1. Introduction
Globally iron and zinc deficiencies are among the most widespread micronutrient deficiencies. While people of all ages are at risk, children and women of reproductive age are at elevated risk of experiencing concurrent deficiencies, especially in low-income countries [
1,
2]. Even mild deficiencies of one or both nutrients may contribute to increased morbidity and mortality [
1]. The first 1000 days of life—the period from conception to the child’s 2nd birthday—are most crucial, since some developmental and functional delays during this period are either irreversible or only partly reversible [
3]. In utero exposure to iron deficiency has been associated with impaired brain maturation of the fetus [
4,
5], while during infancy and childhood, iron deficiency could lead to impaired cognitive and physical functionality and increased risk of mortality [
1,
6]. Zinc is essential for cellular differentiation and maturation and maternal zinc deficiency could lead to growth retardation and other developmental defects of the fetus [
7]. Further, zinc deficient infants and young children are prone to infections and growth retardation [
8].
The major cause of micronutrient malnutrition is a diet consisting mainly of staple foods and lacking in animal sources [
9]. When compounded by exposure to environments laden with pathogens, there is a synergistic worsening of the malnutrition burden [
10,
11]. Although high dose supplementation has been successful in reducing the prevalence of micronutrient deficiencies, programs often only inadequately reach rural or marginalized populations; also high dose iron supplementation has been exposed to criticism in the past decade in malaria endemic areas [
12]. Other interventions such as fortification and biofortification, delivering smaller amounts of micronutrients on a daily basis, might be more effective in reducing the prevalence of micronutrient deficiencies in populations at risk; yet, their effect on the infant during the first 1000 days of life starting from pregnancy until 23 months of age has hardly been investigated.
The objective of this work was thus to evaluate the potential of interventions delivering daily doses of iron and zinc in concentrations up to approximately the Recommended Nutrient Intake (RNI) in diets with low bioavailability [
1] during the first 1000 days of life on child micronutrient status and health.
For this we used data from randomized and quasi-randomized trials of fortification, supplementation or biofortification interventions, in which iron and zinc were provided more than three times a week in concentrations up to approximately the (RNI) for women (iron: 45 mg/day; zinc 20 mg/day) and children 6–23 months (iron: 15 mg/day; zinc: 10 mg/day).
2. Materials and Methods
2.1. Search Strategy
We searched the WHO e-Library of Evidence for Nutrition Actions, the Cochrane Central library, Web of Science, and MEDLINE/PUBMED databases to identify systematic reviews and meta-analyses that investigated the effects of iron and zinc interventions (fortification, supplementation, or biofortification interventions) on nutritional, developmental and health outcomes of children. We restricted the search to reviews published from 2005 to 2015. The following search strategy was adapted for each database: (Fortification OR biofortification OR supplementation) AND (iron OR zinc OR multiple micronutrients OR micronutrient powder) AND (women OR children OR infants OR toddlers). For the relevant topics, 82 potentially useful reviews and meta-analyses published from 2005 to 2015 were identified, the most recent ones conducting their literature searches up until 2014. We searched their reference lists for suitable original studies. Additional searches for original studies published between 2011 and October 2015 were conducted in Web of Science and MEDLINE/PubMed using the following strategies for each age group of interest:
- (a)
Children: (Biofortification OR fortification OR supplementation) AND (iron OR zinc OR micronutrient powder OR multiple micronutrients) AND (children OR infants OR toddlers) AND (trial OR study OR survey OR assessment).
- (b)
Pregnant women: (Biofortification OR fortification OR supplementation) AND (pregnant women OR lactating women OR maternal) AND (iron OR zinc OR micronutrient powder OR multiple micronutrients) AND (trial OR study OR survey OR assessment).
- (c)
Lactating women: (Biofortification OR fortification OR supplementation) AND lactating women AND (zinc OR iron OR micronutrient powder OR multiple micronutrients OR breast milk) AND (trial OR study OR survey OR assessment).
2.2. Inclusion and Exclusion Criteria
Types of trials and interventions: Randomized controlled trials (RCTs) and quasi-experimental studies were included in the review. Studies were eligible if they assessed the impact of iron or zinc supplementation, fortification or biofortification interventions on the micronutrient status, growth or health outcomes of children (details below). Only studies where the daily iron and zinc dose did not exceed 15 mg and 10 mg, respectively for children and 45 mg and 21 mg, respectively for women, were included. We considered micronutrient powders and crushable tablets (foodlets) as fortification, since they are consumed as part of a normal meal. Furthermore, we included studies investigating the effects of iron or zinc supplements, as long as they were within the dose range we specified. We defined supplements as compounds, which are routinely consumed separately from a normal meal, including tablets, pills, drops, capsules, syrups, drinks, biscuits, and lipid-based supplement (LNS). Only studies administering the micronutrients >3 times a week were included.
