Enrique Regidor
email: enriqueregidor@hotmail.com

Salvador De Mateo

Elena Ronda

José Sanchez-Payá

Juan L Gutiérrez-Fisac

Luis De

La Fuente

Cruz Pascual

José Sánchez-Payá

Luis De La Fuente

Jl E Ronda

C Gutierrez-Fisac

Pascual

Heterogeneous trend in smoking prevalence by sex and age group following the implementation of a national smoke-free law

Keywords: smoke-free law, smoking prevalence, Spain

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

Introduction

A large body of evidence has shown that tobacco control policies, especially when they are implemented as part of a comprehensive program, can significantly reduce smoking rates. [START_REF]Tobacco. Guide to Community Preventive Services[END_REF][START_REF] Hopkins | Reviews of evidence regarding interventions to reduce tobacco use and exposure to environmental tobacco smoke[END_REF] Raising the price of tobacco by increasing taxes, and clean indoor air laws, by prohibiting smoking in public places, have been indicated as the policies with the greatest impact on smoking rates. [START_REF] Levy | The effects of tobacco control policies on smoking rates: a tobacco control scorecard[END_REF] Legislation to ban smoking in all indoor public places has been or is currently being introduced in several countries due to the evidence showing the harmful effects of environmental tobacco smoke on non-smokers. [START_REF]Tobacco Smoking[END_REF][START_REF]Health effects of exposure to environmental tobacco smoke: the real report of the California Environmental Protection Agency[END_REF] These types of laws have proved effective in reducing both the amount of and exposure to environmental tobacco smoke. [START_REF]Tobacco. Guide to Community Preventive Services[END_REF][START_REF] Allwright | Legislation for smoke-free workplaces and health of bar workers in Ireland: before and after study[END_REF][START_REF] Thomson | One year of smokefree bars and restaurants in New Zealand: impacts and responses[END_REF][START_REF] Gallus | Effects of new smoking regulations in Italy[END_REF][START_REF] Fong | Reductions in tobacco smoke pollution and increases in support for smoke-free public places following the implementation of comprehensive smoke-free workplace legislation in the Republic of Ireland: findings from the ITC Ireland/UK Survey[END_REF][START_REF] Galán | Impact of the "Tobacco control law" on exposure to environmental tobacco smoke in Spain[END_REF][START_REF] Fichtenberg | Effect of smoke-free workplaces on smoking behaviours: systematic review[END_REF][START_REF]IARC handbooks of cancer prevention: tobacco control[END_REF][START_REF] Evans | The compensating behavior of smokers: taxes, tar, and nicotine[END_REF][START_REF] Chaloupka | The economics of smoking[END_REF][START_REF]Ley de medidas sanitarias frente al tabaquismo y reguladora de la venta[END_REF][START_REF]Indicadores de Salud. La salud de la población española en el contexto europeo y del Sistema Nacional de Salud[END_REF][START_REF]SAS System for windows V8[END_REF][START_REF] Kim | Permutation tests for joinpoint regression with applications to cancer rates[END_REF][START_REF]Statistical Research and Applications Branch[END_REF] Although their main objective is the protection of those involuntarily exposed to tobacco smoke, other indirect effects of these legislative measures that have repeatedly been described are reductions in smoking prevalence, larger numbers of people who quit smoking, and reduced tobacco consumption in smokers . [START_REF]Tobacco. Guide to Community Preventive Services[END_REF][START_REF] Levy | The effects of tobacco control policies on smoking rates: a tobacco control scorecard[END_REF][START_REF] Fichtenberg | Effect of smoke-free workplaces on smoking behaviours: systematic review[END_REF] A reduction in smoking behaviours has been observed in workers subject to restrictions in the workplace. In fact, most investigations that have evaluated the effect of these restrictions have focused on quantifying the effects of smoke-free workplaces on smoking behaviour in employees, whether imposed by public laws or private firms. [START_REF] Fichtenberg | Effect of smoke-free workplaces on smoking behaviours: systematic review[END_REF][START_REF]IARC handbooks of cancer prevention: tobacco control[END_REF] However, compared to the large amount of evidence showing the effects of price on cigarette consumption at the population level, both on prevalence rates and the number of cigarettes consumed by those who continue to smoke, [START_REF] Hopkins | Reviews of evidence regarding interventions to reduce tobacco use and exposure to environmental tobacco smoke[END_REF][START_REF] Levy | The effects of tobacco control policies on smoking rates: a tobacco control scorecard[END_REF][START_REF] Evans | The compensating behavior of smokers: taxes, tar, and nicotine[END_REF][START_REF] Chaloupka | The economics of smoking[END_REF] there is little evidence about the effects of clean indoor air laws on smoking behaviour in the general population. Moreover, according to a recent review of the effect of mandated smoking restrictions, [START_REF]IARC handbooks of cancer prevention: tobacco control[END_REF] the few studies that have assessed smoking behaviour in the general population, before and after implementation of new laws restricting smoking in public and workplaces, were analytically weak and produced mixed results.

On 1 January 2006 Spain introduced a comprehensive smoke-free law covering workplaces, bars and restaurants. [START_REF]Ley de medidas sanitarias frente al tabaquismo y reguladora de la venta[END_REF] Under this law smoking has been completely banned in all indoor workplaces. Smoking is also banned in bars and restaurants larger than 100 square metres, but completely isolated smoking areas can be created, whereas the law authorizes the owners of bars and restaurants smaller than 100 square metres to decide whether or not to allow smoking.

The question addressed in this study is whether this law has been successful in reducing the prevalence of smoking in the Spanish population. We present data on trends in the prevalence of smoking during the period before and after implementation of the law.

Methods

Source of data

The estimates of tobacco consumption were obtained from the General Media Survey conducted by the Association for Research in the Communications Media. Although the main objective of this survey is to identify the use of the communications media -the press, radio and televisionin the Spanish population, the questionnaire also includes questions to explore the use of other products and lifestyles related with leisure time.

The General Media Survey is carried out by personal interview in a representative sample of the non-institutionalised Spanish population aged 15 years or older. Subjects are selected by a random multi-stage stratified procedure, in which the municipalities within each province are grouped into strata depending on population size. First, one municipality is randomly selected within each stratum of each province, then households within each municipality are randomly selected and, finally, one person in each household is selected to answer the survey. Each year three samples of the Spanish population are obtained: January-March, April-June and September-November. The mean sample size in each period is 14,200 subjects -about 44,200 subjects are interviewed per year. In the present study we analysed the 27 surveys carried out between the beginning (January-March) of 2000 and the end (September-November) of 2008.

The response rate ranged between 70% and 75%.

The questionnaire includes questions about the use of blond-tobacco cigarettes, black-tobacco cigarettes, cigars and low-nicotine cigarettes. Respondents are asked whether or not they consume each product and, if so, whether they consume the product at least once a week or less frequently. Since regular smokers may be more sensitive to implementation of a comprehensive smoke-free law, respondents who consumed any of these products at least once a week were considered smokers.

Our study was restricted to persons aged 15 to 64 years because one of the places where the law bans smoking is the workplace, and after age 64 most of the population is retired. Likewise, since previous studies have indicated that the magnitude of the effect of clean indoor air laws varies depending on age, [START_REF] Levy | The effects of tobacco control policies on smoking rates: a tobacco control scorecard[END_REF] separate estimates were made for three age groups: 15-24 years, 25-44 years and 45-64 years, with respective mean sample sizes of 1,800, 4,800 and 3,200 persons interviewed in each survey. The analysis was made separately in men and women since Spain is in the last stage of phase III in the smoking epidemic, in which middle-aged women (45 -64 years) have not yet reversed the rising trend in tobacco consumption. [START_REF]Indicadores de Salud. La salud de la población española en el contexto europeo y del Sistema Nacional de Salud[END_REF]

Statistical analysis

We first estimated the prevalence of smoking in each age-sex group and for each of the 27 periods analysed. The graphic representation of the trend was made by the moving arithmetic average of the prevalence based on three estimates: the one corresponding to each period, the previous period and the subsequent period. The Chi-square statistic was used to test the statistical significance of the difference between the prevalence obtained in the survey before the law was implemented (end of 2005) and the prevalence obtained in the most recent available survey (end of 2008). To take into account the complex sampling design, the estimate of prevalence and of the standard error of the prevalence in each age-sex group and for each of the 27 periods was made using the SAS procedure PROC SURVEYFREQ. [START_REF]SAS System for windows V8[END_REF] We then used the permutation test for joinpoint regression to detect significant changes in the smoking prevalence trend. [START_REF] Kim | Permutation tests for joinpoint regression with applications to cancer rates[END_REF][START_REF]Statistical Research and Applications Branch[END_REF] This procedure chooses line segments that change slope at joinpoints denoting a statistically significant change in trend. The overall statistical significance level was α = 0.05 with a maximum of three joinpoints and four line segments allowed. We fit the models on the log scale, using a weighted least square criterion, where the weight was the standard error of the observed prevalence. In this way the slope of each continuous linear phase is interpreted as the period percent change in the prevalence.

Finally, because the empirical evidence shows that laws restricting smoking in workplaces reduce smoking prevalence in workers, these analyses were also made in the subgroup of employed persons. For this purpose, we selected respondents who, in reply to the question about employment status, said they were working; those who were unemployed, retired, students or home-makers were excluded from this analysis.

Results

Figure 1 depicts the evolution in smoking prevalence in general population -by the moving average -from 2000 to 2008. Table 1 shows a summary form of the trend in smoking prevalence before and after the implementation of the law. From the start of 2000 to the end of 2005, smoking prevalence decreased in all the groups analysed except for women aged 45-64, in whom it increased from 15% to 19%. At the start of 2006 smoking prevalence also declined with respect to the previous period in all groups. However, the prevalence in the final period of 2008 increased with respect to that of the last period of 2005 in all groups, although the difference between the two periods was significant only in women aged 45-64. The largest increases in smoking prevalence were seen in men and women aged 45-64, in whom it rose from 34.8% to 37.8% and from 19.0% to 23.7%, respectively. The results of the statistical analysis with joinpoint regression are shown in figure 2 and table 2.

In men and women aged 15-24 and in women aged 45-64 no significant inflection points were detected between the first period of 2000 and the final period of 2008. In men and women aged 15-24 smoking prevalence declined significantly, whereas in women aged 45-64 years there was a significant increase of 1.70% (p<0.001). The results of the analyses made only in persons who worked showed a similar trend to that observed in the general population (tables 3 and 4).

