MSc Rachel Bakker

BSc Liane E Pluimgraaff

MD Eric Ap Steegers

MD Hein Raat

MD Henning Tiemeier

MD Albert Hofman

MD Vincent Wv Jaddoe
email: v.jaddoe@erasmusmc.nl

Associations of light and moderate maternal alcohol consumption with fetal

Keywords: alcohol consumption, fetal growth, pregnancy, cohort study alcohol consumption, fetal growth, pregnancy, cohort study SD=Standard Deviation, CI=Confidence Interval, OR=Odds Ratio

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.

Methods This study was based on 7333 pregnant women participating in a population-based cohort study. Alcohol consumption habits and fetal growth were assessed in early (gestational age <17.9 weeks), mid-(gestational age 18 -24.9 weeks) and late pregnancy (gestational age ≥25 weeks). We assessed the effects of different categories of alcohol consumption (no; 1 drink per week; 2 to 3 drinks per week; 4 to 6 drinks per week; 1 drink per day, 2 to 3 drinks per day) on repeatedly measured fetal head and abdominal circumference and femur length.

Results

In total, 37% of all mothers continued alcohol consumption during pregnancy, of whom the majority used less than 3 drinks per week. We observed no differences in growth rates of fetal head circumference, abdominal circumference, and femur length between mothers with and without continued alcohol consumption during pregnancy. Compared to mothers without alcohol consumption, mothers with continued alcohol consumption during pregnancy had an increased fetal weight gain (difference 0.61 (95% confidence intervaI: 0.18, 1.04) grams per week). Crosssectional analyses in mid-and late pregnancy showed no consistent associations between the number of alcoholic consumptions and fetal growth characteristics. All analyses were adjusted for potential confounders.

INTRODUCTION

Excessive maternal alcohol consumption during pregnancy is associated with various pregnancy complications such as multiple birth defects [START_REF] Barr | Binge drinking during pregnancy as a predictor of psychiatric disorders on the Structured Clinical Interview for DSM-IV in young adult offspring[END_REF][START_REF] Lazzaroni | Moderate maternal drinking and outcome of pregnancy[END_REF][START_REF] Little | The effects of maternal drinking in the reproductive period: an epidemiologic review[END_REF][START_REF] O'leary | The effect of maternal alcohol consumption on fetal growth and preterm birth[END_REF][START_REF] Warren | Alcohol-related birth defects: an update[END_REF], fetal alcohol syndrome [START_REF] Chiriboga | Fetal alcohol and drug effects[END_REF][START_REF] Sokol | Fetal alcohol spectrum disorder[END_REF] and an increased risk of low birth weight [START_REF] Henderson | Systematic review of the fetal effects of prenatal binge-drinking[END_REF][START_REF] Henderson | Systematic review of effects of lowmoderate prenatal alcohol exposure on pregnancy outcome[END_REF]. Less is known about the effects of light to moderate maternal alcohol consumption [START_REF] Hanson | The effects of moderate alcohol consumption during pregnancy on fetal growth and morphogenesis[END_REF][START_REF] Kramer | Determinants of low birth weight: methodological assessment and meta-analysis[END_REF][START_REF] Little | Moderate alcohol use during pregnancy and decreased infant birth weight[END_REF]. In general, light to moderate alcohol consumption during pregnancy is considered as one or less alcoholic consumption per day on average.

Previous studies focused on the effects of light to moderate alcohol consumption during pregnancy on birth outcomes have shown inconsistent results [START_REF] Lazzaroni | Moderate maternal drinking and outcome of pregnancy[END_REF][START_REF] Little | The effects of maternal drinking in the reproductive period: an epidemiologic review[END_REF][START_REF] Kramer | Determinants of low birth weight: methodological assessment and meta-analysis[END_REF][START_REF] Little | Moderate alcohol use during pregnancy and decreased infant birth weight[END_REF][START_REF] Day | Prenatal exposure to alcohol: effect on infant growth and morphologic characteristics[END_REF][START_REF] Lundsberg | Low-to-moderate gestational alcohol use and intrauterine growth retardation, low birthweight, and preterm delivery[END_REF][START_REF] Mariscal | Pattern of alcohol consumption during pregnancy and risk for low birth weight[END_REF][START_REF] Passaro | The effect of maternal drinking before conception and in early pregnancy on infant birthweight. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood[END_REF]. Most of these studies did not take the effect of potential confounders such as maternal age, smoking habits, weight, height, educational level, ethnicity, and parity into account [START_REF] Mariscal | Pattern of alcohol consumption during pregnancy and risk for low birth weight[END_REF][START_REF] Mcdonald | Cigarette, alcohol, and coffee consumption and prematurity[END_REF][START_REF] Windham | The association of moderate maternal and paternal alcohol consumption with birthweight and gestational age[END_REF][START_REF] Virji | The relationship between alcohol consumption during pregnancy and infant birthweight. An epidemiologic study[END_REF][START_REF] O'callaghan | Maternal alcohol consumption during pregnancy and physical outcomes up to 5 years of age: a longitudinal study[END_REF]. Recently, we observed in a population-based cohort among more than 7000 subjects that only an average maternal alcohol consumption of more than one drink per day is associated with the risk of low birth weight [START_REF] Jaddoe | Moderate alcohol consumption during pregnancy and the risk of low birth weight and preterm birth. The generation R study[END_REF]. To our knowledge, no previous studies focused on the associations of light and moderate maternal consumption with fetal growth characteristics in different trimesters in a population-based prospective cohort design. Such studies would also be able to identify specific critical periods for fetal growth and development.

Therefore, we examined the associations of light to moderate maternal alcohol consumption with fetal growth characteristics measured in different periods of pregnancy. The study was conducted in a population-based prospective cohort among 7333 mothers followed from early pregnancy onwards.

METHODS AND MATERIALS

Design

This study was embedded in the Generation R Study, a population-based prospective cohort study from fetal life until young adulthood [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. The cohort includes 9778 mothers and their children of different ethnicities living in Rotterdam,

The Netherlands and has been described in detail previously [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. Our aim was to enrol women at the routine fetal ultrasound examination in early pregnancy (gestational age <17.9 weeks) but enrolment was allowed until birth of the child.

Assessments in pregnancy, including physical examinations, fetal ultrasound examinations, and questionnaires, were planned in early pregnancy (gestational age <17.9 weeks), mid-pregnancy (gestational age 18 -24.9 weeks) and late pregnancy (gestational age ≥25 weeks). The individual timing of these assessments depended on the gestational age at enrolment [START_REF] Jaddoe | The Generation R Study Biobank: a resource for epidemiological studies in children and their parents[END_REF][START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. The median gestational ages of the early, mid-and late pregnancy measurements for the present study were 13.4 (95% range 9.8 -17.6), 20.5 (95% range 18.5 -23.6), and 30.4 (95% range 28.4 -32.8) weeks, respectively. All children were born between April 2002 and January 2006. Of the total eligible children born in the study area during the enrolment period, 61% was enrolled in the Generation R Study [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. The Medical Ethical Committee of the Erasmus Medical Centre, Rotterdam, has approved the study. Written informed consent was obtained from all participants.

Maternal alcohol consumption

Information about maternal alcohol consumption was obtained by postal questionnaires in early, mid-, and late pregnancy [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. Response rates for these questionnaires were 91%, 80%, and 77%, respectively. In the first questionnaire, the mothers were asked whether they used any alcoholic drinks in the first three months of pregnancy (no; until pregnancy was known; continued after pregnancy was known). This questionnaire was sent to all mothers, including those enrolled after early pregnancy. In the second and third questionnaires, sent in mid-and late pregnancy, respectively, the mothers were asked whether they drank any alcohol in the past two months (no; yes). Of the mothers who reported in the first questionnaire drinking only until pregnancy was known (n = 1974), those who reported alcohol consumption in the second or third questionnaire (n = 988), were reclassified into the 'continued after pregnancy was known' category. The same strategy was used for mothers who reported no alcohol use in the first questionnaire (n = 4174) but who reported drinking in the second or third questionnaire (n = 535). This strategy led to the following groups of alcohol consumption; no alcohol consumption (n=3639), alcohol consumption until the pregnancy was known (n=986), and continued alcohol consumption (n=2708). Mothers who reported any drinking were asked to classify their average alcohol consumption into one of the following six categories: <1 per week; 1 to 3 per week; 4 to 6 per week; 1 per day; 2 to 3 per day; >3 per day. In the Netherlands, the average alcoholic drink (one glass) contains about 12 g of alcohol [START_REF]Nederlands Voedingsstoffenbestand 2006/ Stichting Nederlands Voedingsstoffenbestand[END_REF]. Only 12 mothers used more than three alcoholic drinks per day in early pregnancy. In mid-and late pregnancy none of the mothers drank more than three alcoholic drinks per day. In a separate question we asked the mothers to report whether they had drank more than 6 drinks in one day but these numbers were small (n=23 in mid-pregnancy; n=22 in late pregnancy). Fetal ultrasound examinations were carried out at the research centres in early, mid-, and late pregnancy. These fetal ultrasound examinations were used for both establishing gestational age and assessing fetal growth characteristics [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF].

Fetal growth characteristics

Gestational age was established by fetal ultrasound examination because using the last menstrual period has several limitations, including the large number of women who do not know the exact date of their last menstrual period or have irregular menstrual cycles [START_REF] Tunon | A comparison between ultrasound and a reliable last menstrual period as predictors of the day of delivery in 15,000 examinations[END_REF][START_REF] Altman | New charts for ultrasound dating of pregnancy[END_REF][START_REF] Robinson | The accuracy of radiological estimates of gestational age using early fetal crown-rump length measurements by ultrasound as a basis for comparison[END_REF]. Pregnancy dating curves were constructed for subjects with complete data on gestational age measured by ultrasonography and the last menstrual period [START_REF] Verburg | New charts for ultrasound dating of pregnancy and assessment of fetal growth: longitudinal data from a population-based cohort study[END_REF]. Crown-rump length was used for pregnancy dating up to a gestational age of twelve weeks and five days (crown-rump length less than 65 mm), and biparietal diameter was used for pregnancy dating thereafter (gestational age from twelve weeks and five days onwards, biparietal diameter more than 23 mm) [START_REF] Verburg | New charts for ultrasound dating of pregnancy and assessment of fetal growth: longitudinal data from a population-based cohort study[END_REF].

