Maternal Ferritin Levels during Pregnancy and ADHD Symptoms in 4-Year-Old Children: Results from the INMA–INfancia y Medio Ambiente (Environment and Childhood) Prospective Birth Cohort Study

International Journal of Environmental Research and Public Health Article Maternal Ferritin Levels during Pregnancy and ADHD Symptoms in 4-Year-Old Children: Results from the INMA–INfancia y Medio Ambiente (Environment and Childhood) Prospective Birth Cohort Study Loreto Santa-Marina 1,2,3, Nerea Lertxundi 2,4, Ainara Andiarena 2,4, Amaia Irizar 1,2,5,*, Jordi Sunyer 1,6,7, Amaia Molinuevo 1, Sabrina Llop 1,8, Jordi Julvez 1,7,9 , Andrea Beneito 8, Jesús Ibarluzea 1,2,3,4 , Liher Imaz 3,10 and Maite Ferrin 11,12 1Spanish Consortium for Research on Epidemiology and Public Health (CIBERESP), Instituto de Salud Carlos III, C/Monforte de Lemos 3–5, 28029 Madrid, Spain; [email protected] (L.S.-M.); [email protected]g (J.S.); [email protected] (A.M.); [email protected] (S.L.); [email protected]g (J.J.); [email protected] (J.I.) 2 Biodonostia, Epidemiology and Public Health Area, Environmental Epidemiology and Child Development Group, 20014 San Sebastian, Spain; ner[email protected] (N.L.); ainara.andiar[email protected] (A.A.) 3Public Health Division of Gipuzkoa, Basque Government, 20013 San Sebastian, Spain; [email protected] 4Faculty of Psychology, University of the Basque Country (UPV/EHU), Avenida Tolosa 70, 20018 San Sebastian, Spain 5Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Spain 6Hospital del Mar Research Institute, 08003 Barcelona, Spain 7ISGlobal—Instituto de Salud Global de Barcelona–Campus MAR, PRBB, 08003 Barcelona, Spain 8 Epidemiology and Environmental Health Joint Research Unit, FISABIO–Universitat Jaume I–Universitat de València, 08003 València, Spain; [email protected] 9Institut d0InvestigacióSanitària Pere Virgili (IISPV), Hospital Universitari Sant Joan de Reus, 43204 Reus, Spain 10 Biodonostia, Epidemiology and Public Health Area, Epidemiology of Chronic and Communicable Diseases Group, 20014 San Sebastian, Spain 11 Haringey Child and Adolescent Mental Health Service, Barnet, Enfield and Haringey NHS Mental Health Trust, London N15 3TH, UK; [email protected] 12 Recognition Health, London W1G 9RU, UK *Correspondence: [email protected] Received: 2 October 2020; Accepted: 19 October 2020; Published: 22 October 2020   Abstract: Ferritin status during prenatal brain development may influence the risk of attention deficit and hyperactivity disorder (ADHD) symptoms in childhood. We investigated the association of maternal ferritin in pregnancy and ADHD-like symptoms in offspring. A total of 1095 mother-child pairs from three birth cohorts of the INMA Project (Spain) were studied. Maternal plasma ferritin in pregnancy was measured at 11.57 weeks of gestation. Children 0 s ADHD-like symptoms at ages 4–5 years were assessed using the ADHD Rating Scale-IV. The count model of the zero-inflated Poisson regression model showed a significant inverse association between ferritin (continuous variable) and inattention, β = − 0.19 ( − 0.32, − 0.07), for boys. Comparing ferritin level by tertiles, significant differences were observed between the first tertile ([1.98, 20.92]) and the second ([20.92, 38.79]) and third tertiles ([38.79, 216.5]) (mg/L).The number of symptoms was lower for those in the third tertile, β = − 0.3 ( − 0.55, − 0.5), and for those in the second one, β = − 0.37 ( − 0.6, − 0.14). The model stratification by sex also showed this inverse association for boys only, β = − 0.21 ( − 0.34, − 0.08). No associations Int. J. Environ. Res. Public Health 2020,17, 7704; doi:10.3390/ijerph17217704 www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2020,17, 7704 2 of 17 were found between ferritin level and hyperactivity or total ADHD symptoms. High ferritin levels during pregnancy show a protective association with child inattentive-type ADHD symptoms. Keywords: ferritin status; pregnancy; ADHD