Discussion

Main findings

In men and women aged 25-44 and in women aged 45-64, the trend in smoking prevalence during the three years after the law was passed did not change significantly with respect to the previously observed trend, whereas in men aged 25-44 and 45-64 and in women aged 25-44 a significant inflection point was detected after the implementation of the law, and a rising trend was observed, which reversed the declining trend of the previous years.

Comparisons with other studies and possible explanations

It is possible that the clean indoor air law passed in Spain has not successful in reducing the prevalence of smoking because, although it bans all smoking in workplaces, the limitation is only partial in bars and restaurants. In fact, studies that have evaluated the effects of this law have shown that after it was passed, exposure to environmental tobacco smoke was considerably reduced in workplaces, but the reduction was smaller in bars and restaurants. [START_REF] Galán | Impact of the "Tobacco control law" on exposure to environmental tobacco smoke in Spain[END_REF][START_REF] Jiménez-Ruiz | Study of the impact of laws regulating tobacco consumption on the prevalence passive smoking in Spain[END_REF] Nevertheless, success in applying the law in workplaces does not guarantee a reduction in smoking prevalence, since the trend in workers observed in our study was similar to that of the general population.

According to some authors, policies to control smoking in Spain were among the most permissive in the European Union before this law was passed, [START_REF] Fernández | Lessons learned from tobacco control in Spain[END_REF] and this may have limited their impact on smoking prevalence. However, since 1988 several regulations have been passed in Spain restricting tobacco sales and advertising and limiting the places where smoking is allowed. [START_REF] Regidor | Trends in cigarette smoking in Spain by social class[END_REF][START_REF] Schiaffino | Time trends and educational differences in the incidence of quitting smoking in Spain (1965-2000)[END_REF] Likewise, between 1988 and 2008 the price of tobacco has risen by 400% --as compared to the 120% increase in the general price index. [24] Moreover, during those 20 years the prevalence of smoking in men declined from 57% to 32%, [25-26] while the rising trend in women aged 25-44 was reversed at the beginning of the present decade, which is consistent with a country in the final part of stage three of the smoking epidemic. [START_REF]Indicadores de Salud. La salud de la población española en el contexto europeo y del Sistema Nacional de Salud[END_REF] In addition, during the time covered by the present study, the mean annual increase in the price of tobacco was similar in the periods before and after implementation of the law. Specifically, between 2000 and 2008 the price of tobacco increased by 50% as compared to a 30% increase in the general price index. [24] Few studies have evaluated the effect of clean indoor air laws on the prevalence of smoking in the general population, and in some that have done so, the post-law prevalence was estimated within a year after implementation of the law so the likelihood of observing any potential effect or lack of effect was limited. For example, a study in the Region of Madrid (Spain) found that smoking prevalence in the 6 months after the law's implementation was similar to the prevalence in the 6 months before. [START_REF] Galán | Impact of the "Tobacco control law" on exposure to environmental tobacco smoke in Spain[END_REF] Likewise, two studies in Italy and Scotland found that the prevalence of smoking in the Italian and Scottish populations was unchanged in the year following the introduction of a comprehensive smoke-free law in January 2005 and in March 2006, respectively. [START_REF] Gallus | Smoking in Italy 2005-2006: effects of a comprehensive National Tobacco Regulation[END_REF][START_REF] Haw | Changes in exposure of adult non-smokers to secondhand smoke after implementation of smoke-free legislation in Scotland: national cross sectional survey[END_REF] The authors of the Italian study also estimated the overall prevalence of smoking in the two years following the introduction of the measure and showed that, with respect to the previous two years, the prevalence decreased significantly in men and in the population aged 15-44, but not in women or in the population older than 45 years.

The results of our study are more similar to the findings of the tobacco use surveillance systems in Finland and Ireland based on continuous surveys aimed at the whole population. In Finland, after implementation in March 1995 of the reformed Tobacco Control Act making workplaces totally smoke free, the prevalence of smoking in the population aged 15-64 years dropped from 26% in 1991-93 to 23% in 1994-96, but rose to 25% in 1997-98. [START_REF] Helakorpi | Health Behaviour and Health among Finnish Adult Population, Spring 1999[END_REF] And according to estimates of the Irish Office of Tobacco Control, after the introduction on 29 March 2004 of a comprehensive smoke-free law covering all indoor workplaces, bars and restaurants, the prevalence of smoking in the population declined in the following 12 months from 25.4% to 23.3%, but subsequently increased by the end of the third year to levels similar to those in Ireland before the intervention. [START_REF]Cigarette Smoking Trends[END_REF] After the third year the trends in smoking prevalence by age were heterogeneous: whereas there was a downward trend in persons under age 45, there was a rising trend in the population aged 45 or over.

With the exceptions previously mentioned, the studies to date that have evaluated the impact of the implementation of smoke-free policies on smoking behaviours have been made in the working population after the passage of clean indoor air restrictions that ban the use of tobacco. A systematic review published in 2002 concluded that totally smoke-free workplaces are associated with reductions in the prevalence of smoking of 3.8%. [START_REF] Fichtenberg | Effect of smoke-free workplaces on smoking behaviours: systematic review[END_REF] It is possible that this effect is diluted in studies made in the general population, since not everyone is employed and some employed persons work in the open air. In this regard, a study in Finland on smoking habits among employees showed that, with respect to the previous year, the prevalence of smoking decreased significantly one year after the passage of the reformed Tobacco Control Act in March 1995. [START_REF] Heloma | The short-term impact of national smokefree workplace legislation on passive smoking and tobacco use[END_REF] In the discussion of the results, the authors of this study mentioned that, surprisingly, in the same period there was no significant decline in smoking rates in the general population.

However, we can not rule out the possibility that clean air laws do not really have any effect on the prevalence of smoking, not even among employed people. The findings of the studies conducted are not conclusive. For example, one characteristic of the review studies mentioned is the short interval between the implementation of smoke-free workplace policies and the follow-up survey (a median of 9 months), which limits the conclusions that can be drawn from these investigations.

Likewise, another review published three years before 2002, which included most of the studies in the preceding review, drew different conclusions. [START_REF] Chapman | The impact of smoke-free workplaces on declining cigarette consumption in Australia and the United States[END_REF] According to these authors, design limitations or information biases in some cases, and failure to consider the secular trend in cessation in others, limited the validity of the reduced prevalence of smoking observed in most studies after the implementation of smoking bans in the workplace. On the other hand, the US Task Force on Community Preventive Services concluded in 2001 that the evidence of an effect of banning smoking in indoor public places on the prevalence of tobacco use is inconsistent. [START_REF] Hopkins | Reviews of evidence regarding interventions to reduce tobacco use and exposure to environmental tobacco smoke[END_REF] Finally the most recent review of the effect of mandated smoking restrictions on smoking behaviour concludes that studies of workers subject to restriction in the workplace indicate that new restrictions reduce the number of cigarettes smoked daily, but that whether or not this reduction is sufficient to make smokers less addicted, and therefore more likely to quit in the future, is unknown. [START_REF]IARC handbooks of cancer prevention: tobacco control[END_REF] The results of our study suggest that implementation of smoking bans in the workplace does not achieve a reduction in smoking prevalence in workers, since the results were similar to those of the general population: the trend in smoking prevalence in some population groups was unchanged, but in others the declining trend of previous years was reversed. Even though 60% of the population in Spain works in closed areas where the mandated smoking restriction would exert its effect, [START_REF] Galán | Impact of the "Tobacco control law" on exposure to environmental tobacco smoke in Spain[END_REF] it is not possible to rule out the possibility that the trend observed in the working population as a whole can be attributed to the 40% who work in open spaces.

In the working population aged 15-24 years there was a huge increase in the prevalence of smoking between the first and last period after implementation of the law. Because the sample size was only one-third as large as the total sample size in that age group, this finding must be

The trend in smoking prevalence in workers was also investigated in this study. However, adequate evaluation of the impact of implementation of the smoke-free law on the working population would have required us to distinguish between workers in closed areas and those who work in open spaces, which was not possible. Nor were we able to compare people who were already working in smoke-free environments with workers affected by the law.

As has been pointed out, the findings of studies conducted in the working population show that the effect of smoking restrictions and bans on reducing the amount of tobacco consumed is consistent. Moreover, in Ireland a reduction in the prevalence of smokers of more than 20 cigarettes smoked daily was observed after the introduction of the comprehensive smoke-free law. [START_REF]Cigarette Smoking Trends[END_REF] Unfortunately, the data source used in our study does not collect information on the number of cigarettes consumed by smokers, so we cannot evaluate the possible effect of this in the general population. However, some indirect evidence suggests that this effect may also be

Implication of findings

In summary, three years after the enactment of the clean indoor air law in Spain, the trend in the prevalence of smoking in the different population groups analysed was unchanged or was reversed in comparison with the declining trend observed in previous years. The similarity of our findings to the trend observed in Ireland -the country with the longest series of estimates of smoking prevalence after implementation of a clean indoor air law, suggests that these types of laws, although they reduce exposure to environmental tobacco smoke and the number of cigarettes smoked daily, may not achieve the other objective pursued: reduction of the prevalence of smoking in the population.

  significantly different from zero Joinpoint regression analyses detected one significant inflection point in men aged 25-44 and two significant inflection points in men and women aged 45-64. In men aged 25-44 smoking prevalence declined by 1.15% per period (p<0.001) between the start of 2000 and the end of 2006, and increased by 1.23 (p=0.09) per period between the end of 2006 and the end 2008. In men aged 45-64 years the prevalence of smoking declined by 0.21% per period (p=0.46) between the start of 2000 and the end of 2003, and by 2.44% per period (p=0.007) between the end of 2003 and the start of 2006, but increased by 2.03% (p<0.001) per period between the beginning of 2006 and the end of 2008. Finally, in women aged 25-44 smoking prevalence declined by 0.96% per period (p<0.001) between the start of 2000 and the end of 2004, and by 3.45% (p=0.07) per period between the end of 2004 and start of 2006, but increased by 0.70% (p=0.12) per period between the beginning of 2006 and the end of 2008.

  observed in Spain in the general population. According to data from the Commissioner for the Tobacco Market, [33] the number annual of cigarettes sold per person aged 15 years and older declined by about 5% each year between 2005 and 2008, whereas there was little change in the annual number of cigarettes sold per person between 2000 and 2005.

Figure 1 .

 1 Figure 1. Trend in smoking prevalence (%) by sex and age group in Spain, 2000-2008 (moving average). Note: The vertical line between 2005 and 2006 represents the time when the law was implemented.

Figure 2 Men 15 -

 215 Figure 2 Observed smoking prevalence (%) and best joinpoint model estimates by sex and age group. Spain, 2000-2008. ( observed data; joinpoint estimated data). Note: The vertical line between 2005 and 2006 represents the time when the law was implemented.