Early pregnancy measurements were primarily used to establish gestational age and therefore not included in the growth analyses. Growth characteristics were measured to the nearest millimeter using standardized ultrasound procedures [START_REF]Routine ultrasound screening in pregnancy: protocol[END_REF]. Estimated fetal weight was calculated using the formula of Hadlock with parameters head circumference, abdominal circumference and femur length [START_REF] Hadlock | Estimation of fetal weight with the use of head, body, and femur measurements--a prospective study[END_REF]. Longitudinal growth curves were constructed for all fetal growth measurements [START_REF] Verburg | New charts for ultrasound dating of pregnancy and assessment of fetal growth: longitudinal data from a population-based cohort study[END_REF].

Covariates

Information about educational level, ethnicity and parity was obtained by a questionnaire at enrolment in the study. Maternal smoking habits were assessed in each questionnaire. Maternal distress was assessed by a questionnaire at 20 weeks of gestation using the Brief Symptom Inventory [START_REF] Derogatis | Brief Symptom Inventory (BSI): Administration, scoring and procedures[END_REF]. Maternal anthropometrics, including height and weight, were measured without shoes and heavy clothing during

Population for analysis

In total 8880 mothers were enrolled during pregnancy [START_REF] Jaddoe | The Generation R Study: design and cohort update until the age of 4 years[END_REF]. Those without information about alcohol consumption during pregnancy in the first questionnaire were excluded from the analyses (14%, n = 1241). For the present study, we excluded subjects without fetal growth data in mid-or late pregnancy and twin pregnancies (n = 190). Of the remaining 7449 mothers, those pregnancies leading to fetal deaths (n = 46), missing birth outcomes (n = 69) or with a delivery less than 25 weeks of gestation (n = 1) were excluded, since our interest was in low-risk singleton pregnancies. In our population for analysis, we had 397 mothers with two pregnancies, and 4 mothers with three pregnancies. Since, no differences in results were observed between associations with and without these second and third pregnancies, we included them in the analyses. The associations of maternal alcohol consumption during pregnancy with longitudinally measured fetal growth characteristics were analyzed in the remaining 7333 mothers (Figure 1). Analyses focused on the effects of alcohol consumption levels in mid-and late pregnancy were performed in mothers who were enrolled in early pregnancy (n = 5612) only, leading to 4836 (86%) and 4654 (83%) subjects for mid-and late pregnancy, respectively.

Data analysis

Fetal growth

The associations between alcohol consumption during pregnancy and fetal growth characteristics were analyzed using unbalanced repeated-measurements regression analysis assuming random effects for the intercept and slope. These regression models enable studies on repeatedly measured outcomes, taking account for the correlation between measurements and have an optimal use of available data. Both gestational age independent (difference constant over time) and gestational age dependent (difference not-constant over time) effects were assessed. We used unstructured covariance models. First, we constructed best fitting models using second-degree fractional polynomials of gestational age [START_REF] Royston | The use of fractional polynomials to model continuous risk variables in epidemiology[END_REF]. These models have been described in detail previously [START_REF] Nohr | Does low participation in cohort studies induce bias?[END_REF]. The best fitting fractional polynomial curves were chosen by comparing the deviances and checking the goodness of fit (smallest -2 log likelihood). Subsequently, the correctness of the model was assessed by plotting the SD scores against gestational age. Second, we included alcohol consumption during pregnancy (none; until pregnancy was known; continued after pregnancy was known) to these models as additional intercept as interaction with gestational age. The interactions of alcohol consumption with gestational age were tested with each separate polynomial. Any significant interaction was subsequently included in the final model for analyses. The final models including gestational age and alcohol consumption (alcohol) can be written as:

Head circumference = β 0 + β 1 *

RESULTS

Subject characteristics

As shown in

Fetal growth

The derived fetal growth curves and estimates for the interactions terms of alcohol with each fractional polynomial for the different fetal growth characteristic are given in the supplement (Figure S1 and Table S1, respectively). Figure 2 shows the associations between maternal alcohol consumption categories with estimated differences in fetal growth characteristics (head circumference, abdominal S1). Compared to mothers without alcohol consumption, mothers with continued alcohol consumption during pregnancy had an increased fetal weight gain (difference 0.61 (95% CI: 0.18, 1.04) grams per week).

Dose-response

Table 2 showed an association between using one to three drinks per week with abdominal circumference (increase of 1.02 mm (95% CI: 0.11, 1.92) compared to no drinking) in mid-pregnancy. Drinking four to six drinks per week was associated with a shorter femur length (-0.63 mm (95% CI: -1.15, -0.11)) in mid-pregnancy. After adjustment for potential confounders, no associations of alcohol consumption with fetal growth characteristics in mid-pregnancy remained significant. Also, no significant trends were found. Table 3 shows that in late pregnancy, no significant or consistent effects of alcohol consumption during pregnancy on any fetal growth characteristic was observed. We observed no consistent differences between analyses on the whole cohort using ultrasound for pregnancy dating and subgroup with pregnancy dating on last menstrual period (see supplementary Tables S2 andS3).

Methodological considerations

The strength of this study is the large number of subjects in a prospectively studied cohort. To our knowledge, this is the largest study focused on light to moderate alcohol consumption during pregnancy on fetal growth characteristics in different periods of pregnancy. We were also able to control for many possible confounders. A potential limitation of this study is the missing data on alcohol consumption. No differences in maternal age at enrolment, height and birth weight of the offspring were observed between those with and without information about alcohol consumption. Missing information about alcohol consumption might have led to loss of power. The associations might be underestimated if among mothers without alcohol data the percentage of alcohol consumers would be higher than among mothers without missing alcohol data. However, this seems unlikely since no differences in characteristics between those with and without alcohol consumption data were observed. Among mothers with information about alcohol consumption, we had a limited loss-to-follow-up. Therefore, we do not expect biased results due to loss-to-follow-up [START_REF] Nohr | Does low participation in cohort studies induce bias?[END_REF]. Information on alcohol consumption habits during pregnancy was collected by postal questionnaires. If any, misclassification would be most likely due to underreporting and subsequently lead to underestimation of differences between those not using and using alcoholic consumptions. Previous studies suggested use of biomarkers of alcohol consumption such as carbohydrate-deficient transferrin and gamma-glutamyl transferase. However, currently these biomarkers have a low sensitivity in subjects with light to moderate alcohol consumption [START_REF] Van Pelt | Test characteristics of carbohydrate-deficient transferrin and gamma-glutamyltransferase in alcohol-using perimenopausal women[END_REF]. In our study we had only information about the average number of alcoholic drinks per week, without information on drinking patterns. Our estimation of the amount of alcohol per consumption might be imprecise since both alcohol percentages and serving sizes may differ between mothers.

Gestational age was established by fetal ultrasound examination. This method seems to be superior to use of the last menstrual period [START_REF]Nederlands Voedingsstoffenbestand 2006/ Stichting Nederlands Voedingsstoffenbestand[END_REF]. The major disadvantage of establishing gestational age by ultrasonography is that the growth variation of the fetal characteristics used for pregnancy dating is assumed to be zero.

Therefore, in our study, crown-rump length and biparietal diameter were used for pregnancy dating but not for assessing fetal growth [START_REF] Tunon | A comparison between ultrasound and a reliable last menstrual period as predictors of the day of delivery in 15,000 examinations[END_REF][START_REF] Altman | New charts for ultrasound dating of pregnancy[END_REF]. Since pregnancy dating characteristics and growth characteristics are correlated throughout pregnancy, growth variation in head circumference, abdominal circumference, and femur length may be reduced by dating the pregnancy on the other fetal characteristics. This may have led to underestimation of our effect estimates. However, we expect this effect to be small in our results. First, the longitudinal analyses were focused on fetal growth or change in size during pregnancy within individuals. This change in size is unlikely to have been materially affected by our pregnancy dating method. Second, the analyses assessing the associations of maternal alcohol consumption with fetal growth characteristics in mid-and late pregnancy were restricted to mothers who were enrolled and had their pregnancies dated in early pregnancy (76.5 % of the population for analysis). Since gestational age and fetal growth were not established concurrently, we believe that we minimized the effect of pregnancy dating on growth variation. Our analysis in a subgroup of mothers with pregnancy dating on their last menstrual period, did not show consistent stronger effects. However, results based the last menstrual period shown in Table S2 tended to show larger differences than that in Table 2. This might be due to some underestimation of the main results due to correlation between ultrasound dating and growth characteristics.

