Serum Zinc Levels in Preterm Newborns and its Relation with Retinopathy of Prematurity

Introduction

Prematurity (newborns born before 37 completed weeks of gestational age) is associated with increased morbidity and mortality. According to the Lancet Global Health 2019;7th edition, the estimated global preterm birth rate for 2014 was 10·6% (14·84 million), and India alone contributes 24% of the total preterm births. ¹ With the advent of newer technologies, the survival rate of preterm babies has increased, and hence, the incidence of the associated morbidities. ² Retinopathy of prematurity (ROP) is a preventable cause of blindness in preterm neonates. The risk factors associated with ROP are not fully known but prematurity and associated retinal immaturity at birth represent the major risk. Other risk factors are oxygenation, respiratory distress syndrome (RDS), apnea, bradycardia, heart disease, intraventricular hemorrhage, hypercarbia, acidosis, anemia, and the need for blood transfusion. ³ In preterms with an immature retina, changes in levels of vascular endothelial growth factor, insulin-like growth factor I, oxygen, and reactive oxygen species, retinal blood vessels may grow abnormally which can cause permanent damage to the retina. Thus, antioxidants can prevent retinal damage. ⁴

Zinc is one of the most abundant trace elements in the human body, which is known to have an antioxidant role. Although the reference range of serum zinc has been mentioned for adults, few studies have been done to assess serum zinc levels in preterm neonates. According to Gilles et al, 60% of zinc accretion takes place in the third trimester of pregnancy; hence, preterm infants have a smaller pool of zinc at birth. ⁵ This zinc deficiency might be having some role in the pathogenesis of diseases in preterm neonates which are caused by oxidative damage like ROP. Therefore, we planned this study to assess the level of serum zinc in preterm neonates and the relation of low serum zinc levels (below 70 mcg/dL) with the development of ROP.

Material and Methods

Preterm babies of gestational age 28 weeks to less than 37 weeks (36 weeks 6 days) (gestational age determined using New Ballard Score) were studied from June 2018 to May 2020 in the Department of Pediatrics of a tertiary care institution after taking permission from the institutional ethical committee. ⁶

All the preterm neonates of inborn having gestational age between 28 weeks to less than 37 weeks (36 weeks+6 days) with an age less than or equal to 24 hours were enrolled. We did not include the babies born before 28 weeks of gestation due to high institutional mortality rates amongst extremely preterm babies and thus the inability to complete follow-ups. Also, preterm babies admitted after 24 hours of life and those having lethal congenital malformation were excluded. A total of 455 babies were enrolled out of which 95 were lost due to mortality or loss of follow-up (Figure 1).

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Figure 1.

Study Flowchart.

Informed written consent was obtained from the parents of eligible neonates. Participants’ demographic profiles, maternal history, and birth weight were taken and entered into a pretested case record form. A one-time serum zinc sample was taken at the time of admission of the neonate to eliminate the error which may occur due to feeds or supplements containing zinc. ⁷ For the same 2 ml venous blood was collected in a plain vial and assessment was done by the method of spectrophotometry on the serum collected. Taking the reference value of serum zinc level between 70 and 150 mcg/dL from a previous study by Srinivas et al, a serum zinc level of 70 mcg/dL was taken as the cut-off value. ⁸ We further analyzed the results between 2 groups—one having serum zinc <70 mcg/dL and the other ≥70 mcg/dL.

Post-sampling, all the preterm newborns were kept on exclusive breast milk feeding only (through nasogastric route or paladai or direct breastfeeding based on newborns’ maturity) whenever the newborn’s clinical condition allowed feeding and the same was ensured to be continued even after discharge from the hospital. None of the enrolled newborns received any additional supplementation of zinc (except for the routine prescription of multivitamin drops after discharge with postnatal age 2 weeks or more). Neonates were screened using indirect ophthalmoscopy by a trained ophthalmologist, for the presence or absence of ROP changes at the appropriate postnatal age and on follow-up visits as per the standard guidelines for screening of ROP. ⁹ All neonates were followed up till maturation of the retina was achieved. Participants who had ROP on the first examination or any follow-up were labeled as neonates with ROP. Those participants who did not have ROP changes on the first examination, as well as follow-ups, were labeled as neonates without ROP.

Data regarding the presence or absence of other risk factors of ROP including oxygen supplementation (FiO₂ ≥40% for more than 3 days duration anytime during hospital stay), mechanical ventilation, RDS (clinically diagnosed as the presence of respiratory distress assessed by Silverman Anderson Score of ≥3, requiring positive pressure ventilatory support on day 1 of life), administration of surfactant (implying hypoxemia as well as the risk of rapid changes in blood oxygenation levels in the neonate), antibiotics for more than 7 days and blood transfusion during the stay at the hospital were recorded and analyzed. ¹⁰

Results

A total of 455 newborns were enrolled in the study, out of which 360 newborns completed the follow-up and 95 were lost due to mortality or loss to follow-up. Out of 360, 52% (n = 182) were males and 48% (n = 178) were females. The mean gestational age was 32±2 weeks, while the mean birth weight of the study population was 1.44±0.27 kg.