Table 1 . Pevalence of smoking in Spain before and after implementation of a smoke-free law in January 2006 .

 1

		Smoking prevalence (%) and 95% confidence interval
	Sex and age group	Before smoke-free law January/March September/November	After smoke-free law January/March September/November
		2000	2005*	2006	2008*
	Men
	15-24	36.0 (33.3, 38.7)	31.6 (28.8, 34.7)	29.1 (26.4, 31.8)	32.5 (29.0, 36.0)
	25-44	49.8 (47.9, 51.8)	43.1 (41.3, 44.9)	40.9 (39.1, 42.8)	43.8 (41.6, 45.9)
	45-64	37.0 (34.7, 39.3)	34.8 (32.7, 36.9)	29.8 (27.8, 31.9)	37.8 (35.37, 40.3)
	Women
				28.7 (25.9,
	15-24	35.3 (32.6, 38.1)	29.8 (27.0, 32.6)	31.5)	31.3 (27.8, 34.9)
	25-44	40.8 (38.8, 42.7)	33.2 (31.4, 34.9)	30.9 (29.1, 32.6)	34.1 (32.1, 36.3)
	45-64	15.0 (13.3, 16.6)	19.0 (17.2, 20.7)	17.8 (16.1, 19.4)	23.7 (21.6, 25.9)

* The difference between estimates at the end 2005 and the end 2008 were not statistically significant, except in the subgroup of women aged 45-64

Table 2 . Period percentage change (PPC) in smoking prevalence in Spain by sex and age group

 2

		Trend 1	Trend 2	Trend 3
	Sex and age group	Duration	PPC	Duration	PPC	Duration	PPC
	Men
	15-24	From start
		2000 to	-0.70*
		end 2008
	25-44	From start		From end
		2000 to	-1.15*	2006 to	1.23
		end 2006		end 2008
	45-64	From start		From end		From start
		2000 to	-0,21	2003 to	-2.44*	2006 to	2.03*
		end 2003		start 2006		end 2008
	Women
	15-24	From start
		2000 to	-1.18*
		end 2008
	25-44	From start		From end
		2000 to	-0.96*	2004 to	-3.45
		end 2004		start 2006

Table 3 . Pevalence of smoking in employed persons in Spain before and after implementation of a smoke-free law in January 2006

 3

		Smoking prevalence (%) and 95% confidence interval
	Sex and age group	Before smoke-free law January/March September/November	After smoke-free law January/March September/November
		2000	2005*	2006	2008*
	Men
	15-24	47.0 (43.0, 52.67)	44.6 (40.0, 49.1)	39.2 (34.5, 43.9)	49.2 (43.0, 55.3)
	25-44	50.7 (48.5, 52.9)	42.6 (40.7, 44.5)	39.5 (37.6, 41.4)	43.0 (40.6, 45.4)
	45-64	38.9 (35.7, 41.3)	33.8 (31.4, 36.3)	29.4 (27.0, 31.8)	38.2 (35.2, 41.2)
	Women
				34.6 (29.4,
	15-24	46.4 (40.8, 52.0)	40.9 (35.5, 46.3)	39.9)	43.4 (36.6, 50.1)
	25-44	41.6 (36.6, 50.1)	32.7 (30.6, 34.8)	30.1 (28.1, 32.3)	33.1 (30.6, 35.7)
	45-64	21.6 (17.8, 25.4)	26.0 (22.9, 29.0)	24.0 (21.0, 27.0)	30.6 (27.6, 33.5)
	* Difference between estimates at the end of 2005 and the end of 2008 were not statistically significant

Table 4 . Period percentage change (PPC) in smoking prevalence in employed persons in Spain, by sex and age group

 4

		Trend 1	Trend 2	Trend 3
	Sex and age group	Duration	PPC	Duration	PPC	Duration	PPC
	Men
	15-24	From start
		2000 to	-0.57*
		end 2008
	25-44	From start		From end
		2000 to	-1.25*	2006 to	1,32
		end 2006		end 2008
	45-64	From start		From end		From start
		2000 to	-0,28	2003 to	-2.54*	2006 to	2.32*
		end 2003		start 2006		end 2008
	Women
	15-24	From start
		2000 to	-1.22*
		end 2008
	25-44	From start		From end		From start
		2000 to	-0.75*	2004 to	-2.78*	2006 to	0.68
		end 2004		start 2006		end 2008
	45-64	From start
		2000 to	1.01*
		end 2008

* PPC is significantly different from zero

attributed to the random nature of the estimates. In fact, the period percent change in smoking prevalence from the beginning of 2000 to the end of 2008 was -0.57 in men and -1.22 in women.

Strengths and limitations of study

Based on 27 estimates from the beginning of 2000 to the end of the third year after the smokefree law was enacted, this study has shown the trends in the prevalence of tobacco use in different population groups in Spain. The results of this study, together with Irish Office of Tobacco Control system for monitoring trends in tobacco use, [START_REF]Cigarette Smoking Trends[END_REF] constitute the most relevant information about evolving trends in smoking prevalence in the general population after the implementation of clean indoor air laws.

The definition of tobacco in our study is the same as that used in other places to monitor smoking prevalence. [START_REF] Helakorpi | Health Behaviour and Health among Finnish Adult Population, Spring 1999[END_REF] The estimates obtained are a valid approximation of the prevalence of daily smokers. According to the 2006 National Health Survey, [26] the prevalence of daily smokers in that year in the Spanish population aged 16 and over was 32.2% in men and 22.1% in women, while the estimates obtained for the same year in our data source were 32.7% in men and 21.5% in women.

It might be suggested that smoking prevalence could also be monitored taking into consideration all subjects who smoke, and not just regular smokers. However, when the analyses were made considering all smokers, the results were similar (see table and figure in web site). This is probably because around 95% of smokers, both before and after implementation of the law, were regular smokers.

What is already known on this subject

The few studies that have assessed smoking behaviour in the general population before and after the implementation of new laws restricting smoking in public and workplaces were analytically weak and produced mixed results.

What this study adds

Three years after the introduction of a comprehensive smoke-free law in Spain, the trend in smoking prevalence in some population groups was unchanged, but in others the declining trend of previous years before the implementation of the law was reversed.

The similarity of these findings to the trend observed in Ireland -the country with the longest series of estimates of smoking prevalence after implementation of a clean indoor air law-, suggests that clean indoor air laws, although effective in reducing exposure to second-hand smoke, may not achieve the secondary objective of reducing the prevalence of smoking in the population.


Anita Cj Ravelli

epidemiologist, PhD Miranda Tromp

medical informatician, PhD Martine Eskes

obstetrician, MD, PhD Juliet C Droog

Joris Am Van Der Post

professor of Obstetrics, MD, PhD Kitty J Jager

epidemiologist, MD, PhD Ben Willem Mol

PhD Johannes B Reitsma

Anita Cj

Joris Am Van Der Post

Ethnic differences in stillbirth and early neonatal mortality in the Netherlands

Keywords: Perinatal mortality, ethnicity, stillbirth, late in care, socioeconomic status original research paper, epidemiology

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

What is already known on this topic

-Ethnic minority women have higher perinatal mortality in Western countries.

-Teenage pregnancy, low socioeconomic status and pre-existent diseases are important risk factors for perinatal mortality.

-Women from non-Western ethnic origin have more preterm births and low birth weight newborns.

What this study adds:

-Ethnic differences in foetal and early neonatal mortality exist in the Netherlands and foetal mortality is especially elevated among women of African, South Asian and other non-Western origin.

-Women from other Western or East Asian origin have lower foetal and neonatal mortality risk compared to the Dutch women.

-Living in urbanised areas diminishes the risk on mortality for ethnic minorities and first booking visit after 18 weeks aggravates the risk on mortality. Maternal age, socioeconomic status and pre-existent disease had no effect -In women without risk factors the ethnic risk differences in mortality were even more pronounced.

Introduction

Ethnic disparities in perinatal mortality exist in western countries. 1 2 3 It has been argued that these differences are predominantly determined by socioeconomic disparities, while others state that biological and medical conditions underlay the elevated risks. 4 5 These findings signify that differences in perinatal mortality between ethnic groups are poorly understood. 2 6 7,8 In the Netherlands, 16% of the pregnant women are from non-Western origin. The main ethnic minorities originate from the former Dutch colony Indonesia (independency in 1945), Surinam (independency in 1975) and from Turkey and Morocco as migrant labourers mainly in the sixties. A recent study in the Netherlands showed that the non-Western women had a 40% higher risk on perinatal mortality. [START_REF] Ravelli | Decreasing perinatal mortality in the Netherlands, 2000-2006: a record linkage study[END_REF] Previous studies showed that ethnic specific differences exist in perinatal and infant mortality in the Netherlands. 10 11 12 Ethnic women also experience an increased risk on severe maternal morbidity. [START_REF] Zwart | Severe maternal morbidity during pregnancy, delivery and puerperium in the Netherlands: a nationwide population-based study of 371,000 pregnancies[END_REF] It is unknown if ethnic differences exists in stillbirth on a national level.

The aim of this paper is to describe the differences in foetal and early neonatal mortality between women of different ethnic origin in the Netherlands. Furthermore the aim is to explore the contribution of demographic, socioeconomic, health behavioural and preexistent medical risk factors on foetal and early neonatal mortality among different ethnic groups.

Materials and Methods

Data sources

The Netherlands Perinatal Registry (PRN) contains population based information on pregnancies, deliveries and (re)admissions of newborns. [START_REF]Perinatal Care in the Netherlands[END_REF] The PRN is based on a validated probabilistic linkage of the national midwifery registry (LVR1), the obstetrics registry (LVR2) and the neonatology/ paediatrics registry (LNR). 15 16 The recent linkage of 7-years of 6 obstetric and neonatal registry data allows detailed analysis of a large cohort of 1,3 million births. [START_REF] Ravelli | Perinatal mortality in the Netherlands 2000-2006; risk factors and risk selection[END_REF] The midwifery and obstetrics registries start at the first (booking) prenatal visit. If the child is admitted to a neonatal ward after birth the information is collected in the neonatology registry. Around 96% of all births are included in the perinatal registration.

Study population

The study population comprised nulliparous women who gave birth to singletons during the period 2000-2006. We excluded births before the legal term of 24 weeks gestation and below 500 gram, because there are terminations of pregnancy before 24 weeks which could be distributed unequally among the ethnic groups. [START_REF] Ravelli | Decreasing perinatal mortality in the Netherlands, 2000-2006: a record linkage study[END_REF] Women who conceived after assisted reproductive technology (n=14,766, 2.6%) were also excluded for reason of unequal distribution. The study population existed of 554,234 births including 3135 stillbirths and 1266 early neonatal deaths.

