Retrospective cohort study on the correlation between serum 25(OH)D, vitamin A and neonatal respiratory distress syndrome (NRDS) in premature infants

Abstract

Background

Premature infants are at high risk for neonatal respiratory distress syndrome (NRDS) due to underdeveloped lungs. Understanding serum 25-hydroxyvitamin D [25(OH)D] and vitamin A’s roles may improve early intervention strategies. This study explores the correlation between NRDS and serum 25(OH)D and vitamin A levels in premature infants.

Methods

A retrospective cohort study was conducted involving 148 premature infants. The characteristics of infants diagnosed with NRDS were compared to those without NRDS. Statistical analyses included logistic regression to evaluate the interaction between serum 25(OH)D and vitamin A levels.

Results

Premature infants with NRDS exhibited significantly lower levels of both 25(OH)D and vitamin A compared to those without NRDS. There was no significant difference in serum vitamin A and D deficiency between premature infants whose mothers received and did not receive glucocorticoid treatment before delivery. The risk of developing NRDS was 10.5 times greater in infants with low levels of both vitamins as opposed to those with high levels. Furthermore, the interaction of these two vitamins was shown to contribute an additional 47.54% to the risk of NRDS. A nomogram model created for predicting NRDS risk demonstrated high predictive accuracy with a C-index of 0.961 (95% confidence interval: 0.931–0.990).

Conclusions

The findings suggest that adequate serum levels of 25(OH)D and vitamin A may play a crucial role in mitigating the risk of NRDS in premature infants. Monitoring and intervention strategies regarding these vitamins could enhance clinical outcomes and reduce the incidence of NRDS.

Highlight box.

Key findings

• Premature infants with neonatal respiratory distress syndrome (NRDS) have significantly lower serum levels of both 25-hydroxyvitamin D [25(OH)D] and vitamin A compared to those without NRDS. A synergistic interaction between these two vitamins contributes to an increased risk of NRDS, with the risk being 10.5 times higher in infants with low levels of both vitamins. A predictive nomogram model based on these two serum markers demonstrates a high predictive accuracy (C-index =0.961) for NRDS risk in premature infants.

What is known and what is new?

• It is known that NRDS is primarily caused by immature lung development and surfactant deficiency in premature infants. Previous studies have suggested that both 25(OH)D and vitamin A play roles in lung development and surfactant production. However, the interaction between these vitamins and their combined effect on NRDS risk have not been extensively explored.

• This manuscript adds novel insight into how the interaction of 25(OH)D and vitamin A influences NRDS risk and presents a highly accurate nomogram model for early identification of at-risk infants.

What is the implication, and what should change now?

• The findings suggest that monitoring and correcting serum 25(OH)D and vitamin A deficiencies in premature infants could reduce NRDS risk and improve clinical outcomes. Clinicians should consider including these vitamins as part of early risk assessment and intervention strategies in premature infants to prevent NRDS.

Introduction

At present, clinical studies have confirmed that the lack of pulmonary surfactant and immature lung development are the main pathogenesis of neonatal respiratory distress syndrome (NRDS) in premature infants (1). For premature infants with targeted application of pulmonary surfactant replacement, respiratory support and other methods of treatment, although there is a certain effect, there are still some premature infants with poor prognosis (2,3). Therefore, finding indicators related to NRDS in premature infants may assist in early clinical screening of high-risk groups and find new therapeutic targets. It is reported that 25-hydroxyvitamin D [25(OH)D] and vitamin A are fat-soluble vitamins. Both of them can directly promote the synthesis of pulmonary surfactant protein and phospholipid components in alveolar type II epithelial cells by regulating the expression of related genes, increase the secretion of pulmonary surfactant, promote the development of alveolar capillaries, improve the lung microenvironment, and provide a more suitable physiological basis for the secretion of surfactant, thereby participating in the occurrence and development of lung development and respiratory diseases (4,5). Previous studies have confirmed through logistic regression analysis that 25(OH)D and vitamin A may be involved in the pathological process of NRDS (6,7). However, the interaction and specific relationship between them on the occurrence of NRDS in premature infants are still unclear. In this study, the clinical data of premature infants were retrospectively analyzed to further clarify the correlation between serum 25(OH)D, vitamin A and NRDS. We present this article in accordance with the TRIPOD reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2025-388/rc).

Methods

Research object

The sample size was calculated based on the sample size calculation formula $n=\frac{Z_{\alpha }^{2}p\left(1-p\right)}{{\delta }^2}$. P represented the incidence of NRDS in premature infants. Relevant literature (8) was consulted, and the value was approximately 75.97%. If α was taken as 0.05, then the value of Zα in the two-sided test was 1.96, and δ represented the allowable error of the overall proportion estimate. In this study, 7.28% was taken, resulting in at least 133 total included samples. Considering the inefficiency of about 10%, the final included sample size was at least 148 cases.

The clinical data of 148 premature infants born in The Third Affiliated Hospital of Nanjing Medical University from August 2022 to February 2024 were collected by a retrospective study. Preterm infants with NRDS after birth were included in the NRDS group (n=111), and otherwise included in the non-NRDS group (n=37). Inclusion criteria: (I) singleton; (II) 28 weeks < gestational age <37 weeks; (III) Venous blood collection was completed within 2 hours after birth, and biochemical indicators such as serum 25(OH)D and vitamin A were detected; (IV) mother age ≥20 years old; (V) the clinical management plans for all mothers of premature infants during pregnancy were basically the same; (VI) Complete clinical data. Exclusion criteria: (I) with genetic metabolic diseases, congenital heart disease; (II) with infection, meconium aspiration, asphyxia and other systems seriously affecting the respiratory function of the disease; (III) congenital malformation of thorax and chest wall; (IV) the mother took anticonvulsant and antiepileptic drugs during pregnancy. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Third Affiliated Hospital of Nanjing Medical University (No. [2024] YLJSC010). Informed consent was obtained from the parents or legal guardians of all participants under the age of 16.