symptoms; childhood 1. Introduction Attention deficit/hyperactivity disorder (ADHD) is the most frequent childhood-onset neuropsychiatric condition, with an estimated worldwide prevalence of approximately 5% in school-aged children [1]. ADHD symptoms tend to persist into adulthood in as many as 65% of cases [2]. Despite ADHD being the most studied neuropsychiatric condition in child psychiatry worldwide [ 3 ], the etiological factors [ 4 ] are not well understood [ 5 ]. No specific etiology has been identified for ADHD, and findings are consistent with a multifactorial model [ 6 ]. Indeed, the disorder is likely to be due to a complex combination of environmental, genetic and biological factors. The range of etiologies related to prenatal and perinatal risk factors, genetics and neurobiological deficits that have been proposed may all be involved in the pathophysiology of ADHD [7]. The human brain is highly sensitive to environmental exposure occurring during particular periods of vulnerability [ 8 , 9 ]. In early life, biological brain development is highly active and any factors enhancing or disturbing this process could have permanent effects on brain function [ 10 ]. It is also established that both genetic and a wide range of environmental factors, including physical, biological, psychological and social factors, are able to modulate brain structure and its function by interacting with genes and expression mechanisms (i.e., epigenetic determinants) [11]. In recent years, it has been suggested that iron deficiency (ID) may contribute to behavioral and cognitive dysfunctions [ 12 ]. Iron is an essential trace metal, which plays a central role in many essential brain functions [ 13 ]. Animal models show that early iron deficiency may lead to structural and functional brain abnormalities including alterations in dopamine metabolism, energy metabolism and myelination [ 14 ]. Several case-control studies have reported a significant association between iron deficiency and low scores in tests assessing mental, social and motor development in infants, as well as a lower intelligence quotient [ 15 ], poor learning performance and impaired neuropsychological functions (e.g., poor spatial memory) [13]. Further evidence supports the hypothesis that a lack of brain iron might contribute to the pathophysiology of ADHD [ 16 , 17 ]. Firstly, iron is a cofactor of enzymes necessary for the synthesis and catabolism of the monoaminergic neurotransmitters including dopamine [ 18 ], which has been linked to ADHD [ 7 , 19 ]. Secondly, ID is associated with a decrease in dopamine transporter expression [ 20 ], and the corresponding gene is linked to a genetic vulnerability for ADHD [ 21 ]. Thirdly, ID may lead to dysfunction in the basal ganglia [ 22 ], which again are believed to play a significant role in the pathophysiology and expression of ADHD [5]. The role of serum ferritin levels as a reliable measure of iron stores in body tissues, including the brain, in children with ADHD has been studied in recent years. A meta-analysis [ 23 ] and a systematic review [ 24 ] have found contradictory findings for the relationship between ferritin levels and ADHD, and therefore the extent to which serum ferritin correlates with brain iron levels remains unclear [ 25 , 26 ]. In addition, maternal ferritin levels and their effects on child neuropsychological development have been less studied. A prospective population-based study in a rural province in Vietnam, which enrolled 497 pregnant women at 12–20 weeks of gestation and followed them up with their infants until six months postpartum, found that lower ferritin levels were associated with a poorer cognitive function and worse social and emotional development in the children [ 27 ]. Similarly, a recent study based on health and population register data from the Stockholm Youth Cohort evaluated 532,232 women with ID diagnosed during the first 30 weeks of pregnancy and found that anemia