Outcome variables

Perinatal mortality, foetal and early neonatal mortality were the main outcome variables.

Foetal mortality (stillbirths) was defined as death of the unborn child either antenatal or during labour (per 1000 births). Early neonatal mortality was defined as death up to 7 days of life (per 1000 live births). Perinatal mortality is the sum of foetal and early neonatal mortality divided per 1000 births (including stillbirths) from 24 weeks gestation onwards. Other adverse outcomes were preterm birth (< 37.0 weeks gestational age) low birth weight (<2500 gram) and congenital anomalies.

Definition of ethnicity

The classification of ethnicity was performed by the health care professional on the basis of race and country of birth. Since mid eighties this definition is used in the midwifery and obstetrics registry and is in fact a mixture of ethnicity and race. The Western ethnic groups included ethnic "Dutch" and "other Western" women with Caucasoid ancestry (Belgium, UK, Germany, etc). The non-Western ethnic group was classified in five subgroups. Two different ethnic groups originate from Surinam (a former Dutch colony): the "African" or Creole and the "South Asian" or Hindustani originating from Africa and India respectively. The other subgroups include the "Turkish/Moroccan" women, "East-Asian" women originating mainly from Indonesia (a former Dutch colony) and "other non-Western" women.

Risk factors

Demographic and socioeconomic risk factors were maternal age, urbanisation degree and socioeconomic status (SES). The urbanisation degree and SES indicators were created on the basis of the four digit postcode of the pregnant woman. Urbanisation was based on the number of addresses per square kilometre and categorized in very urban (≥ 2500 adressess/km 2 ), intermediate urban/rural (between 500 and 2500 adressess/km 2 ) and very rural (< 500 adressess/km 2 ). The SES indicators included the data from the Netherlands Institute for Social Research/SCP in three categories (10 th , 10-90, ≥ 90 th percentile) and the mean average household equivalent income for neighbourhoods as low and high (<40%,

≥40%).

Pre-existent medical risk factors were essential hypertension, diabetes mellitus, other endocrine diseases, cardiac disease, systemic diseases, thrombo-embolic disorders and psychiatric disorders as recorded by the midwife or gynaecologist.

Behavioural risk factors considered were the booking visit and smoking during gestation. The timing of the first antenatal or booking visit was measured in weeks of gestational age and categorized in four groups as (<12 weeks (in time), 12-17 weeks, after 18 weeks gestational age (late) and unknown. Heavy smoking registered as "abuse of nicotine" was recorded by the health care professional based on self report of the woman (≥ 20 cigarettes a day).

Statistical analyses

The prevalence of risk factors among the different ethnic groups was determined and differences were tested with a Chi-Square test. The incidence of perinatal, foetal and early neonatal mortality as well as preterm birth, low birth weight and congenital anomalies was described for the different ethnic groups and the differences were tested with a Chi-Square test.

The crude odds ratios (OR) and their 95% confidence interval (95% CI) of the different ethnic groups on foetal and neonatal mortality were calculated using logistic regression modelling.

The reference categories are based on that level of the predictor with the lowest risk of the outcome.

The adjustment (enter method) included all relevant risk factors for mortality: maternal age, urbanization, SES, low income, booking visit, heavy smoking and pre-existing diseases. The effect of each adjustment factor separately on ethnicity was also determined.

A second multivariate model was created including the effect of preterm birth as preterm birth is more prevalent among African and South Asian women and is a strong indicator for mortality. Thirdly, the analyses were repeated in group of women with known ethnicity and without risk factors. For this group, women with age below 20 or above 40 years, low SES, low income level, booking visit ≥ 18 weeks gestation, heavy smoking, pre-existent diseases, gestational age below 32 weeks, congenital anomalies and a birth weight below 2000 gram were excluded. The SAS software, version 9.1, was used (SAS institute Inc, Cary NC).

Results

Table 1 shows the prevalence of risk factors for perinatal mortality among the different ethnic groups. The prevalence of teenage pregnancy, was ten times higher among the African women compared to the Dutch women (2.3%). In addition, African, South Asian, Turkish/Moroccan and other non-Western women lived more often in very urban areas and had a lower social economic status compared to Dutch women. African and other non-Western women were more often late in care. On the contrary heavy smoking was more prevalent in Dutch women. South Asian women more often suffered from diabetes mellitus. Perinatal mortality occurred in 7.9 per 1000 births, with significant ethnic differences (table 2).

Foetal mortality was clearly higher in African, South Asian, other non-Western women and Turkish/Moroccan compared to the Dutch women. Early neonatal mortality was increased among African and other non-Western women mainly caused by deaths within 24 hours after birth (table 2).

African and South Asian women also had the highest risks on preterm births and low birth weight. East-Asian, other Western women and women of unknown ethnicity had a mortality risk that was comparable or even lower than in Dutch women (table 2).

A separate introduction of each risk factor to the model showed that adjustment for urbanisation increased the ethnic differences and adjustment for booking visit after 18 weeks reduced the ethnic differences on both mortality outcomes (table 3). Separate adjustment for age, SES, or pre-existent disease did not alter the ethnic differences on foetal and early neonatal mortality (data not shown).

Table 4 presents the crude and total adjusted odds ratios for different risk factors on foetal and early neonatal mortality. Compared to the Dutch women the foetal mortality risks of both African and South Asian women were 1.7 (95% CI 1.4 to 2.1) and 1.8 (1.4 to 2.3) respectively. The risk for early neonatal mortality was only increased for other non-Western women 1.3 (95% CI 1.0 to 1.8)

The addition of preterm birth did not alter the increased foetal risk for African and South Asian women while the effects for Turkish-Moroccan and other non-Western women increased with OR 1.4 (1.2 to 1.6) and OR 1.5 (1.2 to 1.8) respectively (data not shown). The early neonatal mortality risk for African (OR 1.4 (1.0 to 2.0) women became significant and the odds of other non-Western women (OR 1.5 (1.1 to 2.1) increased (data not shown). When the analysis was limited to the 264.502 (48% of the study group) women without the risk factors, the ethnic differences became even higher. The risk on foetal mortality for African and South Asian women were respectively OR 2.2 (1.1 to 4.0) and OR 2.3 (1.2 to 4.6) (Table 5). In early neonatal mortality the odds ratio for African women was increased to OR 3.1 (1.1 to 8.3). Additional adjustment for age did not change these effects.

Comments <Principal findings>

Important ethnic differences exist in stillbirth and early neonatal mortality among nulliparous women in the Netherlands especially among African, South Asian and other non-Western women. Teenage pregnancy, living in very urban areas, lower social economic status and late in care were more prevalent among all non-Western ethnic groups. Moreover, South Asian women had diabetes mellitus more often. These ethnic differences were more pronounced in foetal mortality risk. Living in urban areas was slightly protective, maternal age, SES and pre-existent medical conditions showed no effect on ethnic differences in mortality.

Booking visist after 18 weeks gestation explained partly the ethnic differences in mortality.

The ethnic differences in mortality became even more visible in women without these risk factors. Women from other Western or East Asian origin have lower foetal and neonatal mortality risk compared to the Dutch women. <Strengths and weaknesses of the study> The nearly complete coverage of all births makes the large linked national perinatal database suitable for analyzing the effect of different ethnic groups on both foetal and neonatal mortality. A study limitation is the definition of ethnicity which is based on the information from the healthcare providers. Turkish and Moroccan women are from different ethnic origin but are registered in one group. The information of the ethnic origin of the father is not recorded and possibly some African women are defined as other non-Western. Another limitation of this study is the possible insufficient adjustment for SES since it was measured on postcode area, an effect of SES could be present if measured on the individual level.

Underreporting by the health care providers of maternal pre-existing risks factors and especially of heavy smoking during pregnancy could also have affected the results, however we have no reason to assume that underreporting is related to ethnicity of the women.

Women with unknown ethnicity had comparable mortality risks as the Dutch women. The percentage of missing values of outcomes and risk factors did not differ between the ethnic groups and were overall below 2%. The 4% missing birth data in the registry are due to non reporting midwives and general practitioners (low risk pregnancies), while 99% of the obstetricians participate in the registry.

The perinatal registry does not contain information on maternal education, body mass index, marital/relationship status and more detailed information about utilization of health care, that might explain the ethnic differences found. Since 2009, this information as well as the country of both parents is included in the revised national perinatal registry. In this study only nulliparous women were included but the ethnic differences are also visible in the total population from 22 weeks onwards including multiple births and pregnancies conceived after assisted reproductive technology. [START_REF] Ravelli | Decreasing perinatal mortality in the Netherlands, 2000-2006: a record linkage study[END_REF] <Relation to other studies > The finding of elevated perinatal mortality among ethnic minorities is in accordance with earlier international studies. 3 8 In the UK, effects on perinatal mortality for African and South Asian women compared to white women were also found, where black women had the largest risks. [START_REF] Balchin | Racial variation in the association between gestational age and perinatal mortality: prospective study[END_REF] The increased risk of African women on neonatal mortality in the first month of life and total infant mortality was also seen in the USA. [START_REF] Hessol | Ethnic differences in neonatal and postneonatal mortality[END_REF] No elevated risk of the East-Asian women compared to the Caucasian women was reported in Norway. [START_REF] Vangen | The heavier the better? Birthweight and perinatal mortality in different ethnic groups[END_REF] The finding on ethnic differences in foetal mortality is new. In previous studies in Europe the differentiation of perinatal mortality in foetal and early neonatal mortality lacks on a national level. Increased perinatal mortality differences among ethnic minorities as was found in other studies. 10 11,20, 21 The healthy migrant effect is a well known phenomenon and this might explain the low foetal and early neonatal mortality in other Western and East Asian women compared to the Dutch women. The higher prevalence of teenage pregnancy among non-Western women is reported as explanation for the elevated perinatal mortality risk. [START_REF] Garssen | Perinatal mortality in the Netherlands Backgrounds on worsening international ranking[END_REF] Our study does not confirm this. The crude mortality risk for teenage African women was even lower than the mortality risk for older African women (data not shown) which was also shown in black newborns in the USA. [START_REF] Hessol | Ethnic differences in neonatal and postneonatal mortality[END_REF] This possibly suggests earlier maturation for procreation of women of African descent. Living in a very urban or rural area has been reported as a risk factor for perinatal mortality. However, we found that living in very urban areas was favourable for non-Western women. Perhaps the easier access (travel distance) and a more adapted health care services to non-Western groups in urban areas plays a role. [START_REF] Larson | Is non-metropolitan residence a risk factor for poor birth outcome in the U.S.?[END_REF] The high prevalence of low SES in non-Western women is another frequently used explanation of the elevated risks among these groups. 24 25 Although low SES is related to more mortality we did not find that low SES explained the elevated mortality risks among the non-Western women as was also found in Denmark. [START_REF] Villadsen | Ethnic disparity in stillbirth and infant mortality in Denmark 1981-2003[END_REF] In the Netherlands there is guaranteed access to maternity care at all levels as part of the national health care system, but this opportunity is possibly not fully taken by women from non-Western origin through possible language, cultural or knowledge barriers. In Norway an audit of stillbirths revealed a strong association of sub-optimal obstetric care in non-Western women compared to Western women. [START_REF] Saastad | Suboptimal care in stillbirths -a retrospective audit study[END_REF] This can be reason of late start of antenatal care and not alarming a caregiver in time, less compliance to treatment or advise and possibly reticent policy of caregivers. [START_REF] Alderliesten | Late start of antenatal care among ethnic minorities in a large cohort of pregnant women[END_REF][START_REF] Essen | Are some perinatal deaths in immigrant groups linked to suboptimal perinatal care services?[END_REF] We found on average that 15% of the pregnant women started prenatal care after 18 weeks while among African and other non-Western women these percentages were doubled. However, in our study the ethnic differences in mortality were also visible in the earlier starters as was also shown in the USA. [START_REF] Healy | Early access to prenatal care: implications for racial disparity in perinatal mortality[END_REF] Language problems are less obvious in the African and South Asian women who both came from Surinam, a former Dutch colony. Women with pre-existing diseases are at elevated risk for perinatal mortality, however adjusting for or even excluding women with pre-existing diseases did not change the ethnic differences on foetal and neonatal mortality.