Maternal alcohol consumption and fetal growth characteristics

Previous studies focused primarily on the associations between maternal alcohol consumption during pregnancy and birth weight and showed inconsistent associations [START_REF] Lazzaroni | Moderate maternal drinking and outcome of pregnancy[END_REF][START_REF] Henderson | Systematic review of the fetal effects of prenatal binge-drinking[END_REF][START_REF] Little | Moderate alcohol use during pregnancy and decreased infant birth weight[END_REF][START_REF] Day | Prenatal exposure to alcohol: effect on infant growth and morphologic characteristics[END_REF][START_REF] Lundsberg | Low-to-moderate gestational alcohol use and intrauterine growth retardation, low birthweight, and preterm delivery[END_REF][START_REF] Mariscal | Pattern of alcohol consumption during pregnancy and risk for low birth weight[END_REF][START_REF] Passaro | The effect of maternal drinking before conception and in early pregnancy on infant birthweight. The ALSPAC Study Team. Avon Longitudinal Study of Pregnancy and Childhood[END_REF][START_REF] Mcdonald | Cigarette, alcohol, and coffee consumption and prematurity[END_REF][START_REF] Windham | The association of moderate maternal and paternal alcohol consumption with birthweight and gestational age[END_REF][START_REF] Jaddoe | Moderate alcohol consumption during pregnancy and the risk of low birth weight and preterm birth. The generation R study[END_REF]. Smaller studies assessing the effect of maternal alcohol consumption during pregnancy on fetal growth retardation [START_REF] Windham | The association of moderate maternal and paternal alcohol consumption with birthweight and gestational age[END_REF][START_REF] Yang | A casecontrol study of maternal alcohol consumption and intrauterine growth retardation[END_REF] also showed inconsistent results. Windham et al. [START_REF] Windham | The association of moderate maternal and paternal alcohol consumption with birthweight and gestational age[END_REF] showed an association of moderate maternal alcohol consumption with an increased risk of fetal growth retardation (odds ratio (OR) = 2.3, 95% CI: 1.2, 4.6) while Yang et al. [START_REF] Yang | A casecontrol study of maternal alcohol consumption and intrauterine growth retardation[END_REF] showed no effect of moderate alcohol consumption on fetal growth retardation. Most studies used low birth weight or fetal growth retardation as outcome measurement and showed conflicting results. Moreover, most of these studies did not adjust for potential confounders [START_REF] Henderson | Systematic review of the fetal effects of prenatal binge-drinking[END_REF][START_REF] Mariscal | Pattern of alcohol consumption during pregnancy and risk for low birth weight[END_REF][START_REF] Mcdonald | Cigarette, alcohol, and coffee consumption and prematurity[END_REF][START_REF] Windham | The association of moderate maternal and paternal alcohol consumption with birthweight and gestational age[END_REF][START_REF] Virji | The relationship between alcohol consumption during pregnancy and infant birthweight. An epidemiologic study[END_REF][START_REF] O'callaghan | Maternal alcohol consumption during pregnancy and physical outcomes up to 5 years of age: a longitudinal study[END_REF]. Examining fetal growth characteristics instead of birth weight enables identification of specific critical periods during pregnancy for the exposure on various patterns of fetal growth and development. In 2006, Handmaker et al. [START_REF] Handmaker | Impact of alcohol exposure after pregnancy recognition on ultrasonographic fetal growth measures[END_REF] examined the effect of maternal alcohol consumption during pregnancy on fetal growth characteristics. This study, comprised of 167 subjects, showed an association between heavy drinking and the ratio of head circumference and abdominal circumference. However, no adjustments were made for potential confounders. We did not find any consistent effects of moderate maternal alcohol consumption on fetal growth characteristics. However, studies in larger numbers of subjects might be able to identify smaller effects. Differences in results from the cross-sectional analyses compared to the repeated measurement analysis can be explained by less power and smaller numbers in the alcohol consumption groups in the cross-sectional analyses. From our results, it seems that any trend for positive associations between alcohol consumption during pregnancy and fetal growth characteristics is explained by socio-demographic and life style related variables. After adjustment, none of the positive associations remained significant. Other potential confounders that might be important include dietary habits and folate acid supplementation [START_REF] Knudsen | Major dietary patterns in pregnancy and fetal growth[END_REF]. However, including maternal folic acid use in early pregnancy into the current analyses did not have an effect on our findings (data not shown).

Previous studies also suggested effects of maternal alcohol consumption on postnatal growth and development. It was shown that the rate of postnatal growth is reduced in children who were prenatally exposed to alcohol. Weight, length and head circumference were negatively affected by alcohol exposure during pregnancy [START_REF] Day | Effect of prenatal alcohol exposure on growth and morphology of offspring at 8 months of age[END_REF].

However, a more recent study showed that moderate maternal alcohol consumption during pregnancy was not associated with either weight or head circumference at the age of five years [START_REF] O'callaghan | Maternal alcohol consumption during pregnancy and physical outcomes up to 5 years of age: a longitudinal study[END_REF]. Also, inconsistent results were found on behavioural A study of Faden et al. [START_REF] Faden | Maternal substance use during pregnancy and developmental outcome at age three[END_REF] showed a higher activity level, a greater difficulty in following instructions and have eating problems among offspring exposed to alcohol during pregnancy. Furthermore, binge drinking during pregnancy was shown to be associated with increased odds for the appearance of psychiatric disorders [START_REF] Barr | Binge drinking during pregnancy as a predictor of psychiatric disorders on the Structured Clinical Interview for DSM-IV in young adult offspring[END_REF]. Our study was focused on the effects of light and moderate maternal alcohol consumption. We also asked the mothers if they drank occasionally more than 6

glasses of alcohol in one day (yes; no), which is considered as binge drinking. In total, 23 mothers in mid-pregnancy and 22 mothers in late pregnancy reported that they drank occasionally more than 6 glasses of alcohol in one day. No differences in fetal growth were observed between binge drinking and non-drinking mothers (data not shown), however, future studies with more power are needed for this analysis.

Although, previous studies did not show consistent associations either of binge drinking during pregnancy with several outcomes, except for neurodevelopmental outcomes [START_REF] Henderson | Systematic review of the fetal effects of prenatal binge-drinking[END_REF], future studies with larger numbers are needed. Whether light to moderate alcohol consumption in this or any other cohort is related to postnatal growth and development needs to be examined in follow-up studies. The effects of alcohol consumption are dependent of the absorption and metabolism in the mother and fetus. This may be partially genetically determined. Therefore, the effects of alcohol consumption in specific groups of women should still be studied.

Conclusion

A. Head circumference

Values reflect difference in fetal growth characteristics of mothers with different alcohol consumption categories and were based on repeated regression models (*P<0.05). The corresponding effect estimates of the differences in the slopes are presented in Table S1. All models are adjusted for maternal age at enrolment, weight at enrolment, height, educational level, parity, ethnicity, fetal gender, smoking habits, maternal stress. S1. All models are adjusted for maternal age at enrolment, weight at enrolment, height, educational level, parity, ethnicity, fetal gender, smoking habits, maternal stress.

Background

  Excessive alcohol consumption during pregnancy has adverse effects on fetal growth and development. Less consistent associations have been shown for the associations of light to moderate maternal alcohol consumption during pregnancy with health outcomes in the offspring. Therefore, we examined the associations of light to moderate maternal alcohol consumption with various fetal growth characteristics measured in different periods of pregnancy.

  research centre at enrolment. Date of birth and gender were obtained from midwife and hospital registries.

  length, estimated fetal weight) between the gestational ages of 18 and 35 weeks. Except for estimated fetal weight, these associations were not significant after adjusting for potential confounders (Table

DISCUSSION

  This population-based prospective cohort study showed no associations of light to moderate maternal alcohol consumption during pregnancy with longitudinally measured fetal head or length growth. Mothers who continued alcohol consumption during pregnancy tended to have a increased fetal weight gain. No consistent associations were observed for the associations between number of alcoholic consumptions and any fetal growth characteristics in different periods of pregnancy.

  development and cognitive processing in children prenatally exposed to alcohol[39,

  Values reflect difference in fetal growth characteristics of mothers with different alcohol consumption categories and were based on repeated regression models (*P<0.05). The corresponding effect estimates of the differences in the slopes are presented in TableS1. All models are adjusted for maternal age at enrolment, weight at enrolment, height, educational level, parity, ethnicity, fetal gender, smoking habits, maternal stress.Values reflect difference in fetal growth characteristics of mothers with different alcohol consumption categories and were based on repeated regression models (*P<0.05). The corresponding effect estimates of the differences in the slopes are presented in TableS1. All models are adjusted for maternal age at enrolment, weight at enrolment, height, educational level, parity, ethnicity, fetal gender, smoking habits, maternal stress.Values reflect difference in fetal growth characteristics of mothers with different alcohol consumption categories and were based on repeated regression models (*P<0.05). The corresponding effect estimates of the differences in the slopes are presented in Table

  alcohol + β 2 *gestational age 2 + β 3 *alcohol*gestational age 2 + β 4 *gestational age 2 *ln(gestational age) + β 5 *alcohol*gestational age. Abdominal circumference = β 0 + β 1 *alcohol + β 2 *gestational age 2 + β 3 *gestational age 2 *ln(gestational age) + β 4 *alcohol*gestational age. Femur length = β 0 + β 1 *alcohol + β 2 *gestational age 3 + β 3 *alcohol*gestational age 3 . -2 *ln(gestational age) + β 4 * gestational age -2 *ln(gestational age) 2 + β 5 *alcohol*gestational age. In these models, 'β 0 ' reflects the intercept and 'β1 * alcohol' tests the difference in intercept between alcohol groups. The other beta's (β 2 β 4 β 5 ), reflect the linear or non linear slope (interaction alcohol consumption with gestational age). These terms test

	Dose-response analyses in mid-and late pregnancy were performed using multiple
	linear regression models. In these analyses also the unexposed fetus (no maternal
	alcohol consumption) was used as reference group. We adjusted the models for the
	following confounders; maternal age, weight, height and distress (all continuous) and
	smoking, educational level, ethnicity, parity, and infant gender (all categorical) [11]. A
	small proportion of mothers participating in this study had a regular menstrual cycle
	(28±4 days) and a known and reliable gestational age based on last menstrual period
	F o r F o r (n=1796). We repeated the analyses focused on the dose-response associations of F o r
	R R R
	e v e v e v
	i e i e i e
	w w w
	O n l O n l O n l
	y y y
	Dose-response
	13 14

age whether the polynomial curves are parallel (whether the groups grow at the same rate as compared to the reference group (no alcohol consumption) between the time points. The main interest for this paper is the beta's which include an interaction with alcohol consumption. Subsequently, we used these models to estimate the estimated differences in fetal growth characteristics between alcohol categories. For all analyses, the unexposed fetus (no maternal alcohol consumption) was used as reference group. We adjusted the models for the following confounders; maternal age, weight, height and distress (all continuous) and smoking, educational level, ethnicity, parity, and infant gender (all categorical)

[START_REF] Kramer | Determinants of low birth weight: methodological assessment and meta-analysis[END_REF]

. Analyses were repeated in Dutch and other European mothers only. Since we found similar results, these associations were not presented separately. All levels of association are presented with their 95% confidence intervals. Analyses were performed using the Statistical Analysis System version 8.2 (SAS, Institute Inc. Gary NC, USA), including the Proc Mixed module for unbalanced repeated-measurements. maternal alcohol consumption in mid-and late pregnancy in this group of women, to exclude underestimation of the effects by ultrasound dating. Tests for trends were performed by using the alcohol consumption categories as continuous variables in the models. All levels of association are presented with their 95% confidence intervals. Analyses were performed using the Statistical Package of Social Sciences version 15.0 for Windows (SPSS Inc, Chicago, IL, USA).

