French D E S I R The

Pascale Study

MD Massin

PhD Céline Lange

MSc Jean Tichet

MD Sylviane Vol

MSc Ali Erginay

MD Martine Cailleau

MD Eveline Eschwège

MD Beverley Balkau
email: beverley.balkau@inserm.fr

PhD D E S I R Study Group

HbA 1c and fasting plasma glucose as predictors of retinopathy at ten years

Keywords: epidemiology, diabetes, fasting plasma glucose, HbA 1c, retinopathy, longitudinal 23/01/2010

Background: Hyperglycaemia is associated with retinopathy, and this cross-sectional relation has been used to define thresholds for defining diabetes. We evaluate the predictive values of HbA 1c and fasting plasma glucose (FPG) for retinopathy, ten years after the baseline examination.

Methods: 700 men and women from the D.E.S.I.R. study were evaluated for retinopathy using a nonmydriatic digital camera. Over the preceding nine years: 235 had diabetes (treated or FPG>7.0 mmol/l at least once), 227 had an impaired fasting glucose (IFG:6.1-6.9 mmol/l) at least once, 238 always had normal glucose levels (<6.1 mmol/l).

Results:

The 44 participants with retinopathy at ten years had higher baseline mean (SD) for FPG and HbA 1c , than those without retinopathy, 7.24mmol/l (2.72) vs 5.90mmol/l (1.22) and 6.4% (1.6) vs 5.7% (0.7) (both P<0.0001). The frequency of retinopathy at ten years, standardized according to the distribution of glycaemia across the entire D.E.S.I.R. population was 3.6%. In our population, FPGs of 6.0mmol/l and 6.5mmol/l had positive predictive values of 8.2% and 15.4% respectively for retinopathy at ten years, and HbA 1c s of 6.0% and 6.5%: 6.0% and 14.8%. After ten years of follow-up, retinopathy was equally frequent in IFG and in those who became diabetic during the study: 8.6% and 6.7% respectively, lower than in those diabetic at baseline 13.9%.

Conclusion:

As the positive predictive values for retinopathy increased sharply from 6.0 mmol/l for FPG, and from 6.0% for HbA 1c , these thresholds are proposed to identify those at risk of retinopathy ten years later.

It is well accepted that the hyperglycaemia accompanying diabetes is associated with microvascular complications, in particular retinopathy, and indeed one of the reasons for choosing the thresholds in the current definition of diabetes was that the frequency of retinopathy and nephropathy started to increase, in cross-sectional studies, above these levels of glucose and HbA 1c . [START_REF]Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus[END_REF][START_REF] Study Group | International Expert Committee report on the role of the A1C assay in the diagnosis of diabetes[END_REF] The determination of thresholds has been done by eye, and in some studies a change point model [START_REF] Ulm | A statistical method for assessing a threshold in epidemiological studies[END_REF][START_REF] Wong | Relation between fasting glucose and retinopathy for diagnosis of diabetes: three population-based cross-sectional studies[END_REF] has been used to identify the glycaemic level where the prevalence of retinopathy changes from a constant to an increasing rate.

However, there is some controversy concerning the actual value of this glycaemic threshold for identifying retinopathy. Wong et al. re-examined the relation between fasting plasma glucose (FPG) and retinopathy in three contemporary studies, and found that while the prevalence of retinopathy increased with FPG concentrations, there was no clear diagnostic cutoff. [START_REF] Wong | Relation between fasting glucose and retinopathy for diagnosis of diabetes: three population-based cross-sectional studies[END_REF] More recently, in Malay adults from Singapore, HbA1c thresholds of 6.6% and 7.0% were identified in relation with mild and moderate retinopathy, respectively, using "optimal values" from receiver operating characteristic (ROC) curves. [START_REF] Sabanayagam | Relationship between glycated haemoglobin and microvascular complications: Is there a natural cut-off point for the diagnosis of diabetes?[END_REF] It is now well established that the non-diabetic population also suffers from retinopathy, [START_REF] Mccartney | The Bedford Survey: observations on retina and lens of subjects with impaired glucose tolerance and in controls with normal glucose tolerance[END_REF][START_REF] Klein | Hypertension and retinopathy, arteriolar narrowing, and arteriovenous nicking in a population[END_REF][START_REF] Stolk | Retinopathy, glucose, and insulin in an elderly population. The Rotterdam Study[END_REF][START_REF] Rajala | Prevalence of retinopathy in people with diabetes, impaired glucose tolerance, and normal glucose tolerance[END_REF][START_REF] Yu | Retinopathy in older persons without diabetes and its relationship to hypertension[END_REF][START_REF] Gabir | Plasma glucose and prediction of microvascular disease and mortality: evaluation of 1997 American Diabetes Association and 1999 World Health Organization criteria for diagnosis of diabetes[END_REF][START_REF] Van Leiden | Blood pressure, lipids, and obesity are associated with retinopathy: the Hoorn study[END_REF][START_REF] Tapp | The prevalence of and factors associated with diabetic retinopathy in the Australian population[END_REF][START_REF] Prevention | The prevalence of retinopathy in impaired glucose tolerance and recent-onset diabetes in the Diabetes Prevention Program[END_REF] albeit at a lower frequency than diabetic patients, and in a milder form, indicating that there may be factors other than glucose which increase the risk of retinopathy.

There have been many more cross-sectional than prospective studies of retinopathy. [START_REF] Wong | Relation between fasting glucose and retinopathy for diagnosis of diabetes: three population-based cross-sectional studies[END_REF] There are few studies of incident diabetes in general populations and most of these studies have focused on either FPG or HbA1C as risk factors for retinopathy, rarely both. Some other risk factors for incident retinopathy have been identified. [START_REF] Nelson | Proliferative retinopathy in NIDDM. Incidence and risk factors in Pima Indians[END_REF][START_REF] Looker | Longitudinal studies of incidence and progression of diabetic retinopathy assessed by retinal photography in Pima Indians[END_REF][START_REF] Cikamatana | Five-year incidence and progression of diabetic retinopathy in a defined older population: the Blue Mountains Eye Study[END_REF][START_REF] Tapp | Longitudinal association of glucose metabolism with retinopathy: results from the Australian Diabetes Obesity and Lifestyle (AusDiab) study[END_REF] The Bedford Study in the UK was one of the earliest to study the presence of retinopathy at seven years, in individuals with "borderline diabetes" at baseline: 2 hour glucose between 6.7 and 11.1 mmol/l, after a 50g oral glucose tolerance test. [START_REF] Mccartney | The Bedford Survey: observations on retina and lens of subjects with impaired glucose tolerance and in controls with normal glucose tolerance[END_REF] One of most complete studies was from the Pima Indians, [START_REF] Mccance | Comparison of tests for glycated haemoglobin and fasting and two hour plasma glucose concentrations as diagnostic methods for diabetes[END_REF] and included almost 1000 individuals, followed over 5 years.