diagnosed up to this point Int. J. Environ. Res. Public Health 2020,17, 7704 3 of 17 in pregnancy was associated with an increased risk of autism spectrum disorder (ASD), ADHD and intellectual disability in offspring [12]. Therefore, the objective of the present study was to evaluate the relationship between prenatal ferritin levels with infant ADHD symptoms. For that, the association between ID during pregnancy measured by ferritin levels in maternal serum and ADHD symptoms in children aged 4–5 years was assessed. Findings might have important implications for clinical practice, regarding prenatal iron supplementation. 2. Materials and Methods 2.1. Subjects The INMA (INfancia y Medio Ambiente, Childhood and Environment) Project [ 28 ], a multicenter birth cohort study, was established between 2003 and 2008 in 3 regions of Spain, namely: Gipuzkoa (Basque Country), Sabadell (Catalonia) and Valencia. Participant recruitment and follow-up procedures have been reported in detail elsewhere (Guxens et al., 2012). A total of 2644 eligible women were recruited during prenatal visits in the first trimester of pregnancy. Inclusion criteria were: ≥ 16 years of age, singleton pregnancy and intention to deliver at the reference hospital; and exclusion criteria were: women having any communication problems that might hinder their participation in the study, fetuses having malformations and pregnancies having been achieved by assisted conception. Women were followed up during pregnancy, and their children were enrolled at birth and followed up until 4–5 years of age. The population finally studied was composed of 1095 pregnant women from the general population resident in Sabadell (N =443), Valencia (N =339) or Gipuzkoa (N =313) and their children at 4 years of age (Figure 1). Int. J. Environ. Res. Public Health 2020, 17, x FOR PEER REVIEW 4 of 22 Figure 1. Maternal ferritin levels (mg/L) were assessed during the first trimester of pregnancy. The study was approved by the ethics committees of the centers involved in the study, and written informed consent was obtained from the parents of all children. 2.2. Ferritin Measurement Maternal whole blood samples were collected during pregnancy (mean [SD] 13.3 [1.5] weeks of gestation) by venipuncture under fasting conditions and stored between −70 and −80 °C until analysis. Maternal plasma ferritin concentrations were quantified in samples from Gipuzkoa and Sabadell cohorts by time-resolved fluorescence immunoassay (DELFIA Ferritin kit A069−101), at the Gipuzkoa Public Health Laboratory, and in samples from the Valencia cohort by immunoturbidimetry (Beckman Coulter AU analysers) at La Fe hospital. Flow chart Exclusion of 859 without ADHD symptoms data Exclusion of 196 without ferritin level. Eligible women with ferritin data in the first trimester of pregnancy: n = 1954 632 Gipuzkoa 651 Sabadell 669 Valencia Women enrolled in the first trimester of pregnancy: n = 2150 638 Gipuzkoa 657 Sabadell 855 Valencia Eligible children with ADHD symptoms data at 4 years old: n = 1095 313 Gipuzkoa 443 Sabadell 339 Valencia Figure 1. Maternal ferritin levels (mg/L) were assessed during the first trimester of pregnancy. The study was approved by the ethics committees of the centers involved in the study, and written informed consent was obtained from the parents of all children. Int. J. Environ. Res. Public Health 2020,17, 7704 4 of 17 2.2. Ferritin Measurement Maternal whole blood samples were collected during pregnancy (mean [SD] 13.3 [1.5] weeks of gestation) by venipuncture under fasting conditions and stored between − 70 and − 80 ◦ C until analysis. Maternal plasma ferritin concentrations were quantified in samples from Gipuzkoa and Sabadell cohorts by time-resolved fluorescence immunoassay (DELFIA Ferritin kit A069–101), at the Gipuzkoa Public Health Laboratory, and in samples from the Valencia cohort by immunoturbidimetry (Beckman Coulter AU analysers) at La Fe hospital. 