<meaning of the study, questions and proposals> The ethnic differences apparent in especially in foetal mortality are remarkable. Our study revealed that excess in perinatal mortality among African, South Asian and other non-Western women could not be explained by the generally supposed explaining risk factors (teenage pregnancy, living in very urban areas and low SES). A possible explanation is that cultural or knowledge barriers affect the access of women to regular care, or affect its effectiveness once women got access to care. Another possibility is a biological working mechanism. It could be relevant for example to evaluate if the optimum duration of pregnancy in the African, South Asian and other non-Western women is different from the Dutch women. [START_REF] Balchin | Racial variation in the association between gestational age and perinatal mortality: prospective study[END_REF] The need for tailored perinatal care for women from African and South Asian ethnic background in case of preconception care, screening and risk factor management should be focus of research. Because there are ethnic differences in foetal and neonatal mortality in the Netherlands, it should be stressed to record race/ethnicity of the patient because of its potential implications for decisional based care paths. [START_REF] Agyemang | Racism in health and health care in Europe: where does the Netherlands stand?[END_REF]

Table 1 Characteristics (%) of the 554,234 pregnant nulliparous women by ethnic group

 1

	Ethnic group	Dutch	Other	Turkish/	African South	East	Other	Unknown Total	Chi
			Western	Moroccan		Asian	Asian	Non-			Square
								Western			p-value
	Pregnant women (number)	460,543	15,007	34,973 11,605 5,853 10,675	12,541	3,037		554,234
	Pregnant women (%)	83.1	2.7	6.3	2.1	1.1	1.9	2.3	0.5 100.0
	Maternal age at birth (years)										<0.0001
	< 20	2.3	4.3	7.7	19.9	6.7	7.4	10.9	6.0	3.5
	20-24	12.6	20.1	43.8	33.6	29.3	22.0	26.0	21.7 15.9
	25-34	74.2	62.4	43.6	40.9	56.7	59.4	52.9	63.7 70.2
	35-39	9.8	11.7	4.2	4.7	6.4	9.7	8.7	7.7	9.3
	>= 40	1.1	1.5	0.9	1.0	1.0	1.6	1.4	0.9	1.1
	mean (year)	29.1	28.5	25.2	24.6	26.7	27.8	26.9	27.6 28.6
	Urbanisation										<0.0001
	Very urban	17.0	35.9	48.7	55.3	55.1	33.6	41.6	32.4 21.7
	urban/rural	64.1	55.6	49.1	40.9	42.0	59.7	51.7	57.1 61.8
	Very rural	18.9	8.5	2.2	3.9	3.0	6.7	6.7	10.5 16.5
	SES										<0.0001
	high	10.4	9.7	2.9	3.8	6.8	9.5	7.2	7.7	9.6
	middle	83.3	74.8	61.4	57.2	59.3	72.7	69.7	76.7 80.3
	low	6.3	15.5	35.7	39.1	33.9	17.9	23.1	15.5 10.1
	Low income	35.3	36.8	61.1	64.1	58.2	42.2	46.8	42.3 38.2 <0.0001
	Booking visit ≥ 18 weeks	13.2	23.0	20.4	32.7	22.8	24.0	28.4	17.4 15.0 <0.0001
	Heavy smoking	0.6	0.4	0.3	0.2	0.1	0.2	0.1	0.4	0.4 <0.0001
	Pre-existent disease
	essential hypertension	0.6	0.3	0.2	0.5	0.5	0.2	0.3	0.2	0.5 <0.0001
	diabetes mellitus	0.2	0.1	0.2	0.3	0.6	0.1	0.1	0.0	0.2 <0.0001
	other endrocrinological diseases	0.4	0.5	0.3	0.2	0.7	0.3	0.4	0.2	0.4 <0.0001
	systemic disease	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.0	0.1	0.03
	cardiac disease	0.1	0.1	0.1	0.1	0.2	0.1	0.1	0.0	0.1	0.46
	trombo-embolitical disorders	0.3	0.1	0.1	0.2	0.2	0.1	0.1	0.1	0.2 <0.0001
	psychiatric disorders	0.3	0.2	0.4	0.4	0.3	0.2	0.3	0.1	0.3	0.002

Table 2 Mortality and other adverse outcome by ethnic group

 2

	Ethnic group	Dutch	Other	Turkish/	African South	East	Other	Unknown	Total	Total	Chi
			Western	Moroccan		Asian	Asian	Non-				Square
								Western
	Number of births	460,543	15,007	34,973 11,605 5,853 10,675	12,541	3,037		554,234 p-value
	Percentage	83%	3%	6%	2%	1%	2%	2%	1%
	Perinatal mortality per 1000 total births	7.6	5.7	9.8	13.9	12.5	6.6	11.4	8.2	7.9	4,401 <0.0001
	Foetal mortality per 1000 total births	5.4	4.5	7.1	10.6	9.9	5.1	8.0	5.9	5.7	3,135 <0.0001
	during gestation	4.3	3.7	5.4	8.4	8.2	4.1	6.9	5.9	4.5	2,508 <0.0001
	during labour	1.1	0.7	1.7	2.2	1.7	0.9	1.0	$	1.1	627	0.0001
	Early neonatal mortality per 1000 live
	births	2.3	1.3	2.7	3.3	2.6	1.5	3.5	2.3	2.3	1,266	0.0008
	within 24hours after birth	1.3	0.7	1.8	2.4	1.6	0.9	2.5	1.3	1.3	742 <0.0001
	early, day 1-6	1.0	0.5	0.9	1.0	1.0	0.6	1.0	1.0	1.0	524	0.694
	Preterm births %	7.8	6.3	5.6	8.9	9.5	6.1	7.2	4.8	7.6	42,299 <0.0001
	Low birth weight %	6.6	5.0	6.1	10.0	14.3	6.2	7.6	5.0	6.7	36,970 <0.0001
	Congenital anomalies %	2.5	2.4	2.6	2.6	2.6	2.3	2.5	1.8	2.5	13,839	0.115
	$

due to low numbers no value

Table 3 Effects of ethnicity on foetal mortality and early neonatal mortality after adjustment for urbanisation or for booking visit

 3

	Foetal mortality	Crude	adjusted for	adjusted for
			urbanisation	booking visit
			OR 95%CI	OR 95%CI
	Ethnicity
	Dutch	1.0	1.0		1.0
	Other Western	0.8 0.7 1.1	0.9	0.7 1.1	0.8 0.6 1.0
	Turkish/Moroccan	1.3 1.2 1.5	1.4	1.2 1.6	1.3 1.1 1.4
	African	2.0 1.7 2.4	2.1	1.8 2.5	1.7 1.4 2.1
	South Asian	1.9 1.4 2.4	2.0	1.5 2.6	1.7 1.3 2.2
	East Asian	0.9 0.7 1.2	1.0	0.7 1.3	0.9 0.7 1.1
	Other non
	Western	1.5 1.2 1.8	1.6	1.3 1.9	1.3 1.1 1.6
	Unknown	1.1 0.7 1.8	1.1	0.7 1.8	1.1 0.7 1.7
	Early neonatal
	mortality	Crude	adjusted for	adjusted for
			urbanisation	booking visit
			OR 95%CI	OR 95%CI
	Ethnicity
	Dutch	1.0 reference	1.0		1.0
	Other Western	0.6 0.4 0.9	0.6	0.4 0.9	0.5 0.3 0.8
	Turkish/Moroccan	1.2 1.0 1.5	1.3	1.0 1.6	1.1 0.9 1.4
	African	1.5 1.1 2.0	1.6	1.1 2.2	1.3 0.9 1.7
	South Asian	1.1 0.7 1.9	1.2	0.7 2.0	1.1 0.6 1.8
	East Asian	0.7 0.4 1.1	0.7	0.4 1.1	0.6 0.4 1.0
	Other non
	Western	1.5 1.1 2.1	1.6	1.2 2.2	1.3 1.0 1.8
	Unknown	1.0 0.5 2.2	1.1	0.5 2.2	1.0 0.5 2.1

Table 4 Crude and total adjusted odds ratio's (95% CI) of risk factors for foetal and early neonatal mortality

