European Journal of Cardiovascular Medicine

Anaemia is a common disease affecting an estimated 1.6 billion people globally. Anaemia is defined as a haemoglobin (Hb) concentration which falls two standard deviations below the mean for the age of the patient [1]. The prevalence of anaemia remains high in late infancy and early childhood even though breastfeeding rates have increased along with improvements in public health and development of iron-fortified foods [2]. Anaemia is a global public health concern, especially afflicting adolescent girls, women 15-49 years of age, pregnant women, and children in low- and middle-income countries. The WHO estimates that in 2019, 30% (571 million) women aged 15-49 years, 37% (32 million) pregnant women, and 40% (269 million) children 6-59 months of age were affected by anaemia[1]. In India, the prevalence of nutritional anaemia was found to be 65% in infant and toddlers, 60% between 1 to 6 years of age, 88% among adolescent girls and 85% among pregnant women, the most common etiology being iron deficiency anaemia (IDA) [3].

Prevalence of anaemia at 6 months was more frequent in preterm infants compared to term infants [4], the most common form of anaemia being IDA [5]. It was found that around 26% of premature infants had iron deficiency (ID) during the first year of life [6]. Iron deficiency is defined as the decrease of the total content of iron in the body and occurs before fall in hemoglobin [7].

Iron is an essential component of many proteins such as haemoglobin and myoglobin . Iron functions as an enzymatic cofactor for aconitase, NADH dehydrogenase, succinate dehydrogenase, and α-glycerophosphate dehydrogenase.[8] It is a component of numerous heme-containing enzymes, including catalase, xanthine oxidase, and glutathione peroxidase. It is an essential factor in various processes such as neuronal myelination, metabolism, neurotransmission and neurogenesis as studied in mice models which ultimately affects behaviour, memory, learning and development  [9].

Iron deficiency occurs when deficiency is sufficiently severe to reduce erythropoiesis which may be the result of excessive loss, decreased iron sources or rarely decreased absorption. ID in infants remains largely underdiagnosed due to difficulty in blood sampling in infants and necessity of tests only when a significant clinical event is present. Many of the symptoms of ID such as, irritability, poor feeding, fatigue and lethargy are nonspecific and are not identified in infancy [10]. Infancy-onset iron deficiency is linked to compromised mental and physical development, primarily in the areas of language, balance, and coordination [11]. ID and IDA is associated with impaired neurocognitive function, and it is seen that the association exists even after its successful treatment which makes early identification of ID extremely important [12]. Infants with IDA processed information slower at 12 months of age than iron sufficient infants of same age group [13]. Between 12 to 23 months, infants with IDA did not catch up in motor development, even though these infants were on iron therapy which highlights the significance of early identification and starting iron supplementation early in infancy [10].

The neonatal brain has a higher metabolic rate, and develops continuously during fetal period and infancy, as a result of which ID during this critical period is associated with unfavourable neurodevelopmental outcomes. Comparing premature and term infants, it is found that premature infants are associated with IDA than term infants as these infants are at higher risk due to inadequate erythropoietin production, decreased reserves, increased blood sampling during the neonatal period, rapid growth and other clinical events such as haemorrhage and haemolysis [14].

Latent iron deficiency occurs when reticuloendothelial macrophage iron stores are depleted. The serum iron level drops and TIBC increases without a change in haematocrit. This stage may be detected by a routine check of transferrin saturation [15]. Hence transferrin saturation index detects iron deficiency even before drop in haemoglobin or haematocrit which can prevent subtle neurological abnormalities and makes it a useful biomarker [15].

When the infant is 4-6 months of age, the stores can become low or depleted. This is exacerbated when there are inadequate iron stores due to low birth weight and prematurity; increased requirements from rapid growth and erythropoiesis [16]. This period coincides with the latter part of the brain growth spurt and with the evolution of fundamental mental and motor processes [17]. Human breast milk initially contains relatively high amounts of iron, but the level decreases, independent of maternal iron status, to approximately 0.3 mg/L after 5 months of lactation [15]. After 6 months of age breast-feeding does not protect against iron deficiency and a supplemental source of dietary or medicinal iron is required for optimal iron nutrition, hence it becomes important to evaluate the iron status in the period of time between 5 to 6 months [8] in a developing country like India. Therefore, it is emphasized that there is a need for evaluation for ID in both term and preterm babies at 5 to 6 months of age. With this background, the present study aimed to estimate the prevalence of iron deficiency in preterm and exclusively breastfed term infants at chronological age of 5 to 6 months in a tertiary care hospital for early identification of ID in this age group.