Types of participants: For interventions administered to the mother, we included studies that provided interventions to pregnant women or lactating women, regardless of their health status. For interventions administered to children, we were interested in effects on young children, thus only studies where over 50% of participating children were 6–23 months old were included. Only studies involving apparently healthy children were considered, with the exception that studies including malnourished (underweight, stunted, wasted) children and children suffering from anemia or deficiencies of iron and zinc were included. We did not include therapeutic studies, such as short term zinc supplementation studies to treat acute diarrhea.
2.3. Study Design and Comparison Groups
The control groups of the included fortification trials either received unfortified foods or regular diets. Studies were also included if both the control and intervention groups received the same fortified foods, but either with different iron or zinc concentrations, or if the control group received an identical micronutrient compound given to the intervention group, but without iron or zinc. For supplementation, the control groups of the included trials either received no supplements, placebo, a lower concentration of iron or zinc or different micronutrients identical to intervention group preparations, except that they excluded zinc and/or iron.
Studies were only included in meta-analyses if the data for outcomes of interest were presented in a manner that allowed inclusion in the meta-analysis (i.e., data could only be used when presented as mean (SD or SE), mean (95% CI), median (95% CI), or median (range), but not when reported as median (IQR)). Where studies did not report on the average daily micronutrient intake, but instead stated the micronutrient concentration per 100 mL/100 g or the daily micronutrient intake in mg/kg body weight, we calculated the average intake as appropriate.
2.4. Outcome Measures
Only child outcomes were of interest, even when interventions were administered to pregnant or lactating women. For example, with regard to effects of prenatal iron supplementation on anemia, maternal anemia was not the focus of this analysis; rather, the infant/child anemia was of relevance even if the mother received the intervention.
Outcomes evaluated include: (1) hemoglobin (Hb) concentration (g/dL); (2) anemia (%; defined as Hb <110 g/L); (3) serum ferritin concentration (µg/L); (4) iron deficiency (%; defined as serum ferritin <10 µg/L or <12 µg/L); (5) iron deficiency anemia (%; defined as hb <105 g/L or <110 g/L and serum ferritin <10 µg/L or <12 µg/L); (6) serum or plasma zinc (µmol/L); (7) zinc deficiency (%; defined as serum zinc <10.7 µmol/L); (8) birth outcomes (birth weight in g; prevalence of low birth weight in %, defined as weight <2500 g); (9) infant anthropometric measures (height for age z-score (HAZ); weight for age z-score (WAZ); weight for height z-score (WHZ); stunting (≤−2 HAZ scores), wasting (≤−2 WHZ scores) underweight (≤−2 WAZ scores)); (10) mental and motor development (Bayley mental development index (MDI); Bayley psychomotor development index (PDI)); (11) morbidity (diarrhea, fever and respiratory infection).
2.5. Data Synthesis and Statistical Analysis
From each eligible study, we extracted all data that would allow the estimation of the effects of interest. For example, for zinc studies, data were extracted for groups assigned to zinc alone compared with placebo, as well as data from groups assigned to more than one nutrient including zinc compared with a group assigned to the identical nutrient combination but excluding zinc. Where the total number of comparisons was sufficiently large, we examined the influence of combining such comparisons into one meta-analysis, and where possible, conducted sub-group analyses examining the possibility of nutrient interactions. Additionally, where studies had more than one group assigned to the nutrient of interest (for instance, different nutrient dosages) but only one suitable comparison group, we included such data as separate estimates for the meta-analysis. For that, we divided the comparison group into two groups (or more, when required) with smaller sample sizes, so the same children were not involved in more than one comparison. To examine if this decision affected the results, we conducted sensitivity analyses combining all relevant control groups of affected studies into a single comparison group, and combining all relevant intervention groups into a single group, for some outcomes. As results were very similar, we chose to continue analyses with the sub-groups. When studies reported outcomes at multiple time points, we selected only the results reported at the study end (or the latest time point) for the meta-analyses. Most studies were individually randomized trials, but where cluster randomized trials were eligible for inclusion, we estimated design effects to adjust for the influence of intra-cluster correlations on the precision of estimates as appropriate. To maximize the number of studies that could contribute to meta-analyses of continuous outcomes, we converted medians (reported with ranges) and geometric means (reported with standard deviations, standard errors or confidence intervals) to arithmetic means and standard deviations using methods developed by Hozo et al. [
13] and Higgins et al. [
14] and examined the effect that combining transformed and untransformed estimates had on pooled results. Studies were excluded from meta-analyses if published reports presented insufficient information for estimating desired effect estimates and variances.
