicle count (aFc), serum aMH level (ng/ml, Kit Beckman-Coulter, Immunotech) and day 3 serum FSH level (iU/l). in case of suspected diminished ovarian reserve (Dor, FSH>10 iU/l), the couple was not included. in case of suspected tubal pathology, a laparoscopy was proposed to women.

The management of unexplained infertility consisted to start with gonadotropin iUi and then, if unsuccessful, to propose iVF. The maximum number of iUi before undergoing iVF was determined in the center depending on the woman age: up to 4 iUi up to 38-yearsold, up to 2 after 38-years-old.

Prognostic factors

The prognostic factors that we aimed to study were: age, Body Mass index (BMi) and smoking status for both man and woman, infertility duration, type of infertility (primary or secondary), aFc, day 3 serum FSH level (UI/L), serum AMH level (Beckman-Coulter kit, ng/mL), woman Chlamydiae trachomatis serology, a diagnostic laparoscopy in the initial workup for infertility. We also assessed the number of iUi cycles, pregnancy outcomes (with iUi or iVF), and spontaneous pregnancies with the time to conceive.

Statistical analysis

The main analysis compared prognostic factors between two groups of couples: the couples with a lB after iUi, and the couples follicle-stimulating hormone (FSH) level, serum anti-Müllerian hormone (aMH) level and antral follicular count (aFc) remains controversial. [START_REF]Fertility: assessment and treatment for people with fertility problems[END_REF][START_REF]Practice committee of the american Society for reproductive Medicine. Diagnostic evaluation of the infertile female: a committee opinion[END_REF][9][START_REF]Arrêté du 22 juin 2015 définissant les règles de bonnes pratiques applicables à la stimulation ovarienne y compris lorsqu'elle est mise en oeuvre indépendamment d'une technique d'assistance médicale à la procréation[END_REF] laparoscopy should be performed in case of suspicion of tubal or pelvic pathology. [START_REF]Fertility: assessment and treatment for people with fertility problems[END_REF][START_REF]Practice committee of the american Society for reproductive Medicine. Diagnostic evaluation of the infertile female: a committee opinion[END_REF][9] Chlamydiae trachomatis serology is not systematically recommended; in case of seropositivity, tubal abnormalities are suspected and a laparoscopy should be performed. 9 Several management of unexplained infertility are proposed: expectant management, intrauterine insemination (iUi) with ovarian stimulation and then, if unsuccessful, in vitro fertilization (iVF), or starting the treatment with iVF. [START_REF] Kamath | Demographics of infertility and management of unexplained infertility[END_REF][START_REF] Shah | Unexplained infertility: an update and review of practice[END_REF][START_REF] Steures | collaborative effort on the clinical evaluation in reproductive Medicine. intrauterine insemination with controlled ovarian hyperstimulation versus expectant management for couples with unexplained subfertility and an intermediate prognosis: a randomised clinical trial[END_REF][START_REF] Van Rumste | cecerM. longterm outcome in couples with unexplained subfertility and an intermediate prognosis initially randomized between expectant management and immediate treatment[END_REF][START_REF] Gianaroli | Best practices of ASRM and eSHre:a journey through reproductive medicine[END_REF][START_REF] Bensdorp | Prevention of multiple pregnancies in couples with unexplained or mild male subfertility: randomised controlled trial of in vitro fertilisation with single embryo transfer or in vitro fertilisation in modified natural cycle compared with intrauterine insemination with controlled ovarian hyperstimulation[END_REF][START_REF] Tjon-Kon-Fat | is iVF-served two different ways-more cost-effective than iUi with controlled ovarian hyperstimulation?[END_REF][START_REF] Veltman-Verhulst | intra-uterine insemination for unexplained subfertility[END_REF][START_REF] Eshre Capri | Intrauterine insemination[END_REF][START_REF] Van Rumste | iVF with planned single-embryo transfer versus iUi with ovarian stimu-Vol. 69 -No. ?? MiNerVa GiNecoloGica 7 discontinuation[END_REF][START_REF] Aboulghar | controlled ovarian hyperstimulation and intrauterine insemination for treatment of unexplained infertility should be limited to a maximum of three trials[END_REF][START_REF] Mcdonnell | Intrauterine insemination or in-vitro fertilisation in idiopathic subfertility and male subfertility: a randomised trial and cost-effectiveness analysis[END_REF][START_REF] Neumann | a randomized clinical trial to evaluate optimal treatment for unexplained infertility: the fast track and standard treatment (FASTT) trial[END_REF][START_REF] Pandian | in vitro fertilisation for unexplained subfertility[END_REF][START_REF] Goldman | a randomized clinical trial to determine optimal infertility treatment in older couples: the Forty and over Treatment Trial (ForT-T)[END_REF][START_REF] Pashayan | cost-effectiveness of primary offer of iVF vs. primary offer of iUi followed by iVF (for iUi failures) in couples with unexplained or mild male factor subfertility[END_REF][START_REF] Custers | couples with unexplained subfertility and unfavorable prognosis:a randomized pilot trial comparing the effectiveness of in vitro fertilization with elective single embryo transfer versus intrauterine insemination with controlled ovarian stimulation[END_REF][START_REF] Quaas | Diagnosis and treatment of unexplained infertility[END_REF][START_REF] Brandes | Unexplained infertility: overall ongoing pregnancy rate and mode of conception[END_REF] There is no international consensus on the best strategy for the management of unexplained infertility. For example, the Guidelines of the National institute for Health and care excellence on infertility advise couples to try to conceive for a total of 2 years, including up to one year before their infertility investigations, before considering iVF. [START_REF]Fertility: assessment and treatment for people with fertility problems[END_REF] in France, the choice between iUi or iVF remains to the physician, depending on prognostic factors such as age and infertility duration, without clear cutoffs. 9 However, these arT options are greatly different in terms of cost, hardship and risk for the woman. Thus, the aim of our study was to identify live birth's (lB) prognostic factors of couples with unexplained infertility in order to define the best infertility treatment.