Table 1

 1

	, of all mothers, 50% (n=3639) did not use alcoholic drinks in
	pregnancy, 13% (n = 986) stopped alcohol consumption after their pregnancy was
	known and 37% continued alcohol consumption during pregnancy (n = 2708). Age of
	the mothers in the whole cohort ranged from 15.3 to 46.3 years and was significantly
	higher in mothers who continued alcohol consumption after pregnancy was known
	compared to mothers who did not use alcohol during pregnancy. The percentages of

high educated and Dutch or other European mothers were highest among those who continued alcohol consumption. Of all pregnancies, 5.5% (n = 401) were second or third pregnancies in the study. Since, no differences in results were observed between associations with and without these second and third pregnancies, we included them in the analyses. Mean birth weight of the children was 3417 (95% range 635 -5310) grams. Gestational age at birth ranged from 25.3 to 43.6 weeks with a median of 40.1 weeks. Of all singleton live births, 5.0% were born before a gestational age of 37 weeks. Analyses were repeated in Dutch and other European mothers only. Since we found similar results, these associations were not presented separately.

TABLE 1 . Maternal characteristics according to alcohol consumption during pregnancy category (n = 7333 mothers) No alcohol consumption

 1

	Our results suggest that in a Western population-based cohort moderate maternal
	alcohol consumption during pregnancy does not adversely affect fetal growth. Future
	studies are needed to examine whether the effects differ among subjects with
	different absorption and metabolism patterns and to examine the effects on organ

TABLE 2 . Dose-response associations between maternal alcohol consumption during pregnancy and fetal growth characteristics in mid-pregnancy (18 -24.9 weeks) (n = 5612 mothers) Difference in head circumference (mm)

 2

	Alcohol consumption	Number of subjects	Crude	Adjusted
	None	n=3081	Reference	Reference
	< 1 drink/week	n=1235	-0.00 (-0.41, 0.40)	-0.31 (-0.74, 0.12)
	1 to 3 drinks/week	n=327	0.50 (-0.20, 1.20)	0.32 (-0.40, 1.04)
	4 to 6 drinks/week	n=43	-0.10 (-1.94, 1.74)	-0.08 (-1.98, 1.81)
	1 drink/day	n=19	0.92 (-1.84, 3.68)	1.33 (-1.36, 4.02)
	2 to 3 drinks/day	n=3	5.14 (-1.79, 12.08)	6.77 (-0.01, 13.55)
			Ptrend = 0.21	Ptrend = 0.59
		Difference in abdominal circumference (mm)
	Alcohol consumption	Number of subjects	Crude	Adjusted
	None	n=3092	Reference	Reference
	< 1 drink/week	n=1237	-0.01 (-0.52, 0.53)	-0.54 (-1.10, 0.02)
	1 to 3 drinks/week	n=329	1.02* (0.11, 1.92)	0.54 (-0.40, 1.48)
	4 to 6 drinks/week	n=41	-1.70 (-4.14, 0.75)	-2.11 (-4.64, 0.43)
	1 drink/day	n=19	2.29 (-1.29, 5.86)	1.97 (-1.54, 5.48)
	2 to 3 drinks/day	n=3	6.02 (-2.96, 15.00)	5.88 (-2.95, 14.71)
			Ptrend = 0.13	Ptrend = 0.94
		Difference in femur length (mm)
	Alcohol consumption	Number of subjects	Crude	Adjusted
	None	n=3088	Reference	Reference
	< 1 drink/week	n=1235	-0.01 (-0.21, 0.02)	-0.03 (-0.15, 0.10)
	1 to 3 drinks/week	n=329	-0.05 (-0.25, 0.15)	0.00 (-0.21, 0.21)
	4 to 6 drinks/week	n=43	-0.63* (-1.15, -0.11)	-0.36 (-0.91, 0.19)
	1 drink/day	n=19	-0.48 (-1.26, 0.30)	-0.53 (-1.31, 0.25)
	2 to 3 drinks/day	n=3	-0.42 (-2.39, 1.55)	-0.26 (-2.22, 1.69)
			Ptrend < 0.05	Ptrend = 0.26

Difference in estimated fetal weight (grams) Alcohol consumption Number of subjects Crude Adjusted

		F o r
		R
		e v
		i e
		w
	None < 1 drink/week	n=3078 n=1228	Reference -0.80 (-3.44, 1.85) O n l	Reference -1.98 (-4.82, 0.86)
	1 to 3 drinks/week 4 to 6 drinks/week	n=329 n=41	4.17 (-0.38, 8.71) -10.04 (-22.36, 2.28) y	3.29 (-1.46, 8.04) -9.78 (-22.59, 3.04)
	1 drink/day	n=19	4.94 (-13.09, 22.97)	3.07 (-14.65, 20.78)
	2 to 3 drinks/day	n=3	23.98 (-21.31, 69.28)	25.55 (-19.05, 70.14)
			Ptrend = 0.54	Ptrend = 0.97
	* P < 0.05. Values are regression coefficients (95% confidence interval) and reflect the difference for each growth
	characteristic.
	Crude model: adjusted for gestational age. Adjusted model: Crude model + maternal age, weight, height,
	smoking, distress, educational level, ethnicity, parity, and infant gender.
	P-values for trend were based on multiple linear regression models with alcohol consumption categories as a
	continuous variable.
		29

TABLE 3 . Dose-response associations between maternal alcohol consumption during pregnancy and fetal growth characteristics in late pregnancy (≥ 25 weeks) (n = 5612 mothers) Difference in head circumference (mm) Alcohol consumption Number of subjects Crude Adjusted

 3

	None	n=2890	Reference	Reference
	< 1 drink/week	n=1227	0.73* (0.11, 1.34)	-0.37 (-1.04, 0.30)
	1 to 3 drinks/week	n=396	1.48** (0.50, 2.45)	0.28 (-0.74, 1.30)
	4 to 6 drinks/week	n=43	-0.53 (-3.32, 2.27)	-0.76 (-3.65, 2.12)
	1 drink/day	n=16	-1.45 (-6.00, 3.11)	-2.74 (-7.23, 1.74)
	2 to 3 drinks/day	n=4	1.41 (-7.68, 10.50)	1.64 (-8.33, 11.62)
			Ptrend < 0.01	Ptrend = 0.61
		Difference in abdominal circumference (mm)
	Alcohol consumption	Number of subjects	Crude	Adjusted
	None	n=2904	Reference	Reference
	< 1 drink/week	n=1235	0.42 (-0.45, 1.28)	-0.22 (-1.18, 0.75)
	1 to 3 drinks/week	n=398	1.37 (0.00, 2.74)	0.03 (-1.44, 1.50)
	4 to 6 drinks/week	n=43	-2.97 (-6.90, 0.97)	-3.13 (-7.29, 1.02)
	1 drink/day	n=16	-1.13 (-7.54, 5.29)	-2.52 (-8.99, 3.94)
	2 to 3 drinks/day	n=4	11.95 (-0.85, 24.74)	10.99 (-3.38, 25.36)
			Ptrend = 0.16	Ptrend = 0.56
		Difference in femur length (mm)
	Alcohol consumption	Number of subjects	Crude	Adjusted
	None	n=2909	Reference	Reference
	< 1 drink/week	n=1238	-0.00 (-0.15, 0.15)	-0.07 (-0.23, 0.10)
	1 to 3 drinks/week	n=397	-0.07 (-0.30, 0.16)	-0.09 (-0.34, 0.16)
	4 to 6 drinks/week	n=43	-0.49 (-1.15, 0.18)	-0.40 (-1.10, 0.31)
	1 drink/day	n=16	0.06 (-1.03, 1.14)	-0.28 (-1.38, 0.82)
	2 to 3 drinks/day	n=4	-0.54 (-2.71, 1.62)	-0.60 (-3.04, 1.84)
			Ptrend = 0.36	Ptrend = 0.18

Difference in estimated fetal weight (grams) Alcohol consumption Number of subjects Crude Adjusted

  P < 0.05, ** P < 0.01. Values are regression coefficients (95% confidence interval) and reflect the difference for each growth characteristic. Crude model: adjusted for gestational age. Adjusted model: Crude model + maternal age, weight, height, smoking, distress, educational level, ethnicity, parity, and infant gender. P-values for trend were based on multiple linear regression models with alcohol consumption categories as a continuous variable.

	None < 1 drink/week 1 to 3 drinks/week 4 to 6 drinks/week 1 drink/day 2 to 3 drinks/day * F n=2899 n=1233 n=397 n=43 n=16 n=4 o r R e v i Reference 3.44 (-8.65, 15.54) 13.65 (-5.39, 32.70) -47.71 (-102.37, 6.95) -12.29 (-101.46, 76.88) 122.26 (-55.71, 300.23) Ptrend = 0.46 e w O n l y	Reference -5.44 (-18.84, 7.97) -1.14 (-21.57, 19.28) -46.04 (-103.69, 11.61) -38.14 (-127.88, 51.59) 116.76 (-82.74, 316.26) Ptrend = 0.36

FIGURE 1. Flow chart of participants included for analysis.

