Patients and Study Design This was an experimental study with pre-test/post-test control groups, double-blind with randomization block technique. This study was conducted from March to December 2019 in the obstetrics and neonatology unit of Universitas Sumatera Utara Hospital, Malahayati Islamic Hospital and Royal Maternity Hospital in Medan. Research subjects were pregnant mothers with their newborns who met the inclusion criteria. The study samples were divided into two groups and administered either zinc supplementation 20 mg/day or placebos, respectively, for 12 weeks. The zinc tablets or placebos were given in the morning after meals. All subjects were also given iron and folic acid tablets in accordance with the Indonesian government program. Subjects were healthy pregnant mothers in their second or third trimester, 20-35 years old, with a height of \>150 cm and no indication of cons following this study, as determined by obstetrician and gynaecology specialists; subjects were enrolled with a consecutive sampling technique. Births involving twins, congenital abnormalities, prematurity (gestation age \<37 weeks) or stillbirth were excluded.
Informed consent was given by all subjects' legal guardians following the provision of sufficient information prior to the study.
This study was approved by the Health Research Ethical Committee, School of Medicine, Universitas Sumatera Utara in accordance with the principles of the Helsinki Declaration (No.179/TGL/FK USU-RSUP HAM/2019).
Methods The parameters assessed in this study were maternal serum zinc levels, cord blood osteocalcin and birth length measurements. Maternal serum zinc levels were measured twice with 6 ml of vein blood each time, both during initial antenatal care and after 12 weeks of supplementation. Samples were subsequently centrifuged for 15 minutes at 3000 rpm. Specimens were processed by the inductively coupled plasma-mass spectrometry (ICP-MS) method using Agilent 7700 analyser (Santa Clara, USA, 2014). Normal serum zinc levels were defined based on a cut-off value of ≥ 56 µg/dL in accordance with Second National Health and Nutrition Examination Survey data from 1976-1980 (NHANES II).
Cord blood osteocalcin levels were measured with 6 ml cord blood samples prior to delivery, which were subsequently centrifuged for 15 minutes at 1000 rpm using Cobas e601 analyser (Roche Diagnostic, Mannheim, Germany) and N-Mid Osteocalcin (Abbexa) reagent. Osteocalcin was measured using the enzyme-linked immuno-sorbent assay (ELISA) method, and in accordance with Prodia clinical laboratory standards.
Neonatal birth length was performed with a SECA 232 digital baby scale for length with an accuracy of 0.1 cm. Maternal zinc intake was calculated in accordance with the NutriSurvey 2007 Indonesian version.
Statistical Analysis Descriptive analyses, correlation tests, and linear regression tests were performed using SPSS version 23.0 (SPSS Inc., Chicago, Illinois, USA). Statistical significance was considered at a p-value \< 0.05 with a 95% confidence interval.