 4

						Early neonatal	Early neonatal
	Risk factors	Foetal mortality	Foetal mortality		mortality	mortality
			crude	total adjusted *1		crude	total adjusted *
		n=3.135/554.234			n=1.266/551.099
		Odds	95%CI	Odds	95%CI	Odds	95%CI	Odds	95%CI
	Ethnicity
	Dutch	1.0 reference	1.0 reference	1.0	reference	1.0 reference
	Other Western	0.8 0.7 1.1	0.8 0.6 1.0	0.6	0.4 0.9	0.5	0.3 0.8
	Turkish/Moroccan	1.3 1.2 1.5	1.3 1.1 1.4	1.2	1.0 1.5	1.1	0.9 1.4
	African	2.0 1.7 2.4	1.7 1.4 2.1	1.5	1.1 2.0	1.2	0.9 1.7
	South Asian	1.9 1.4 2.4	1.8 1.4 2.3	1.1	0.7 1.9	1.1	0.7 1.8
	East Asian	0.9 0.7 1.2	0.9 0.7 1.1	0.7	0.4 1.1	0.6	0.4 1.0
	Other non Western	1.5 1.2 1.8	1.3 1.1 1.6	1.5	1.1 2.1	1.3	1.0 1.8
	Unknown	1.1 0.7 1.8	1.1 0.7 1.7	1.0	0.5 2.2	1.0	0.5 2.1
	Age (years)
	<20	1.5 1.2 1.7	1.1 0.9 1.3	1.6	1.3 2.1	1.3	1.0 1.7
	20-24	1.3 1.2 1.4	1.1 1.0 1.2	1.2	1.0 1.4	1.1	0.9 1.3
	25-34	1.0 reference	1.0 reference	1.0	reference	1.0 reference
	35-39	1.3 1.2 1.5	1.3 1.2 1.5	1.2	1.0 1.5	1.2	1.0 1.5
	>= 40	1.9 1.5 2.5	1.9 1.4 2.4	1.5	1.0 2.4	1.5	0.9 2.3
	Urbanisation
	Very urban	1.0 0.9 1.1	0.8 0.7 0.9	0.9	0.8 1.1	0.9	0.7 1.0
	urban/rural	1.0 reference	1.0 reference	1.0	reference	1.0 reference
	Very rural	1.1 0.9 1.1	1.0 0.9 1.1	1.1	0.9 1.3	1.1	0.9 1.2
	SES
	high	1.0 reference	1.0 reference	1.0	reference	1.0 reference
	middle	1.3 1.2 1.5	1.3 1.1 1.5	1.2	1.0 1.4	1.2	0.9 1.4
	low	1.6 1.4 1.9	1.4 1.2 1.7	1.2	1.0 1.6	1.2	0.9 1.7
	Low income	1.2 1.1 1.3	1.1 1.0 1.2	1.0	0.9 1.2	1.0	0.8 1.1

Total adjusted for age, urbanisation, SES, low income, booking visit, smoking and pre-existent diseasesTable 5

 5 Crude odds ratio's (95% CI) for ethnicity on foetal and early neonatal mortality for 264502 women without risk factors * Women with age below 20 or above 40 years, low SES, low income level, booking visit ≥ 18 weeks gestation, heavy smoking, pre-existent diseases, gestational age below 32 weeks, congenital anomalies and a birth weight below 2000 gram were excluded.

		Foetal mortality		Early neonatal mortality
			crude			crude
	Ethnicity
	Dutch	1.0	Reference	1.0	Reference
	Other Western	0.8	0.5	1.5	1.2	0.5	3.0
	Turkish/Moroccan	1.4	0.8	1.7	1.2	0.6	2.6
	African	2.2	1.1	4.0	3.1	1.1	8.3
	South Asian	2.3	1.1	4.6	$
	East Asian	0.9	0.4	1.7	$
	Other non Western	0.8	0.4	1.6	$
	$ due to low numbers no value

*

Acknowledgements

The authors would like to thank all Dutch midwives, obstetricians, neonatologist and other perinatal health care providers for the registration of perinatal information. We also like to thank the Foundation of the Netherland Perinatal Registry (www.perinatreg.nl) for the permission of using the registry data. In the PRN registry the LVR1, LVR2 and LNR registries from the Royal Dutch Midwifery Organization (KNOV), the Dutch Association for Obstetrics and Gynaecology (NVOG) and the Pediatric Association of the Netherlands (NVK) are combined to one perinatal database.

The Dutch Perinatal Registry has given permission for the analysis of the

Funding

None

data. Anonymous registry data: no ethical approval needed.

Competing interests

None


Richard G Brown
email: richard.g.brown@kcl.ac.uk

PhD Sabine Landau

PhD; John V Hindle

FRCP Jeremy R Playfer

MD, FRCP Michael Samuel

MD; Kenneth C Wilson

PhD Catherine S Hurt

PhD Rachel Anderson

BSc Joanna Carnell

Lucy Dickinson

BSc Grant Gibson

BSc Rachel Van Schaick

Katie Sellwood

BSc Bonnita A Thomas

David J Burn

MD; for the PROMS-PD Study Group

Depression and anxiety subtypes in Parkinson's disease

Keywords: Parkinson's disease, depression, anxiety, heterogeneity, latent class analysis

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche

INTRODUCTION

As well as causing of distress, depression in Parkinson's disease has been associated with greater functional disability, [START_REF] Brown | Depression and disability in Parkinson's disease: A follow-up of 132 cases[END_REF] rate of physical and cognitive decline, [START_REF] Starkstein | A prospective longitudinal study of depression, cognitive decline, and physical impairments in patients with Parkinson's disease[END_REF] dementia risk, [START_REF] Tandberg | The occurrence of depression in Parkinson's disease. A communitybased study[END_REF] and mortality, [START_REF] Hughes | Mortality in Parkinson's disease and its association with dementia and depression[END_REF] and reduced quality of life. [START_REF] Schrag | What contributes to quality of life in patients with Parkinson's disease?[END_REF] Anxiety is also a prominent feature of PD [6;7] although implications for outcome are less well understood. Evidence for the efficacy of pharmacotherapy remains weak and often inconsistent, [8;9] although there is a growing number of trials of pharmacological and psychological interventions for depression (e.g. see http://ClinicalTrials.Gov). Trials are needed in PD anxiety although there is some evidence that anxiety symptoms improve in depressed PD patients treated with the SSRI citalopram. [START_REF] Menza | Citalopram treatment of depression in Parkinson's disease: the impact on anxiety, disability, and cognition[END_REF]. Although depression and anxiety symptoms can occur in isolation in PD, they frequently co-exist. [11;12] While some have suggested that this reflects the co-morbidity of distinct pathophysiological substrates, [START_REF] Negre-Pages | Anxious and depressive symptoms in Parkinson's disease: the French cross-sectionnal DoPaMiP study[END_REF] others argue that anxious-depression represents a specific and common depressive subtype in PD. [START_REF] Walsh | Parkinson's disease and anxiety[END_REF] The relationship between depression and anxiety has important implications for treatment and clinical trials. Non-PD patients with Major Depression (MD) with marked anxiety (anxious-depression) show poorer outcomes than those with MD alone when treated with SSRI's and when switched/augmented with other agents. [START_REF] Fava | Difference in treatment outcome in outpatients with anxious versus nonanxious depression: a STAR*D report[END_REF] If anxious-depression is common in PD, similar reduced treatment efficacy may be anticipated, while combining subtypes could produce aggregate results that do not adequately inform the response to treatment of either subgroup.

Broader clinical heterogeneity of PD has been examined using empirical approaches such as cluster analysis (CA) or latent class analysis (LCA). [START_REF] Van Rooden | The identification of Parkinson's disease subtypes using cluster analysis: a systematic review[END_REF] In contrast to pre-specified clinical or theoretically defined subgroups, data-driven methods seek to identify clusters of patients defined by low intra-group but high inter-group differences across a set of selected variables. Applying similar methods to depression and anxiety related symptoms may identify empirical subgroups that then become available for validation and further study. Differences in pathophysiology and other factors involved in symptom onset and maintenance could guide the development of more targeted treatment. The present study describes the first use of a data-driven approach to explore possible heterogeneity in depressive and anxiety-related symptoms in patients with PD.

METHODS

Eligibility and recruitment

Patients were recruited over a 12-month period from specialist PD or movement disorder outpatient clinics (Ethics ref 07/MRE01/9) as part of a longitudinal study (PROMS-PD) (UKCRN ID 2519).

Inclusion criteria were a clinical diagnosis of idiopathic PD, [START_REF] Hughes | Accuracy of clinical diagnosis of idiopathic Parkinson's disease: A clinicopathological study of 100 cases[END_REF] the ability to provide informed consent at entry into the study, and living within two hours travel time of a study research centre. Exclusion criteria were the presence of sensory loss or communication difficulty (including inadequate command of English) sufficient to interfere with assessment. Patients with cognitive impairment or severe psychiatric disorder were excluded only if these interfered with the capacity to consent. The eligibility criteria were deliberately chosen to include as broad a range of patients as possible in terms of age and clinical severity.

Assessments

Participants were assessed over two sessions, typically in their own home. Where possible patients were assessed in their motor 'on' state. Depressive, anxiety and related symptoms were assessed using a semistructured interview based on the Geriatric Mental State (GMS), [START_REF] Copeland | A semi-structured clinical interview for the assessment of diagnosis and mental state in the elderly: the Geriatric Mental State Schedule. I. Development and reliability[END_REF] an instrument designed to assess psychopathology in older adults including those with significant cognitive impairment. Items were rated 0=absent or 'normal'; 1=present but not prominent (mild to moderate intensity, infrequent or fleeting), 2=prominent (severe, frequent or persistent). Symptoms were rated as reported or observed. [START_REF] Marsh | Provisional diagnostic criteria for depression in Parkinson's disease: report of an NINDS/NIMH Work Group[END_REF] Selfreported depression and anxiety was assessed using the Hospital Anxiety and Depression Scale (HADS), [START_REF] A S Zigmond | The Hospital Anxiety and Depression Scale[END_REF] with subscale scores of ≥11 indicating significant symptoms. Motor function and disability was assessed using the Unified Parkinson's Disease Rating Scale (UPDRS) [START_REF] Fahn | members of the UPDRS development committee. Unified Parkinson's Disease Rating Scale[END_REF] Parts II and III, Hoehn and Yahr Scale [START_REF] Hoehn | Parkinsonism: onset progression and mortality[END_REF] and Schwab and England Scale. [START_REF] Schwab | Projection technique for evaluating surgery for Parkinson's disease[END_REF] The ratio of tremor to non-tremor motor symptoms was calculated as previously described. [START_REF] Lewis | Heterogeneity of Parkinson's disease in the early clinical stages using a data driven approach[END_REF] Cognition was assessed using the Addenbrooke's Cognitive Examination -Revised (ACE-R), [START_REF] Mioshi | The Addenbrooke's Cognitive Examination Revised (ACE-R): a brief cognitive test battery for dementia screening[END_REF] with a score of ≤83 indicating significant impairment. [START_REF] Reyes | Addenbrooke's Cognitive Examination validation in Parkinson's disease[END_REF] Annual assessments are planned for up to 4 years.