Materials and methods

The study protocol was approved by the French committee of ethics for research in obstetrics and Gynecology in January 2016 (ceroG 2015-GYN-1204). informed consent was obtained from all participants.

Patients

We conducted a retrospective observational study in a single university fertility center in France. The data were collected from the medical records of couples. We included all the couples managed for unexplained infertility and who underwent their first IUI between January 2011 and June 2015.

P R O O F M I N E R V A M E D I C A Vol. 69 -No. ??
MiNerVa GiNecoloGica 3 was 17.6±8.5. Mean value of women's basal FSH level was 6.5±1.7 iU/l, mean serum level of aMH was 4.0±2.7 ng/ml. Chlamydiae trachomatis antibody was tested in 52 women (39%), and 6 women were seropositive. Ten women (7.5%) had a diagnostic laparoscopy, and no pelvic pathology was found. among the 133 couples included, 50 couples had a lB after iUi, most of the time after the second iUi cycle. Twenty couples had a spontaneous pregnancy leading to a lB. Mean time to spontaneous pregnancy was 3.7±1.6 years. For the remaining 63 couples, iVF was decided after several failed iUi. of these 63 couples, 21 dropped-out from infertility treatments, before starting iVF. Seventeen couples had a pregnancy after iVF (fresh and frozenthawed embryo transfer) with lB. it was the first IVF attempt for two thirds of couples. Prognostic factors of the 50 couples with a pregnancy after iUi were compared with those of the 63 couples who should have started iVF, without any difference (Table i).

Moreover, the 20 couples who finally had a spontaneous pregnancy were compared with the childless couples despite iUi and iVF, without any prognostic factor being highlighted (Table ii). Finally, cumulative lB rate (lBr), including spontaneous and arT pregnancies, for couples who were managed for unexplained infertility was 65.7% (N.=87).

Discussion

in the management of unexplained infertility, the choice between iUi and iVF is still who should undergo iVF after iUi failure. a secondary analysis compared two groups of couples: couples with a spontaneous pregnancy and lB, and the childless couples despite time trying to conceive spontaneously associated to arT management (iUi±iVF).

iBM SPSS Statistics version 20.0 (inc., il., USa) software was used for data analysis. Statistical significance was defined as P<0.05. Quantitative data were expressed as mean±standard deviation (SD) or as medians with range (minimum, maximum), qualitative data were expressed as numbers and percentages. Qualitative data were compared using the Pearson's χ 2 Test or Fisher's exact Test when available. The relationship between a dichotomous variable and a quantitative variable was assessed using the Student t test or the nonparametric Mann-Whitney Test. if the qualitative variable had more than two categories, the F Test aNoVa was used.

Results

Between January 2011 and July 2015, 133 couples with unexplained infertility were included, corresponding to 320 gonadotropin iUi cycles. The average number of iUi by couple was 2.4±1.2. At the first cycle, the mean age was 31.6±4.6 years for women, 34.9±6.2 years for men. Primary infertility was observed on 67% of couples. The mean infertility duration at first cycle was 3.1±1.6 years. Current smoking was observed on 32% of women and 40% of men. Mean BMI was 24.7±5.6 kg/m 2 for women, 24.4±3.4 kg/m 2 for men. Mean aFc [START_REF] Kr | eunice Kennedy Shriver National institute of child Health and Human Development reproductive Medicine Network. Predictors of pregnancy and livebirth in couples with unexplained infertility after ovar-2[END_REF] Tjon-Kon-Fat et al. published a secondary analysis of a randomized controlled trial including 116 couples with unexplained or mild male The couples were randomized to one cycle of iVF with elective single embryo transfer with subsequent frozen-thawed embryo transfers or 3 cycles of iUi. Female age and infertility duration were not associated with better ongoing pregnancy chances after iVF compared to iUi. only when prewash total motile count was lower than 110 (x10 6 spermatozoa/ml), the probability of ongoing pregnancy was higher in women al-debated. our study wanted to assay lB's prognostic factors in these infertile couples in order to counsel them the best as possible: iUi prior iVF or starting immediately with iVF? our study did not show any predictive factor of lB after iUi in these couples. lBr after iUi was 38%. Most of the time, 2 iUi cycles were required to be pregnant. in 2014, Kaser et al. published a secondary analysis of 2 trials, FaSTT and ForT-T, including 603 infertile women aged 21 to 42 years, randomized to conventional or accelerated treatment with iUi and/or iVF (2717 total cycles). Women were stratified in 4 groups according to basal FSH and estradiol. Women with FSH of 10-15 IU/L and estradiol ≥40 pg/ mL on day 3 testing were unlikely to achieve lB after iUi. [START_REF] Kaser | When is clomiphene or gonadotropin intrauterine insemination futile? Results of the Fast Track and Standard Treatment Trial and the Forty and over Treatment Trial, two prospective randomized controlled trials[END_REF] However, FSH of 10 iU/l is highly suggestive of Dor, and could explain the "unexplained" infertility. in our study, we excluded women with hormone profile suggesting a DOR. The difficulty of analyzing the literature is due to differences in the definition of "unexplained" infertility and the paraclinic exams that have led to this diagnosis. ideally, we should use "unexplained infertility" only with a normal complete workup including pelvic ultrasound, HSG, hormone profile, assessment of ovarian reserve, a Chlamydia trachomatis serology and for male a normal semen analysis. [START_REF] Steiner Az | reproductive Medicine Network. Chlamydia trachomatis immunoglobulin G3 seropositivity is a predictor of reproductive outcomes in infertile women with patent fallopian tubes[END_REF] in addition, diagnostic laparoscopy is not routinely performed for these women. Devranoglu et al. published a cross-sectional study including couples with unexplained infertility, with women at the age ≤36 years with