2.3. Child and Family Characteristics Data on maternal and child characteristics were collected through two questionnaires administered during face-to-face interviews at different points in the first and third trimester of pregnancy, at birth, at 14 months after birth and when children reached 4–5 years of age. We used data on maternal country of origin (Spain/other), maternal social class (manual or lower class [IV and V] and non-manual/skilled or higher class [I–III]), maternal education (primary or lower, secondary and university), maternal age, maternal mental health (mental problems, yes/no), maternal intelligence quotient, parity, smoking during pregnancy (yes/no), alcohol intake during pregnancy (yes/no), pre-pregnancy body mass index (BMI) (underweight =BMI <18.5; normal weight =BMI 18.5–25; overweight =BMI 25–30; and obesity =BMI >30 kg/m 2 ) and breastfeeding (weeks of any breastfeeding). Anthropometric measures at birth and the sex of the infant were obtained from the child’s medical records. Deliveries before 37 weeks of gestation were defined as preterm births. Gestational age was calculated from the date of the last menstrual period reported at recruitment and confirmed using ultrasound examination at week 12 of gestation. Whole blood samples were collected by venipuncture of cord vessels before the placenta was delivered for the measurement of mercury in the newborn. At 14 months of age, various types of data related to the children were collected including daycare attendance, birth order among siblings (first/not first), whether they were living with their parents (both/only one) and the number of people in the household. The exact age of the child was also recorded at the time of the assessment of ADHD-like symptoms. 2.4. ADHD Symptoms Child ADHD symptoms at 4 years of age were assessed in the period 2008–2013 using the ADHD Rating Scale-IV developed by DuPaul, Power and Anastopoulos [ 29 ] completed by the child’s classroom teacher, which reflects the Diagnostic and Statistical Manual of Mental Disorders 4th edition Criteria for ADHD (ADHD-DSM-IV; [ 30 ]). The scale comprises 18 ADHD symptoms and is designed to evaluate inattention (9 symptoms) and hyperactivity/impulsivity (9 symptoms). Each symptom is rated using a 4-point scale: 0 =“not at all”, 1 =“just a little”, 2 =“pretty much” and 3 =“very much”. Scores were summed to provide continuous measures of inattention, hyperactivity/impulsivity and total ADHD symptoms. The presence of symptoms in the clinical range was estimated by coding ratings of 0 and 1 as “symptom absent” and ratings of 2 and 3 as “symptom present”. The good psychometric characteristics of this measurement in the INMA project have been reported previously [31]. 2.5. Statistical Analysis First, we compared socio-demographic characteristics of mothers and children across the three cohorts studied (Gipuzkoa, Sabadell and Valencia). The difference in the mean number of symptoms was analyzed using the Student’s t-test (for two samples) and one-way analysis of variance (more than two samples). Due to the excess of zeros in symptom ratings, the association between ferritin level and symptoms was analyzed using zero-inflated Poisson regression models. This type of model has two parts, a Poisson count model and a logit model for predicting excess zeros. The level of ferritin was entered in the models in two ways: as a continuous variable and categorized in tertiles (Tertile 1: [1.98, 20.92]; Int. J. Environ. Res. Public Health 2020,17, 7704 5 of 17 Tertile 2: (20.92, 38.79]; and Tertile 3: (38.79, 216.5] (mg/L)) to study the trend across the tertiles. Initially, variables with a p-value ≤ 0.20 in the bivariate analysis were added to the base models (ferritin level and ADHD symptoms). These variables were maintained in the models if they changed the magnitude of the main effects by more than 10% or were significant. The final model was adjusted for: cohort (Gipuzkoa, Sabadell or Valencia), maternal