Dividing the FPG and 2 hour glucose concentrations and HbA1c levels into deciles, they showed a sharp increase in incident retinopathy in the two highest decile groups.

The aim of this report was to study the frequency of retinopathy in individuals 10 years after baseline, according to baseline levels of FPG and HbA 1c , and to evaluate positive predictive values for retinopathy at various levels of these two glycaemic parameters. The secondary objective was to evaluate potential risk factors associated with retinopathy, at baseline, and over the nine years preceding the evaluation of retinopathy.

METHODS

PARTICIPANTS

The men and women in this study participated in the D.E.S.I.R. cohort (Data from an Epidemiological Study on the Insulin Resistance Syndrome), and they were recruited in 1994-1996 from consultants in ten French Social Security health examination centres in the central western part of France. [START_REF] Balkau | Proposed criteria for the diagnosis of diabetes: evidence from a French epidemiological study (D.E.S.I.R.)[END_REF] At entry into the study, participants were aged 30 to 65 years, and they were re-invited to health examinations three, six and nine years after inclusion. After the year-nine examination, the 321 individuals who had been treated for diabetes or who had a FPG ≥ 7.0 mmol/l at some time during the study, were invited to attend a special examination on microvascular complications of diabetes, in particular an examination of the retina: 237 (74%) participated, 17 had died. For comparison, two other groups, matched by age, sex and examination centre with those in the diabetes group, were also invited to the retinal examination: group 1, impaired fasting glucose (IFG: FPG 6.1-6.9 mmol/l) at any time during the study; group 2, normal glucose levels (FPG < 6.1 mmol/l) during the entire study. There were 246 individuals in the IFG group (68% participation, 9 had died) and 249 (56% participation, 3 had died) in the normal FPG group. In comparison to the 237 diabetic individuals who participated in the study, the 94 who did not participate had higher glucose (but not HbA 1c ) levels and higher systolic blood pressure at inclusion, but did not differ according to age, or sex; no differences were seen between those who did or did not participate in the IFG or the normal FPG groups. At the year-10 examination, eight of the participants selected as IFG were treated for diabetes, so they were analysed in the diabetes group. We excluded from this analysis, the 18 individuals for whom the photographs were not interpretable for either eye, and we excluded a further 15 participants for whom only one eye was interpretable; they had similar baseline characteristics to those included in the analysis, excepting those excluded were older (P < 0.001); 10 were in the diabetes group, 11 in the IFG group, and 12 in the normal FPG group.

Our analysis thus included 700 participants: 235 with diabetes (145 treated for diabetes of whom 36 were treated for diabetes at baseline, 90 had at least one FPG > 7.0 mmol/l); 227 with IFG at some time during the study; 238 with normal glucose levels during the entire study.

The D.E.S.I.R. study was approved by the Kremlin Bicêtre Hospital ethics committee, and participants signed an informed consent.

METHODS OF MEASUREMENTS AT BASELINE

Blood pressure was taken in a supine position after 5 minutes of rest and waist circumference (the smallest circumference between the lower rib and the iliac crests), weight and height were measured in lightly clad participants, and the BMI calculated.

All biochemical measurements were from one of four health centre laboratories at La Riche, Blois, Chartres or Orleans. FPG was measured by the glucose-oxidase method, using a Technicon RA100 (Bayer Diagnostics, Puteaux, France) or a Specific or a Delta (Konelab, Evry, France). HbA 1c was measured using an automated HPLC ion-exchange analyser from Hitachi/Merck-VWR or a DCA 2000 automated immunoassay system from Bayer Diagnostics, Puteaux, France. To adjust for differences between laboratories, both glucose and HbA 1c data were standardized in age and sex strata, with respect to reference data assayed in the La Riche laboratory. Serum insulin was quantified centrally, by Micro particle Enzyme Immunoassay with an IMX automated analyser from Abbott (Rungis, France). Marburg), and were only available on 206 participants at baseline and at the three year follow-up, but on all participants at the six and nine year follow-up examinations. The inter-laboratory variability was assessed monthly on normal and pathological values for each biologic variable, the coefficients of variation for laboratories were lower than 6% over the inclusion period.

Albuminuria was determined from a single-void sample by nephelometry (BNA Behring, Rueil-Malmaison, France) in participants without dip-stick positive hematuria. Micro-or macro-albuminuria was defined as albuminuria above 20 mg/l or dip-stick positive proteinuria.

Participants completed a self-administered questionnaire on whether they had a family history of diabetes, and drug treatment for diabetes, hypertension or lipids. The presence of clinical retinopathy was not recorded at baseline.

RETINAL PHOTOGRAPHY

The Topcon TRC-NW6 camera (Topcon, Rotterdam, The Netherlands) is a non-mydriatic digital retinal camera which allows color photographs to be taken without papillary dilation, using semiautomatic guidance for peripheral fixation. Images were captured in true colour (24 bits) at a resolution of 1490 x 960 pixels. Retinal photographs were taken by a qualified orthoptist who was specifically trained for this study. The room was well-darkened and three photographs taken: one centered on the macula, one on the optic disc and the third one temporal to the macula. The right eye was always photographed first, after 5 minutes of adaptation to the dark. Participants were asked to close their eyes for a few seconds between each photograph. After an interval of 2-3 minutes, the left eye was photographed. The orthoptist, who was blinded to the glycaemic status of the participant, viewed each digital image immediately, and repeated the image acquisition process if the original image was unsatisfactory.

23/01/2010 7/18 EVALUATION OF RETINAL PHOTOGRAPHS Images were displayed on a 21-inch monitor (resolution 1280 × 1024 × 24 bits). All photographs were graded for quality and retinal characteristics. Image processing with IMAGEnet™ 2000 software (Topcon, Rotterdam, The Netherlands) was used to enhance grading accuracy.

All photographs were graded by a trained observer, according to a simplified version of the Wisconsin protocol, [START_REF] Aldington | Methodology for retinal photography and assessment of diabetic retinopathy: the EURODIAB IDDM complications study[END_REF] and a sample of 60 photographs was chosen and graded a second time by the same observer and also by a second observer, for intra-and inter-observer reproducibility. Retinal characteristics are shown for the worst eye; individuals with microaneurysms, hemorrhages, exudates, cotton-wool spots, intra microvascular abnormalities (IRMAs), venous bleedings or new vessels were classified as having retinopathy.

The intra-and inter-observer reproducibility for retinopathy grading was 97% and 95%. As stated above, 33 participants (4.7%) had ungradeable photographs in at least one eye and were excluded from the analysis STATISTICAL METHODS All analyses used SAS (version 9.1.3, SAS Institute, Cary, NC). For statistical analysis, the logarithms were taken of variables which were skewed: glucose, HbA 1c , triglycerides, insulin, CRP. Men and women were analysed together. Baseline characteristics were compared by t-and ²-tests, according to whether or not participants had any retinopathy ten years after baseline. Further, the areas under the curve over the 9 years of the study, were calculated for putative risk factors, and compared between those with and without retinopathy, by t-tests; when there was missing data, it was imputed by the average of neighbouring observations, or if data was missing at the nine-year follow-up, the six-year values were used.