Statistical analysis

Latent Class Analysis (LCA) can identify unobserved (latent) subgroups of cases that share similar symptoms from a set of multivariate categorical data. Model-based CA methods such as LCA avoid a criticism of conventional heuristic CA approaches such as linkage that do not provide a formal basis for PAGE 6 determining the optimal number of clusters or indeed the existence of more than a single cluster. LCA provides inferences for statistically comparing the fit of solutions across different numbers of clusters, and is also better able to handle missing data. We employed LCA of binary symptom variables using M-Plus software. [START_REF] Muthen | Mplus User's Guide[END_REF] Subjects were allocated to classes based on the maximum estimate of a posteriori probability of cluster membership (Maximized Allocation Probability method). Individual GMS symptoms were dichotomized as 'prominent' (score=2), or 'absent or present but not prominent' (score<2), to provide a classification based on the presentation of symptoms with clear clinical significance. Symptoms with a frequency of less than 2% (N<10) were excluded. In total 28 items were identified as relevant to phenotyping (see fig 1).

Preliminary assessment of validity was conducted by examining the association between the defined classes and other variables not included in the LCA. One-way analysis of variance (ANOVA) with paired comparisons (Tukey's HSD, P<0.05) was used to determine whether LCA classes could be discriminated on continuous demographic and clinical variables, and χ 2 for categorical data. Finally, multinomial logistic regression was used to examine clinical and demographic predictors of class membership. This latter analysis included a range of dichotomized measures associated with broad clinical heterogeneity in previous studies. [START_REF] Van Rooden | The identification of Parkinson's disease subtypes using cluster analysis: a systematic review[END_REF]

RESULTS

Sample

941 patients were invited to participate of whom 525 consented but 12 subsequently withdrew before completing the initial assessment. Complete assessment was obtained from 513. The sample had a wide range of disease durations and severities although the majority were in Hoehn and Yahr stages II-III (table 1) with a non-tremor dominant motor profile. [START_REF] Carter | Living with a person who has Parkinson's disease: The spouse's perspective by stage of disease[END_REF] Motor symptom onset preceded the age of 50 in 18.7%. Approximately 30% of participants showed evidence of significant cognitive impairment on the ACE-R.

On the HADS 13% reported marked depressive symptomatology and 22% anxiety. Mild to moderate symptoms (HADS subscale score 8-10) were reported in a further 22% and 23% respectively. Almost all (94.9%) were taking antiparkinsonian drugs, and 24% reported taking antidepressant and/or anxiolytic medication.

PAGE 7

Figure 1 shows the ranked profile of 28 GMS items. The most frequent prominent symptoms were worry (33.5%), subjective tension and restlessness (33.2%) and fatigue (27.6%). Symptoms such as excessive energy, belief about punishment and suicidal thinking were rare (<2%, data not shown) and not included in the LCA.

Empirically defined patient subgroups

Seven LCA solutions were evaluated, with a 4-class solution providing the best overall fit across a range of information indices (table 2). The median allocation probability was 99% with 90% of the sample allocated to their final class with a probability of 80% or more. The largest class (class 4) included 59.4% of the sample, class 3 22%, and classes 1 and 2 8.9% and 9.0% respectively.

Interpreting classes from cluster-based methods is a largely non-empirical process and involves visually identifying those features which most clearly (a) define an individual class and (b) distinguish it from other classes. Figure 2a and 2b shows the symptom probability profiles for the 28 variables for each of the individual class. Features of classes 1 and 2 were sad and/or empty mood, loss of interest, slow thinking, loss of confidence, lack of energy and poor concentration. In contrast, loss of enjoyment was rare and symptoms of hopelessness, worthlessness and guilt relatively uncommon, particularly in class 2. In addition, and in contrast to class 2, patients in class 1 also showed high probabilities of prominent worry and tension/restlessness, irritability, general anxiety/panic, specific fear and autonomic symptoms of anxiety. Class 3 was marked by a profile of prominent worry and tension/restlessness, and moderate probabilities of other severe anxiety-related symptoms but with a low probability of depressive symptomatology. Finally, class 4 was characterized by overall low probability of prominent symptoms. Entropy: a measure of classification quality, the larger the better, should be approximately 0.9

PAGE 10
Table 3 shows the main demographic and clinical characteristics of the 4 classes. All variables showed highly significant differences (P < 0.001) including when age was used as a covariate. Patients in class 4

tended to be older, with a high proportion of males, later age at onset and shorter disease duration, with lower levels of motor symptoms, disability and medication. Non-tremor symptoms were least common in this class. Class 2 was a similar to class 4 in terms of age, age of onset, duration of PD and level of medication, but showed more cognitive and motor impairment and disability, and somewhat higher levels HADS depression and anxiety. This class also displayed the most pronounced non-tremor dominant symptom profile. Classes 1 and 3 were the youngest with more advanced disease and higher mean levels of antiparkinsonian medication than classes 2 and 4. Class 1 in particular showed early mean age of onset and long disease duration. Class 1 patients also showed greater disability and twice the level of significant cognitive impairment than class 3, and markedly increased levels and rates of anxiety and depression.

Predictors of class membership

Class membership was assessed using multinomial logistic regression with a set of dichotomised demographic and clinical variables: age (<70/≥70 years), gender (female/male), age at diagnosis (<55/≥55 years), PD duration (<5/≥5 years), levodopa equivalent daily dose (LEDD) (<600/≥600mg/day), Schwab and England (<80/≥80), UPDRS III (<23/≥23), ACE-R (<83/≥83). Class 4 was the reference category.

Duration of PD did not independently predict class membership in any model and was therefore not included in the final analysis.

Relative to class 4, membership of class 1 was significantly (P<0.05) predicted by younger age (Odds Ratio (OR)=2.6, 95% confidence interval=1.0-6.6), PD onset less than 55 years (OR=3.

DISCUSSION

This study provides the first detailed investigation of affective heterogeneity in PD using a broad range of indicator variables associated with (although not specific to) the psychiatric constructs of depression and anxiety. The results supported a four class solution with empirically defined subgroups that appear distinctive in terms of clinical and demographic features. We should not equate such subgroups with syndromal disorders, nor probabilistically defined class membership with diagnosis. Nevertheless, it is useful to suggest provisional clinically meaningful labels to the classes. Patients in the largest group (class 4) showed low levels of prominent symptomatology and can be labeled 'Psychologically healthy', although the high symptom threshold does not rule out some problems. Class 3 showed prominent anxiety-related features with a profile similar to Generalized Anxiety Disorder (GAD) [START_REF]American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders Fourth Edition (Text Revision) (DSM-IV-TR)[END_REF] and can be labeled 'Anxious'. The profiles of classes 1 and 2 were more complex. Both showed elevated probabilities of core depressive symptoms of sad or empty mood and loss of interest, along with other supporting features such as poor concentration, slow thinking and fatigue, although loss of enjoyment were largely absent, and feelings of worthless, hopelessness and self-blame evident only in class 1. Patients in class 1 had a higher probability of prominent depression-related symptomatology but also showed marked prevalence of subjective tension, worry and general anxiety or panic and irritability in class 1, suggesting the label 'Anxious-depressed'. Class 2, together with depressive features also showed high levels of cognitive impairment and may reflect a 'Depressed' or possibly 'Apathetic-depressed' subtype.

A recent systematic review and meta-analysis. [START_REF] Van Rooden | The identification of Parkinson's disease subtypes using cluster analysis: a systematic review[END_REF] summarized studies using similar approaches to define broad clinical heterogeneity in PD. Two studies [22;28] independently identified four subgroups of patients labeled 'Young onset', 'Tremor dominant', 'Non-tremor dominant' and 'Rapid disease progression'. Only the 'Non-tremor dominant' group (including akinesia and postural instability and gait symptoms) was associated with increased levels of depression. A young onset cluster, with typically slow rate of progression was associated with low levels of depression, although another study [START_REF] Schrag | Heterogeneity of Parkinson's disease[END_REF] found the opposite result. Other studies, not included in the systematic review, have examined non-motor heterogeneity. One study [START_REF] Kulisevsky | Prevalence and correlates of neuropsychiatric symptoms in Parkinson's disease without dementia[END_REF] analysed 10 broad psychiatric and behavioural symptoms of the Neuropsychiatric inventory (NPI) [START_REF] Cummings | The Neuropsychiatric Inventory: comprehensive assessment of psychopathology in dementia[END_REF] identifying a large 'low-total NPI' subgroup and four others exhibiting predominant apathy, psychosis, depression or anxiety. However, substantial levels of

PAGE 13
depression and particularly anxiety were evident in all of the groups except for the first. Another study considered depression, anxiety, apathy, daytime somnolence and cognition, [START_REF] Mavandadi | Use of latent variable modeling to delineate psychiatric and cognitive profiles in Parkinson disease[END_REF] and defined four subgroups with the largest showing good cognitive and psychiatric function. The remainder showed cognitive impairment, psychiatric impairment or both. No evidence of heterogeneity was found within the psychiatric symptoms themselves, although the use of a single global index for each precluded detailed assessment.

Cluster-based methods are subject to a variety of methodological factors that can influence the robustness and therefore potential validity of the solutions. As noted, LCA has advantages over traditional CA methods in providing a range of indices to inform decisions about the optimal number of clusters. The present results strongly supported a 4-class solution over one with more or fewer subgroups. The high allocation probabilities further suggest that patients were assigned unequivocally to one of the 4 classes.

Ideally, we would have liked to replicate the classification in an independent sample to further confirm its reliability, as done in one similar study using cluster analysis. [START_REF] Kulisevsky | Prevalence and correlates of neuropsychiatric symptoms in Parkinson's disease without dementia[END_REF] Unfortunately, our sample size, although large, was insufficient given the number of parameter estimates being modelled (N=115).

Face validity was high and the subgroups clinically interpretable. Validity was further supported by the associations of the classes with a range of variable not included in the LCA. These converged on evidence from previous studies describing associations between depression and broad clinical and motor phenotypes. Our results support a link between psychopathology and non-tremor dominant motor phenotype, with the 'Healthy' subgroup showed the highest proportion of tremor symptoms and the 'Apathetic-depressed' group the least. Younger age of onset emerged as a distinguishing feature in class 3 but particularly in class 1, along with female gender and higher LEDD. However, unlike the apparently benign psychiatric profile of the 'Young onset' groups of some previous studies [22;28] younger age of onset in the present study was associated with marked anxiety (classes 1 and 3). The present study also suggests that at least some patients with younger onset (class 1) experience significant depressive symptomatology, particularly those with longer duration, advanced disease and greater disability, similar to a previous report. [START_REF] Schrag | Heterogeneity of Parkinson's disease[END_REF] An important question is whether subtypes remain stable or patients move between subtypes. Pending longitudinal study, a partial answer can be found in evidence on the duration of the individual symptoms PAGE 14

that define class membership (see supplementary information). Almost all prominent symptoms reported had been present for most days in the past 6 months in over 80% of patients, and for many, most days in the past 2 years. Although not proving that the subtypes are stable, it suggests that that the symptoms on which they are based are persistent in this sample.