	Prenatally included mothers
	N=8880
			N=1241 excluded, due to missing information
			on alcohol consumption
	Women with information on
	alcohol consumption
	N=7639
			N= 236 excluded, due to twin pregnancies
			(N=190) and fetal deaths (N=46)
	Singleton live births
	N=7403
	Total population for analysis F o r	N=70 excluded, due to missing birth weights or gestational age at birth less than 25 weeks
	Longitudinal analyses N=7333	R
	Cross sectional analyses Mid-pregnancy analysis N=4836	e v
	Late pregnancy analysis N=4654		i e
			w
			O n l
			y

FIGURE 2. Differences in fetal growth measures between alcohol consumption categories.

ACKNOWLEDGEMENTS

The Generation R Study is conducted by the Erasmus Medical Centre, Rotterdam in close collaboration with the School of Law and Faculty of Social Sciences of the Erasmus University Rotterdam, the Municipal Health Service Rotterdam area, the Rotterdam Homecare Foundation and the Stichting Trombosedienst & Artsenlaboratorium Rijnmond (STAR), Rotterdam. We gratefully acknowledge the contribution of general practitioners, hospitals, midwives and pharmacies in Rotterdam.

FUNDING

This work was supported by the Erasmus Medical Centre, Rotterdam, the Erasmus University Rotterdam and the Netherlands Organization for Health Research and Development (Zon Mw).


MD Norbert Gattermann
email: gattermann@med.uni-duesseldorf.de

MD Eliezer A Rachmilewitz
email: rachmilewitz@wolfson.health.gov.il

Iron overload in MDSpathophysiology, diagnosis and complications

Keywords: iron overload, myelodysplastic syndromes, oxidative stress, nontransferrin-bound iron, labile plasma iron, iron chelation

Many patients with myelodysplastic syndromes (MDS) become dependent on blood transfusions and develop transfusional iron overload, which is exacerbated by increased absorption of dietary iron in response to ineffective erythropoiesis. However, it is uncertain whether there is an association between iron accumulation, clinical complications and decreased likelihood of survival in MDS patients. Here we discuss our current understanding of the effects of transfusion dependency and iron overload in MDS, indicate our knowledge gaps, and suggest that particular emphasis should be placed on further characterizing the role of redox-active forms of labile iron, which may be as important as the total iron burden.

Introduction

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal bone marrow disorders characterized by ineffective hematopoiesis and peripheral blood cytopenias. Genomic instability of the MDS clone often leads to disease progression, which results in overt leukemia in 0-32% of patients depending on MDS subtype [START_REF] Germing | Prospective validation of the WHO proposals for the classification of myelodysplastic syndromes[END_REF]. Eighty percent of patients have a hemoglobin level of less than 10 g/dl at diagnosis, and most of these will become transfusion dependent [START_REF] Sanz | Two regression models and a scoring system for predicting survival and planning treatment in myelodysplastic syndromes: a multivariate analysis of prognostic factors in 370 patients[END_REF]; the development of iron overload (IO) is unavoidable in these patients. Moreover, iron accumulation in MDS patients is an ongoing process as ineffective erythropoiesis provides a signal that stimulates intestinal iron absorption. The molecular nature of that signal remained elusive for decades, but there is now evidence that ineffective erythropoiesis is associated with increased secretion of growth differentiation factor 15 and/or TWSG1 [START_REF] Tanno | High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin[END_REF][START_REF] Tanno | Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells of the iron regulatory protein hepcidin[END_REF] by maturing erythroblasts, leading to suppressed hepcidin production in the liver [START_REF] Winder | Urinary hepcidin excretion in patients with myelodysplastic syndrome and myelofibrosis[END_REF]. Since hepcidin downregulates iron absorption in the duodenum, lack of hepcidin causes unrestrained intestinal iron uptake. Although this mechanism contributes to iron overload in MDS, it is not the main cause, and rarely leads to serum ferritin (SF) levels above 1000 ng/ml at diagnosis [START_REF] Gattermann | Clinical consequences of iron overload in myelodysplastic syndromes and treatment with chelators[END_REF]. The main cause of iron overload in MDS is chronic transfusion therapy.

Most of our knowledge regarding the detrimental effects of transfusional iron overload stems from the experience in -thalassemia major. The question is whether a similar association between iron accumulation, clinical complications and decreased likelihood of survival is also true of MDS patients. Here we discuss current understanding of the effects of transfusion dependency and iron overload in MDS, indicate the gaps in our knowledge, and suggest that particular emphasis should be placed on further characterizing the role of redox-active forms of labile plasma iron (LPI) and of labile cellular iron (LCI).

Pathophysiology of iron overload

Intestinal iron absorption is controlled by hepcidin, a peptide hormone that generally adapts duodenal iron absorption to the demands of erythropoiesis and regulates the release of iron from macrophages [START_REF] Ganz | Hepcidin, a key regulator of iron metabolism and mediator of anemia and inflammation[END_REF][START_REF] Nemeth | Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization[END_REF]. Iron is stored in ferritin, an intracellular iron storage protein primarily found in the liver and in the macrophage system. During infection and other inflammatory states, hepcidin production is increased, leading to a decrease in both intestinal iron absorption and macrophage iron release. The resulting decrease in transferrin saturation helps to deprive circulating microorganisms of iron, but also restricts iron availability for erythropoiesis, thereby causing anemia of chronic inflammation.

Circulating iron is bound to transferrin and when the binding capacity of transferrin is exceeded, non-transferrin-bound iron (NTBI) species appear in the plasma [START_REF] Hershko | Non-specific serum iron in thalassemia: an abnormal serum iron fraction of potential toxicity[END_REF]. The portion of NTBI with the weakest binding to plasma biomolecules is labile plasma iron (LPI), which is redox-active and able to permeate organs such as the heart and liver, where it contributes to the generation of reactive oxygen species (ROS) [START_REF] Cabantchik | LPI-labile plasma iron in iron overload[END_REF]. While hydrogen peroxide and superoxide are comparatively non-toxic and can actually function as physiological signaling molecules, their reaction with unliganded or incompletely liganded iron ions creates more damaging oxygen radicals, in particular the extremely reactive hydroxyl radical (OH•) [START_REF] Kell | Iron behaving badly: inappropriate iron chelation as a major contributor to the aetiology of vascular and other progressive inflammatory and degenerative diseases[END_REF]. While LPI is detected almost exclusively in pathological conditions, labile cellular iron (LCI) is a normal component that is regulated to serve the cellular iron requirements and to prevent an excess of redox-reactive iron that may trigger cellular damage. High LCI levels catalyze increased ROS generation through the Haber-Weiss and Fenton reactions, which may eventually overwhelm the cell antioxidant capacity and deplete cellular antioxidants like reduced glutathione (GSH), resulting in tissue oxidative damage and organ dysfunction (Fig 1).

Decreased life expectancy in transfusion-dependent MDS: identifying the culprit

Transfusion dependency is strongly associated with decreased survival in MDS patients (Fig. 2), as shown by Malcovati et al who demonstrated a dosedependent impact of transfusion requirements on overall and leukemia-free survival [START_REF] Malcovati | Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO criteria: a basis for clinical decision making[END_REF]. These observations were recently corroborated in a large, retrospective, multicenter analysis from Spain [START_REF] Sanz | Independent impact of iron overload and transfusion dependency on survival and leukemic evolution in patients with myelodysplastic syndrome[END_REF]. However, the association between transfusion dependency and decreased life expectancy in patients with MDS might have several causes. Patients may develop clinical complications resulting from iron overload due to inadequate iron chelation therapy; however, transfusion dependency could merely indicate a more severe bone marrow disease with complications independent of iron overload.

As the severity of bone marrow disease is at least partly determined by karyotype anomalies, one would expect transfusion dependency to lose its prognostic value when the karyotype is taken into account. However, several studies have shown that this is not the case. Transfusion dependency is a risk factor that is independent of cytogenetic risk groups in MDS [START_REF] Malcovati | Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO criteria: a basis for clinical decision making[END_REF], suggesting that the prognostic influence of transfusion dependency is not solely based on the severity of the underlying bone marrow disease but also on an additional component, which is most likely to be the effects of iron overload. A recent multivariate analysis [START_REF] Sanz | Independent impact of iron overload and transfusion dependency on survival and leukemic evolution in patients with myelodysplastic syndrome[END_REF] showed that the prognostic impact of IO was independent of the World Health Organization (WHO) classification-based scoring system (WPSS) which already incorporates transfusion dependency [START_REF] Malcovati | Time-dependent prognostic scoring system for predicting survival and leukemic evolution in myelodysplastic syndromes[END_REF]. Therefore, even after transfusion requirement had been taken into account, IO with SF levels above 1000 ng/ml remained an independent prognostic factor, both for overall and leukemia-free survival. This issue is portrayed in Figure 3 which points out that transfusion dependency clearly has a negative effect on overall survival because it is a consequence of severe bone marrow disease, reflecting the complications of chronic anemia, infections and bleeding. In addition, transfusion dependency causes iron overload, thereby creating a new medical problem which has its own negative impact on survival. This impact is not restricted to patients with low-risk MDS, but was also documented in refractory anemia with excess blasts (RAEB)-1 and even in RAEB-2 [START_REF] Sanz | Independent impact of iron overload and transfusion dependency on survival and leukemic evolution in patients with myelodysplastic syndrome[END_REF].

Chee et al concluded that neither SF nor number of red blood cell (RBC) transfusions affect overall survival in patients with refractory anemia with ring sideroblasts (RARS) [START_REF] Chee | Neither serum ferritin nor number of red blood cell transfusions affect overall survival in refractory anemia with ringed sideroblasts[END_REF]. However, since 72% of the RARS patients had received only a mean of 23 units of packed red cells, and since diTucci had demonstrated that at least 100 units are required before cardiac T2* becomes abnormal [START_REF] Di Tucci | Myocardial iron overload assessment by T2* magnetic resonance imaging in adult transfusion dependent patients with acquired anemias[END_REF], the absence of more evidence of cardiac failure in the study by Chee et al. is not surprising. Moreover, because autopsies were not performed and almost half of the patients who died had no cause of death available, the results of that study have to be interpreted with caution [START_REF] Bennett | MDS Foundation`s Working Group on Transfusional Iron Overload. Consensus statement on iron overload in myelodysplastic syndromes[END_REF].