As this study population was selected to provide three groups, matched by age, sex and examination centre with those in the diabetic group, it is not representative of the entire D.E.S.I.R. cohort. Thus, we standardized the frequencies of ten-year retinopathy according to the selection criteria for this study, glycaemic status, using the baseline distributions of fasting plasma glucose and HbA1c in these three glycaemic groups. The 10 year prevalence of retinopathy is given according to six classes of 0.5 mmol/l for FPG, and six classes of 0.5% for HbA 1c ; positive predictive values are shown by units of 0.1 mmol/l and 0.1% respectively. Confidence intervals were calculated for frequencies, and they were compared between groups by ²-tests. The receiver operating characteristic (ROC) curve was drawn and the c-statistic which is equivalent to the area under the ROC curve (AROC) was calculated.

The presence of retinopathy ten years after baseline was modelled by logistic regression, with the logarithms of FPG and HbA1c as independent variables. These relations were linear, as the addition of a squared term did not improve the model; these models were additionally adjusted on age, sex, BMI, systolic blood pressure, and hypertensive treatment. Pearson correlation coefficients were determined between FPG and HbA1c.

RESULTS

Over 70% of the participants with diabetes at nine years were men, thus in this study, 70% were men due to gender-matching (Table 1). The average age at baseline was 52 years. A total of 44 participants were classified as having retinopathy: 19 with diabetes (5 treated at baseline, 15 treated at the examination, 4 with fasting plasma glucose ≥7.0 mmol/l during the study), 19 with IFG and 6 with normal glucose levels throughout the 9 years of the study. In most participants, retinopathy was mild, limited to microaneurysms and/or haemorrhages. Those with retinopathy were more often treated for diabetes at baseline than those without retinopathy (11% vs 5%, P = 0.06) (Table 1), and they had a higher baseline mean FPG and HbA 1c (P < 0.0001), with a trend for a higher systolic blood pressure, 142 vs 137 mmHg (P = 0.06), and a higher percentage were treated for hypertension, 36% vs 20% (P < 0.008). Of the six participants who were always normoglycaemic but who had signs of retinopathy, five were women; they had similar age, BMI, blood pressures, and lipid parameters, as the other 38 23/01/2010 9/18 participants with retinopathy. None of these six had cardiovascular disease, nor microalbuminuria; five had a few microaneurysms and/or retinal haemorrhages, and one cotton wool spots.

Over the nine year follow-up, the area under the curve analysis showed that FPG and HbA1c were the factors most associated with retinopathy at 10 years (P < 0.0001), but triglyceride levels was higher in those with retinopathy (1.68 vs 1.42 mmol/l, P < 0.04, Table 2). CRP was analysed on only 206 of the participants, and showed a marginal relation with retinopathy; the average areas under the curve for years six and nine, when data was available for all participants, were 3.4 vs 2.7 mg/l (P < 0.01) for those with and without retinopathy.

The overall frequency of retinopathy at ten years, standardized to the baseline glucose status in the study, was 3.6%, and there was a sharp increase in frequency in the group with the highest baseline glucose levels, FPG > 7.0 mmol/l (Fig. 1A), with a highly significant difference in frequency between the last two glucose groups (P < 0.0001). For the 0.5% HbA 1c groups, the frequency of retinopathy also increased in a similar fashion (Fig. 1B), with a statistically significant difference between the last two HbA 1c groups (P < 0.05). When individuals with drug treated diabetes at baseline were excluded from the analysis, the relations were little changed (Fig. 1A and1B).

For the positive predictive values (Fig. 2A and2B), there were essentially two parts to the relations, a very gradual increase, then a higher slope after around 6.0 mmol/l for FPG and around 6.0% for HbA 1c . These could be considered as potential thresholds for defining those at risk of later retinopathy.

A FPG of 6.0 mmol/l had a positive predictive value of 8%, a negative predictive value of 97%, sensitivity and specificity of 28% and 88% and a positive likelihood ratio of 2.6. For an HbA 1c of 6.0%, the corresponding values were 6%, 98%, 19%, 92% and 1.8. For a glucose threshold of 6.5 mmol/l, these values were 15%, 97%, 19%, 96%, 4.8 and for an HbA 1c of 6.5%: 14%, 97%, 9%, 98%, 4.8. Once again, little change was observed when patients with diabetes at baseline were excluded. The ROC curves are fairly shallow for both FPG and HbA1c (Fig. 3), and the area under the curve was 64% for both measures. The frequencies (95% CI) of ten-year retinopathy were 2.5% (0.5-4.5) in participants who were always normoglycaemic, higher in those IFG at some time during the ten-year follow-up 8.6% (5.0-12.2) (P < 0.0001), similar to the 7.9% (4.4-11.3) (P < 0.01 in comparison to normoglycaemic) in participants diabetic at the end of the follow-up (Fig. 4). The frequency of retinopathy in the 36 individuals treated for diabetes at baseline was 13.9% (2.6-25.2), in comparison to a frequency of 6.7% (3.2-10.3) in the 199 participants who became diabetic during the ten years of the study.

The Pearson correlation coefficients between the logarithms of FPG and HbA1c was quite high, 0.73. In univariate regression models, FPG was more closely associated with retinopathy than HbA1c (² Wald Statistics were 26.75 and 18.81 respectively, both P < 0.0001). There was no improvement in adding HbA1c to the model with FPG alone. Both FPG and HbA1c remained predictive of 10-year retinopathy (P < 0.0001, 0.0003) after adjusting for age, sex, hypertensive treatment, systolic blood pressure, none of which were significant. Using the area under the ROC curve as a measure to discriminate between those with and without 10-year retinopathy, the areas were 64% for FPG alone as well as for HbA1c alone or for the combination of risk factors: age, sex, hypertensive treatment, systolic blood pressure; thus all three models discriminate equally. Adding FPG to the basic risk factors, the area to increased 72%, while adding HbA1c the increase was lower at 69%, and adding HbA1c to the model with FPG and the risk factors the area remained at 72%, thus addition of HbA1c did not improve the discrimination.

COMMENT

The positive predictive values for retinopathy, increased very slowly for both baseline FPG and HbA 1c at lower values, and then increased more rapidly at the values we might consider indicative of diabetes risk: approximately 6.0 mmol/l for FPG and 6.0% for HbA 1c . It is more difficult to discern a threshold after which the frequency of retinopathy increased, as the frequency can only be presented by grouping the glycaemic measures into intervals. The positive predictive values provide more 23/01/2010 11/18 information about possible thresholds. Further, we emphasise the use of the positive predictive values as they are more clinically relevant to find thresholds to predict those who will have retinopathy in the future -rather than sensitivity, which is the probability of having an FPG or an HbA1c above a given threshold, given the individual had 10-year retinopathy.