A criticism of cross-sectional studies is that empirically distinct clusters may simply reflect observations of patients at different stages of a homogenous but progressive disease. [START_REF] Van Rooden | The identification of Parkinson's disease subtypes using cluster analysis: a systematic review[END_REF] A number of facts suggest that this is not the case with the present findings. The between-group differences were modest compared to the range observed within the classes, with the 'Anxious' and 'Depressed' subgroups showing almost identical mean disease durations and levels of disability. Also, the logistic regression analysis failed to indicate a role of disease duration in predicting subtype. In contrast, age of onset emerged as a more significant influence, with younger onset (independent of duration) associated with the two anxiety-related subtypes.

The high level of anxiety-related symptomatology in this sample, and specifically in two of the subgroups, is consistent with emerging evidence. [6;7;33] Even when sub-syndromal, GAD-like symptoms of subjective tension, worry and irritability can be a significant source of distress, particularly if chronic, but may be missed without adequate screening. Anxiety-related symptoms are a common feature of depressive disorder although the depressive symptoms are generally held to be primary and the target of treatment. However, anxiety symptoms may turn out to have a high prominence in some PD patients with depression, [START_REF] Merschdorf | Psychopathological symptoms of depression in Parkinson's disease compared to major depression[END_REF] while symptoms such as anhedonia and guilt may be less characteristic with implications for assessment and diagnosis. If anxious-depression is common in PD it would also have important implications for management and outcome. In the elderly, remission rates of co-morbid depression and GAD is only 27% over 3 years compared to 41% for depression and 48% for GAD. [START_REF] Schoevers | Depression and generalized anxiety disorder: co-occurrence and longitudinal patterns in elderly patients[END_REF] In a major trial of citalopram (STAR*D), time to remission was longer for anxious-depression, remission rates were significantly lower and reported side-effects higher. [START_REF] Fava | Difference in treatment outcome in outpatients with anxious versus nonanxious depression: a STAR*D report[END_REF] The broad phenotypic similarity of patients in classes 1 and 3, suggests that these two subtypes may be closely associated. Longitudinal study will test the hypothesis that persistent GAD-like anxiety symptoms are a risk factor for anxious-depression. If so, then early identification and treatment of the chronic anxiety symptoms such as worry and tension may be indicated.

PAGE 15

Heterogeneity of affective disturbance in PD may have implications for pathophysiology. Apatheticdepressive subtype, associated with non-tremor dominant PD and cognitive dysfunction, may have a mainly dopaminergic substrate with involvement of the ventral striatum and associated prefrontal regions, plus higher burden of cortical pathology in addition to the more typical nigro-striatal changes. [START_REF] Selikhova | A clinico-pathological study of subtypes in Parkinson's disease[END_REF] This subtype may have contributed to the previously reported associations between depression and rate of cognitive decline and dementia risk.[2;3] In contrast, non-dopaminergic mechanisms may be the major factor in the anxious-depressed and anxious subtypes. Evidence of serotinergic involvement in depression in PD is inconsistent, [START_REF] Brooks | Imaging non-dopaminergic function in Parkinson's disease[END_REF] (perhaps due to heterogeneity) although there is some support for the involvement of the noradrenergic system in PD depression, [START_REF] Remy | Depression in Parkinson's disease: loss of dopamine and noradrenaline innervation in the limbic system[END_REF] with monoamine uptake ([ 11 C]RTI 32) in noradrenergic areas related to the severity of the anxiety but not depressive symptoms.

Demonstrating heterogeneity of depression in PD has implications for clinical trials. If studies combine patients with potentially different pathophysiology the outcome may be different response rates to treatment. This dilution of effect may account, at least in part, for the apparently poor overall response rates observed with conventional SSRI-based antidepressants in PD. [START_REF] Weintraub | Antidepressant studies in Parkinson's disease: a review and meta-analysis[END_REF] Selecting homogeneous samples with specifically targeted treatment may provide more reliable and useful evidence to focus clinical management.

The present study used convenience sample of outpatient clinic attenders. The rates of significant selfrated depressive (13%) and anxiety-related (22%) symptomatology from the HADS were comparable to that reported in two recent validation studies with figures of 17% and 20%, [START_REF] Marinus | Evaluation of the hospital anxiety and depression scale in patients with Parkinson's disease[END_REF] and 14% and 22% [START_REF] Rodriguez-Blazquez | Psychometric Attributes of the Hospital Anxiety and Depression Scale in Parkinson's Disease[END_REF] respectively, and with very similar mean scores. However, the purpose of the present study was not to define the prevalence of the subtypes (the absolute values for which should be treated with caution) but to identify their characteristics and associated features. Studies in different populations may produce different prevalence rates of the 4 subtypes, just as the rate of the 'rapid disease-progression and old ageat-onset' clinical subtype described in other studies has varied from 6 to 64%. [START_REF] Van Rooden | The identification of Parkinson's disease subtypes using cluster analysis: a systematic review[END_REF] Such variability in prevalence, however, does not challenge the validity of the subtype.

In conclusion, this study suggests that models of depression and anxiety as distinct unitary conditions in PD may be inaccurate. GAD-like anxiety symptoms were amongst the most common in the sample and occurred both independently and in association with significant depressive symptoms. The distinctive PAGE 16 clinical and demographic profiles suggest that anxious-depression may represent a subtype distinct from depression. Such heterogeneity may need to be considered when carrying out research into the pathophysiological substrate of affective disorder in PD and in planning current management and future clinical trials. Frequencies of selected symptoms from the GMS, rated present but not prominent (white bar) or prominent (grey bar) Figure 2a.

Symptom profiles (predicted probabilities) for class 1 (N=44) (--) and class 2 (N=46) (--) (NB Symptoms are organized for ease of interpretation rather than by frequency).

Figure 1 .

 1 Figure 1.

Figure 2b .

 2b Figure 2b. Symptom profiles (predicted probabilities) for class 3 (--) (N=113) and class 4 (--) (N=310)

Table 1 .

 1 Demographic, social and clinical characteristics at the time of assessment (N=513)

	Variable

Table 2 .

 2 Latent Class Analysis: Model fit indices comparing different cluster solutions (well fitting

	indices are underlined)
				Number of clusters (k)
	Index	1	2	3	4	5	6	7
	AIC 16718	8059	7883	7810	7805	7790	7832
	BIC 16863	8300	8247	8297	8416	8523	8689
	VLMR LR-	n.a.	<0.0001	0.051	0.115	0.845	0.262	0.380
	test (p-value)
	Entropy	1.00	0.895	0.874	0.885	0.797	0.834	0.856
	Estimated	100%	33.3%	13.7%	8.9%	8.2%	4.5%	15.4%
	class probabilities		66.7%	24.9%	9.0%	7.0%	4.5%	8.0%
				61.4%	22.7%	19.4%	4.8%	5.6%
					59.4%	27.2%	19.3%	13.3%
						38.2%	23.5%	8.2%
							43.2%	45.9%
								3.5%

AIC: Akaide's information criterion: lower values indicate better model fit, known to favor complex solutions BIC: Bayesian information criterion: lower values indicate better model fit, known to select less complex solutions VLRM: Vuong-Lo-Mendell-Rubin Likelihood Ratio Test: significance test to compare the null hypothesis of k-1 clusters with the alternative of k clusters

Table 3 .

 3 Demographic, social and clinical characteristics of the four latent classes (Mean(SD)/%).

	Variable	Class 1	Class 2	Class 3	Class 4	Paired
		(N=44)	(N=46)	(N=113)	(N=310)	differences
	Demographic and social
	• Age (Years)	61.4 (9.0)	70.3 (10.2)	65.2 (9.2)	69.4 (10.4)	13<24
	• Gender (% Female)	40.9%	37.0%	52.2%	27.4%	-
	Parkinson's disease, symptoms and treatment
	• Age at PD onset (Years)	51.6 (10.8)	62.9 (13.0)	57.6 (10.9)	63.2 (11.7)	1<234, 3<24
	• Duration (Years)	9.6 (8.2)	7.6 (6.2)	7.7 (6.1)	6.1 (5.4)	1>4
	• UPDRS-III (Total score)	33.0 (14.6)	30.5 (10.6)	27.0 (10.9)	24.9 (11.7)	1>34, 2>4
	• Tremor/non-tremor ratio	0.52 (0.59)	0.40 (0.36)	0.48 (0.47)	0.73 (0.88)	4>3,2
	• % Hoehn and Yahr stage ≥III	68.3%	54.3%	60.2%	28.5%	-
	• Schwab and England	64.1 (24.0)	71.7 (18.1)	73.7 (15.4)	79.5 (15.7)	4>123, 3>1
	• LED (mg/day)	1129 (1171)	728 (439)	1017 (763)	694 (608)	1>24,3>4
	Cognitive function
	• ACE-R (Total score)	83.1 (13.3)	81.0 (11.9)	89.1 (8.7)	86.7 (10.3)	12<3, 2<4
	• % below cutoff (≤83)	34.1%	52.2%	16.8%	26.0%	-
	• MMSE (Total score)	26.8 (3.5)	26.6 (2.6)	28.6 (2.0)	28.0 (2.3)	1<3, 2<34
	Depression and Anxiety
	• HADS-Depression	10.7 (4.1)	8.8 (6.7)	6.7 (3.0)	5.2 (3.2)	12>34, 3>4
	• % above cutoff (≥11)	50.0%	33.3%	9.9%	6.4%	-
	• HADS-Anxiety	12.5 (3.8)	8.4 (3.8)	9.6 (3.8)	5.4 (3.8)	1>234, 23>4
	• % above cutoff (≥11)	66.7%	25.6%	34.2%	10.7%	-
	• Total score	23.2 (6.5)	17.2 (5.1)	16.3 (5.8)	10.6 (6.1)	1>234, 23>4

ACKNOWLEDGEMENTS

The authors acknowledge support from: Parkinson's UK; NIHR Biomedical Research Centre for Mental Health at the South London and Maudsley NHS Foundation Trust (SLaM) and Institute of Psychiatry, King's College London; NIHR Dementias and Neurodegenerative Diseases Research Network (DeNDRoN); Wales Dementias and Neurodegenerative Diseases Research Network (NEURODEM Cymru); NIHR Mental Health Research Network (MHRN); British Geriatric Society.