P R O O F M I N E R V A M E D I C A Vol. 69 -No. ?? MiNerVa GiNecoloGica 5
study, only 10 women (7.5%) had a diagnostic laparoscopy, while it may be discussed of the infertility work-up despite normal HSG. Indeed, in unexplained infertility, endometriosis stage 1 or 2, pelvic adhesions or tubal disease are found in 49% to 90.5% of women. [START_REF] Boujenah | laparoscopy in arT?[END_REF] among the 133 couples included, iUi management failed for 63 couples. a third of these couples stopped infertility treatment after these failures, while an iVF treatment should have been recommended to them. This highlights the high rate of drop-out in infertility treatments, corroborating data from other studies on the subject. [START_REF] Verberg | Why do couples drop-out from iVF treatment? a prospective cohort study[END_REF][START_REF] Van Dessel | couples dropping out of a reimbursed intrauterine insemination program: what is their prognostic profile and why do they drop out?[END_REF][START_REF] Lande | Why do couples discontinueunlimited free iVF treatments?[END_REF][START_REF] Troude | Medical factors associated with early iVF[END_REF] in our study, the mean infertility duration was 3.1±1.6 years, with a minimum and a maximum of 1 and 8 years respectively, which is relatively long. added to the time required to perform several iUi and the burnout due to repeated failures, this could contribute to discouraging some couples. So do not let the couples waste their time and let them start directly infertility management by iVF or right after two or three failed iUi cycles.

Conclusions

Prognostic factors for pregnancy in the context of unexplained infertility do not seem to exist. [START_REF] Dinelli | Prognosis factors of pregnancy after intrauterine insemination with the husband's sperm: conclusions of an analysis of 2,019 cycles[END_REF] This study suggests the interest to start the treatment with up to 2 gonadotropin iUi attempts for any couple whose infertility is considered unexplained because iUi is less invasive and less expensive than iVF.

Moreover, chance of spontaneous pregnancy in these couples should be considered. indeed, it has been shown that the diagnosis of infertility and its treatment could lead to sexual dysfunction and less frequent sexual intercourse. [START_REF] Oddens | Psychosocial experiences in womenfacing fertility problems--a comparative survey[END_REF][START_REF] Salvatore | Psychopathology, personality, and marital relationship in patients undergoing in vitro fertilization procedures[END_REF][START_REF] Fernandez | .[impact of infertility and assisted reproductive techniques on sexuality[END_REF][START_REF] Reder | Does sexuality still have a place for couples treated with assisted reproductive techniques?[END_REF][START_REF] Shoji | Sexual satisfaction of infertile couples assessed using the Golombok-Rust Inventory of Sexual Satisfaction (GriSS)[END_REF][START_REF] Piva | a literature review on the relationship between infertility and sexual dysfunction: does fun end with baby making?[END_REF][START_REF] Tao | The impact of infertility on sexuality: a literature review[END_REF] It is important to track sexual dysfunction and reassure the couple on natural conception. located to iVF. This study included a heterogeneous population, with men with abnormal semen analysis. [START_REF] Tjon-Kon-Fat | can we identify subfertile couples that benefit from immediate in vitro fertilisation over intrauterine insemination? eur[END_REF] a recent retrospective cohort study by Geisler et al. included 851 couples with unexplained infertility, mild endometriosis, one patent Fallopian tube, mild male infertility or ovulatory dysfunction, up to 3 cycles iUi. at each attempt, more than 70% couples were couples with unexplained infertility. independent associates of successful outcome factors were lower age and multiparity, for all the couples and the subgroup of unexplained infertility. The unadjusted cumulative pregnancy rate for lB per cycle started, over three cycles, was 34.9%, which is near from our lBr of 37.6%. in this study, more than 90% of total lBs with IUI is achieved during the first 2 cycles, corroborating our conclusion. [START_REF] Geisler Me | intrauterine insemination-No more Mr. N.i.c.e. guy? eur[END_REF]

Limitations of the study

The strength of our study was to be very restrictive on the couples' inclusion criteria. We did not include couples with mild male infertility, even with relatively mild sperm abnormalities, neither women with a suspicion of Dor (FSH>10 iU/l). [START_REF]Practice committee of the american Society for reproductive Medicine. Diagnostic evaluation of the infertile female: a committee opinion[END_REF] So, we have included a very homogeneous population of couples whose infertility seemed truly unexplained. The very restrictive inclusion criteria, however, lead to include only a small population, even if the data have been studied over many years. The lack of significant results could be correlated with a lack of study power. One can also criticize the retrospective design of the study, the lack of randomization, and the long study period during which management changes could have taken place. Chlamydiae trachomatis seropositivity is associated with a lower natural conception rate and a lower likelihood of pregnancy after iUi, even with patent fallopian tubes. [START_REF] Steiner Az | reproductive Medicine Network. Chlamydia trachomatis immunoglobulin G3 seropositivity is a predictor of reproductive outcomes in infertile women with patent fallopian tubes[END_REF][START_REF] Coppus | chlamydia trachomatis igG seropositivity is associated with lower natural conception rates in ovulatory subfertile women without visible tubal pathology[END_REF] in our study, less than half of women (39%) were tested for Chlamydiae trachomatis, and we are not able not analyze this factor's reliably. The systematic prescription of the Chlamydiae trachomatis serology before iiU in our arT center is the result of recent management changes. Moreover, in our

Table I .

 I -Prognostic factors of live birth after IUI.