Baseline hypertension was associated with later retinopathy, and over the nine year follow, higher triglyceride and CRP levels were associated with 10-year retinopathy.

The strengths of this study are the long-term follow-up, the nine-year characterisation of risk factors and the method to detect retinopathy, using a non-mydriatic digital camera, with three photographs per eye, which is more sensitive in detecting diabetic retinopathy than ophthalmoscopy.

We were not able to study grades of severity of retinopathy, because of their low frequency, even among those treated for diabetes at baseline. As retinopathy was not evaluated at baseline, and there was no information recorded about clinical retinopathy, we are not able to study of the incidence of retinopathy. Our study included volunteers for a free health check-up, who agreed to participate in the nine-year D.E.S.I.R. study, and then for an additional examination to study diabetic microvascular disease. This type of self-selection is a common limitation in studies in the general population, but should not change the relations between the glycaemic parameters or other risk factors and the outcome. Our sample size only permits us find those risk factors with a strong association with retinopathy.

The American Diabetes Association and the World Health Organisation used a FPG of 7.0 mmol/l or higher to define diabetes, [START_REF]Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus[END_REF][START_REF]Definition and diagnosis of diabetes mellitus and intermediate hyperglycaemia[END_REF] based on three pivotal studies which showed that signs of retinopathy were rare below a FPG <7.0 mmol/l, but that the prevalence increased substantially above this level. These studies suffered from poor methodology to detect diabetic retinopathy, based either on ophthalmoscopy or a one field photograph. Re-evaluating this threshold in three contemporary studies, using multiple retinal photographs and a validated retinal grading, Wong et al. found that the prevalence of retinopathy increased with FPG concentration, without a clear diagnostic cut-off. [START_REF] Wong | Relation between fasting glucose and retinopathy for diagnosis of diabetes: three population-based cross-sectional studies[END_REF] They also found that signs of retinopathy occur in 7-13% of the population below the FPG concentration of 23/01/2010 12/18 7.0 mmol/l. For HbA1c, a large study from Malaysia recommended a threshold value of between 6.6 and 7.0% for diagnosing diabetes. [START_REF] Sabanayagam | Relationship between glycated haemoglobin and microvascular complications: Is there a natural cut-off point for the diagnosis of diabetes?[END_REF] Most reports on prevalent and incident retinopathy only study glycaemic factors. Other factors have been shown to be associated with prevalent retinopathy: age, hypertension, hypertensive medications, insulin, BMI, cholesterol, triglycerides, C-peptide, urinary albumin creatinine ratio, inflammation and endothelial dysfunction. [START_REF] Sabanayagam | Relationship between glycated haemoglobin and microvascular complications: Is there a natural cut-off point for the diagnosis of diabetes?[END_REF][START_REF] Klein | Hypertension and retinopathy, arteriolar narrowing, and arteriovenous nicking in a population[END_REF][START_REF] Yu | Retinopathy in older persons without diabetes and its relationship to hypertension[END_REF][START_REF] Van Leiden | Blood pressure, lipids, and obesity are associated with retinopathy: the Hoorn study[END_REF][START_REF] Van Hecke | Inflammation and endothelial dysfunction are associated with retinopathy: the Hoorn Study[END_REF] Incident retinopathy was associated with waist-hip ratio in the Hoorn study. [START_REF] Van Leiden | Risk factors for incident retinopathy in a diabetic and nondiabetic population: the Hoorn study[END_REF] In the D.E.S.I.R. study, at baseline, only hypertensive treatment was related with retinopathy at 10 years, but for the cumulated effects over the 9 years of the study, evaluated by the area under the curve over this period, there was only a significant association with higher levels of triglycerides. Marginal associations (P < 0.1) were also seen with BMI, waist circumference, insulin, systolic blood pressure, HDL-and LDL-cholesterol and CRP.

There are few longitudinal studies of retinopathy, and they all show the relation with either glucose or HbA 1c levels. In the largest single study of incident retinopathy, diagnosed from fundus photographs, the incidence increased from 15 cases per 10 000 person years for FPG below 5.0 mmol/l to around 30 cases for glucose between 5.0 and 7.0 mmol/l and then increased rapidly for three glucose classes within the diabetic range. [START_REF] Ito | Importance of OGTT for diagnosing diabetes mellitus based on prevalence and incidence of retinopathy[END_REF] In the D.E.S.I.R. study we were able to show a significant difference in retinopathy frequency at ten years, between FPG < and > 7.0 mmol/l at baseline. In the Pima Indian study, [START_REF] Mccance | Comparison of tests for glycated haemoglobin and fasting and two hour plasma glucose concentrations as diagnostic methods for diabetes[END_REF] five-year incident retinopathy increased at FPG and HbA1 values around 7.0 mmol/l and 7.0%. Incident retinopathy over 9.4 years in the Hoorn Study, [START_REF] Van Leiden | Risk factors for incident retinopathy in a diabetic and nondiabetic population: the Hoorn study[END_REF] was more than two fold higher in the diabetic patients than in the normoglycaemic The incidence of retinopathy was more than three-fold higher in the upper third of the population, with HbA 1c > 5.8%, than in the remainder of the population. In the D.E.S.I.R. study, the ten-year prevalence of retinopathy was 3.3% for HbA 1c < 6.0% and 6.8% for those with a higher HbA 1c > 6.0%. In the ARIC study, 26 the 3-year incidence of any retinopathy was three times higher above the upper quartile of the FPG distribution at baseline (6.3 mmol/l). The recent publication from Australia, [START_REF] Wong | Relation between fasting glucose and retinopathy for diagnosis of diabetes: three population-based cross-sectional studies[END_REF] showed the five-year incidence of any retinopathy increased at approximately 8.0 mmol/l for FPG. It is difficult to compare actual glucose and HbA 1c levels where the frequency of retinopathy increases in these different studies, as there were no doubt differences in the accuracy of these assays both for glucose, but especially for HbA 1c .

The standardization of the HbA 1c assay results should improve this situation for future studies. [START_REF]Consensus statement on the worldwide standardization of the hemoglobin A1C measurement: the American Diabetes Association, European Association for the Study of Diabetes, International Federation of Clinical Chemistry and Laboratory Medicine, and the International Diabetes Federation[END_REF] HbA 1c and FPG at baseline were both related with the presence of retinopathy ten years later, and the levels where the positive predictive values increase provide a rationale for the choice of thresholds for the definition of hyperglycaemia associated with 10-year retinopathy. We propose that that thresholds of 6.0 mmol/l for FPG and 6.0% for HbA 1c could be used to define those who are at risk of retinopathy; this is in agreement with our observation of a risk of retinopathy within the IFG range (FPG > 6.1 mmol/l). Factors other than glucose measures play only a minor role in retinopathy.