Increased risk of leukemic transformation in MDS patients with iron overload: is there a link?

Both the Italian and the Spanish groups reported that iron overload is related to the risk of leukemic transformation [START_REF] Sanz | Independent impact of iron overload and transfusion dependency on survival and leukemic evolution in patients with myelodysplastic syndrome[END_REF][START_REF] Malcovati | Predicting survival and leukemic evolution in patients with myelodysplastic syndrome[END_REF]. Although this could be due to severe bone marrow disease, the Spanish study showed that the predictive value of IO was independent of the WPSS. Why should iron overload affect the frequency of leukemic transformation? It has been shown that endogenous production of ROS may be elevated in myeloid malignancies as a consequence of the underlying genetic changes [START_REF] Rassool | Reactive oxygen species, DNA damage, and error-prone repair: a model for genomic instability with progression in myeloid leukemia?[END_REF]. Increased ROS can drive a cycle of genomic instability leading to DNA double-strand breaks and error-prone repair, promoting the acquisition of further genomic changes [START_REF] Sallmyr | Genomic instability in myeloid malignancies: increased reactive oxygen species (ROS), DNA double strand breaks (DSBs) and errorprone repair[END_REF]. Increased LCI, generated by dysregulated cellular iron metabolism or increased LPI, may further exacerbate oxidative stress and thus aggravate the genomic instability of the pre-leukemic clone, thereby promoting clonal evolution towards acute leukemia [START_REF] Pullarkat | Objectives of iron chelation therapy in myelodysplastic syndromes: more than meets the eye?[END_REF].

On the other hand, a recent matched-pair analysis from the Düsseldorf MDS Registry, including 93 patients with various types of MDS undergoing long-term chelation therapy and 93 matched patients receiving supportive care only, did not show a significant difference in the frequency of acute myeloid leukemia transformation between the groups (10% vs. 12% [2 years after diagnosis] and 19% vs. 18% [5 years after diagnosis], respectively) [START_REF] Fox | Matched-pair analysis of 186 MDS patients receiving long-term iron chelation therapy or transfusion therapy only[END_REF]. These results do not indicate that the risk of leukemic transformation is decreased by iron chelation.

However, achieving such an effect may require constant elimination of LPI by effective chelation, in order to suppress oxidative stress. LPI levels were not measured in this retrospective analysis but should be included in prospective studies.

Oxidative stress in MDS

The concept of oxidative stress in MDS (Fig. 4) is supported by laboratory investigations. Oxidized pyrimidine nucleotides were identified in the progenitorenriched bone marrow CD34+ compartment from MDS patients but not in CD34-MDS cells or CD34+ cells from normal subjects [START_REF] Peddie | Oxidative DNA damage in CD34+ myelodysplastic cells is associated with intracellular redox changes and elevated plasma tumour necrosis factor-α concentration[END_REF]. MDS CD34+ blood cells also had oxidized pyrimidine nucleotides compared with MDS CD34-cells. The authors suggested that intracellular ROS production plays a role in the pathogenesis of ineffective hemopoiesis and, consequently, that there is a rationale for therapeutic use of antioxidants in MDS. When four antioxidant enzymes were studied in normal, MDS and acute myeloid leukemia bone marrow cells [START_REF] Bowen | Antioxidant enzyme expression in myelodysplastic and acute myeloid leukemia bone marrow: further evidence of a pathogenetic role for oxidative stres?[END_REF], their expression was most frequently increased in MDS/acute myeloid leukemia granulocytes, but less so in CD34+ cells. The authors concluded that the CD34+ compartment in MDS is under oxidative stress, with maturing cells being selected according to their augmented antioxidant defense. Another study showed that patients with low-risk MDS also had increased oxidative DNA damage in more differentiated CD34-bone marrow cells [START_REF] Novotna | Oxidative damage in bone marrow cells of patients with low-risk myelodysplastic syndrome[END_REF]. The authors suggested that oxidative damage contributes to genomic instability and disease progression in low-risk MDS. However, besides increased oxidative stress, impaired DNA repair capacity may also contribute to irreversible oxidative DNA damage [START_REF] Jankowska | Base excision repair dysfunction in a subgroup of patients with myelodysplastic syndrome[END_REF]. Using flow cytometry techniques, evidence of oxidative stress was found in RBC, platelets and granulocytes from patients with MDS. ROS were higher while reduced GSH was lower in RBC and platelets compared with normal cells. In neutrophils, no difference was found in ROS, while the GSH levels were lower. A correlation (r = 0.6) was found between serum ferritin levels of the patients and the ROS in their RBC and platelets [START_REF] Ghoti | Oxidative stress in red blood cells, platelets, and polymorphonuclear leukocytes from patients with myelodysplastic syndromes[END_REF].

ROS-induced damage may be an important trigger of the increased apoptotic activity that occurs in hematopoietic precursors of MDS patients. The anti-oxidant N-acetylcysteine significantly reduced apoptosis in an in-vitro assay of the clonogenic potential of MDS progenitors, suggesting a useful role for ameliorating oxidative stress in MDS patients [START_REF] Cortelezzi | Efficacy of N-acetylcysteine and alltrans retinoid acid in restoring in vitro effective hemopoiesis in myelodysplastic syndromes[END_REF]. Oxidative stress in MDS may partly be related to labile, redox-active iron. Elevated levels of NTBI have been observed in the plasma of patients with MDS, even in patients not receiving transfusions [START_REF] Cortelezzi | Non-transferrin-bound iron in myelodysplastic syndromes: a marker of ineffective erythropoiesis?[END_REF].

Furthermore, LCI was found to be increased in MDS erythroid cells [START_REF] Ghoti | Changes in parameters of oxidative stress and free iron biomarkers during treatment with deferasirox in iron-overloaded patients with myelodysplastic syndromes[END_REF].

Significant changes in intra-and extracellular free iron species and oxidative stress parameters were demonstrated during treatment of iron-loaded MDS patients with the oral iron chelator deferasirox. In RBC, there was a significant decrease in LCI, mean levels of ROS, and membrane lipid peroxidation, with a concomitant increase in GSH during deferasirox therapy [START_REF] Ghoti | Changes in parameters of oxidative stress and free iron biomarkers during treatment with deferasirox in iron-overloaded patients with myelodysplastic syndromes[END_REF].

Iron-related oxidative stress may be aggravated by mitochondrial abnormalities in MDS, which may lead to further mitochondrial impairment [START_REF] Gattermann | The Role of Mitochondria in MDS[END_REF]. Mitochondrial DNA (mtDNA) is particularly susceptible to oxidative damage. Gao et al recently demonstrated that after 3-5 days of exposure to high iron, rat cardiac myocytes exhibited damage to mtDNA reflected by diminished amounts of near full-length 15.9-kb polymerase chain reaction product with no change in the amounts of a 16.1-kb product from a nuclear gene [START_REF] Gao | Mitochondrial DNA damage in iron overload[END_REF]. With the loss of intact mtDNA, cellular respiration declined and mRNAs for three electron transport chain subunits and 16S mRNA encoded by mtDNA were decreased, whereas no decrements were found in four subunits encoded by nuclear DNA. The authors concluded that longterm damage to cells and organs in iron-overload disorders involves interactions between iron and mitochondrial ROS resulting in cumulative damage to mtDNA, impaired synthesis of respiratory chain subunits and respiratory dysfunction.

Finally, ROS may also be involved in mediating the suppression of hepcidin in MDS patients with iron overload [START_REF] Choi | ROS mediate the hypoxic repression of the hepcidin gene by inhibiting C/EBP alpha and STAT-3[END_REF] because they can repress the hepcidin gene by preventing C/EBPalpha and STAT-3 binding to the hepcidin promoter. Therefore, it was not surprising that serum hepcidin levels increased after amelioration of oxidative stress parameters by deferasirox treatment [START_REF] Ghoti | Decrease in intra-and extra-cellular free iron species and oxidative stress parameters and increase in serum and urinary hepcidin during treatment with deferasirox in iron loaded patients with MDS[END_REF].

Clinical consequences of iron overload in MDS

As most patients with MDS are elderly, comorbidities may render them more susceptible to iron toxicity; it is therefore important to evaluate whether ironrelated complications can diminish their life expectancy.

Hepatic complications

Transfusion-dependent MDS patients usually develop hepatic IO as documented by magnetic resonance imaging (MRI) (Table I). Although MDS patients generally do not live long enough to develop end-stage liver cirrhosis, a recent Japanese study suggests that hepatic damage contributes to non-leukemic death [START_REF] Takatoku | Retrospective nationwide survey of Japanese patients with transfusion-dependent MDS and aplastic anemia highlights the negative impact of iron overload in morbidity/mortality[END_REF].

Patients dying from cardiac or hepatic failure had received more than twice the number of RBC units than those who died from other causes. Thirty-seven of 38 MDS patients who died from cardiac or hepatic failure had a SF level of ≥1000 ng/ml.

Diabetes

A retrospective, age-matched analysis in a large US Medicare database showed that diabetes occurred significantly more frequently in patients with MDS than in the overall Medicare population (40% vs 33.1%; p<0.01) [START_REF] Goldberg | Incidence and clinical complications of myelodysplastic syndromes among United States medicare beneficiaries[END_REF] and patients with MDS requiring RBC transfusions had a greater prevalence than those who were independent of transfusions (44,4% vs. 37.1%; p=0.1). Iron overload, attributable to transfusion therapy and/or ineffective erythropoiesis may have contributed to pancreatic endocrine insufficiency. In contrast to cardiac failure, it is difficult to invoke concomitant anemia as a confounding factor in the development of diabetes.