	live birth after iUi, N.=50 30.8±4.4 34.6 ±6.1 O O F at least unilateral tubal patency at HSG: 105 iUi failure, N.=63 P value 32.1 ±4.7 0.1 34.5±5.9 0.9 women with a day 3 FSH≥10 IU/L (170 IUI A cycles) were compared to 95 women with a normal FSH (170 iUi cycles too). hcG posi-tivity, clinical and ongoing pregnancy rates I C did not differ between women with normal and elevated FSH levels (P=0.2, 0.3 and 0.4, D respectively). Thus, according to this study, in women under 36 years, basal serum FSH E was not a prognostic factor for pregnancy af-ter iUi. 31 Hansen et al. published a secondary M analysis of a prospective, randomized, mul-ticenter clinical trial including 900 couples R Women's age (y), mean (SD) Men's age (y), mean (SD) P M with unexplained infertility who underwent iUi treatment, up to 4 cycles. ovarian stimu-lation was performed with clomiphene citrate, letrozole, or gonadotropins. Woman's age, V A infertility duration, level of income and prior pregnancy loss were significant predictors of R lB. in this study, the FSH level was not stud-ied in contrast to aMH that was not correlated E with the LBR, confirming the data from our I N study.
	Women's BMI (kg/m 2 ), mean (SD)	25.7±5.9	24.3±5.8	0.2
	Men's BMI (kg/m 2 ), mean (SD)	24.3±3.6	24.5±3.7	0.8
	Female smokers, (N.)%	18 (36)	20 (31.7)	0.7
	Male smokers, (N.)%	18 (36)	28 (44.4)	0.4
	Primary infertility, (N.)%	31 (62)	45 (71.4)	0.3
	infertility duration (y), mean (SD)	3.2±1.7	3.1±1.5	0.9
	FSH (Ui/l), mean (SD)	6.2±1.9	6.8±1.6	0.05
	aMH (ng/ml), mean (SD)	4.2±3.0	4.1±2.5	0.9
	aFc, mean (SD)	18.2±9.5	17.1±7.8	0.6
		MiNerVa GiNecoloGica		???? 2017

Table II .

 II -Prognostic factors of LBR after spontaneous pregnancy.

		Spontaneous pregnancy and live birth, N.=20	childless after iUi±iVF, N.=46	P value
	Women's age (y), mean (SD)	32.1±4.5	32.1±5.1	1
	Men's age (y), mean (SD)	36.8±7.3	35.1±6.2	0.3
	Women's BMI (kg/m 2 ), mean (SD)	23.3±3.7	24.2±5.8	0.5
	Men's BMI (kg/m 2 ), mean (SD)	24.5±2.0	24.5±4.0	1
	Female smokers, (N.) %	4 (20)	16 (34.8)	0.2
	Male smokers, (N.) %	6 (30)	22 (47.8)	0.2
	Primary infertility, (N.) %	13 (65)	35 (76.1)	0.4
	infertility duration (y), mean (SD)	2.6±1.2	3.1±1.5	0.3
	FSH (Ui/l), mean (SD)	6.4±1.4	6.8±1.7	0.4
	cFa, mean (SD)	18.2±9.6	17.0±8.6	0.7
	aMH (ng/ml), mean (SD)	3.4±2.2	3.8±2.0	0.6


BS Sanghee Moon
email: smoon@kumc.edu

PhD Maud Ranchet
email: maud.ranchet@ifsttar.fr

PhD, MPH, MBA Abiodun Emmanuel Akinwuntan
email: aakinwuntan@kumc.edu

PhD Mark Tant
email: mark.tant@bivv.be

MD David Carr
email: dcarr@wustl.edu

MD Mukaila Raji
email: muraji@utmb.edu

PhD Hannes Devos
email: hdevos@kumc.edu

The impact of advanced age on driving safety in older adults with medical conditions

Keywords: oldest-old, driving safety, fitness-to-drive, motor vehicle crash

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

Introduction

A rapid increase in the older population in the western world is driven by a longer life expectancy and aging of baby boomers [1]. Globally, it is anticipated that the number of adults aged 60 and older will increase by 56% from 901 million in 2015 to 1.4 billion in 2030, especially older adults aged 80 and older will increase by 61% from 125 million to 202 million during the same period [START_REF]United Nations: World population ageing[END_REF]. For instance, in the United States, the number of licensed drivers aged 65 years and older is projected to rise from 40 million (15%) in 2015 to 56 million (20%) by 2025 [START_REF]Traffic safety facts -older population -2015 data[END_REF][START_REF]Highway Loss Data Institute: Estimating the effect of projected changes in the driving population on collision claim frequency[END_REF][5]. This growing number of older drivers, especially those with medical conditions, may pose considerable traffic safety concerns in all western industrialized countries. Motor vehicle crashes (MVCs) are the second leading cause of unintentional injury-related fatalities among 55 to 64 (middle-aged), 65 to 74 (young-old), 75 to 84 (old-old), and 85 and older (oldest-old) [START_REF]National Center for Injury Prevention and Control: Web-based Injury Statistics Query And Reporting System (WISQARS)[END_REF]. Involvements in fatal MVCs per miles traveled begin to increase in older drivers aged 70 and peak after age 80 and older [START_REF] Cicchino | Trends in older driver crash involvement rates and survivability in the United States: An update[END_REF]. Although older drivers start to show higher fatality due to MVCs [START_REF]National Center for Injury Prevention and Control: Web-based Injury Statistics Query And Reporting System (WISQARS)[END_REF][START_REF] Mcgwin | Relations among chronic medical conditions, medications, and automobile crashes in the elderly: A population-based case-control study[END_REF], only few studies have focused particularly on MVC in the oldest-old drivers [START_REF] Meuser | Motor-vehicle crash history and licensing outcomes for older drivers reported as medically impaired in Missouri[END_REF].

Several factors for the higher involvement in fatal MVCs in advanced age have been postulated. First, the disability rate and physical frailty increase with age [START_REF] Mcgwin | Relations among chronic medical conditions, medications, and automobile crashes in the elderly: A population-based case-control study[END_REF][START_REF] Lin | Trends in US older adult disability: Exploring age, period, and cohort effects[END_REF]. The frailty due to atherosclerosis and osteoporosis raises the likelihood of fatal chest and head injuries [START_REF] Lotfipour | Fatality and injury severity of older adult motor vehicle collisions in orange county, California, 1998-2007[END_REF].