smoking during pregnancy (yes/no), alcohol intake during pregnancy (g/day), pre-pregnancy BMI and social class (manual/non-manual), the child’s sex, age at the time of the test and whether they were living with parents (both/only one) and the week of sample collection for ferritin analysis. For the count model of the association of inattention symptoms with ferritin levels, the interaction between ferritin and sex was significant, and hence results are shown separately for boys and girls. The final model was also stratified by cohort and sex of the child. Statistical analyses were conducted using R version 3.6.1. 3. Results Overall, 1095 eligible mother-child pairs were included in this study (Figure 1). The socio-demographic characteristics of mothers and children across the three cohorts (Gipuzkoa, Sabadell and Valencia) are summarized in Table 1. The Valencian cohort had higher levels of ferritin and an earlier week of blood sample collection than the Gipuzkoa and Sabadell cohorts. Mothers recruited in Gipuzkoa were the oldest, breastfed for the longest and had the highest level of education and social class, lowest BMI and lowest percentage of foreign mothers. Regarding lifestyle during pregnancy, Valencia had the highest rates of smoking and alcohol consumption among the mothers. In addition, children from Valencia had the highest levels of Hg in umbilical cord blood. The mean ages of mothers and children at assessment of ADHD-like symptoms were 30.9 years and 4.9 years, respectively. Compared with women excluded, those included in the analysis were older, had higher levels of education and social class, breastfed for longer and tended to smoke less during pregnancy (Supplementary Table S1). The mean maternal ferritin level was 35.9 mg/L (standard deviation: 26.81 mg/L). In the univariate analyses, the total ADHD symptom score was higher in boys than girls. Further, total ADHD symptom scores were higher in children of mothers with higher BMI, lower alcohol intake, worse mental health and lower education and social class, who smoked and did not breastfeed (Table 2). Considering ferritin levels as a continuous variable, the count model of the zero-inflated Poisson regression model showed a significant inverse association between ferritin levels and inattention symptoms, β = − 0.19 ( − 0.32, − 0.07), in boys. In addition, comparing ferritin levels by tertiles for boys, there were significant differences between the first and second tertile, β = − 0.3 ( − 0.55, − 0.05), and the first and third tertile, β = − 0.37 ( − 0.6, − 0.14), with a significant trend (p=0.002). In contrast, no significant association was found between ferritin level and hyperactivity/impulsivity or total ADHD symptom scores (Table 3). The stratification of the count model by the sex of the child also showed an inverse association between ferritin level and inattention symptoms for boys, both when taking ferritin levels continuously, β = − 0.21 ( − 0.34, − 0.08), and also when comparing the individuals by tertiles: the expected change in the number of symptoms was lower for those in the second tertile, β = − 0.32 ( − 0.57, − 0.06), and for those in the third one, β = − 0.4 ( − 0.63, − 0.16) (p for trend 0.001). In contrast, the count model for inattention showed a positive association for the girls in the third tertile of ferritin levels, β =0.58 (0.02, 1.13), compared with those in the first one (p for trend 0.012). Similarly, when considering the total number of ADHD symptoms, a negative association was observed for the boys in the third tertile, β = − 0.22 ( − 0.39, − 0.06). The zero-inflation model for ADHD symptoms showed a positive association, β =0.6 (0.05, 1.16), for girls in the second tertile and a positive association was also observed when taking ferritin level as a continuous variable, β=0.39 (0.08, 0.69) (Table 4). Int. J. Environ. Res. Public Health 2020,17, 7704 6 of 17 Table 1. Descriptive characteristics of the