  Total cholesterol, HDL-cholesterol and triglycerides were assayed by a DAX 24 (Bayer Diagnostics, Puteaux, France) or a KONE (Evry, France); LDL-cholesterol was determined by the Friedwald equation. C reactive protein (CRP) levels were assessed by immunonephelemetric method (Dade Behring 23

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Frequency (SE) of retinopathy ten years after baseline for all participants , and without those treated for diabetes at baseline , according to: A. baseline fasting plasma glucose concentrations (mmol/l); B. baseline HbA 1c levels (%)

Figure 4 .

 4 Figure 4. Frequency (95% CI) of retinopathy ten years after baseline, according to glucose status during the 10 year study.

Table 1 .

 1 Characteristics at baseline (mean (SD), n (%)) associated with the absence or presence of retinopathy at ten years. The D.E.S.I.R. Study

		No retinopathy	Retinopathy	P -value*
		n=656	n=44
	Age	52 (8)	53 (8)	0.5
	Men	476 (73%)	28 (64%)	0.2
	Family history of diabetes	140 (21%)	11 (25%)	0.6
	Treated for diabetes at baseline	31 (5%)	5 (11%)	0.06
	Treated for diabetes at 10 years	130 (20%)	15 (34%)	0.03
	BMI (kg/m²)	26.3 (3.9)	27.2 (4.0)	0.1
	Waist circumference (cm)	90 (11)	92 (11)	0.2
	Fasting plasma glucose* (mmol/l)	5.90 (1.22)	7.24 (2.72)	0.0001
	HbA 1c * (%)	5.7 (0.7)	6.4 (1.6)	0.0001
	Fasting serum insulin* (pmol/l)	56.2 (39.4)	62.2 (31.6)	0.1
	Systolic Blood Pressure (mmHg)	137 (16)	142 (17)	0.06
	Diastolic Blood Pressure (mmHg)	83 (10)	84 (10)	0.6
	Hypertensive treatment	128 (20%)	16 (36%)	0.008
	Triglycerides* (mmol/l)	1.44 (1.67)	1.61 (1.23)	0.2
	HDL-cholesterol (mmol/l)	1.53 (0.40)	1.49 (0.41)	0.5
	LDL-cholesterol (mmol/l)	3.75 (0.91)	3.90 (0.77)	0.3
	Lipid treatment	88 (13%)	4 (9%)	0.4
	CRP* (mg/l) n=206	2.71 (4.10) (n=193)	2.66 (2.50) (n=13)	0.9
	Micro or Macro albuminuria	86 (15%)	9 (23%)	0.2
	*logarithms used for statistical analyses

Table 2

 2 Average (SD) of area under the curve per year over the 9 years of follow-up for various characteristics associated with the absence or presence of retinopathy at ten years.

	The D.E.S.I.R. Study
		No retinopathy	Retinopathy	P -value*
		n=656	n=44
	BMI (kg/m²)	26.8 (4.1)	27.8 (3.6)	0.1
	Waist circumference (cm)	92 (11)	94 (10)	0.1
	Glucose* (mmol/l)	6.11 (1.15)	7.30 (2.23)	0.0001
	HbA 1c * (%)	5.9 (0.7)	6.5 (1.4)	0.0001
	Insulin* (pmol/l)	67.8 (41.9)	77.7 (48.7)	0.1
	Systolic Blood Pressure (mmHg)	140 (15)	143 (18)	0.1
	Diastolic Blood Pressure (mmHg)	83 (8)	84 (9)	0.4
	Triglycerides* (mmol/l)	1.42 (0.86)	1.68 (1.01)	0.04
	HDL-cholesterol (mmol/l)	1.49 (0.35)	1.40 (0.34)	0.1
	LDL-cholesterol (mmol/l)	3.63 (0.67)	3.79 (0.65)	0.1
	CRP*(mg/l) n=206 (n=193 & 13)	2.5 (2.6)	3.2 (1.9)	0.07

*logarithms used for statistical analyses

Acknowledgments

The D.E.S.I.R. study has been supported by INSERM contracts with CNAMTS, Lilly, Novartis Pharma and Sanofi-Aventis; by INSERM (Réseaux en Santé Publique, Interactions entre les déterminants de la santé, Cohortes Santé TGIR), the Association Diabète Risque Vasculaire, the Fédération Française de Cardiologie, La Fondation de France, ALFEDIAM, ONIVINS, Ardix Medical, Bayer Diagnostics, Becton Dickinson, Cardionics, Merck Santé, Novo Nordisk, Pierre Fabre, Roche, Topcon. The D.E.S.I.R. Study Group. INSERM U780: B. Balkau, MA. Charles, P. Ducimetière, E. Eschwège; INSERM U367: F. Alhenc-Gelas; CHU D"Angers: Y. Gallois, A. Girault; Bichat Hospital: F. Fumeron, M. Marre; CHU de Rennes: F. Bonnet; CNRS UMR8090, LILLE: P. Froguel; Centres d"Examens de Santé: Alençon, Angers, Caen, Chateauroux, Cholet, Le Mans, Tours; Institute de Recherche Médecine Générale: J. Cogneau; General practitioners of the region; Institute inter-Regional pour la Santé: C. Born, E. Caces, M. Cailleau, J.G. Moreau, F. Rakotozafy, J. Tichet, S. Vol. We thank Nathalie Robert for training Astrid Germain the study orthoptist, Marina Grinsztein for coding the retinal photographs and Lilly France for providing financial support for this project.

Duality of interest

The other authors declare no duality of interest. Lilly France provided financial support for this study, but they were not involved in the analysis or interpretation of the data.

23/01/2010


Maria De Agostini
email: maria.deagostini@inserm.fr

Seta Kazandjian

Céline Cavezian

Joseph Lellouch

Sylvie Chokron

Visual Aesthetic Preference: Effects of Handedness, Sex, and Age-Related Reading/Writing Directional Scanning Experience

Keywords: Aesthetic preference, development, vision, hemispheric specialization, handedness, sex, reading/writing habits

Most studies of visual aesthetic preference report that right-handers prefer pictorial arrangements possessing left-to-right directionality and/or containing the region of greatest weight or interest on the right side. However, visual aesthetic preference has also been linked to directional scanning depending on the individual's reading/writing habits. The present study aims to assess the respective role of biological factors, related to the functional specialization of the two cerebral hemispheres, indexed by handedness, and cultural factors (age-related reading/writing habits) in visual aesthetic preference. For this purpose, we tested the effects of handedness, sex and age on visual aesthetic preference in 40 children and 40 adults. Results revealed effects of handedness, sex, and age as well as a relationship between directional scanning, reading/writing habits and handedness. The question of a dynamic model of cerebral specialization based on interplay between cerebral plasticity and cultural/environmental factors is raised.

