The relationship between umbilical cord blood vitamin A levels and late preterm infant morbidities: a prospective cohort study

The aim of this study is to explore the association between umbilical cord blood (UCB) vitamin A levels and late preterm infants morbidities. We conducted a prospective cohort study of 208 late-preterm infants(from 34 0/7 to 36 6/7 weeks gestational age) between January 1, 2014 and June 30, 2015. UCB specimens were collected shortly after birth, and vitamin A levels were determined by enzyme-linked immunosorbent assay. Prevalence of low UCB vitamin A level < 0.7 μmol/L was 37.5% in late preterm infants. In comparison to vaginal delivery, cesarean section was associated with UCB vitamin A level < 0.7 μmol/L (P < 0.001). Nevertheless, UCB vitamin A levels did not correlate with gestational age, birth weight, and gender. UCB vitamin A level < 0.7 μmol/L was not an independent risk factor for hospitalization, oxygen supplementation, hyperbilirubinemia, sepsis, and respiratory distress syndrome. Conclusions: Low umbilical cord blood vitamin A levels are common among late-preterm infants. Cesarean section delivery is associated with low umbilical cord blood vitamin A level. Low umbilical cord blood vitamin A levels at birth do not increase morbidity of late-preterm infants, including hyperbilirubinemia, sepsis, and respiratory distress syndrome. What is Known: • Late preterm infants have a higher morbidity and mortality rates when compared to term infants. • Low plasma vitamin A levels increase the risk of preterm infants’ morbidity. What is New: • Late preterm infants commonly have low level of umbilical cord blood vitamin A. • Low umbilical cord blood vitamin A level at birth appears to be not associated with the morbidity of late-preterm infants. • Cesarean section is associated with low umbilical cord blood vitamin A level < 0.7 μmol/L compared with vaginal delivery. What is Known: • Late preterm infants have a higher morbidity and mortality rates when compared to term infants. • Low plasma vitamin A levels increase the risk of preterm infants’ morbidity. What is New: • Late preterm infants commonly have low level of umbilical cord blood vitamin A. • Low umbilical cord blood vitamin A level at birth appears to be not associated with the morbidity of late-preterm infants. • Cesarean section is associated with low umbilical cord blood vitamin A level < 0.7 μmol/L compared with vaginal delivery.


Introduction
Late preterm infants (with gestational age between 34 0 / 7 and 36 6 / 7 weeks) are common among preterm infants. Late preterm infants have a higher morbidity of apnea, respiratory distress, kernicterus [1], and mortality rates when compared to term infants [2,3]. Vitamin A, a kind of fat-soluble vitamin, has several physiological functions. It plays an essential role in retina growth and differentiation, and the lung. Also, vitamin A maintains epithelial integrity, bone development, and immunity and promotes the maturation of type II alveolar epithelial cells [4,5]. Vitamin A aberration is associated with many diseases, such as night blindness, dry eye, corneal softening, corneal ulcers, immune dysfunction, and respiratory infections. Several studies have reported vitamin A insufficiency to be associated with morbidity of premature infants [4]. With vitamin A deficiency occurring during pregnancy, pulmonary surfactant is poorly produced with reduced phospholipid and protein content [5]. This, in turn, leads to respiratory distress syndrome (RDS). Serum retinol insufficiency increases the risk of severe RDS and adverse pulmonary outcomes, such as bronchopulmonary dysplasia (BPD) in preterm infants < 1250 g or < 29 weeks' gestation [6]. Moreover, Esteban-Pretel et al. have suggested that vitamin A deficiency can adversely affect alveolar function and increase the risk of lung disease [7]. Preterm neonates born with low vitamin A levels have an increased risk of developing chronic lung disease and hyperbilirubinemia [8,9]. Also, vitamin A deficiency can reduce both cellular and humoral immunity in intestinal mucosa and increase gut permeability [10,11]. However, vitamin A supplementation can ameliorate intestinal mucosal injury induced by butyric acid and necrotizing enterocolitis (NEC) [12][13][14].
Insufficient serum vitamin A level may increase the risk of RDS and adverse pulmonary outcomes in very low birth weight infants [6,15]; no study has yet investigated the connection between vitamin A levels and late preterm infant morbidity. This study was based on the premise that low UCB vitamin A levels can increase late preterm infant morbidities. We performed analysis on obtained UCB specimens to determine vitamin A levels and ascertain the relationship between UCB vitamin A levels and late preterm infant morbidities.