Cardiac failure

Cardiac problems seem to be the most frequent and serious complication of IO in MDS [START_REF] Jaeger | Iron overload in polytransfused patients with MDS: use of L1 for oral iron chelation[END_REF][START_REF] Schafer | Clinical consequences of acquired transfusional iron overload in adults[END_REF]. In the above-mentioned US Medicare population, significantly more MDS patients (73.2%) suffered cardiac-related events during 3-year follow-up, which exceeded the Medicare population (54.5%; p<0.01). Moreover, a cardiac event occurred significantly more often in transfused than in non-transfused patients (82.4% vs. 67.1%; p<0.001). Nevertheless, it is difficult to assess the impact of myocardial IO because old age, cardiac comorbidities, and the effects of chronic anemia also contribute to cardiac abnormalities in patients with MDS [START_REF] Oliva | Hemoglobin level threshold for cardiac remodelling and quality of life in myelodysplastic syndrome[END_REF].

The frequency and magnitude of cardiac iron overload in MDS is inconsistent.

The available studies employing MRI are given in Table 1, which shows that only a minority of MDS patients have cardiac iron deposition detectable by MRI [START_REF] Chacko | Myocardial iron loading by magnetic resonance imaging T2* in good prognostic myelodysplastic syndrome patients on long-term blood transfusions[END_REF][START_REF] Di Tucci | Myocardial iron overload assessment by T2* magnetic resonance imaging in adult transfusion dependent patients with acquired anemias[END_REF][START_REF] Glanville | MRI evidence of cardiac iron accumulation in myelodysplasia and unusual anemias[END_REF][START_REF] Konen | No evidence for myocardial iron overload in multitransfused patients with myelodysplastic syndrome using cardiac magnetic resonance T2* technique[END_REF]. Almost 40 years ago, Buja and Roberts performed a post-mortem study of cardiac iron deposits in 131 transfused adult patients, excluding thalassemia and sickle cell anemia. Among those who had received more than 75 units of blood, the majority showed cardiac iron deposits (Fig. 5). Grossly visible cardiac iron deposits were always associated with cardiac dysfunction and usually chronic heart failure [START_REF] Buja | Iron in the heart. Etiology and clinical significance[END_REF].

A cardiac MRI study conducted in MDS reported that all patients with evidence of cardiac IO (T2* <20 ms) died of heart failure within a few months [START_REF] Di Tucci | Myocardial iron overload assessment by T2* magnetic resonance imaging in adult transfusion dependent patients with acquired anemias[END_REF]. The authors concluded that myocardial iron deposition in MDS should be prevented rather than treated, because iron-mediated mechanisms may cause cardiac tissue damage before cardiac iron levels are detectable by MRI.

There are data from animal studies suggesting that cardiac function may be more vulnerable to iron overload than liver function. Wood and colleagues [START_REF] Wood | Cardiac iron determines cardiac T2*, T2, and T1 in the gerbil model of cardiomyopathy[END_REF] correlated T2* data drawn from studies of cardiac iron in a gerbil model with studies of left ventricular ejection fraction (LVEF) in patients with thalassemia major. This analysis indicated that myocardial damage sufficient to impair LVEF occurs at tissue iron concentrations >2 mg/g dry weight of tissue (Fig. 6). By comparison, hepatic iron concentrations >22 mg/g dry weight are associated with the development of fibrosis and cirrhosis [START_REF] Bassett | Value of hepatic iron measurements in early hemochromatosis and determination of the critical iron level associated with fibrosis[END_REF]. Therefore, cardiac dysfunction may occur at lower tissue iron concentrations than liver dysfunction [START_REF] Wood | Cardiac iron determines cardiac T2*, T2, and T1 in the gerbil model of cardiomyopathy[END_REF].

How to diagnose iron overload in MDS

Iron overload in MDS is generally diagnosed by measuring serum ferritin levels, as this methodology is widely available and inexpensive. This approach is associated with a number of limitations as measurements become imprecise at high SF levels and ferritin is an acute-phase reactant that is increased during infection, inflammation and malignancy. Therefore, concomitant testing of Creactive protein is recommended. Nevertheless, serial SF assessment, performed in the same testing laboratory, can provide a reliable assessment of increasing or decreasing total body iron burden. In MDS patients undergoing myeloablative hematopoietic stem cell transplantation, it has recently been shown that elevated SF levels were strongly correlated with the number of RBC units transfused, i.e. the magnitude of iron loading, and that a single pre-transplantation measurement of SF level was sufficient for prognostic modelling [START_REF] Alessandrino | Prognostic impact of pre-transplantation transfusion history and secondary iron overload in patients with myelodysplastic syndrome undergoing allogeneic stem cell transplantation: a GITMO study[END_REF]. Transfusion history, though, is an important indicator of iron overload in patients with MDS, and the number of transfusions a patient receives will help to guide decisions about initiating chelation therapy. Establishing methods of monitoring individual patient transfusion burden will facilitate this process, and guidelines from the MDS Foundation suggest this may include encouraging the use of a personal RBC transfusion diary, obtaining blood bank transfusion tracking data and requesting automatic alerts to physicians after a patient receives 20 units of blood [START_REF] Bennett | MDS Foundation`s Working Group on Transfusional Iron Overload. Consensus statement on iron overload in myelodysplastic syndromes[END_REF]. On average, patients with MDS reach a SF level of 1000 ng/ml after receiving 21 RBC units [START_REF] Malcovati | Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO criteria: a basis for clinical decision making[END_REF]. It is important to be aware that, while SF levels correlate well with hepatic IO in thalassemia, an association with cardiac iron overload does not appear to exist and therefore SF levels cannot predict impending cardiac iron overload [START_REF] Wood | Onset of cardiac iron loading in pediatric patients with thalassemia major[END_REF].

If iron overload is suspected based on elevated serum ferritin, a transferrin saturation >55% in men or >50% in woman supports the diagnosis; transferrin saturations above 70%-80% are associated with the appearance of LPI [START_REF] Piga | High nontransferrin bound iron levels and heart disease in thalassemia major[END_REF]. The diagnosis of iron overload can be confirmed by measuring liver iron concentration (LIC) by T2*MRI, superconducting quantum interference device (SQUID) or liver biopsy, although the latter is rarely performed in MDS patients as there is an increased risk of bleeding due to thrombocytopenia and/or platelet dysfunction. Similar to SF levels, LIC does not appear to reflect cardiac iron loading, since a long latent period relative to hepatic iron loading predates the development of myocardial iron loading in transfusion-dependent MDS patients. The data obtained in thalassemia major suggests that there is a threshold of LIC and SF above which cardiac iron starts to accumulate. The SF threshold appears to be around 1500 ng/ml [START_REF] Jensen | Evaluation of myocardial iron by magnetic resonance imaging during iron chelation therapy with deferrioxamine: indication of close relation between myocardial iron content and chelatable iron pool[END_REF].

Since cardiac iron measurement by T2*MRI requires considerable expertise, it is often replaced by assessment of cardiac function using echocardiography. While this method is useful to diagnose ventricular dysfunction, it is also prone to interobserver variability. In the context of secondary hemochromatosis, echocardiography is a crude method because a decrease in LVEF is a late event in the development of cardiac IO. The emphasis should be on preventing rather than diagnosing iron-related organ dysfunction. Accordingly, measurements of NTBI and/or LPI at diagnosis and during follow-up may be helpful in assessing the duration and intensity of exposure to redox-active plasma iron, which in turn may correlate with the risk of clinical complications. As yet, only a few laboratories have established the methodology for measuring NTBI/LPI, but it is hoped that accurate and reproducible methods will become more widely available in the near future.

Is there a survival benefit of iron chelation in MDS?

If iron overload causes clinical complications that decrease the likelihood of survival in MDS, iron chelation therapy should achieve the opposite. This idea was first supported by a small retrospective series from Canada [START_REF] Leitch | Improved survival in patients with myelodysplastic syndrome receiving iron chelation therapy[END_REF], then corroborated by a larger study from France [START_REF] Rose | Does iron chelation therapy improve survival in regularly transfused lower risk MDs patients? A multicenter study by the GFM[END_REF], and recently underscored by a matched-pair analysis from the Düsseldorf MDS Registry [START_REF] Fox | Matched-pair analysis of 186 MDS patients receiving long-term iron chelation therapy or transfusion therapy only[END_REF]. These studies consistently show that patients with lower-risk MDS who receive iron chelation therapy fare significantly better than those who remain untreated. However, one must be aware of a problem that is common to all these retrospective studies. As the decision to chelate was not randomized, it is impossible to exclude that patients may have been more likely to receive iron chelation if, based on unmeasurable factors, their physicians considered them to have a good prognosis. This potential bias can only be avoided by a prospective randomized placebo-controlled trial. Such a trial was recently started and is projected to recruit 630 MDS patients at 126 centers worldwide.