Introduction

There is some evidence of a relationship between handedness and cognitive performance particularly between handedness and perceptual asymmetries [START_REF] Annett | Handedness and Brain Asymmetry[END_REF] but the link between handedness and aesthetic preference remains a controversial. Most studies have reported that right-handers but not left-handers prefer pictorial arrangements possessing left-to-right directionality (for example, an image depicting a horse with a rightward gallop) and/or containing the region of greatest weight or interest on the right side (for example, a scene with a landscape with the most important objects on the right side; [START_REF] Beaumont | Lateral organization and aesthetic preference: The importance of peripheral visual asymmetries[END_REF][START_REF] Freimuth | The influence of lateral organization on the evaluation of paintings[END_REF][START_REF] Levy | Lateral dominance and aesthetic preference[END_REF][START_REF] Mead | The roles of handedness and stimuli asymmetry in aesthetic preference[END_REF]. In left-handers, [START_REF] Levy | Lateral dominance and aesthetic preference[END_REF] found no evidence of systematic preference, whereas [START_REF] Mclaughlin | Aesthetic preference in dextrals and sinistrals[END_REF] did find that this group tended to prefer asymmetric paintings whose area of interest was in the left half of the picture.

The two main explanations proposed to account for this right hemispatial visual preference in right-handers involve hemispheric activation. One proposition has been that when the focus of interest is lateralized to the right side of the image, the left hemisphere (LH) is activated, which corrects for the imbalance produced by the over-activation of the right hemisphere (RH) induced by pictorial viewing [START_REF] Levy | Lateral dominance and aesthetic preference[END_REF][START_REF] Goldstein | Perception[END_REF]. In contrast, other authors have suggested that the rightward position of the focus of interest of an image would attract the viewer's gaze, leaving most of the picture in the left visual field and allowing the RH to analyze the pictorial information [START_REF] Beaumont | Lateral organization and aesthetic preference: The importance of peripheral visual asymmetries[END_REF].

Recently it was demonstrated that far from being solely dependent on hemispheric specialization, visual aesthetic preference can be influenced by visuo-perceptual and/or motoric exploratory strategies. When left-to-right and right-to-left readers were presented opposite-facing picture pairs possessing directionality, the participants' preference was related to reading/writing directionality [START_REF] Nachson | Effects of directional habits and handedness on aesthetic preference for left and right profiles[END_REF][START_REF] Chokron | Reading habits influence aesthetic preference[END_REF].

Nevertheless, it is interesting to note that if reading/writing habits seem to determine the preference for pictures expressing directionality, they do not modulate preference for landscape pictures with no directional information. For these non-directional pictures, a rightward preference was expressed by right-handed viewers irrespective of their reading habits, from leftto-right or from right-to-left [START_REF] Chokron | Reading habits influence aesthetic preference[END_REF]. These findings raise the question of an interaction between cultural factors (picture directionality related to reading/writing directionality) and cerebral dominance (rightward preference for pictures without manifest directionality regardless of the viewer's reading/writing habits) in aesthetic preference tasks.

However, using drawing tasks instead of perceptual ones, several authors have demonstrated that handedness and reading/writing habits may influence the preferred directionality [START_REF] Reed | A further experimental investigation of the relative speeds of left and right-handed writers[END_REF][START_REF] Gesell | The development of handedness[END_REF][START_REF] Rice | Excellence of production and types of movements in drawing[END_REF][START_REF] Singh | Reading/writing vs. handedness influences on line length estimation[END_REF][START_REF] Vaid | Stroke direction asymmetry in figure drawing: Influence of handedness and reading/writing habits[END_REF]. For example, [START_REF] Shanon | Graphological patterns as a function of handedness and culture[END_REF] compared right-and left-handers with different reading habits (left-to-right or right-to-left) using graphomotor and drawing tasks. He showed that directionality of right-handers with either reading habit seems determined by biological factors, whereas left-handers are more influenced by environmental factors and exhibit compromises when these are in conflict with the biological factors.

Although a motor component is undoubtedly implicated in directional drawings [START_REF] Picard | Impact of manual preference on directionality in children's drawings[END_REF], it is very likely that a perceptuo-motor integration is reflected by the 'directionality' preference. [START_REF] Takala | Asymmetries of the Visual Space[END_REF] has thus noticed that the perceptual object itself appears to acquire the quality of 'directedness', with the feeling by some participants of being able to draw a picture only in a given direction, the opposite direction seeming 'wrong'. As [START_REF] Alter | A cerebral origin for "directionality[END_REF] pointed out, directionality in drawing could be related to a perceptual ordering or directional scanning bias.

In children, directionality expressed in visuo-motor tasks is generally related to handedness. Drawing a horizontal line is one of the tasks that particularly differentiate between right-and left-handed children: right-handers are predominantly right-directed whereas lefthanders predominantly show a left directionality for horizontal line drawing [START_REF] Scheirs | Relationships between direction of movements and handedness in children[END_REF]. In other visuo-motor tasks, such as drawing a profile of a human face, a predominant lateral direction is usually found in right-handed adults (profiles facing to the left) but not in left-handed adults [START_REF] De Agostini | The influence of handedness on profile and line drawing directionality in children, young, and older normal adults[END_REF]. The few studies that investigated the perceptual aspects of directionality or hemispatial predominance in children show that these lateral biases are related to handedness. A natural left-to-right trend (e.g., seen prior to reading education) in directionality exists only in right-handed children [START_REF] Dreman | Directionality trends as a function of handedness and of reading and writing habits[END_REF].

The influence of another biological factor studied in this experiment is the sex effect. In the 1970s, a large debate on possible sex differences in functional brain lateralization was a central point in neuropsychological literature. A difference in language and visuospatial lateralization has been suggested with greater hemispheric specialization seen in males than in females, at least in right-handers [START_REF] Kimura | Sex and Cognition[END_REF]. Consistent with this hypothesis, Cela-Conde and colleagues (2009), using magnetoencephalography to record brain activity during an aesthetic preference task, found a bilateral activation in parietal regions in women, whereas it was lateralized in the right hemisphere in men. However, some reviews of literature data tend to minimize this sex difference, suggesting this could account only for 1% to 2% of the variance in laterality [START_REF] Hiscock | Is there a sex difference in human laterality? II. An exhaustive survey of visual laterality studies from six neuropsychology journals[END_REF].

In the present paper we test for the effect of both age and handedness, in males and females, on visual aesthetic judgment. As mentioned earlier, using a visual aesthetic preference paradigm, we were recently able to demonstrate that healthy, right-handed children and adults significantly prefer pictures possessing the same directionality as their reading/writing habits [START_REF] Chokron | Reading habits influence aesthetic preference[END_REF]. Indeed, right-handers (children and adults) with left-to-right reading/writing habits significantly prefer pictures with a left-to-right directionality over their mirror representation. This left-to-right preference might be due both to their functional organization (as right-handers) and to their left-to-right scanning when reading.

