TSH Mediated the Effect of Iodized Salt on Child Cognition in a Randomized Clinical Trial

Objectives: This study examines the hormonal mediators of the effect of iodized salt in pregnancy on child cognition. Methods: Sixty districts across 6 zones in the Amhara region of Ethiopia were randomly allocated to a control or intervention arm of early market access to iodized salt. Twenty-two villages per arm were randomly selected for this sub-study. A total of 1220 pregnant women who conceived after the intervention began were enrolled and assessed for their iodine and iron status. Data were collected once on the household socio-demographic status and iodized salt use, and maternal urinary iodine during pregnancy. Then, infants’ diet, urinary iodine level, cognitive development (Bayley III), serum hormonal levels, iron status, and inflammation markers were measured between 2 and 13 months of age. Results: The median maternal urinary iodine concentration was adequate and significantly higher in the intervention mothers than that of the controls (163 vs 121 µg/L, P <.0001). Intervention children compared to the control children had lower thyroid-stimulating hormone (TSH) (mean: 2.4 ± 1.0 µIU/mL vs 2.7 ± 1.0 µIU/mL, effect size = 0.18, P <.01) and thyroglobulin (Tg) (41.6 ± 1.0 ng/mL vs 45.1 ± 1.0 ng/mL, effect size = 0.14, P <.05). There was an interaction between the intervention and iron stores such that cognition was higher with iron (effect size = 0.28, 100 vs 94 IQ points). TSH was a partial mediator (12%) of the effect of the intervention on child cognition (Sobel z-score = 2.1 ± 0.06, P <.05). Conclusion: TSH partially mediated the effect of the iodized salt intervention on child cognition. The broader study was a randomized cluster trial, with districts defining clusters and units of randomization. 9 Sixty districts within 6 zones in the Amhara Region of Ethiopia were selected by a random selection process. Thirty of the districts were randomly assigned to the intervention arm and 30 to the control arm, and 1 village was randomly selected from each district to be part of the evaluation. For this specific data collection, 22 of the 30 districts per study arm were randomly chosen. The initial report of this paper has been published. 10 Participants were recruited by trained students and Health Extension Workers (HEW) within each selected village where they conducted a census of all pregnant women. Eligible women were those who: (i) became pregnant at least 1 month after the salt iodization intervention had started, (ii) had not received iodine capsules in the past year, (iii) spoke Amharic, (iv) were 18 years or older, (v) had lived in the selected village for at least a year, and (vi) planned to continue living in the selected village for at least another year to allow follow-up. Eligible mothers were enrolled after signing the informed consent form. This paper is part of a larger study evaluating the effect of iodized salt on children’s cognitive development. Two previous intervention trials with pregnant women in moderate iodine-deficient areas gave an effect size of 0.02 and 1.38 (95% CI: 0.45, 2.32) for mental development in children.11,12 This study’s group sample size was calculated with a small effect size (d) of 0.2 and power of 0.8 using the formula: n = [2 (Zα/2 + Zβ) 2]/d2; where n = number per group, α = 0.05, β = 0.2, Zα/2 = 1.96 and Zβ = 0.84. 13 The sample size was 393 per group. The intra-cluster correlation (ICC) was assumed to be 0.04 and the variance inflation factor (VIF) = 1 + (cluster size−1) × ICC. 14 From a preliminary study, the average number of pregnant women per village was 30. A conservative cluster size of 18 was used, then accounting for the cluster: VIF = 1 + ((18–1) × 0.04) = 1.68. Hence the adjusted sample size per group was 393 × 1.68 = 660. An adjusted cluster size of 30 would give a minimum of 22 (660/30) clusters per group. A total of 660 pregnant women per intervention arm were required. Subsequently, only 1220 pregnant women were available and recruited for data collection from which 1035 of their children were involved in the final data collection. The intervention involved the facilitation of iodized salt supply to the intervention villages (see previous paper for more details10,15). As salt was gradually being iodized in the country, the main distributors were contacted and assisted by the project to supply iodized salt first to the intervention markets. Staff at the salt monitoring offices within each district were trained and provided with rapid test kits to monitor and stop the channel of non-iodized salt into intervention markets and villages. A salt monitoring officer was hired and trained by the project to monitor samples of salt present in the market areas of the intervention communities. He also monitored the quality of samples of the salt supplied by salt distributors, those sold within the market, and those used in the village. Salt in control markets and villages was left to market forces but periodically sampled for information purposes. Iodized salt reached intervention communities 4 to 6 months before control communities. 15 The research assistants involved in data collection were blinded to which of the communities were intervention or control. Data was collected at recruitment (during pregnancy), a field visit post-recruitment was done to collect blood from mothers who were still pregnant, and the final data collection was done 2 to 13 months after the mothers delivered their babies. Refer to the consort flow diagram in our previous publication. 10 A household salt was sampled in salt bags and urine specimen of the pregnant women were collected using urine cups and transferred into urine tubes to assess iodine status. 16 The urine samples in tubes were preserved with 29.2 g/L boric acid 17 and were subsequently sent to the project office and frozen at −20°C, and then transferred to the Ethiopian Public Health Institute (EPHI) for urinary iodine analysis using the Sandell-Kolthoff reaction. 