Guidelines for the management of iron overload in MDS

Guidelines for the management of iron overload in MDS are currently based on a low level of evidence and usually make inferences from the more established data obtained in thalassemia major. Nevertheless, a number of consensus statements and practice guidelines have been developed by various groups to outline best practice [START_REF] Bennett | MDS Foundation`s Working Group on Transfusional Iron Overload. Consensus statement on iron overload in myelodysplastic syndromes[END_REF][START_REF] Alessandrino | Evidence-and consensusbased practice guidelines for the therapy of primary myelodysplastic syndromes. A statement from the Italian Society of Hematology[END_REF][START_REF] Arrizabalaga | Guía clínica de quelación del paciente con síndrome mielodisplásico [Clinical guide to chelation therapy for patients with myelodysplastic syndrome (Spanish guidelines)[END_REF][START_REF] Bowen | Guidelines for the diagnosis and therapy of adult myelodysplastic syndromes[END_REF][START_REF] Gattermann | Consensus statement on iron overload in myelodysplastic syndromes[END_REF][START_REF]NCCN Clinical Practice Guidelines in Oncology v.1: Myelodysplastic Syndromes[END_REF][START_REF] Suzuki | Japanese epidemiological survey with consensus statement on Japanese guidelines for treatment of iron overload in bone marrow failure syndromes[END_REF][START_REF] Valent | Iron overload in myelodysplastic syndromes (MDS)diagnosis, management, and response criteria: a proposal of the Austrian MDS platform[END_REF][START_REF] Wells | Iron overload in myelodysplastic syndromes: a Canadian consensus guideline[END_REF]. These guidelines vary somewhat in their recommendations for initiating iron chelation therapy and strategies for the ongoing management of IO [START_REF] Gattermann | Overview of guidelines on iron chelation therapy in patients with myelodysplastic syndomes and transfusional iron overload[END_REF]. Overall, they favor starting treatment if more than 20 blood transfusions have been given and SF levels exceed 1000-2000 ng/ml, with the goal to maintain SF levels below 1000 ng/ml. There is general agreement that the MDS patients most likely to benefit from chelation therapy are those who have a good survival expectancy according to the WHO classification system [START_REF] Brunning | Myelodysplastic syndromes/neoplasms, overview[END_REF] and the IPSS [62]. Most of the previous guidelines recommend deferoxamine (DFO) for iron chelation, while those published more recently also recommend the use of deferasirox [START_REF] Gattermann | Overview of guidelines on iron chelation therapy in patients with myelodysplastic syndomes and transfusional iron overload[END_REF].

Efficacy of chelation therapy in MDS

Data on the efficacy of chelation therapy in MDS are limited to a relatively small number of studies. One of the earliest studies followed 11 patients for up to 60 months of treatment with DFO and observed an improvement in hematopoietic output [63]. A greater than 50% reduction in transfusion requirement was noted in seven patients (64%), while five (45.5%) became completely transfusion independent. Neutrophil and platelet counts improved in 78% and 64% of the patients, respectively. SF levels decreased clearly in nine patients (82%), while LIC assessed by MRI decreased in all the patients although the decrease was minor in five patients. The best hematopoietic responses were observed in patients with the greatest efficacy based on change in LIC. However, the demanding continuous parenteral DFO treatment may lead to difficulties with compliance, particularly in elderly patients with MDS.

The first commercially available alternative to DFO was deferiprone, an oral chelator given three times a day. A pilot study evaluating oral deferiprone in three MDS patients did not observe decreased SF levels in any patient and was stopped prematurely due to the concerns over the risk of agranulocytosis [START_REF] Jaeger | Iron overload in polytransfused patients with MDS: use of L1 for oral iron chelation[END_REF]. A larger study in 18 patients with MDS reported a 25% median decrease in SF (from 3113 ng/ml at baseline) during 1 year of treatment [START_REF] Kersten | Long-term treatment of transfusional iron overload with the oral iron chelator deferiprone (L1): a Dutch multicenter trial[END_REF]. Agranulocytosis developed in one patient and recurred on rechallenge, thus necessitating treatment withdrawal.

More recently, once-daily oral deferasirox has become commercially available and has demonstrated efficacy in decreasing iron burden in patients with MDS [START_REF] List | Two-year analysis of efficacy and safety of deferasirox (Exjade) treatment in myelodysplastic syndrome patients enrolled in the US03 study[END_REF][START_REF] Metzgeroth | Deferasirox in MDS patients with transfusion-caused iron overload -a phase-II study[END_REF][START_REF] Porter | Relative response of patients with myelodysplastic syndromes and other transfusion-dependent anemias to deferasirox (ICL670): a 1-yr prospective study[END_REF][START_REF] Gattermann | Deferasirox in iron-overloaded patients with transfusion-dependent myelodysplastic syndromes: Results from the large 1-year EPIC study[END_REF]. Doses of 10-30 mg/kg/day maintain or decrease SF and LIC in a doseand iron intake-dependent manner and have also been show to produce sustained reductions in LPI levels [START_REF] List | Two-year analysis of efficacy and safety of deferasirox (Exjade) treatment in myelodysplastic syndrome patients enrolled in the US03 study[END_REF][START_REF] Porter | Relative response of patients with myelodysplastic syndromes and other transfusion-dependent anemias to deferasirox (ICL670): a 1-yr prospective study[END_REF][START_REF] Gattermann | Deferasirox in iron-overloaded patients with transfusion-dependent myelodysplastic syndromes: Results from the large 1-year EPIC study[END_REF]. The 1-year EPIC (Evaluation of Patients' Iron Chelation with Exjade) deferasirox clinical trial enrolled the largest cohort of MDS patients (n=341) evaluated for iron chelation therapy [START_REF] Gattermann | Deferasirox in iron-overloaded patients with transfusion-dependent myelodysplastic syndromes: Results from the large 1-year EPIC study[END_REF]. Overall median SF decreased significantly at 1 year (-253 ng/ml; P = 0.002) and decreases occurred irrespective whether patients had previously received chelation therapy or were chelation naïve; changes in SF were reflective of dose adjustments and ongoing iron intake. The discontinuation rate in MDS patients was 48.7%.

Greenberg et al. [START_REF] Greenberg | Prospective assessment of effects of iron-overload parameters of deferasirox therapy in patients with myelodysplastic syndromes[END_REF] recently reported the results of a prospective study evaluating the effects of deferasirox on liver iron concentration (LIC), LPI and pharmacokinetics (PK) along with SF values in MDS patients with IPSS Low-and Intermediate-1 risk profile and evidence of iron overload. Twenty-four heavily transfused MDS patients were enrolled in a planned 52 weeks of therapy. PK studies showed dose-proportional total drug exposure. The data demonstrated that deferasirox was well tolerated and effectively reduced LIC, LPI and SF in the iron-overloaded patients with MDS who completed 24 and 52 weeks of therapy despite ongoing receipt of RBC transfusions.

Similar to observations with DFO, deferasirox has been shown to improve hemoglobin levels and reduce transfusion requirements in a small population (n=5) of patients with MDS and primary myelofibrosis [START_REF] Di Tucci | Correction of anemia in a transfusion-dependent patient with primary myelofibrosis receiving iron chelation therapy with deferasirox (Exjade, ICL670)[END_REF][START_REF] Messa | Deferasirox treatment improved the hemoglobin level and decreased transfusion requirements in four patients with the myelodysplastic syndrome and primary myelofibrosis[END_REF].

It should be noted that deferasirox treatment is less well tolerated in the elderly MDS population when compared with young thalassemia patients. Diarrhea is the most common side effect, which can usually be managed by dose reduction but sometimes necessitates discontinuation of the drug. Anecdotal reports suggest that diarrhea is diminished when deferasirox is taken in the evening. A significant rise in serum creatinine occurs in approximately 25% of MDS patients, but progressive increases can be avoided with appropriate dose reductions. The observed safety profile of deferasirox in MDS may be related to pre-existing comorbidities, concomitant medication use and the advanced age of the MDS patient population.

Discussion

The discussion about the importance of iron overload in MDS underscores the need for a prospective trial investigating the potential survival benefit of iron chelation therapy. We suggest that until results of such a trial become available, one should not ignore the potential impact of unliganded or inadequately liganded 'free iron' that may cause cell and tissue damage. Chelation experience in thalassemia major suggests that substantial clinical benefit can be derived from detoxification of redox-active iron species, as supported by an experimental model of heart cells [START_REF] Glickstein | Intracellular labile iron pools as direct targets of iron chelators: a fluorescence study of chelator action in living cells[END_REF][START_REF] Glickstein | Action of chelators in iron-loaded cardiac cells: Accessibility to intracellular labile iron and functional consequences[END_REF] and the beneficial effects of deferasirox on arterial function [START_REF] Cheung | Effect of deferasirox (ICL670) on arterial function in patients with beta-thalassaemia major[END_REF]. Moreover, Piga et al recently demonstrated a link between NTBI and heart disease in thalassemic patients with transfusional iron overload [START_REF] Piga | High nontransferrin bound iron levels and heart disease in thalassemia major[END_REF], as NTBI levels were significantly higher in patients with heart disease than those without. All patients with heart disease had transferrin saturation above 70% and measurable NTBI levels. Conversely, no patients without NTBI and/or with transferrin saturation of less than 70% had preclinical or clinical evidence of heart disease. The authors support the concept of a threshold level of excess iron (indicated by a transferrin saturation of 70-80%) beyond which the normal regulatory mechanisms protecting against iron toxicity are overwhelmed.

We hope that in the near future treatment decision and monitoring will be supported by LPI and LCI measurements. Measuring the toxic forms of iron in plasma and cells at diagnosis and follow-up, preferentially in parallel with oxidative stress parameters, and correlating the results with clinical complications of IO, with and without iron chelation therapy, will enable a more comprehensive and meaningful assessment of iron overload. Measuring LPI may also help to assess patient compliance.

We feel that, currently, initiating chelation therapy should be tailored to the individual patient, based on the type of MDS and its prognosis, patient's age and comorbidities, transfusion history, measurements of iron burden, and the intensity of the current transfusion regimen. Though it may be difficult to recruit patients, a well-designed prospective trial is required to corroborate the current practice guidelines supporting the use of chelation therapy in transfusion-dependent patients with lower-risk MDS.

Table I. MRI detection of cardiac and liver iron overload in MDS

Fig 1 .

 1 Fig 1.Pathophysiology of iron overload. Adapted from:[START_REF] Cohen | Transfusion and iron chelation therapy in thalassemia and sickle cell anemia[END_REF]

Fig 2 .Fig. 3 Fig 4 .

 234 Fig 2. Impact of transfusion dependency on survival in MDS[START_REF] Malcovati | Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO criteria: a basis for clinical decision making[END_REF]

Fig 5 .Fig 6 .

 56 Fig 5.Relationship between transfusions and cardiac iron in MDS. Based on data from[START_REF] Buja | Iron in the heart. Etiology and clinical significance[END_REF]