study sample. GIPUZKOA SABADELL VALENCIA p-Value * TOTAL N=313 N =443 N =339 N =1095 Variables N (%) Mean (SD) N (%) Mean (SD) N (%) Mean (SD) N (%) Mean (SD) Child characteristics Sex Female 157 (50.16) 217 (48.98) 166 (48.97) 0.939 540 (49.32) Male 156 (49.84) 226 (51.02) 173 (51.03) 555 (50.68) Missing 0 (0) 0 (0) 0 (0) 0 (0) Preterm No 301 (96.17) 425 (95.94) 320 (94.4) 0.168 1046 (95.53) Yes 11 (3.51) 12 (2.71) 18 (5.31) 41 (3.74) Missing 1 (0.32) 6 (1.35) 1 (0.29) 8 (0.73) Sibling order Not first 143 (45.69) 188 (42.44) 147 (43.36) 0.669 478 (43.65) First 170 (54.31) 255 (57.56) 192 (56.64) 617 (56.35) Missing 0 (0) 0 (0) 0 (0) 0 (0) Lives with mother/father Both 295 (94.25) 426 (96.16) 332 (97.94) 0.488 1053 (96.16) Only one 3 (0.96) 5 (1.13) 7 (2.06) 15 (1.37) Missing 15 (4.79) 12 (2.71) 0 (0) 27 (2.47) Number of people who they live with Missing 15 (4.79) 2.58 (0.72) 12 (2.71) 2.57 (0.79) 0 (0) 2.58 (0.76) 0.997 27 (2.47) 2.57 (0.76) Child’s age at the time of the test Missing 119 (1.74) 4.41 (0.22) 1 (0.09) 4.44 (0.26) 0 (0) 5.92 (0.32) <0.001 20 (1.83) 4.90 (0.75) Breastfeeding Missing 13 (4.15) 29.33 (20.14) 1 (0.23) 27.48 (19.64) 0 (0) 23.04 (19.3) <0.001 14 (1.28) 26.6 (19.82) Hg (µg/l) in umbilical cord Missing 35 (11.18) 9.6 (5.83) 117 (26.41) 8.35 (6.53) 95 (28.02) 15.34 (11.6) <0.001 247 (22.56) 10.77 (8.65) Maternal characteristics Ferritin level (log scale) Missing 0 (0) 3.28 (0.74) 0 (0) 3.25 (0.79) 0 (0) 3.45 (0.68) <0.001 0 (0) 3.32 (0.75) Maternal blood collection week Missing 0 (0) 13.33 (1.31) 2 (0.45) 13 (1.8) 0 (0) 8.09 (4.23) <0.001 2 (0.18) 11.57 (3.58) Alcohol intake during pregnancy Missing 0 (0) 0.19 (0.47) 0 (0) 0.37 (1.07) 0 (0) 0.41 (1.19) 0.009 0 (0) 0.33 (0.98) Pre-pregnancy BMI Missing 0 (0) 22.9 (3.35) 0 (0) 23.87 (4.71) 0 (0) 23.8 (4.43) 0.004 0 (0) 23.57 (4.29) Age Missing 0 (0) 31.58 (3.48) 1 (0.23) 30.66 (4.1) 0 (0) 30.64 (4.06) 0.002 1 (0.09) 30.92 (3.94) Mental health (14 months after birth) Missing 39 (12.46) 9.23 (3.33) 27 (6.09) 9.9 (3.95) 339 (100) −0.021 405 (36.99) 9.64 (3.72) Parity Missing 0 (0) 0.53 (0.64) 2 (0.45) 0.49 (0.65) 0 (0) 0.5 (0.64) 0.705 2 (0.18) 0.51 (0.64) Smoking during pregnancy No 269 (85.94) 379 (85.55) 264 (77.88) <0.001 912 (83.29) Yes 34 (10.86) 58 (13.09) 75 (22.12) 167 (15.25) Missing 10 (3.19) 6 (1.35) 0 (0) 16 (1.46) Country of birth Spain 305 (97.44) 402 (90.74) 318 (93.81) 0.001 1025 (93.61) Other 8 (2.56) 39 (8.8) 21 (6.19) 68 (6.21) Missing 0 (0) 2 (0.45) 0 (0) 2 (0.18) Social class No manual 190 (60.7) 247 (55.76) 157 (46.31) <0.001 594 (54.25) Manual 123 (39.3) 196 (44.24) 182 (53.69) 501 (45.75) Missing 0 (0) 0 (0) 0 (0) 0 (0) Education level Primary 34 (10.86) 104 (3.48) 90 (26.55) <0.001 228 (20.82) Secondary 116 (37.06) 190 (42.89) 155 (45.72) 461 (42.1) University 162 (51.76) 146 (32.96) 94 (27.73) 402 (36.71) Missing 1 (0.32) 3 (0.68) 0 (0) 4 (0.37) * One-way ANOVA for mean differences and chi-squared test for categorical variables. Int. J. Environ. Res. Public Health 2020,17, 7704 7 of 17 Table 2. Percentiles 75–90–95, mean and standard deviation for inattention, hyperactivity and ADHD symptoms. Inattention Hyperactivity ADHD Percentiles Mean sd p* Percentiles Mean sd p* Percentiles Mean sd p* Variables Child characteristics Sex Female 0 1 2 0.33 1.13 <0.001 0 1 3 0.43 1.23 <0.001 0 2.1 5 0.76 1.97 <0.001 Male 1 4 6 0.89 1.98 1 3 5 0.76 1.76 2 6 9 1.65 3.3 Preterm No 0 2 5 0.61 1.63 0.768 0 2 4 0.6 1.54 0.362 1 4 8 1.21 2.76 0.848 Yes 1 2 3 0.68 1.54 1 2 2 0.46 0.87 2 3 5 1.15 1.89 Sibling order Not first 0 2 4.15 0.63 1.61 0.884 0 2 4 0.59 1.48 0.871 1 4 7 1.21 2.66 0.997 First 0 2 5 0.61 1.67 0 2 4 0.6 1.57 1 5 8 1.21 2.84 Lives with mother/father Both 0 2 5 0.61 1.63 0.760 0 2 4 0.59 1.53 0.020 1 4 7.4 1.2 2.75 0.342 Only one 0 0 2.1 0.47 1.81 0 0.6 1.3 0.2 0.56 0 1.2 3.8 0.67 2.09 Number of people who they live with ≤2 0 2 4 0.56 1.55 0.318 0 2 3.15 0.57 1.49 0.693 1 4 7.15 1.13 2.63 0.415 >2 0 2 5 0.67 1.73 0 2 4 