Study Design

Across-sectional study was conducted in thePaediatricsDepartment of Sri Ramachandra Medical Centre, Sri Ramachandra Institute of Higher Education and Research (SRIHER), Chennai, Tamil Nadu.The human Institutional Ethical Committee (IEC) of SRIHER approved the study proposal before the commencement of the study (Clearance number: CSP-MED/18/AUG/45/127). Written informed consent was obtained from the parents, and the study was conducted according to the National Ethical Guidelines for Biomedical Research Involving Children, Indian Council of Medical Research (ICMR), 2017.

Selection criteria

Preterm infants (birth <37 completed weeks of gestation) and term (birth 37 to 41 completed weeks of gestation) exclusively breastfed infants who attended the Outpatient Department at chronological age between 5 to 6 months were included in the study. Exclusions from the study were babies who had undergone blood transfusions, had acute or chronic infections, had congenital abnormalities, or had serious systemic disorders such as necrotizing enterocolitis, sepsis, seizures or intraventricular haemorrhage.

Sample size

Considering mean hemoglobin  of preterm newborns as 9.59±1.50 and among terms  10.55±0.90 according to the study by Yamada RT et al [18], minimum sample size was calculated by using open epi software considering confidence interval of 95% with 90% power .The sample size was calculated to38 inpreterm and term infants.

Data collection

The demographic details, anthropometric measurements, feeding pattern, iron supplementation taken by infant were collected in a data collection form. Anthropometric measurements were charted using the International Fetal and Newborn Growth Consortium for the 21^st Century and classified into appropriate for gestational age (AGA), small for gestational age (SGA) and large for gestational age (LGA) [19]. Socioeconomic status was calculated according to modified Kuppuswamy socioeconomic scale 2023 [20]. Mothers were classified into , those who took iron  supplementation for at least  90 days  in the form iron folic acid tablets,one tablet per day (each containing 60 mg elemental iron and 500 mcg folic acid) and those who did not take iron supplements or took it for less than 90 days [21]. For each infant, 2 ml of blood was drawn by venepuncture in plain vial. Serum iron and total iron binding capacity (TIBC) was estimated using Ferene and Ferrozine method respectively. Transferrin saturation (TS) index was calculated (serum iron /TIBC X 100) to identify iron deficiency(ID). TS index less than 16 was considered ID [22], [8].

Statistical analysis

Data was recorded in excel sheet. All outcomes were analysed by Statistical Package for the Social Sciences (SPSS) version 20.0 (IBM Corporation, Armonk, NY). Continuous variables were expressed as mean + SD. Categorical variables were expressed as percentage or number of cases and compared using Chi-square test or Fishers exact test. Independent t test and Logistic regression was applied. Statistical significance was considered at p values <0.05.

In the present study, the total number of infants enrolled was 91, out of which 42 (46.2%) were terms and 49 (53.8%) were preterm. The number of males and females were similar in our study (Table 1).

Table 1: Demographic details of  study participants.

Note: Abbreviations: SGA: Small for gestational age; LGA: Large for gestational age; AGA: Appropriate for gestational age according to Intergrowth-21Charts (2024)Socioeconomic status based on Modified Kuppuswamy Socioeconomic status scale

Among the infants enrolled majority belonged to appropriate for gestational age (AGA) group both in term and preterm.However, 39% were small for gestational age (SGA) among preterm and 14.3% were SGA among terms (Table 1).Among term iron deficient infants 26 (81.25%) were appropriate for gestational age (AGA) and this was statistically significant (p=0.003).

Table 2 shows the descriptive statistics of the study participants. The mean maternal age was 29.45 in preterms and 27.81 in terms with a mean haemoglobin at delivery being 11.1 in preterms and 12 in terms.