As a matter of fact, biological directionality and reading/writing habits lead to the same directional bias in aesthetic preference only for right-handers, at least in readers of left to right languages. However, for left-handed individuals, the reported right-to left directional bias in left to right readers [START_REF] Karev | Directionality in right, mixed and left handers[END_REF][START_REF] De Agostini | The influence of handedness on profile and line drawing directionality in children, young, and older normal adults[END_REF] may change over time as a function of learning to read from left-to-right. Reading education has been found to shift directional preferences among children. [START_REF] Nachson | Directional preference of bilingual children[END_REF] found that Israeli school children with a native right-to-left reading language demonstrated shifts in directional preference to leftto-right with increased exposure to written English. Furthermore, bi-literate children who learned English and Hebrew concurrently showed a different directional pattern compared with monoliterate English or Hebrew reading children. Thus, with greater exposure to reading a leftto-right language, the directional preference is likely to also strengthen. By testing both the effect of age and handedness on visual aesthetic preference as well as that of sex, we aim to investigate the interaction between biological and cultural factors in such processing. In this way, if the performance of left-handers changes with age, it may reflect the growing effect of reading/writing directionality on visual scanning. In the present experiment, we chose images of objects with directionality that are identified as static, moving or that possess no directionality (: e.g., landscapes) in order to assess the effect of these dimensions on visual aesthetic preference.

METHOD Participants

Forty French children aged from 7 to 10 years [m=8.9 (0.6)] and 40 French adults aged from 20 to 30 years [m=22.9, (2.9)], neurologically normal, participated in the study. In each age group there were 20 males and 20 females. Half of them were strongly right-handed and the remaining half were strongly left-handed. The children's hand preference was assessed using an instrument reported in De Agostini and Dellatolas (1988), and that of the adults by the questionnaire reported in [START_REF] Dellatolas | Mesure de la préférence manuelle par auto-questionnaire dans la population française adulte[END_REF]. Children were tested in their school following informed consent from parents. Adult participants provided informed consent.

Materials

The Aesthetic Preference Task administered is composed of 50 stimuli. For each stimulus there are two mirror-image representations, positioned vertically on a sheet (21x29.6 cm). Stimuli were classified into three series as illustrated in Figure 1. Series 1 contained 20 pictures representing static objects (e.g. an asymmetric lamp); series 2 contained 20 pictures representing moving objects (e.g., a goose); series 3 contained 10 pictures representing landscapes (e.g., a sun umbrella on a beach).

For series 1 and 2, objects were located in the center of the sheet and they expressed a left-to-right or right-to-left directionality. For series 3, the most salient aspect of the landscape was positioned on the left ("left-landscape") or right ("right-landscape") half of the sheet. The stimulus pairs were displayed in a random order. For each series, in half of the pairs the object with a left-to-right directionality (or right preponderance for landscapes) was on the top of the sheet; in the remaining half of the pairs it was on the bottom.

Procedure

Participants were presented with 50 mirror-image pairs of stimuli located one above the other and were asked to indicate which stimulus was more interesting to look at or more aesthetically pleasing. Equivalence was not allowed. Participants' responses were coded as rightward preference when the chosen item was an object that either faced to the viewer's right or had the area of interest on the right of the landscape. The scores in each task (pictures series) are expressed in percentage of rightward preference: they varied from 0 (all leftward preference) to 1 (all right preference). A score of 0.5 revealed equal rates of rightward and leftward preference.

RESULTS

A linear mixed model analysis was carried out, with age, sex and handedness as betweensubjects variables, and picture series as a within-subject variable. Results showed two highly significant main effects: series (static figures, m=0.61; moving figures, m=0.61; landscapes figures, m=0.43; F(2,144)=26.80; p<.0001) and handedness (right-handers, m=0.63, left-handers m=0.47; F(1,144)=20.94; p<.0001). The effects of sex (males, m=0.53; females, m=0.57; p=.17) and age (children, m=0.52; adults, m=0.58; p=0.09) were not significant at the .05 level.

Nevertheless, the interpretation of the differences of main means is not simple because of the presence of significant 2-and even 3-order interactions between the variables (series by age: F(2,144)=2.90; p=.05; sex by manual preference by series: F(2,144)=3.08; p<.05), which shows that the data have a complicated structure and makes separate task analysis necessary. The means are quasi identical for static and moving figures but very different from landscapes figures. A mixed model analysis including only static figures and moving figures showed that not only the main series effect but also its interactions with the other factors were far from statistical significance. This strongly suggests that moving and static figures behave similarly. So, we combined moving and static figures scores to create a new score (directional figures), a percentage of the right preference of the 40 items (Table 1). Separate analyses of variance were conducted on the directional figures and the landscape stimuli, with age, sex, and handedness as between-subjects variables.

In the ANOVA analysis for the directional figures, a significant handedness effect, F(1,72)=15.09, p=.0002, was found (right-handers=0.69, left-handers=0.54) as was a significant age effect, F(1,72)=6.27, p = .01 (with the mean score for children being 0.56 and that for adults, 0.66). There was no effect of sex, p=.85 (males =0.61; females=0.62). Moreover, the interaction of handedness by sex was significant, F(1,72)=7.44, p=.003. As such, the data for males and females were subjected to separate analyses of variance.

In males, there was a significant effect of age (children, m=0.55; adults, m=0.67; F(1,36)=5.28; p<.03) but no effect of handedness (right-handers, m=0.63; left-handers, m=0.59; p=.40). It must be noted that the interaction handedness by age was very close to significance (F(1,36)=3.65; p=.06). In right-handed males no effect of age was found (children, m=0.62; adults, m=0.64; p=.71) whereas in left-handed males this effect was significant (children, m=0.48; adults, m=0.70; t(18)=-2.61; p=.01). In females, the ANOVA analysis showed a strong effect of manual preference (right-handers, m=0.74; left-handers, m=0.49; F(1,36)=20.55; p<.0001), no significant effect of age (children, m=0.58; adults, m=0.65; p=.21) and no interaction at all, p=.73 (Figure 2). In summary, for the directional figures, the results are different in males and females. The handedness effect is strongly present in females (girls and women) with more right preference in right-handers and no age effect. In males, the handedness effect was present in children but not in adults; in fact the only participant group showing an age effect was the left-handed males group, whose right preference increased significantly with age.

In the ANOVA analysis for the landscape figures, significant handedness (right-handers, m=0.51; left-handers, m=0.35; F(1,72)=8.08; p<.01) and sex effects (males, m=0.36; females, 0.5 : No directional preference m=0.49; F(1,72)=4.89; p<.05) were found, but not an age effect (children, m=0.44; adults, m=0.42; p=.69). The interaction handedness by sex by age was significant (F(1,72)=6.61; p=.01) leading again to a separate analysis for males and females.