0.61 1.55 1 4 7.55 1.27 2.87 Child´s age at the time of the test ≤4.50 0 1 3 0.46 1.4 0.001 0 2 3 0.49 1.7 0.018 1 3 5 0.95 2.39 0.001 >4.50 0 3 5.65 0.79 1.87 0 3 4.65 0.72 1.37 1 5 9 1.51 3.11 Breastfeeding (weeks) No 1 4.7 6 1.02 2.17 0.010 0 3 5 0.78 1.83 0.028 2 8 10 1.8 3.54 0.007 0–16 0 2 5 0.67 1.67 1 3 5 0.79 1.91 1 5 8.35 1.47 3.13 16–24 0 2.5 5 0.66 1.79 0 2 4 0.57 1.4 1 5 6.5 1.24 2.92 >24 0 2 3.7 0.49 1.39 0 2 3 0.48 1.28 1 4 6 0.97 2.24 Hg (µg/l) in umbilical cord ≤8,2 0 2 5 0.64 1.690 0.608 0 2 3 0.58 1.58 0.791 1 4 7.75 1.22 2.87 0.653 >8.2 0 2 4 0.59 1.590 0 2 4 0.55 1.46 1 4 7 1.14 2.68 Maternal characteristics Cohort Gipuzkoa 0 1 3 0.42 1.29 0.012 0 1 2 0.35 1.18 0.004 0 3 5 0.77 2.22 0.002 Sabadell 0 2 5 0.62 1.62 1 2 4 0.69 1.6 1 4.8 7.9 1.31 2.8 Valencia 0 3 6 0.8 1.92 0 3 4.1 0.7 1.69 1 5 9 1.5 3.1 Ferritin level (log scale) ≤3.37 0 2 5 0.61 1.67 0.955 0 2 4 0.62 1.49 0.636 1 4 8 1.23 2.75 0.819 >3.37 0 2 4.8 0.62 1.61 0 2 4 0.57 1.57 1 4.6 7 1.19 2.77 Maternal blood collection week ≤12 0 2 5 0.67 1.72 0.300 0 2 4 0.61 1.53 0.824 1 5 8 1.27 2.82 0.462 >12 0 2 4 0.56 1.55 0 2 4 0.59 1.53 1 4 7 1.15 2.7 Alcohol intake during pregnancy ≤0.0201 0 3 5.8 0.71 1.850 0.019 0 2 4 0.67 1.65 0.052 1 5 9 1.39 3.12 0.013 >0.0201 0 2 4 0.49 1.300 0 2 3 0.5 1.34 1 4 6 0.99 2.18 Pre-pregnancy BMI Underweight 0 2 2.9 0.51 1.1 <0.001 0 1 2.9 0.49 1.39 0.073 0.5 4 4.9 1 2.01 <0.001 Normal weight 0 2 4 0.49 1.44 0 2 4 0.54 1.42 1 4 6 1.03 2.46 Overweight 0 3 6 0.78 1.92 1 2 3.4 0.69 1.68 1 5 9.4 1.47 3.31 Obese 2 6 7.2 1.42 2.44 1 4 5.6 0.96 2.04 4 8.2 10.6 2.37 3.76 Age ≤31 0 2 5 0.64 1.64 0.664 0 2 4 0.64 1.56 0.295 1 5 7 1.27 2.77 0.404 >31 0 2 5 0.59 1.64 0 2 4 0.54 1.49 1 4 8 1.13 2.76 Mental health (14 months after birth) ≤9 0 1 3 0.39 1.15 0.015 0 2 3 0.46 1.2 0.265 1 3 5 0.85 2 0.040 >9 0 2 5 0.68 1.8 0 2 3 0.58 1.55 1 4 8 1.26 2.98 Int. J. Environ. Res. Public Health 2020,17, 7704 8 of 17 Table 2. Cont. Inattention Hyperactivity ADHD Percentiles Mean sd p* Percentiles Mean sd p* Percentiles Mean sd p* Parity 0 0 2 5 0.61 1.67 0.921 0 2 4 0.6 1.57 0.833 1 5 8 1.22 2.84 0.954 ≥1 0 2 4.05 0.62 1.6 0 2 4 0.59 1.48 1 4 7 1.21 2.65 Smoking in pregnancy No 0 2 4 0.54 1.54 0.003 0 2 3.45 0.53 1.45 0.006 1 4 6 1.08 2.6 0.001 Yes 1 4.4 6 1.06 2.11 1 4 5 0.97 1.93 2 7 10 2.03 3.51 Country of birth Spain 0 2 5 0.6 1.61 0.305 0 2 4 0.59 1.51 0.430 1 4 7.8 1.19 2.72 0.292 Other 1 2.3 6.3 0.87 2.08 1 2 5 0.76 1.81 2 5 7.3 1.63 3.38 Social class Non-manual 0 1 3 0.44 1.31 <0.001 0 2 3 0.54 1.38 0.158 1 3 6 0.98 2.36 0.002 Manual 0 3 6 0.83 1.94 0 2 4 0.67 1.69 1 5 9 1.5 3.15 Education level Primary 0.25 5 7 1.03 2.25 <0.001 0.25 2 4 0.7 1.68 0.283 2 6.3 10 1.73 3.41 0.001 Secondary 0 2 4 0.61 1.52 0 2 4 0.62 1.54 1 5 6 1.23 2.64 University 0 1 3 0.39 1.28 0 2 3 0.51 1.41 0 3 5 0.9 2.41 *t-test for mean comparison (2 samples) or ANOVA (more than two samples). Table 3. Zero-inflated Poisson regression models for the association between inattention, hyperactivity and ADHD symptoms with ferritin levels (mg/L log scale) in continuous scale and in tertiles. Tertile 1 [1.98, 20.92] (mg/L) Tertile 2 (20.92, 38.79] (mg/L) Tertile 3 (38.79, 216.5] (mg/L) p for Trend ** Continuous Beta CI (95%) Beta CI (95%) Beta CI (95%) Beta CI (95%) Inattention Count model *1 Male *** 1 (Ref.) −0.3 (−0.55, −0.05) −0.37 (−0.6, −0.14) 0.002 −0.19 (−0.32, −0.07) Female *** 1 (Ref.) −0.11 (−0.64, 0.42) 0.35 (−0.07, 0.77) 0.066 0.09 (−0.13, 0.31) Zero-inflation model *21 (Ref.) 0.19 (−0.24, 0.63) 0.19 (−0.24, 0.63) 0.488 −0.04 (−0.26, 0.19) Hyperactivity Count model *11 (Ref.) −0.07 (−0.29, 0.16) −0.11 (−0.33, 0.11) 0.327 −0.03 (−0.15, 0.09) Zero-inflation model *21 (Ref.) 0.24 (−0.16, 0.64) 0.17 (−0.23, 0.56) 0.411 0.11 (−0.11, 0.32) ADHD Count model *11 (Ref.) −0.09 (−0.24, 0.06) −0.09 (−0.23, 0.05) 0.239 −0.02 (−0.1, 0.05) Zero-inflation model *21 (Ref.) 0.36 (0, 0.71) 0.12 (−0.22, 0.47) 0.515 0.13 (−0.06, 0.33) Models adjusted by