The mean serum zinc level was 114.99±54.47 mcg/dL. The proportion of newborns, who developed ROP changes, was 26% (n = 95), and out of them, 73% had stage 1, 17% had stage 2, 7% had stage 3, 2% had stage 4, and 1% had stage 5 ROP changes. Out of 360 newborns, 40 newborns had serum zinc levels <70 mcg/dL, and among them, 42% (n = 17) developed ROP changes (p = .02). In the group with serum zinc levels ≥70 mcg /dL, 24% (n = 78) developed ROP. The distribution of ROP according to various gestational ages, birth weights, and serum zinc levels is shown in Table 1. The mean serum zinc level in newborns with ROP was 111.65±49.54 mcg/dL and in newborns without ROP changes was 115.60±56.91 mcg/dL and it was not statistically significant. A significant association was found between gestational age and birth weight with ROP (Table 2).

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Table 1.

Retinopathy of Prematurity in Different Gestational Ages, Birth Weights, and Serum Zinc Levels.

Variables	ROP Absent	ROP Present	Total
Age (Gestational Weeks)
28–30	5 (62%)	3 (38%)	8
30–32	28 (56%)	22(44%)	50
32–34	74(70%)	32(30%)	106
34–36	158 (80%)	38(20%)	196
Weight (kilogram)
< 1.5	151(69%)	68(31%)	219
1.5–2	101 (80%)	25(20%)	126
2–2.5	13 (87%)	2(13%)	15
Serum Zinc Levels (mcg/dL)
<70	23(58%)	17(42%)	40
≥70	242(76%)	78(24%)	320

Abbreviation: ROP, retinopathy of prematurity.

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Table 2.

Association of Gestational Age and Birth Weight with ROP.

Parameter (Mean±SD)	ROP Absent	ROP Present	p Value
Gestational age (weeks)	33.31 ± 2.04	32.59 ± 1.99	<.01
Birth weight (kg)	1.46 ± 0.28	1.37 ± 0.25	<.01

Abbreviation: ROP, retinopathy of prematurity.

Note: Statistical test used—Student t test.

On univariate analysis, oxygen supplementation, RDS, use of surfactant, antibiotic use for more than 7 days, and serum zinc level <70 mcg/dL had a statistically significant association with the development of ROP (Table 3). All the factors found significant in univariate analysis, were included in the multiple logistic regression model. After adjusting for other factors, the independent predictors for ROP were gestational age, use of surfactant, and serum zinc level <70 mcg/dL (Table 4).

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Table 3.

Risk Factors with ROP Changes.

Risk Factor	ROP Present (n = 95)	ROP Absent (n = 265)	Total (n = 360)	p Value
Serum zinc <70 mcg/dL	17 (18%)	23 (8%)	40	.02*
O2 supplementation (>3 days)	77 (81%)	173 (65%)	250	.004*
Antibiotics >7 days	80 (84%)	196 (74%)	276	.0478*
Surfactant administration	15 (16%)	15 (6%)	30	.004*
Mechanical ventilation	08 (8%)	20 (8%)	28	.824
RDS	79 (83%)	185 (70%)	264	.01*
Blood transfusion	05 (5%)	10 (4%)	15	.554
Sepsis	17 (18%)	104 (39%)	121	.21
Shock	6 (6%)	50 (19%)	56	.65
IVH	1 (1%)	8 (3%)	9	1.0

Abbreviation: ROP, retinopathy of prematurity; RDS, respiratory distress syndrome; IVH, intraventricular hemorrhage.

Notes: Statistical test used—chi-square test and Fischer exact test. *indicates p < .05.

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Table 4.

Multivariate Logistic Regression Analysis of Independent Factors for Prediction of ROP.

Factors	Exp (β)/Odds Ratio [95% CI]	p Value
Gestational age	0.89 [0.79- 0.98]	.03*
Zn level (<70 mg/dl)	2.22 [1.10-4.48]	.02*
O2 supplementation	1.75 [0.75-4.14]	.19
Antibiotics >7 days	1.11 [0.52-2.38]	.78
Surfactant	2.37 [1.08-5.20]	.03*
RDS	1.21 [0.53-1.73]	.64

Abbreviations: RDS, respiratory distress syndrome.

Notes: Statistical test used—multivariate logistic regression analysis. *indicates p < .05.