Second, older drivers may experience age-related changes known to affect driving. Such changes include declines in cognitive, visual, motor, and sensory functions to respond timely and accurately to a constantly changing environment [START_REF] Anstey | Cognitive, sensory and physical factors enabling driving safety in older adults[END_REF]. Lastly, older drivers may have more difficulty making left-hand turns against opposing traffic, which may increase the risk of MVC [START_REF] Sifrit | Intersection crashes among drivers in their 60s, 70s and 80s[END_REF][START_REF] Andrea | The assessment of safe and unsafe turns by young and dlder drivers[END_REF]. Furthermore, the old-old and oldest-old have not been considered separately in most studies despite potential differences in driving related characteristics. A previous study examining driving competence in the oldest-old showed potential differences in cognitive skills associated with on-road driving performance [START_REF] Hollis | The driving competence of 90-year-old drivers: from a hospital-based driving clinic[END_REF]. Hence, there is a need to understand how advanced age influences all aspects of safe driving, including MVCs, traffic violations, and fitness-to-drive, especially among those with medical conditions. Such understanding will inform driving-related public policy and assist physicians in their decision-making process of fitness-todrive.

Determining fitness-to-drive often requires physician's involvement [START_REF]National Highway Traffic Safety Administration: Clinician's guide to assessing and counseling older drivers (3rd edition)[END_REF]. Our previous study investigated individuals with various medical conditions [START_REF] Moon | Comparison of unsafe driving across medical conditions[END_REF]. We found that physicians were more likely to issue a favorable recommendation compared to a formal fitness-to-drive decision by driving evaluation specialists [START_REF] Moon | Comparison of unsafe driving across medical conditions[END_REF]. Yet, it remained unclear whether advanced age affects the degree of agreement between fitness-to-drive recommendations by referring physicians and comprehensive fitness-to-drive decisions by driving evaluation specialists.

The primary aim was to examine the impact of advanced age on driving safety with a particular focus on the oldest-old with reported medical conditions. A secondary aim was to compare agreements in fitness-to-drive recommendations between physicians and driving evaluation specialists involving the oldest-old with medical conditions.

Method

Study participants

This population-based retrospective study included individuals aged 55 and older with medical conditions. They were referred to the Center for Evaluation of Fitness to Drive and Car Adaptations (CARA) of VIAS Institute between January 2, 2013 and December 30, 2014, because they or a third party raised driving safety concerns. Individuals were referred for a driving evaluation (1) because of a change in their medical status; (2) to extend the validity period of their driver's license; (3) to request a new driver's license category; or (4) because of a mandated referral by medical experts, courts, or insurance companies. CARA is the only official entity in Belgium to make decisions about fitness-to-drive and car adaptations [START_REF] Van Den Meerschaut | Rijgeschiktheid[END_REF]. The CARA database contains individual's demographic, clinical, and medical information collected from CARA in Brussels and 46 CARA clinical hubs across Belgium. We excluded individuals without a comprehensive fitness-to-drive decision record, and used the 2013 records when the individual revisited CARA in 2014.

The study ethics was approved by the Institutional Review Board of the University of Kansas Medical Center.

Age group

Individuals were categorized into four age groups: 55 to 64 (middle-aged); 65 to 74 (young-old); 75 to 84 (old-old); and 85 and older (oldest-old). We adopted the definition of the World Health Organization and other governmental organizations to describe the oldest-old [START_REF]World Health Organization: Global health and ageing[END_REF][START_REF] Jacobsen | America's aging population[END_REF][START_REF] Tomassini | The demographic characteristics of the oldest old in the United Kingdom[END_REF].

Fitness-to-drive procedure

Fitness-to-drive was assessed through a two-tier legal procedure [START_REF] Devos | Evaluation and rehabilitation of driving ability in people with neurological conditions[END_REF]. The first tier involved an appraisal by referring physicians (e.g., primary care physician). Individuals with medical conditions that may adversely affect driving were obliged to discuss their wish to continue driving to a physician of choice. This physician might appraise the fitness-to-drive or might seek assistance from doctor-specialists or the CARA center. If drivers showed any functional deficits known to affect driving, they were obliged to seek a fitness-to-drive advice from CARA (second tier). During this first-tier procedure, the individual and the referring physician filled out a driving referral form of demographic (e.g., age, sex, etc.), medical (e.g., primary diagnosis, presence of comorbidities, etc.), and driving (e.g., numbers of MVCs and traffic violations) information.

In the second tier, the CARA team of occupational and physical therapists with expertise in on-road evaluation, neuropsychologists, and physicians conducted a comprehensive fitness-todrive evaluation consisting of medical, visual, and on-road evaluations. When the medical referral form indicated that there were psychiatric or cognitive complaints, individuals were also evaluated by neuropsychologists. Of note, driving evaluation procedures varied depending on individual's medical condition, but always included a practical on-road evaluation and basic visual sensory screen conducted by trained occupational therapists or physical therapists. Each on-road evaluation course was standardized and included segments of residential driving and urban driving.

Driving safety outcome measures

The study used four main outcome measures for driving safety: (1) first-tier fitness-todrive recommendation by referring physician; (2) final-tier comprehensive fitness-to-drive decision by CARA; (3) number of self-reported MVCs in the past five years; and (4) number of self-reported traffic violations in the past five years.

For fitness-to-drive recommendation and comprehensive decision, referring physicians and CARA team used the same three-class categorization: (1) fit-to-drive without restrictions; [START_REF]United Nations: World population ageing[END_REF] fit-to-drive with restrictions; and (3) unfit-to-drive. Restrictions included time (e.g., no nighttime driving), speed (e.g., no highway driving), or distance (e.g., driving in limited area only).

For analysis purpose, we dichotomized the three-class categorization into 'fit-to-drive' (continue to drive with or without restrictions) or 'unfit-to-drive', similar to our previous work [START_REF] Moon | Comparison of unsafe driving across medical conditions[END_REF].