cohort, smoking during pregnancy, alcohol intake during pregnancy, pre-pregnancy BMI, social class, sex and age of the child, living with mother/father and weeks (ferritin extraction). * 1 Count model coefficients (Poisson with log link). * 2 Zero − inflation model coefficients (binomial with logit link). ** pfor the trend between tertiles. *** Model with interaction term between sex and ferritin. Int. J. Environ. Res. Public Health 2020,17, 7704 9 of 17 Table 4. Zero-inflated Poisson regression models for the association between inattention, hyperactivity and ADHD symptoms with ferritin levels (log scale). Tertile 1 Tertile 2 Tertile 3 p Trend ** Continuous [1.98, 20.92] (mg/L) (20.92, 38.79] (mg/L) (38.79, 216.5] (mg/L) Beta CI (95%) Beta CI (95%) Beta CI (95%) Beta CI (95%) Stratified by sex Inattention Boys Count model *11 (Ref.) −0.32 (−0.57, −0.06) −0.4 (−0.63, −0.16) 0.001 −0.21 (−0.34, −0.08) Zero-inflation model *21 (Ref.) 0.09 (−0.46, 0.63) −0.27 (−0.8, 0.25) 0.27 −0.14 (−0.43, 0.15) Girls Count model*11 (Ref.) 0.04 (−0.61, 0.69) 0.58 (0.02, 1.13) 0.012 0.15 (−0.09, 0.39) Zero-inflation model *21 (Ref.) 0.37 (−0.36, 1.09) −0.32 (−1.13, 0.49) 0.311 −0.38 (−1.17, 0.42) Hyperactivity Boys Count model *11 (Ref.) −0.09 (−0.37, 0.19) −0.16 (−0.44, 0.11) 0.244 −0.03 (−0.18, 0.12) Zero-inflation model *21 (Ref.) 0.16 (−0.37, 0.68) 0 (−0.52, 0.51) 0.981 −0.03 (−0.32, 0.25) Girls Count model *11 (Ref.) −0.02 (−0.44, 0.39) −0.07 (−0.49, 0.35) 0.727 −0.1 (−0.33, 0.13) Zero-inflation model *21 (Ref.) 0.41 (−0.22, 1.04) 0.48 (−0.16, 1.12) 0.143 0.34 (−0.02, 0.7) ADHD Boys Count model *11 (Ref.) −0.14 (−0.32, 0.03) −0.22 (−0.39, −0.06) 0.008 −0.07 (−0.16, 0.02) Zero-inflation model *21 (Ref.) 0.25 (−0.23, 0.73) −0.05 (−0.52, 0.41) 0.778 −0.02 (−0.27, 0.24) Girls Count model *11 (Ref.) 0.02 (−0.31, 0.34) 0.17 (−0.13, 0.47) 0.237 0.02 (−0.14, 0.19) Zero-inflation model *21 (Ref.) 0.6 (0.05, 1.16) 0.49 (−0.05, 1.04) 0.077 0.39 (0.08, 0.69) Stratified by cohort Inattention Gipuzkoa Count model *11 (Ref.) −0.18 (−0.82, 0.47) 0.18 (−0.35, 0.71) 0.41 0.22 (−0.04, 0.48) Zero-inflation model *21 (Ref.) 0.8 (−0.15, 1.75) 0.05 (−0.82, 0.91) 0.873 0.16 (−0.31, 0.63) Sabadell Count model *11 (Ref.) −0.35 (−0.72, 0.03) −0.22 (−0.55, 0.1) 0.116 −0.16 (−0.31, 0) Zero-inflation model *21 (Ref.) 0.38 (−0.29, 1.05) 0.03 (−0.6, 0.66) 0.887 −0.03 (−0.36, 0.3) Valencia Count model *11 (Ref.) −0.24 (−0.64, 0.15) −0.39 (−0.76, −0.02) 0.043 −0.29 (−0.51, −0.06) Zero-inflation model *21 (Ref.) −0.44 (−1.22, 0.35) −0.6 (−1.35, 0.14) 0.123 −0.27 (−0.71, 0.18) Hyperactivity Gipuzkoa Count model *11 (Ref.) −0.07 (−0.77, 0.62) 0.06 (−0.67, 0.78) 0.932 0.23 (−0.11, 0.57) Zero-inflation model *21 (Ref.) 0.65 (−0.3, 1.6) −0.1 (−1, 0.8) 0.838 0.1 (−0.4, 0.59) Sabadell Count model *11 (Ref.) −0.25 (−0.6, 0.11) −0.31 (−0.64, 0.02) 0.053 −0.12 (−0.28, 0.03) Zero-inflation model *21 (Ref.) 0.24 (−0.36, 0.85) −0.09 (−0.69, 0.51) 0.814 −0.04 (−0.35, 0.27) Valencia Count model *11 (Ref.) 0.06 (−0.36, 0.48) 0.06 (−0.36, 0.48) 0.904 0.01 (−0.25, 0.27) Zero-inflation model *21 (Ref.) −0.02 (−0.87, 0.82) 0.59 (−0.17, 1.36) 0.07 0.4 (−0.03, 0.82) ADHD Gipuzkoa Count model *11 (Ref.) −0.06 (−0.48, 0.36) 0.37 (−0.01, 0.74) 0.045 0.33 (0.14, 0.53) Zero-inflation model *21 (Ref.) 0.85 (0.06, 1.63) 0.27 (−0.48, 1.02) 0.412 0.28 (−0.13, 0.69) Sabadell Count model *11 (Ref.) −0.29 (−0.53, −0.06) −0.28 (−0.5, −0.07) 0.005 −0.1 (−0.21, 0) Zero-inflation model *21 (Ref.) 0.39 (−0.14, 0.92) 0.03 (−0.49, 0.55) 0.857 0.08 (−0.19, 0.35) Valencia Count model *11 (Ref.) 0.09 (−0.16, 0.35) −0.11 (−0.35, 0.14) 0.244 −0.07 (−0.21, 0.08) Zero-inflation model *21 (Ref.) −0.04 (−0.68, 0.6) 0.07 (−0.55, 0.69) 0.788 0.12 (−0.24, 0.49) Models adjusted by cohort, smoking during pregnancy, alcohol intake during pregnancy, pre-pregnancy BMI, social class, sex and age of the child, living with mother/father and weeks (ferritin extraction). 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