This single-blind randomized controlled trial, using the randomization box, was conducted in the Department of Obstetrics and Gynecology and the Intensive Care Unit of Qaem Hospital, affiliated to Mashhad University of Medical Sciences, Mashhad, Iran, between January 21st, 2019 and August 23rd 2019. An informed consent was obtained from each participant by a single researcher prior to study entrance. The study protocol was approved by the Ethics Committee of Mashhad University of Medical Sciences (IR.MUMS.fm.REC.1395.52) and was registered in the Iranian Registry of Clinical Trials (IRCT20190814044529N1; 21/01/2020).
All singleton pregnant women with a gestational age of less than 34 weeks and at risk of preterm delivery, normal prenatal ultrasound screening results, and no significant medical and obstetrical conditions regarding the mother (such as cardiovascular disorders, diabetes mellitus, epilepsy, and asthma) were included in the study.
The exclusion criteria was as follows: a gestational age of ≥ 34 weeks, neonatal death at birth, major congenital anomalies, severe perinatal asphyxia, necrotizing enterocolitis, stillbirth, respiratory distress except for neonatal respiratory distress syndrome, and neonatal abstinence syndrome.
Pregnant mothers at risk of preterm labor were assigned to the intervention and control arms of the study based on simple randomization with respect to the inclusion criteria. Initially, 50,000 units of 25(OH) D was injected intramuscularly for mothers in the intervention group. The principal researcher evaluated the neonates' outcome including respiratory distress, unaware of group assignments. The collected data were classified as groups A and B in the SPSS software, and a statistical analyst who was blind to group assignments interpreted the data. Due to the lack of similar studies in the literature, this study is regarded as a pilot study. A sample consisting of at least 20 cases and 20 controls was considered to fulfill the comparisons. Therefore, in total 175 mothers were enrolled in the study.
Medical data relevant to the study were documented in the patients' medical records. Maternal data included age, parity, antenatal corticosteroids and vitamin D supplements consumption, and gestational age at the time of admittance and delivery. The neonatal data consisted of gestational age, gender, birth weight, Apgar scores at 1 and 5 minutes, hospitalization in the neonatal intensive care unit, the need for ventilation support, surfactant replacement therapy for RDS, length of hospital stay, morbidities and mortalities due to prematurity such as necrotizing enterocolitis and infection (based on positive blood culture).
Pregnant women in the intervention group who had not taken any 25(OH) D supplements during pregnancy were injected with a single dose of 50,000 units of intramuscular 25(OH)D within 72 hours of preterm delivery.
A 1.5 ml blood sample was taken from both neonates and mothers after delivery to check the vitamin D level. The prepared samples were kept at -20°C and sent to the central laboratory of Qaem Hospital for analyses. Vitamin D level was measured in ng/mL by the ELISA method using an Elisa Reader (RT2100c, Germany) and Elisa Washing devices. A 25(OH) D level of less than 30 ng/mL was considered as vitamin D deficiency and a level ≥ 30 ng/mL was considered as the sufficient value. Subjects with vit D deficiency were divided into three groups according to their serum 25(OH) D level: severe deficiency (≤ 10 ng/mL), moderate deficiency (10.1–20 ng/mL), and mild deficiency (20.1–30 ng/mL). Other related laboratory studies based on the local neonatal guidelines including the quantitative assessment of CRP, CBC, and blood culture were also performed.
All hospitalized neonates with respiratory problems underwent chest x-rays. The radiographic findings used for confirming the diagnosis of neonatal respiratory distress syndrome included bilateral and relatively symmetric diffused ground glass opacities with low volumes and a bell-shaped thorax, air bronchograms, and absence of hyperinflation.
The criteria for diagnosing respiratory distress syndrome were: respiratory distress findings including tachypnea (> 60 /min), intercostal muscle retraction, grunting and nasal flaring; chest radiographic findings as mentioned previously, the need for respiratory support under nasal CPAP with a PEEP > 8 cmH2O and/or FiO2 > 30, and abnormal arterial blood gas analysis.
Patients and Methods
This study was a clinical trial conducted on 175 pregnant women with a GA of less than 34 weeks at birth. Among them, 25 cases from the intervention group were placed under nasal continuous positive airway pressure (NCPAP) therapy with FiO2 of 30% and PEEP of 5 cmH2O at the time of admission. Neonates in whom respiratory support, including increasing PEEP to > 8 cmH2o with Fio2 > 30, surfactant administration and extubation and or endotracheal intubation had been introduced met the inclusion criteria. All subjects were monitored continuously by routine observation and pulse oximetry. Neonatal variables including blood gas analysis (pH, PO2, PCO2), oxygen saturation, gender of the infant, birth weight and gestational age were recorded. The neonatal primary outcome measure in the study group was respiratory distress syndrome and its incidence in neonates born less than 34 weeks of gestation. Diagnosis of sepsis was based on the observation of clinical signs and positive blood culture. Diagnosis of necrotizing enterocolitis was based on the Bell staging criteria, including systemic symptoms severity and radiographic findings.
Statistical analysis was performed by the SPSS software (IBM SPSS Statistics, Version 21). Independent t-test or Mann-Whitney test were used to compare quantitative variables between the two groups. Quantitative variables within each group were compared with paired sample t-test. To compare qualitative variables between the two studied groups, Chi-square test and Fisher's exact tests were used. Data are presented as mean ± standard deviation (SD). A P-value < 0.05 was considered as statistically significant.