Table 2: Maternal and infant characteristicsamong preterm and term Infants (N=91)

Abbreviations: BMI: body mass index; APGAR score: Appearance / Pulse Rate / Grimace / Activity / Respiration

Iron deficiency appears to be more prevalent in term infants (76.2%) compared to preterm infants (59.2%), but this was not statistically significant (p=0.085). (Table 3).

Preterm infants have a higher mean transferrin saturation (15.2%) compared to term infants (11.69%), which indicates less iron deficiency in preterm than terms and this difference in transferrin saturation between preterm and term infants was statistically significant (p value -0.022). (Table 3)

Table 3: Comparison of iron deficiency in term and preterm infants

Abbreviations TSI: Transferrin Saturation Index. *Chi-square test , +independent t test

Among iron deficient term infants ( TSI <16 ) , 71.87 % belonged to lower socioeconomic status and this was statistically significant (p =0.00001).

Among the iron deficient preterms , 3 (10 %)  mothers  and among iron deficient term 15 (46.88%) mothers had moderate anemiarespectivelywhich were also statistically significant (p=0.006) .

Among iron deficient infants 13 (45.83%) preterms and 2 (6.25 %) terms were twins and this was statistically significant.(p=0.004).

It was found that there was no significant correlation between ID in preterm and term in comparison with their sex, , mother’s status of consumption of iron supplementation (taken for at least 90 days), birth weight and current weight of the infants.( Table 4 ). The lab results were discussed with the parents.

Table 4. Maternal and Infant Characteristics among Iron Deficient Infants(TSI<16)

Values expressed as n (%) Abbreviations: TSI: transferrin saturation index; SGA: Small for gestational age; LGA: Large for gestational age; AGA: Appropriate for gestational age; ELBW: Extremely low birth weight; VLBW: Very low birth weight; LBW: Low birth weight. p value calculated using Chi square test

In preterm infants already on iron supplementation, but with iron deficiency, doses were increased appropriately and term infants who had ID were also started on iron supplementation.

Anaemia is a serious global public health problem affecting around 40% of children under 5 years of age (1) with ID being the commonest cause of anaemia. In our study, 91 infants were enrolled out of which 49 were preterm and 42 were term and their iron status was studied at 5 to 6 months of chronological age.

Comparing the various population characteristics in the current study, it was found that the mean maternal age was 29.5 years in preterm and 27.81 years among terms. This was similar to a study done by Yamada where mean maternal age was higher in preterm than terms (25.96 in late preterm and 24 in terms) [18], which was attributed to the fact that IVF and ICSI conception was higher among preterm which was associated with advanced maternal age [23].

In the same study by Yamado et al in Brazil it was seen that, exclusively breastfed late-preterm newborns had greater reductions in haemoglobin/haematocrit and lower iron stores at a corrected gestational age of one month post-term than  term newborns  [18]. In our  study , 76.2 % terms were found to have iron deficiency in contrast to the above study  though this  was not statistically significant (p=0.85).

Our study evaluated iron deficiency at 5 to 6 months a period associated with introduction complimentary feeding and decreased levels of iron in breast milk [24]. To the best of our literature no other study has assessed the iron deficiency in term and preterm infants during this crucial period of growth and development .Preterm infants have a higher mean transferrin saturation (15.2%) compared to exclusively breast-fed term infants (11.69%) and this difference was statistically significant (p=0.022) indicating that iron deficiency is more prevalent among exclusively breastfed term (Table 3). This is in contrast to fact that preterms are prone for iron deficiency owing to decreased iron stores and increased blood sampling in NICU.[14].

Transferrin saturation index(TSI) helps identifying iron deficiency even before iron deficiency anaemia[15] sets in which is indicated by a drop in hemoglobin . A decrease in TSI occurs even before hemoglobin decreases [8] , hence TSI was used as the parameter to assess iron deficiency in our study .Early identification of iron deficiency helps in preventing neurological disabilities (10) which  is essential for the growth and development of the infant .