Discussion

ROP is a major complication of premature birth. Per se, it is known to be caused due to oxidative damage of the premature retina. ⁵ Oxygen supplementation in preterm neonates with poor anti-oxidant systems leads to the formation of reactive oxygen species, which contribute to mitochondrial damage and the release of vascular growth factors, causing abnormal vasoproliferation of the immature retina. Thus, there can be a role of antioxidants in preventing the development of ROP. ¹⁰

Several studies have been done to analyze the effect of anti-oxidants like vitamin A, vitamin E, Lutein, and zinc on ROP but other than vitamin A and vitamin E none have shown any conclusive result.^11–14 A meta-analysis of randomized controlled trials on the effect of vitamin E on ROP was done by T.N. Raju et al and they found a significant reduction in the incidence and severity of ROP after Vitamin E supplementation. ¹⁵ Like Vitamin E, Zinc is also known to have antioxidant properties. It is siphoned to the fetus in late gestation; hence, preterm neonates are born with a smaller zinc pool. ⁵ Various studies have shown that the incidence of diseases such as neonatal sepsis, NEC, and BPD, which have similar pathogenesis to ROP, have reduced significantly with zinc supplementation.^{15, 16} Hence, the hypothesis that zinc deficiency might play a role in the pathogenesis of ROP was made. Limited studies have been done to assess zinc levels in preterm neonates and to see the relationship between zinc and ROP in the past.^17–19 Our study has analyzed the relationship between serum zinc levels and ROP as the primary outcome.

The overall prevalence of ROP was 26%; although different studies have shown the variable prevalence in different parts of the world ranging from 6% by Riazi et al to 47% by Minghua et al.^{20, 21} This variation may be because of the variable sample size and availability of healthcare facilities in different parts of the world which affect the survival of preterm neonates.

In our study, the mean serum zinc level was (114.99 ± 54.47 mcg/dL). It was similar to serum zinc levels found in preterm babies in studies done by Jyotsna et al (83.45±16.74 mcg/dL), and Jeswani et al (94.32±17.79 mcg/dL).^{22, 23} Taking the reference range of serum zinc levels between 70 and 150 mcg/dL as mentioned in previous studies ⁶ we studied the newborns into 2 groups—one with serum zinc levels < 70 mcg/dL and ≥ 70 mcg/dL. Out of 40 neonates who had serum zinc levels <70 mcg/dL 42% developed ROP changes while among the group who had serum zinc levels ≥70 mcg/dL only 24% developed ROP changes (p = .02). Although no difference was observed while comparing the mean or median of serum zinc levels in the groups with or without ROP changes. Similar to our results, Agrawal et al in their nested case-control study found zinc levels in cord blood and serum of neonate at 40 weeks postmenstrual age to be low in the babies who developed ROP in comparison to those who did not develop ROP. However, low zinc levels failed to emerge as a significant risk factor in multivariate analysis in their study. ¹⁸ Also, Terrin et al in a randomized controlled trial, done at 7 days of life of preterm neonates, found a reduction in the incidence of ROP with zinc supplementation, although it was statistically insignificant. ¹⁷

Contrary to these results, Yang et al in their study found higher zinc levels in cases than in the control. ¹⁹ They had taken serum zinc levels in babies between the 3rd and 7th days of life. Surprisingly, the zinc levels in their control group were low thereby they concluded relatively higher levels in cases than control. However, all these studies have taken zinc as a secondary outcome and compared to our study their sample size was less.

In our study, the risk factors which came out to have a significant association with ROP in univariate analysis were oxygen supplementation (p = .004), RDS (p = .01), use of surfactant (p = .004), antibiotic use for more than 7 days (p = .0478), and serum zinc less than 70 mcg/dL (p = .02), other than birth weight and low gestational age which are known to have a significant association with ROP. On multivariate analysis, the independent predictors for the development of ROP came out to be low gestational age, low serum zinc levels (<70 mcg/dL), and use of surfactant. Hence, our study affirmed low serum zinc level (<70 mcg/dL) to be a statistically significant independent risk factor for the development of ROP. Although to establish a role of zinc deficiency in the pathogenesis of ROP, more large-scale studies are needed to be conducted, which may also suggest a preventive role of zinc supplementation in preterm low birth weight babies.

Limitations

Due to the higher institutional mortality rates in gestational age less than 28 weeks, babies less than 28 weeks could not be taken in the study. Nonavailability of maternal serum zinc level, nonuniform group size, and absence of serial zinc measurements were also the limitation of our study.

Conclusion

We found low serum zinc levels (<70 mcg/dL) as an independent risk factor for the development of ROP apart from known risk factors including gestational age, birth weight, and surfactant administration. However, further larger studies are needed to establish a causative association between low serum zinc levels and ROP as well as to know the efficacy of maternal zinc supplementation to prevent the development of ROP.