Study designs and patients
We conducted a prospective cohort study between January 1, 2014, and June 30, 2015 with late preterm infants delivered at Wenling Maternal and Child Health Care Hospital. The hospital is an upper second-class hospital located in Wenling city, Taizhou city, Zhejiang province, China. All singleton late preterm infants were eligible for the study. Newborns that had significant congenital malformations or chromosomal abnormality or congenital genetic and metabolic diseases or severe congenital heart diseases were excluded. Kendall's sample size calculation principle yields sample sizes 5-10 times the number of variables [16]. In our study, there were 20 variables. Sample size was set at 210; thus, (20 × 10 = 200).

Procedure
Firstly, we collected UCB specimens to determine vitamin A levels. We then assessed neonatal morbidities during followup, investigated the relationship between UCB vitamin A levels and late preterm infant morbidities, and finally, ascertained whether UCB vitamin A levels could predict for late preterm infant morbidities. Institutional Review Board approval was obtained.
Obstetricians or midwives selected 3-mL umbilical blood samples from the maternal end of the umbilical cord vein after umbilical cords had been clamped and cut in the delivery or operating room. Samples were preserved in anticoagulant brown tubes and were prepared by centrifugation at 1000×g for 15 min at 4°C. Supernatants were collected and stored securely in -20°C environment until assayed. Vitamin A levels were measured with enzyme-linked immunosorbent assay (ELISA) Vitamin A kits (Affandi, A097404-96T) in duplicate according to the manufacturer's instructions (Shanghai Fu Sheng Industrial Co., Ltd., Shanghai, China). Both intraassay and inter-assay reproducibilities of the vitamin A kits were 10%. The detection limit for vitamin A was 10-320 ng/ mL.
The following conditions were recorded in study participants during follow-ups: jaundice, sepsis, RDS, BPD, NEC, and death. Pediatricians with extensive clinical experience conducted daily check-ups of obstetric newborns. Those patients suffering from neonatal diseases were admitted to the Department of Neonatology for the treatment until they were discharged from the hospital. Those without abnormalities adhered to the necessary follow-ups before being discharged. Follow up started from birth to when late preterm infants were discharged from the hospital. All clinical data was recorded through both prospectively in real time and hospital electronic medical record system. Clinicians collecting data were blinded to lab results, and the lab investigators were also blinded to clinical data.

Definitions
Bilirubin levels are interpreted according to infants' age in hours. Hyperbilirubinemia is defined as neonatal jaundice that needs phototherapy [17,18]. Oxygen supplementation refers to various forms of respiratory support, including mechanical ventilation, continuous positive airway pressure (CPAP), bi-level positive airway pressure (BiPAP), head net, and nasal catheter. Sepsis is defined as systemic inflammatory response syndrome (SIRS) in the presence of or as a result of a suspected or proven infection. SIRS is defined as the presence of at least two of the following four criteria, one of which must be abnormal temperature or leukocyte count: (1) core temperature of > 38.5°C or < 36°C, (2) tachycardia or bradycardia, (3) mean respiratory rate 2 standard deviation (SD) above normal for age or mechanical ventilation for an acute process not related to underlying neuromuscular disease or the receipt of general anesthesia, and (4) leukocyte count elevated or depressed for age (not secondary to chemotherapy-induced leukopenia) or 10% immature neutrophils [19]. Suspected infection is defined as clinical symptoms and signs consistent with infection without isolation of a causative organism. Proven infection is defined as clinical symptoms and signs consistent with infection confirmed by positive cultures from blood, or cerebrospinal fluid, and or urine [19,20]. RDS is determined based on classic signs of respiratory distress, as well as exclusion of other causes of respiratory failure. RDS diagnosis is confirmed radiologically in cases of reduced lung volume, reticulogranular lung consolidation, and air bronchograms [21]. BPD is identified when there is clinical evidence of BPD, combined with oxygen therapy at 28 days of life [22]. NEC is clinically and radiographically diagnosed using modified Bell's criteria [23]. UCB vitamin A level is divided into two ranges, low vitamin A level < 0.70 μmol/L and high vitamin A level ≥ 0.7 μmol/L [24].

Statistical analysis
The distribution of data was analyzed by the Kolmogorov-Smirnov test. Normal distributed data are expressed as mean ± SD. Continuous variables were compared between 2 groups using Student's t test. Non-normally distributed data were expressed as medians (interquartile range) and data compared between 2 groups using Mann-Whitney U test. Countable data were expressed as number (%). Data were compared between groups using chi-square or Fisher's exact test. Variables with P value < 0.1 in the univariate analysis were selected for the multivariable analysis. Binary logistic regression analysis by the enter method was used to investigate whether risk factors were independently associated with outcome variables. Pearson correlation analysis was used to evaluate multicollinearity for categorical variables. Correlation coefficient (r) < 0.85 indicates that categorical variables have no collinearity. Goodness of fit was evaluated by pseudo-R-squares. Results were described as odds ratios (ORs) and 95% confidence intervals (CIs). P value < 0.05 was considered statistically significant. All data were analyzed by IBM Statistical Package for the Social Sciences (SPSS), version 23 (IBM Corporation).