Berglund et al found that preterm and term infants with normal birth weights were at significant risk of developing iron deficiency(ID) (36%) and IDA (9.9%) at 6 months (p=0.001), which was in contrast to our study where 24 % of  preterm and 31% term has normal birth weight with no statistical significance(p=0.752)

Among iron deficient term infants ( TSI <16 ) , 71.87 % belonged to lower socioeconomic status and was statistically significant (p =0.00001) . However this was in contrast to a study done by Lozoff et al where there was no  statistical significance between socioeconomic status and iron deficiency[25]

In a cross-sectional study to verify the influence of breastfeeding and maternal anaemia on haemoglobin concentration at 6 months in term infants, Teixera [25] found that maternal anaemiadid have an influence (P<0.05) on the haemoglobin levels of 6-month-old infants this was similar to our study where  among the iron deficient preterms  and term infants 3 (10 %) and 15 (46.88%) mothers had moderate anemia[21]and this was statiscally significant (p=0.006)

Miled [26] compared the risk of anaemia due to ID in mothers and infants of twin and single pregnancies and it was noted that haemoglobin and ferritin levels were lower in twins at 3 months and 6 months , this was similar to our study where among iron deficient infants ,13 (45.83%) preterms and 2 (6.25 %) terms were twins and this was statistically significant.(p=0.004).

Frie et al found that term babies who were supplemented with iron at 6 months showed better neurological outcomes than their non-iron supplemented counterparts [27], hence emphasizing the need for evaluation at 5 to 6 months of age. In the current study 59.2% of preterm who were iron deficient received iron supplementation and while 76.2% of terms who were iron deficient were started on iron supplementation, which may have improved their later neurological outcome

Friel [27] noted in exclusively breastfed infants who received iron supplements between the ages of 1 and 6 months had higher mean corpuscular volume and higher Hb concentration at 6 months of age compared to their counterparts who were not given supplements. Therefore, for better neurodevelopmental, cognitive, and behavioural outcomes in their growing years, it has been suggested that exclusively breastfed term infants receive iron supplementation, starting at 4 months of age, and continuing until appropriate iron-containing complementary foods have been introduced [28]. For preterm infants, iron supplementation should be started at one chronologic month and continued until 12 months chronologic age [28]. 

Iron deficiency appears to be more prevalent in term infants (76.2%) compared to preterm infants (59.2%), though not statistically significant (p=0.085). Preterm infants have a higher mean transferrin saturation (15.2%) compared to term infants (11.69%), and the difference in transferrin saturation between preterm and term infants is statistically significant(p=0.022).

Among iron deficient term and preterm infants there is a significant correlation with respect to socioeconomic status (p=0.00001),  maternal anemia(p=0.006), birth anthropometry(p=0.03) and twin gestation(p=0.004) . It was found that there was no significant correlation between ID in preterm and term in comparison with their sex, mother’s status of consumption of iron supplementation (taken for at least 90 days), birth weight and current weight of the infants.

Author Recommendations:

Based on the study findings, it is recommended to screen for ID in all term babies at 5 to 6 months and start them on 1 mg/kg/day of iron supplementation if ID is found and continued until appropriate iron-containing complementary foods have been introduced. It is also recommended to assess the iron status of preterm who are on iron supplements, by laboratory investigations and treat them appropriately if found to be iron deficient. It is always a good practice to check iron intake of preterm as they grow and modify iron supplementation as per weight periodically and continue up to one year of age.

Strengths of Study

The iron status of preterm and term infants on exclusive breast feeds at 5 to 6 months ,a period associated with start of complimentary feeds and decreased iron content in mother’s milk is poorly understood. There is paucity of literature which looks at iron deficiency rather than iron deficiency anemia in preterm infants and exclusively breastfed term at 5 to 6 months of age. Hence our study uses Transferrin Saturation index to detect iron deficiency even before iron deficiency anaemia sets in at 5 to 6 months of age which helps in early correction and prevention of the consequences of iron deficiency anaemia.

Limitations of the study:

Longitudinal follow up of iron deficient babies for later neurodevelopmental outcomes was not done. Since these babies were not followed up, the effect of iron supplementation, on later neurodevelopmental outcomes was not studied. Cord haemoglobin and other iron parameters were not checked at birth, hence could not be compared with values at 5 to 6 months chronological age.

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