For continuous outcomes, we estimated pooled mean differences or standardized mean differences and confidence intervals, as appropriate, while for categorical outcomes, we estimated pooled risk ratios and confidence intervals. Study-level effect estimates were pooled using the random effects meta-analysis method by DerSimonian and Laird [
15]. We assessed heterogeneity among studies, and used the method proposed by Higgins et al. to measure the inconsistency (
I2) of effect estimates across studies [
16]. Heterogeneity among study estimates was considered to be substantial if the
I2 exceeded 50%. For outcomes having at least 10 comparisons per variable of interest, we explored sources of heterogeneity by conducting pre-specified sub-group analyses and meta-regressions, to examine whether effects were modified by the study-level factors: micronutrient doses provided, type of intervention (whether fortification or supplementation), study quality and baseline micronutrient status. Funnel plots were constructed for visual assessment of the variability of individual study estimates and to evaluate the possibility of publication bias, and when appropriate, Egger’s tests were used to examine if effect estimates varied with study sample size.
All analyses were conducted using the metafor package [
17] of the R statistical program (R version 3.1.3 (2015-03-09), The R Foundation, Vienna, Austria, 2015).
2.6. Assessment of Quality and Risk of Bias
We assessed study quality in three areas—random sequence generation, adequacy of blinding of study participants and personnel and completeness of outcomes assessment—but did not exclude studies based on the assessment of quality. Studies were categorized as being of the ‘highest quality’ if interventions were randomly assigned, both participants and study personnel were adequately blinded to the intervention assignment, and if outcomes were assessed in at least 75% of the enrolled study population. Intermediate quality trials were randomized trials for which only one of the remaining two criteria was suboptimal, lowest quality trials were randomized trials for which both the remaining criteria were suboptimal, and quasi-experimental trials (although rarely included) were given the very lowest rating. The effect of pooling results from studies of different quality was examined in sub-group analyses where possible.
The quality of the evidence resulting from each analysis was assessed using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) method [
18]. Domains included: risk of bias within studies (internal validity), inconsistency or heterogeneity of results across studies, indirectness or use of proxy populations, interventions or outcomes measures, imprecision (large variability and wide confidence intervals), number of studies and risk of publication bias. The evidence was judged as high quality if further research studies were unlikely to change the pooled estimate obtained; moderate quality if further research could alter the current estimate; low quality if further research was needed to confirm the magnitude and direction of the true effect; and very low quality if there was great uncertainty about the validity of the pooled estimate. It should be noted that because the GRADE method considers several domains and not just internal study validity, confidence in pooled estimates could be ‘low’ or ‘very low’ even if all studies that contributed to the estimate were well conducted.
4. Discussion
We found that interventions delivering iron and zinc in concentrations up to the RNI during the 1000 days window from the prenatal period to the first 2 years of a child’s life can have positive impacts on the iron and zinc status of young children. In particular, when interventions were conducted in children aged 6–23 months old, their hemoglobin levels, and iron and zinc status improved. For both nutrients, supplementation triggered a significantly stronger response than fortification even when doses were similar. For other outcomes assessed—birth weight, growth in childhood, psychomotor development—findings are less conclusive, partly due to the limited number of studies.
We show that low dose iron and zinc interventions providing no more than 45 mg/day of iron and 20 mg/day of zinc during pregnancy may not have an impact on children’s birth weights and prevalence of low birth weight. Similar to our results, other meta-analyses found no effect of zinc on birth weight and prevalence of low birth weight [
108,
109]. Most of the studies included in those analyses did not meet our inclusion criteria because they gave higher doses of zinc than we were interested in. For iron, our results are also in accordance with a recent meta-analysis, which only observed beneficial effects at iron doses exceeding our threshold [
110]. However, due to the scarcity of studies having follow-up data extending beyond the perinatal period, it is unclear if any growth advantages of routine iron and zinc ingestion during pregnancy could manifest later in infancy or early childhood.
We found that the use of iron in dietary doses (no more than 15 mg/day) during infancy and early childhood had positive effects on hemoglobin levels, anemia prevalence, and iron status (serum ferritin, ID and IDA prevalence), which agrees with a systematic review published in 2012 assessing the effect of micronutrient-fortified milk and cereal foods on infants and children [
111] and a meta-analysis of supplementation trials in children 6–23 month of age, published in 2013 [
112]. We did not find an effect of iron interventions on child growth or morbidity, suggesting that food-based iron interventions at doses investigated here have no beneficial impact on the occurrence of childhood morbidities. Limited effects of iron can be expected regarding mental and motor development until the age of 2 years (we focused on the Bayley’s scales for mental and motor development due to more consistent reporting).