Participants in the cohort study
The study cohort comprised 210 late preterm infants between January 1, 2014 and June 30, 2015. Two patients were excluded due to insufficient umbilical cord blood samples. Therefore, the study population included 208 late preterm infants. The flow diagram of participants is shown in Fig 1.

Clinical characteristics of late preterm infants
The mean gestational age and birth weight of the samples were 35.68 ± 0.74 weeks and 2613.32 ± 425.57 g, respectively. The median UCB vitamin A level of late preterm infants was 0.754 μmol/L (0.656, 0.892). Prevalence of low UCB vitamin A level < 0.7 μmol/L was 37.5% in late preterm infants. The clinical characteristics of UCB vitamin A low and high levels are summarized in Table 1.

Potential factors affecting UCB vitamin A levels
We analyzed various potential factors that may affect UCB vitamin A levels, such as gestational age (< 35 weeks and ≥ 35 weeks), birth weight (< 2500 g and ≥ 2500 g), delivery mode, and gender (Table 1). In comparison to vaginal delivery, cesarean section was associated with UCB vitamin A level < 0.7 μmol/L. Gender, gestational age < 35 weeks, and birth weight < 2500 g were not independent risk factors of UCB vitamin A level < 0.7 μmol/L in late preterm infants (Table 2).

Relationship between UCB vitamin A levels and late preterm infant morbidities
We observed relationship between UCB vitamin A levels and late preterm infant morbidities, such as 1 min Apgar score ≤ 7, hospitalization, oxygen supplementation (CPAP, BiPAP, and head net), hyperbilirubinemia, sepsis, SIRS, suspected infection, proven infection, and RDS. UCB vitamin A level < 0.7 μmol/L was not an influence factor of 1 min Apgar score ≤ 7, hospitalization, oxygen supplementation (CPAP, BiPAP, and head net), hyperbilirubinemia, sepsis, SIRS, suspected infection, proven infection, and RDS before and after adjusting confounding factors, such as gender, mode of delivery, gestational age, and birth weight (Table 3).