In males, a significant effect of handedness was found (right-handers, m=0.46; lefthanders, m=0.28; F(1,36)=6.08; p<.02), but not an age effect (children, m=0.38; adults, m=0.36; p=.83). The interaction handedness by age was significant (F(1,36)=6.08; p<.02). In fact, there was a nearly significant age effect in left-handers (children, m=0.20; adults, m=0.36; t(18)=-1.86; p=.07) and no effect in right-handers (children, m=0.55; adults, m=0.36; p=.11). In females, the handedness effect (right-handers, m=0.56; left-handers, m=0.42; p=0.11) and the age effect (children, m=0.51; adults, m=0.48; p=0.73) were not significant and no interactions were found, p=.20 (Figure 3). In summary, for the landscape figures, the results were different for males and females. In females, no handedness or age effect was found. In males, the interaction handedness by age shows that while no age effect was found in right-handers, a trend was present in the left-handed group whose right preference increased with age. The same interaction is present for variables directional and landscape figures in the left-handed male group. However, if right preference increases for both variables with age, for landscape figures, left preference is still predominant in adults.

Discussion

In the present paper we tested for the effects of both age and handedness in male and female participants on visual aesthetic judgments. The interaction between biological factors, such as cerebral lateralization (comparing right-/left-handers and males/females), and cultural factors, like reading/writing directional scanning experience (comparing children and adults) were explored. We also tested for the effect of stimuli characteristics using pictures with directionality (with static and moving objects) and without directionality (landscapes).

The present study confirms the effect of stimulus characteristics on the visual aesthetic preference task, found in a previous paper [START_REF] Chokron | Reading habits influence aesthetic preference[END_REF]. In this previous work, in right-handers with different directional reading/writing habits, we found a significant effect of reading/writing habits on aesthetic preference, with viewers preferring pictures with the same directionality as their reading/writing habits. In contrast, we found no significant effect of reading/writing habits for landscape images. In the present paper, the results for directional images of static and moving objects were very similar, showing that preference was determined more by the directionality than by the static or mobile characteristics of the pictures. However the visual preference manifested for the landscape images was again different from the preference for directional pictures. In this way, the present results confirm earlier results and emphasize the fact that directional pictures and landscapes may be differentially processed.

Our results highlight the effect of the biological factors of handedness and sex on visual aesthetic judgment and a cultural effect (age-related reading/writing direction) at least in the lefthanded male group. Indeed, according to our hypothesis, based on the fact that biologicallybased directional preferences and reading/writing habits have the same directionality, in pictures with directionality all right-handed groups, children and adults, males and females, show a preference for stimuli depicting objects with a rightward directionality, i.e., in the same direction as the one used when reading. The age effect, which could also reflect the reading/writing directional scanning experience effect, was expected in left-handers, who present opposite biological/writing directionalities at an early stage of reading/writing. This significant change in visual preference for directional pictures was observed at least in left handed males who, from childhood to adulthood, significantly switch their preference for pictures with leftward directionality to pictures with a left-right directionality (according to their reading direction). We cannot exclude that this change stems from a natural maturational brain process in left-handers [START_REF] Geschwind | Cerebral lateralization. Biological mechanisms, associations and pathology: I. A hypothesis and a program for research[END_REF], but our results suggest that it could be seen as resulting from learning to read and write with a left-to-right direction.

In left-handers, if an atypical language cerebral representation has often been described [START_REF] Szaflarski | Language lateralization in left-handed and ambidextrous people: fMRI data[END_REF], the influence of familial sinistrality (not assessed in the current study) seems more important than the sex influence per se [START_REF] Hécaen | Cerebral organization in lefthanders[END_REF][START_REF] Bryden | Patterns of cerebral organization[END_REF]. Nevertheless, the interaction observed in the present study between sex and manual preference for directional images suggests that the environmental influence of reading/writing habits significanlty affects only the male participants' judgements. Left-handed adult female participants do no show any directional preference, as left-handed girls do. These results emphasize again the contribution of biological factors in visuospatial organization: females, at least left-handed females, would be less sensitive to the environmental influence than males.

It is by now well established that right-and left-handers, at least as groups, differ in cerebral lateralization of functions [START_REF] Annett | Handedness and Brain Asymmetry[END_REF]. Recently some studies have suggested that handedness should be considered on a continuum, based on method of measure, rather than as a dichotomy between right-handed and left-handed individuals [START_REF] Bourne | Examining the relationship between degree of handedness and degree of cerebral lateralization for processing facial emotion[END_REF]. In this study, given the greater variability in lateralization in left-versus right-handers, only strong right-or lefthanded individuals were tested. It is possible that our left-handed population has distinctive lateralization characteristics based on this methodological choice.

Differences in lateralization according to sex have also been found suggesting a greater hemispheric specialization in male compared to female right-handed adults [START_REF] Kimura | Sex and Cognition[END_REF]. More recently some studies have considered the interaction between handedness and sex. [START_REF] Bourne | Examining the relationship between degree of handedness and degree of cerebral lateralization for processing facial emotion[END_REF], with a chimeric faces test, found a relationship between handedness and lateralization for men only. Along the same lines, in the present study the expected effect of directional acquired habits and handedness on lateralization was found only in men.

In summary, the current findings confirm previous studies among French and Israeli readers and raise the question of an interaction between cultural factors and cerebral dominance in visuospatial organization. The results demonstrate that despite biological factors associated with hemispheric specialization and the early presence of functional asymmetries (e.g., handedness) both of which influence scanning direction, environmental or cultural factors appear to contribute to the aesthetic preference of images. In this report we also highlight that the contribution of culture on aesthetic preference does not manifest to the same extent for lefthanded children and adults. In addition to age, a fourth factor -sex -intervenes in aesthetic preference judgments. Left-handed females appeared to be less influenced by their reading direction compared to left-handed males. In conclusion, culture, in the form of reading/writing directional scanning, does play an important role in the way one scans and represents visuospatial information. The extent of this influence and its possible effect on hemispheric specialization remains to be investigated.

Indeed, these results stress the need of complementary studies, especially in participants differing in manual preference and reading/writing habits, to disentangle biological and cultural factors more thoroughly. As discussed in previous papers [START_REF] Chokron | On the origin of free-viewing perceptual asymmetries[END_REF]Chokron, Kazandjian and De Agostini, in press;[START_REF] Kazandjian | Paying attention to reading direction[END_REF], experimental studies dealing either with cognitive/perceptual biases and/or with hemispheric specialization only scarcely take into consideration the interaction between age, sex, manual preference and cultural influence (such as reading and writing direction). However, as the present findings underscore, we may need to study these factors concurrently in order to understand how nature and nurture interact in the perception and representation of the external world.

Figure 1 :

 1 Figure 1: Examples of stimuli used in Aesthetic Preference Task.

Figure

  Figure 2 : Series with directional figures

Figure

  Figure 3 : Series with landscape figures

Table 1 :

 1 Mean scores (from 0 to 1) for right preference in Directional and Landscape images, according to group age, manual preference, and sex.

	Males	Females