Categorization of medical conditions

The primary medical condition was defined as the principal diagnosis that individuals and their referring physicians reported on their driving referral form. These medical conditions were classified into ten different medical categories: (1) neurological conditions (e.g., stroke, multiple sclerosis, etc.) (Supplementary Table 1); (2) psychiatric conditions (e.g., psychotic disorder, schizophrenia, etc.); (3) musculoskeletal conditions (e.g., amputation, fracture, etc.); (4) visual conditions (e.g., cataract, glaucoma, etc.); (5) vestibular or hearing conditions (e.g., loss of balance, etc.); (6) cardiovascular or pulmonary conditions (e.g., hypertension, atrial fibrillation, etc.); (7) liver or renal conditions (e.g., renal insufficiency, etc.); [START_REF] Mcgwin | Relations among chronic medical conditions, medications, and automobile crashes in the elderly: A population-based case-control study[END_REF] sleep disorders (e.g., sleep apnea, etc.); (9) diabetes mellitus; and (10) substance abuse (e.g., alcohol, cannabis, etc.). The categorization criteria were adopted from the National Highway Traffic Safety Administration of the US Department of Transportation [START_REF] Dobbs | Medical conditions and driving: A review of the scientific literature[END_REF] and other classifications [START_REF]Driver & Vehicle Licensing Agency: Assessing fitness to drive: A guide for medical professionals[END_REF][START_REF]Austroads: Assessing fitness to drive for commercial and private vehicle drivers[END_REF].

Statistical analysis

Kolmogorov-Smirnov test was used to assess the normality. Kruskal-Wallis tests (KW) or chi-squared tests (χ 2 ) were utilized for group comparisons with an alpha level at 5%. Bonferroni correction for multiple comparisons (p = 0.008 or 0.05/6) was used for post-hoc tests including Mann-Whiney U tests and χ 2 .

To investigate the effect of age on driving safety outcomes, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using separate logistic regression models for each of the four main outcome measures. The middle-aged (55 to 64 years old), which was closest to the median age (55 years old) of the entire CARA database [START_REF] Moon | Comparison of unsafe driving across medical conditions[END_REF], was used as a reference group in each model.

The agreement between physician's recommendation of fitness-to-drive and comprehensive fitness-to-drive decision was calculated using the prevalence-and bias-adjusted kappa (PABAK). The interpretation of PABAK followed Landis and Koch's interpretation [START_REF] Landis | The measurement of observer agreement for categorical data[END_REF].

Substantial agreement reflected PABAK between 0.61 and 0.80. Almost perfect agreement reflected PABAK between 0.81 and 1.00. Statistical analyses were conducted using SPSS 23 (IBM Corp., Armonk, NY) and SAS Enterprise Guide 6.1 (SAS Institute Inc., Cary, NC).

Results

Demographic and medical characteristics

Among 6,584 individuals who visited CARA for a driving evaluation between 2013 and 2014, 3,425 individuals were aged 55 and older (Table 1). Among those, 40% were middle-aged (n = 1,386), followed by 30% young-old (n = 1,013), 23% old-old (n = 803), and 7% oldest-old (n = 223). The age range of the oldest-old was from 85 to 95 years old. In all groups, males represented the majority, representing more than 70% of the database. The percentage of males in the young-old and old-old were significantly higher compared with the middle-aged (post-hoc p < 0.008). Individuals were mostly referred to CARA to extend their driver's license, followed by change in medical conditions, mandated referral, and request for a new driver's license. Of particular importance was the significantly high percentage of old-old (n = 178; 26%) and oldestold (n = 41; 22%) who were mandated by court, law enforcement, medical experts, or insurance companies to seek a fitness-to-drive evaluation (post-hoc p < 0.008). Moreover, insurance companies referred significantly more individuals in the old-old and oldest-old, while those in the middle-aged and young-old were primarily referred by medical experts (χ 2 = 61.32; p < 0.001).

The distribution of primary medical conditions significantly differed across age groups (p < 0.001) (Table 1). The oldest-old showed a significantly lower percentage of neurological conditions than the old-old and middle-aged (post-hoc p < 0.008). Per cardiovascular or pulmonary conditions, the oldest-old had a significantly higher percentage than other age groups (post-hoc p < 0.008). Per visual conditions, the percentage in the oldest-old was the highest among all groups (post-hoc p < 0.008). The percentage of musculoskeletal conditions was not significantly different across groups. The results of the post-hoc analyses are shown in Table 1.

Although the proportions of medical conditions across groups were significantly different, neurological conditions represented the major medical category that made individuals referred for a driving evaluation. To illustrate, stroke was the most common neurological condition across all age groups. The second most common neurological condition in the middleaged and young-old groups was multiple sclerosis, whereas it was dementia including Alzheimer's dementia in the old-old and oldest-old groups. Parkinson's disease was the third most common disease in the young-old and old-old groups (Supplementary Table 1).

Fitness-to-drive, MVCs, and traffic violations across age groups

Overall, most drivers were safe drivers. For all groups, more than 80% of drivers were considered fit-to-drive according to physicians or CARA (Figure 1). Yet, the fit-to-drive rate of physician's recommendation and comprehensive decision by CARA decreased with advancing age (both p < 0.001) (Figure 1). Physicians judged 93% of individuals in the oldest-old as fit-todrive, which was considerably lower compared with the fit-to-drive rate of the middle-aged (98%) and young-old (98%) (post-hoc p < 0.008). Likewise, the CARA team judged a significantly lower percentage of oldest-old (81%) as fit-to-drive compared with the middle-aged (92%) and young-old (91%) (post-hoc p < 0.008). No significant differences between oldest-old and old-old groups were observed in physician's recommendation and comprehensive fitness-todrive decisions (Figure 1).

The oldest-old (30%) and old-old (26%) reported significantly more MVCs in the past five years compared with the middle-aged (13%) and young-old (13%) (post-hoc p < 0.008) (Figure 1). The number of traffic violations did not differ between age groups (p = 0.88).

Logistic regression models were employed for each outcome measure. Significant ORs and CIs were noted in the following (p < 0.05):

Per the physician's fitness-to-drive recommendation, an increase of one decade in the oldest-old increased the odds of being declared unfit-to-drive with 4.47 (95% CI 2.20-9.10) compared with the middle-aged. Likewise, the odds of being declared unfit-to-drive by the physician was 2.09 (95% CI 1.16-3.98) times greater than the middle-aged.

A similar pattern was observed for the comprehensive decision. Compared with the middle-aged, the odds of being declared unfit-to-drive was 2.74 (95% CI 1.87-4.03) in the oldest-old and 1.82 (95% CI 1.38-2.41) in the old-old.