Discussion
In this study, we analyzed UCB vitamin A levels, and its relationship with some medical morbidities in late preterm infants. The median UCB vitamin A level of late preterm infants was 0.754 μmol/L (0.656, 0.892). Our median UCB vitamin A level was higher than a previous study where UCB vitamin A level of 0.55 ± 0.17 μmol/L was reported [25]. However, that study did not mention the birth weight of late preterm infants. In another study, UCB vitamin A level less than 1.05 μmol/L was measured in 22/56 and levels below 0.7 μmol/L only measured in 2/56 for gestational age < 33 weeks [8]. These results were inconsistent. Two possible influence factors were the different gestational age and birth weight of study participants. Nevertheless, preterm infants are prone to low vitamin A level [25]. Our results showed the prevalence of low UCB vitamin A levels < 0.7 μmol/L in late preterm infants to be high (37.5%). Several reasons could have accounted for this. For example, fetuses cannot synthesize vitamin A, and thus, primarily depend on maternal placental supply in late pregnancy [26]. Also, retinol-binding protein (RBP) manufactured in the fetal liver during the final quarter of pregnancy plays a vital role in fetal vitamin A transportation. Premature infants are unable to synthesize enough RBP to transport vitamin A [27,28]. This results in preterm being susceptible to having low vitamin A level.
We compared the effect of various factors on vitamin A levels, including gestational age, birth weight, delivery mode, and gender. Late preterm infants delivered via cesarean section is more likely to be low UCB vitamin A level < 0.7 μmol/L than those delivered via the vagina ( Table 2). Cesarean section was associated with low UCB vitamin A level < 0.7 μmol/L. Vaginal deliveries without anesthesia are more stressful than cesarean deliveries, and this stress can increase maternal corticosteroids [29]. Corticosteroids increase placental cord concentrations of vitamin A [30], hence lower and higher UCB vitamin A levels in cesarean section and vaginal deliveries, respectively. Contrastingly, Gonzalez-Corbella et al. reported no significant difference in vitamin A levels between vaginal and cesarean deliveries [29]. We Data were analyzed by univariate and multivariate logistic regression analysis (N = 208) UCB umbilical cord blood, OR odds ratio, CI confidence interval, CPAP continuous positive airway pressure, BiPAP bi-level positive airway pressure, SIRS systemic inflammatory response syndrome, RDS respiratory distress syndrome, N number of study participants a Adjusted for gender, mode of delivery (cesarean section, vaginal), gestational age(< 35 weeks, ≥ 35 weeks), birth weight(< 2500 g, ≥ 2500 g) Data were analyzed by multivariate logistic regression analysis UCB umbilical cord blood, OR odds ratio, CI confidence interval believe their inconsistent results were associated with the small sample size of the study (35 vaginal and 21 cesarean deliveries). Thus, based on our results, we are of the view that UCB vitamin A levels are significantly lower in cesarean section than vaginal delivery. This is may be another proof that vaginal delivery is a generally preferred mode of delivery than caesarean section. Additionally, vitamin A levels in our study were not associated with birth weight, gestational age, and gender in late preterm infants. These results were in line with previous studies [8,29]. Retinol is significantly higher in the serum of mothers giving preterm birth as a result of preterm premature rupture of membranes [31]. Thus, preterm premature rupture of membranes could affect vitamin A levels; nonetheless, the exact mechanisms is yet to be comprehensively elucidated. Vitamin A acts on lung's retinoic acid receptors to upregulate the transcription and expression of surfactant protein-B gene. This, in turn, promotes the synthesis of pulmonary surfactant [32]. Besides, mild vitamin A deficiency has been shown to delay fetal lung maturation in animal studies. Surfactant proteins-A, B, and C messenger RNAs-were decreased by 46%, 32%, and 28%, respectively, in vitamin A-deficient fetuses [5]. Kiatchoosakun et al. suggested that vitamin A supplementation could reduce intubation time and oxygen supplementation, while also shortening the length of hospital stay [33]. However, another study reported no difference in the duration of oxygen therapy between vitamin A supplementation and control groups [34]. In our study, we have not found vitamin A level < 0.7 μmol/L to be an independent risk factor for hospitalization, oxygen supplementation, and RDS in late preterm infants (Table 3). Our result was consistent with a previously reported study [8]. Vitamin A can promote lung surfactant synthesis and thus enhance lung maturation. However, this might not have a significant effect on late preterm infants who have relatively matured lungs than other preterm infants. Having said that, further studies are warranted to clarify this.
Vitamin A can regulate specific and non-specific immunity and protect against bacterial infection as an antioxidant [35]. Plasma vitamin A concentrations are decreased during airway infection in premature neonates [36]. In our study, however, UCB vitamin A level < 0.7 μmol/L was not an independent risk factor for sepsis, SIRS, suspected infection, and proven infection (Table 3). We also found UCB vitamin A level < 0.7 was not an independent risk factor for hyperbilirubinemia in late preterm infants (Table 3). To the best our knowledge, there are limited studies reporting an association between vitamin A concentration and hyperbilirubinemia in late preterm infants. As such, larger sample studies based on specific population are needed to corroborate this relationship. In addition, none of the neonates in this study suffered from BPD, NEC, and death. Thus, the relationship between UCB vitamin A levels and BPD, NEC, or death in late preterm infants requires further study.
Our study has some limitations. For example, the number of study participants was small. Besides, the study did not include the umbilical cord samples of term infants as control group of the study, although we did a subgroup analysis. Also, we did not measure other vitamins or micronutrients, which might have an impact on the results.
In conclusion, late preterm infants commonly have low level of umbilical cord blood vitamin A. Cesarean section delivery is associated with low umbilical cord blood vitamin A level in late preterm infants. On the basis of our results, there is no association between low vitamin A levels and morbidity of late preterm infants, including hyperbilirubinemia, sepsis, and respiratory distress syndrome.
Authors' contributions E Tao: conception and design, acquisition of data, analysis and interpretation of data; drafting the article; final approval the version to be published; funding acquisition. C Chen, Y Chen, L Cai: acquisition of data; final approval the version to be published. T Yuan: conception and design; revise the article; final approval the version to be published; funding acquisition.
Funding information The work of the authors is supported by National Natural Science Foundation of China (grant no. 81571466) and Science and Technology Foundation of Taizhou City, Zhejiang Province, China (grant no. 1902ky159).

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Informed consent Written informed consent was obtained from the mother or legal guardian of each neonate before inclusion into the study.
Ethical approval The study was approved by the medical ethical committee of Wenling Maternal and Child Health Care Hospital (approval no. 2013-01218).