16 The hemoglobin (Hb) of a sample of women who were available and still pregnant during a post-recruitment field visit was measured using the HemoCueTM 201+ portable photometer. Those below the cutoff of 110 mg/dL were considered anemic. 18 Final data collection was done 2 to 13 months after women gave birth. Home visits were made by research assistants to collect data on the socio-demographic background of households, hygiene practices, and water and sanitation practices. Household salt samples were taken and checked for iodine using the rapid test kits (MBI Kits International, Chennai, Tamil Nadu, India). Randomly selected 20% of salt samples that tested positive for iodine were taken to EPHI to analyze iodine concentrations by titration method using the modified Sandell-Kolthoff reaction. 16 Children’s mental development was assessed using the Bayley Scales of Infant and Toddler Development (version III) by trained psychology graduate students, overseen by clinical psychologists. 19 Four subscales of the Bayley tests were used, namely cognitive, expressive language, receptive language, and fine motor skills. A drop of capillary blood was used to test the Hb of the children by the azide methemoglobin method with a HemoCueTM 201+ portable photometer. 20 Venous blood was also taken to evaluated thyroid biomarkers. Children’s urine samples were collected with urine bags and stored in urine tubes at −20°C. All urine samples were analyzed in duplicate. Levels less than 100 μg/L for children and 150 μg/L for pregnant women were considered inadequate. The laboratory participated in the Centers for Disease Control and Prevention’s Ensuring the Quality of Urinary Iodine Procedures (EQUIP) standardization program for quality assurance related to iodine testing. 21 The procedure used to collect data on children’s thyroid biomarkers was as follows. Venous blood was taken in vacutainer tubes and then centrifuged. The serum was then transferred into mineral-free cryogenic vials, labeled, and transported back to the project office in ice packs to keep them frozen. The vials were stored at −20°C before being transported to EPHI and stored at −80°C before serum analysis. The serum was analyzed for markers of iron status including serum ferritin and soluble transferrin (sTfR), thyroid hormones including T3, T4, TSH, and Tg, and inflammation markers, namely serum C-reactive protein (CRP) and alpha 1-acid glycoprotein (AGP). The thyroid hormones, ferritin, and CRP were analyzed using the electrochemiluminescence immunoassay (ECLIA); sTfR and AGP were analyzed using the immunoturbidimetric method. Quality control was ensured by strictly following the analytic procedures, and running the controls, and duplicates, with every run to control procedural and instrumental performance. The details of the procedures and quality control used in analyzing the serum samples were published elsewhere. 22 A total of 1220 respondents were enrolled (651 intervention and 569 control) and 1035 (84.8%) were included in the final analysis. The consort flow diagram was published in an earlier publication. 10 Main outcomes and related variables (hemoglobin, urinary iodine levels, mental development scales) were first checked for normality and skewed variables were transformed (squared to transform negatively skewed variables and log to the base 10 to transform positively skewed variables). The results of the analysis using transformed and original variables did not vary. World Health Organization cut-off points for deficiency were used to classify the serum indicators for Hb (110 mg/dL 17 ) and ferritin (12 ng/mL 23 ). The cutoff for CRP (5 mg/L) and sTfR (8.3 mg/L) were obtained from other published works.24,25 A cutoff of below 2.5th percentile score was used for low levels of T3 and T4, and above 97.5th percentile score was used for high TSH and AGP. The cutoff for Tg (18 ng/mL) was obtained from the manufacturer and the calculated TfR-F index (sTfR ÷ log Ferritin, the lower the better) used a cutoff of 7.7 (using sTfR of >8.3 mg/L and ferritin of <12 ng/mL) to define the deficiency of iron stores. 25 Group differences of the socio-demographic variables were analyzed with cluster-adjusted PROC MIXED analyses from SAS 9.4 to identify covariates of the main outcomes (mental development and serum hormonal variables). Cluster-adjusted logistic regression analyses were used to analyze dichotomous serum variables. Cluster-adjusted PROC MIXED was used to analyze the treatment effect, adjusting for identified covariates and conventional covariates from the literature (household assets, water and sanitation, maternal education, child age, and hygiene for mental development indicators; household assets, child sex, age, hygiene, CRP, and AGP for serum outcome indicators). Mixed models were used to analyze the interaction between serum indicators and intervention on Bayley scores. Modifiers tested include Hb (low: <110 mg/dL), serum T3 (low: <1.55 ng/ml), T4 (low: <7.57 µg/dL), TSH (high: >6.68 µIU/mL), TG (low: <18 ng/mL), ferritin (low: <12 ng/mL), sTfR (high: >8.30 mg/L), TfR-F index (high: >7.70), AGP (high: >1.20 g/L), and CRP (high: >5.00 mg/L). Only significant interactions are presented in results tables before running stratified analyses to obtain separate effect sizes. Mediation analyses were conducted to examine serum hormonal levels as potential mediators of the effect of the intervention on mental development; that is, to be an intermediate in the relationship between the 2 variables. First, conditions to support mediation were examined to establish that there was an association between the intervention and mental development and between hormones and mental development. Secondly, the Sobel test was used to check the significance of the mediation effect (Figure 1). 26 Bootstrapping mediation analysis in R was then used to calculate the percentage mediated. Finally, means were reported with standard deviations, and mixed models were reported with least-squares means and standard error. TSH mediation of the intervention effect on cognition. Abbreviations: Cognitive: outcome variable; Cognitive⟶Intervention: regression of Cognitive on Intervention; Intervention: independent variable; TSH: mediator.