The odds of MVC also increased with advanced age. Compared with the middle-aged, the OR of MVC in the oldest-old was 2.79 (95% CI 1.88-4.12) and 2.35 (95% CI 1.81-3.05) in the old-old. No differences in OR were observed for traffic violations.

The oldest-old also differed from the old-old regarding driving safety outcomes.

Compared to the old-old, the odds of being declared unfit-to-drive in the oldest-old was 2.13 (95% CI 1.06-4.27) by the referring physician and 1.51 (95% CI 1.02-2.22) by the CARA team.

Agreement between physician's fitness-to-drive recommendation and comprehensive fitness-to-drive decision

Across all groups, the agreement between physician's recommendation and comprehensive fitness-to-drive decision was substantial to almost perfect (Table 2). Yet, the percentage of agreement decreased with aging. The percentage of agreement was the lowest in the oldest-old. Of all disagreed cases, referring physicians were more likely to issue a favorable fitnessto-drive recommendation compared with the comprehensive fitness-to-drive decision. The percentage of overestimation by referring physicians ranged from 85% in the old-old to 91% in the oldest-old and young-old. The PABAK agreement between referring physicians and CARA assessors decreased with advanced age. The agreements in the oldest-old (PABAK = 0.72, 95% CI 0.66-0.79) and old-old (PABAK = 0.78, 95% CI 0.74-0.81) were only substantial, whereas those in the young-old (PABAK = 0.86, 95% CI 0.83-0.89) and middle-aged (PABAK = 0.88, 95% CI 0.85-0.91) were almost perfect (Table 2).

Discussion

This study investigated driving safety in the older population in a medical fitness-to-drive setting with a special focus on the oldest-old aged 85 and older. The main findings include: (1) the oldest-old are a distinct and unique group of drivers who not uncommonly had medical conditions that affect driving safety; (2) advanced age negatively affects several outcomes of driving safety; and (3) advanced age negatively affects fitness-to-drive agreements between referring physicians and driving evaluation specialists.

The oldest-old reported more cardiopulmonary and visual conditions compared with any other age group, yet they also presented fewer neurological conditions compared to the old-old.

Although this results seem counterintuitive, it is likely that adults with neurological conditions cease driving prior to 85 years old. Neurological conditions impair motor, visual, and cognitive abilities and may be accompanied with comorbidities that can directly impact their fitness-todrive [START_REF] Uc | Driving and neurodegenerative diseases[END_REF]. In addition, it is possible some of the oldest-old could have neurological diseases such as dementia, but were either not diagnosed or lacked insight into their presence [START_REF] Bradford | Missed and delayed diagnosis of dementia in primary care: Prevalence and contributing factors[END_REF]. Meanwhile, the percentage of cardiopulmonary and visual conditions increased with aging, suggesting older adults continued to drive with these conditions. For instance, nearly half of the cardiovascular conditions in this database were hypertension or fibrillation [START_REF] Moon | Comparison of unsafe driving across medical conditions[END_REF]. These conditions, in the absence of serious or major complications, may not affect motor and cognitive skills needed to drive safely, even in the oldest-old. Likewise, mild visual conditions such as reduced visual acuity may be remediated using corrective glasses. Mild visual field defects do not preclude safe driving either since drivers can be taught to use panoramic mirrors, compensatory head and eye scanning, or other driving strategies [START_REF]National Highway Traffic Safety Administration: Clinician's guide to assessing and counseling older drivers (3rd edition)[END_REF]. It is also possible that oldest-old drivers were more likely to be referred to CARA because of age-related comorbidity other than neurological conditions.

Despite the majority of older individuals with medical conditions being safe drivers, advanced age reduced their fitness-to-drive. Individuals with advanced age were more likely to receive unfit-to-drive decisions by referring physicians and driving evaluation specialists. The oldest-old showed greater odds of being declared unfit-to-drive by their physician and CARA team compared with the old-old. Decreased fitness-to-drive and higher risks of unsafe driving in older adults were also reported in previous studies [START_REF] Vaa | Impairments, diseases, age and their relative risks of accident involvement: Results from meta-analysis[END_REF][START_REF] Marshall | The role of reduced fitness to drive due to medical impairments in explaining crashes involving older drivers[END_REF]. Furthermore, we found that the oldest-old and old-old age groups reported more MVCs than other age groups. Since this study lacked data on population numbers or mileage, comparisons with studies that investigated the impact of aging on crash rates per population or per miles traveled is not possible. Tefft's study [START_REF] Tefft | Motor vehicle crashes, injuries, and deaths in relation to driver age: United States[END_REF] found that the crash rate per miles traveled in older age groups (60 and above) decreased with advanced age whereas the crash rate per population increased with aging. Future studies need to investigate relative crash rates per population or per miles traveled to compare how older population with medical conditions differs from general older population.

Percentage agreements between fitness-to-drive decisions by referring physicians and driving evaluation specialists at CARA decreased from 92% in the middle-aged to 82% in the oldest-old. In previous studies involving neurological conditions, the percentage agreements were 43% in individuals with Alzheimer's disease [START_REF] Ranchet | Fitness-to-drive disagreements in individuals with dementia[END_REF], 64% in Parkinson's disease [START_REF] Ranchet | Comorbidity in Drivers with Parkinson's Disease[END_REF], 73% in stroke [START_REF] Ranchet | Fitness-to-drive agreements after stroke: medical versus practical recommendations[END_REF], and 88% in multiple sclerosis [START_REF] Ranchet | Agreement between physician's recommendation and fitness-to-drive decision in multiple sclerosis[END_REF]. In these past studies, the percentage agreement decreased with reduced fit-to-drive rates on comprehensive fitness-to-drive decisions. Likewise, the reduced fitness-to-drive in the oldest-old may have resulted in more disagreement between the medical and comprehensive fitness-to-drive recommendations. Another study suggested agerelated bias of driving evaluation specialists may result in the increased percentage of negative fitness-to-drive decisions [START_REF] Meuser | Motor-vehicle crash history and licensing outcomes for older drivers reported as medically impaired in Missouri[END_REF]. Referring physicians were more likely to overestimate the fitness-to-drive, which was consistent with previous studies suggesting that physicians are hesitant to give an unfavorable recommendation due to fear of harming the doctor-patient relationship [START_REF] Devos | Shifting up a gear: Considerations on assessment and rehabilitation of driving in people with neurological conditions. An extended editorial[END_REF], ambiguous fitness-to-drive criteria [START_REF] Devos | Shifting up a gear: Considerations on assessment and rehabilitation of driving in people with neurological conditions. An extended editorial[END_REF], and potential lawsuits [START_REF] Bacon | American Academy of Neurology position statement on physician reporting of medical conditions that may affect driving competence[END_REF]. Awareness of these factors implies a need to develop more objective and clinician-friendly tools for fitness-to-drive evaluation.