We conducted sub-group analyses stratified by type of intervention (fortification/ supplementation), intervention dose, a measure of baseline micronutrient status, as well as study quality. Sub-group analyses demonstrated that iron administered as fortificants significantly increased serum ferritin and hemoglobin levels, although to a lesser extent than supplements. This could be due to the presence of absorption inhibitors in fortified foods. For biofortification, the magnitude of the effect on iron and zinc status is unclear as to date no biofortification trials have been conducted with a focus on the 1000 days window. Yet, we would expect comparable effects as observed from fortification since both approaches deliver the micronutrients together with other dietary components. Sub-group analysis stratified by dose showed that daily iron doses as low as 6–8 mg increased serum ferritin levels. Larger effect sizes were observed for higher daily iron doses (8–10 mg), however, such doses might be difficult to attain for infants by dietary diversification or biofortification interventions alone. Surprisingly, baseline iron status did not explain much of the heterogeneity of the findings in our analysis. This is somewhat in contrast to previously published data [
113,
114] and could be ascribed to other factors influencing iron absorption and utilization in contexts of high exposure to inflammation [
115]. But also, the categorization of studies into iron deficient versus iron sufficient population had to be done in a relatively crude manner.
We found that zinc supplementation at doses no higher than 10 mg/day increased the serum zinc concentrations of children and reduced the risk of suffering from zinc deficiency, whereas the effect of zinc administered in fortification trials, although in comparable concentrations as supplemental zinc, had no significant effect on serum zinc levels. We did not find any modification of the zinc intervention effects by child baseline zinc status. Sub-group analysis stratified by dose showed that daily zinc doses of 4–6 mg increased serum zinc by 0.9 µmol/L while larger effect sizes were observed for higher zinc doses 7–10 mg, which might be difficult to reach in children 6–23 months old through dietary means alone. Effects of zinc were significant for WHZ and WAZ, although the differences remained rather small, compared with controls. Zinc interventions probably do not have a measurable effect on the occurrence of respiratory infections and fever and effects on diarrhea are inconclusive.
Lastly, we found that, when zinc and iron were given together, their beneficial effects on serum zinc and serum iron levels were weaker than when each nutrient was given alone, suggesting that iron and zinc compete for absorption from the gut, a finding that has previously been posited [
116].
Possible limitations of the review should be noted. First, many results exhibit large amounts of heterogeneity, which could not be explained by the factors we explored, and which are probably due to the differences in study design, the different types of interventions and reporting. Thus, the pooled estimates of some of the effects shown may be imprecise and results have to be interpreted with caution. Second, none of the studies meeting our inclusion criteria covered the full 1000 days window meaning that the impacts of the micronutrients could not be assessed holistically across the different life stages, but only in a compartmentalized manner. Thus, no statements can be made about whether a holistic approach covering the full 1000 days (iron and zinc interventions starting during pregnancy and continuing until 2 years of age) would have a synergistic or additive effect on certain outcome measures.
To illustrate this using ferritin levels and the Psychomotor Development Index, the absorption and utilization of dietary iron is affected by the subject’s iron status [
117,
118]. Thus, if an iron intervention given to pregnant women suffering from moderate to severe dietary iron deficiency anemia has a positive effect on the fetus’s iron status, infants born to such women would have a better iron status than control peers of the same age whose mothers received no iron [
119]. Continuing the higher iron regime into infancy could actually decrease iron absorption/utilization by children in the intervention group, assuming they had benefited from exposure to prenatal use, while children in the prenatal control group who start receiving iron after birth may more efficiently absorb and utilize the little iron available. Taken together, it is possible that only small differences in iron status would be observed between the two groups at the age of two years. In contrast, with regard to the Psychomotor Development Index, one could imagine that a 1000-day intervention could lead to a synergistic effect, since some early neurodevelopmental constraints are partly irreversible [
120]. Thus, a child exposed to prenatal iron would be primed for a better start early in life. Third, only few studies assessed the inflammatory status of the children and reported prevalence of inflammation and therefore it was not possible to stratify studies by level of inflammation. During inflammation the human body down regulates iron absorption, and thus studies with a high proportion of subjects suffering from inflammation would be expected to show less effects of iron on iron status. Also, in those populations, an effect of iron on hemoglobin and anemia prevalence could be masked by anemia of inflammation in the same subject.
To conclude, providing dietary or relatively low daily doses of iron and zinc to young children could be beneficial for their iron and zinc status, indicating that food based approaches can be useful tools to reduce the prevalence of iron and zinc deficiencies. However, it is questionable if an intervention would affect child outcomes throughout the whole 1000 days period, not least because of breast milk iron and zinc homeostasis [
121,
122,
123,
124]. More research assessing the impact of iron and zinc holistically over the different life stages (pregnancy, lactation, and early childhood) is required to understand the potential role of interventions provided throughout the 1000 days window.