The main strength of this study is the comparison of four real-world outcomes of driving safety in older adults with medical conditions, including more than 200 drivers aged 85 and above. In addition, we did not employ stringent inclusion and exclusion criteria to obtain the representative sample of older adults with medical conditions that interfered with driving. Thus, the group of older individuals studied in this study was inherently heterogeneous in their medical diagnosis, comorbidities, reasons for referral, and their performance on on-road and off-road testing.

The main limitation of the study includes a number of missing variables, inherent in a retrospective study design. However, the separate analysis of the oldest-old drivers provides a unique view on how advanced age affects driving safety. In addition, this study focused on older adults with medical conditions referred to driving evaluation, which may reduce the generalizability of the results to general population. The results of practical driving evaluation could be biased since the on-road assessors might not blinded to patient's medical condition or other assessment at CARA. However, this may not cause a confounding result since the comprehensive fitness-to-drive decision was decided under consideration of all aspects of inoffice and on-road assessments at CARA. Also, we found that insurance companies were more likely to refer individuals aged 75 and older compared with younger individuals, which needs to be confirmed by comparing insurance policies across different age groups. Insurance companies in other countries may not have similar approaches in pursuing fitness to drive evaluations and thus, may also limit the generalizability of these findings.

Our findings call for particular attention to treat the oldest-old as a separate entity from the old-old with specific medical conditions that challenge their fitness-to-drive. Physicians, healthcare providers, and policymakers need to be particularly vigilant to the driving safety of the oldest-old, since they are the fastest growing segment of the aging population [START_REF]United Nations: World population ageing: 1950-2050[END_REF] and their increased frailty often leads to fatal injuries when MVCs occur [START_REF] Lotfipour | Fatality and injury severity of older adult motor vehicle collisions in orange county, California, 1998-2007[END_REF]. To fully understand the effect of advanced age on driving outcomes, longitudinal studies are warranted to evaluate visual, cognitive, and motor predictors of fitness-to-drive in the oldest-old. Also, the oldest-old may have a unique combination of comorbidities, which can be an important factor affecting fitness-to-drive [START_REF] Ranchet | Fitness-to-drive agreements after stroke: medical versus practical recommendations[END_REF]. Future studies should examine the effect of comorbidities on driving safety using standardized comorbidity scales (e.g., Cumulative Illness Rating Scale for geriatrics and Charlson Comorbidity Index) [START_REF] De Groot | How to measure comorbidity[END_REF], which will provide additional insights on the role of comorbidities in driving safety [START_REF] Papa | Comorbidities and crash involvement among younger and older drivers[END_REF]. This study utilized self-reported MVC and traffic violation records, which might cause an underestimation of the actual numbers. However, police or insurance-reported MVC records might also be underreported since minor MVCs are often unreported [START_REF] Anstey | Comparison of self-reported crashes, state crash records and an on-road driving assessment in a population-based sample of drivers aged 69-95 years[END_REF]. Lastly, our findings regarding traffic violations may not be generalizable to other countries with different traffic regulations and enforcement levels, since we used data collected in Belgium.

This population-based study investigated driving safety in the older population with medical conditions. The majority of older adults were safe drivers, yet the risk of unsafe driving increased with advanced age. The oldest-old are a unique group of drivers with medical conditions that may affect their driving safety.

  Fit-to-drive rate in physician's fitness-to-drive recommendation and comprehensive fitness-to-drive recommendation across age groups. (B) Involvement rate in MVCs and traffic violations in the past 5 years across age groups. Abbreviation: MVC = motor vehicel crash. Age group: 55-64 = middle-aged; 65-74 = young-old; 75-84 = old-old; 85+ = oldest-old. Missing variables: physician recommendation (n = 334); MVC in past 5 years (n = 1011); and traffic violation in past 5 years (n = 973). * Post-hoc analysis (p < 0.008):-Physician's fitness-to-drive recommendation (MA>ODO, YO>ODO).-Comprehensive fitness-to-drive recommendation (MA>OO, MA>ODO, YO>OO, YO>ODO).-MVC in the past 5 years (MA<OO, MA<ODO, YO<OO, YO<ODO).-Traffic violation in the past 5 years (not significant).

Figure 1 .

 1 Figure 1. Outcomes of unsafe driving across age groups

Funding sources

This study was supported by the VIAS institute, Brussels, Belgium.

 43) 108 (48) Values are presented as median (Q1-Q3) or frequency (%). Abbreviation: χ 2 = chi-squared test; KW = Kruskal-Wallis test; MVC = motor vehicle crash. a Missing variable: reason for application (n = 516). b Age group: 55-64 = middle-aged; 65-74 = young-old; 75-84 = old-old; 85+ = oldest-old. c Mandated referral (insurance / medical expert / court or law enforcement): middle-aged (26% / 69% / 5%); young-old (23% / 71% / 6%); old-old (77% / 22% / 1%); oldest-old (82% / 18% / 0%). b Overestimation: the referring physician judged a driver fit-to-drive, but the driver received unfit-to-drive decision by the driving evaluation specialists at CARA. c Underestimation: the referring physician judged a driver unfit-to-drive, but the driver received fit-to-drive decision by the driving evaluation specialists at CARA.