Research method

Diagnostic method of NRDS

According to the relevant content of “European Consensus Guidelines on the Management of Respiratory Distress Syndrome - 2019 Update” (9), the diagnosis of NRDS was made: shortly after birth, respiratory distress occurred and aggravated progressively, main manifestations were shortness of breath (>60 beats/min), cyanosis, inspiratory three-concave sign (suprasternal fossa, intercostal space, subxiphoid inspiratory depression), etc. In severe cases, it manifested as superficial breathing, irregular breathing rhythm, apnea and limb relaxation. Physical examination showed a flat chest, abnormal sounds can be heard in both lungs, breathing sounds weakened, alveolar exudation can be heard when the fine wet rales. Chest X-ray showed that the transmittance of both lungs decreased generally, and diffuse uniform fine-grained reticular shadows could be seen. Under the background of diffuse atelectasis, clear inflatable dendritic bronchial shadows were seen. Both lung fields were white, and the lung-liver boundary and lung-heart boundary disappeared. Blood gas analysis suggested respiratory acidosis and hypoxemia.

Data collection of premature infants

The general data of premature infants were collected through the hospital electronic medical record system. This included gender, gestational age, birth weight, 1-min Apgar score, 5-min Apgar score, blood routine indicators (hemoglobin, white blood cells, red blood cells, platelets), high-sensitivity C-reactive protein, 25(OH)D, vitamin A levels, acceptance of treatment with pulmonary surfactants within 5 minutes after birth. Specific detection methods for blood routine and 25(OH)D and vitamin A levels: 2 tubes of 0.3 mL venous blood were collected within 2 hours after birth. One tube was used to detect the level of blood routine indexes by xn350 automatic blood cell analyzer, and the detection method was Coulter principle. Another tube was centrifuged and the serum was taken. The level of high-sensitivity C-reactive protein was estimated by Pumen automatic biochemical analyzer. The detection method was immunoturbidimetry. The levels of 25(OH)D and vitamin A were detected by liquid chromatography-liquid chromatography/mass spectrometry [Meikang Shengde Medical Technology (Suzhou) Co., Ltd., Suji Zhun 2019221449, Model: Altis Plus]. All blood collection, centrifugation and testing processes were carried out in a weak light environment. Both the blood samples and the separated serum samples were stored in the dark at 2–8 ℃ and the testing was completed within 4 hours. The detection method was mass spectrometry. 25(OH)D deficiency was defined as 25(OH)D level <15 ng/mL, 25(OH)D insufficiency was defined as 25(OH)D level 15–20 ng/mL, and 25(OH)D sufficiency was defined as 25(OH)D level >20 ng/mL (10), vitamin A deficiency was defined as A level of vitamin A <200 ng/mL, and vitamin A deficiency was defined as A level of vitamin A between 200 and 300 ng/mL, and vitamin A adequacy was defined as a level of vitamin A >300 ng/mL (11).

Mother-related data collection

General information of mothers of premature infants was collected through the hospital electronic medical record system, including age, pregnancy, parity, pre-pregnancy weight, height, prenatal body mass index, pregnancy disease, prenatal completion of ≥1 glucocorticoids treatment, amniotic fluid properties, mode of delivery, etc.

Statistical analysis

The data were analyzed by SPSS 25.0, and the count data were analyzed by the test, expressed as n (%). The measurement data conforming to normal and skewed distribution were tested by t and Mann-Whitney U test, respectively, and expressed as mean ± standard deviation, median (interquartile range). The relative excess risk due to interaction (RERI), the synergy index (SI) and the attribution percentage (AP) were used to analyze the interaction of the above two indices on NRDS. The nomogram was drawn by R software and rms package, and the consistency index (C-index) was calculated to evaluate the predictive efficacy of the prediction model constructed by the above two indices and other main indices. Two-sided test level α=0.05.

Results

Clinical data

Compared non NRDS group, the gestational age of premature infants in the NRDS group was smaller, the birth weight, 1- and 5-min Apgar scores, serum 25(OH)D and vitamin A levels were lower, the proportion of pregnant women who completed ≥11 course of glucocorticoids before delivery was higher, and the proportion of sufficient serum 25(OH)D and vitamin A was lower (P<0.001, Tables 1,2).

Table 1. Comparison of clinical data of both groups of premature infants.

Item	NRDS group (n=111)	Non NRDS group (n=37)	χ2/Z/t	P
Gender			0.15	0.70
Male	62 (55.86)	22 (59.46)
Female	49 (44.14)	15 (40.54)
Fetal age, weeks	34.00 (33.00, 35.00)	36.00 (35.00, 36.00)	5.495	<0.001
Birth weight, g	2,250 (1,900, 2,600)	2,800 (2,500, 3,050)	6.070	<0.001
1-min Apgar score	8.00 (7.00, 8.00)	9.00 (8.00, 9.00)	6.025	<0.001
5-min Apgar score	9.00 (8.00, 9.00)	9.00 (9.00, 10.00)	5.340	0.002
Leucocyte, ×109/L	10.90 (8.69, 14.01)	11.30 (10.03, 15.73)	1.503	0.13
Erythrocyte, ×1012/L	4.65±0.51	4.60±0.47	0.568	0.57
Hemoglobin, g/L	172.00 (158.00, 184.00)	169.00 (158.50, 182.50)	0.246	0.81
Thrombocyte, ×109/L	271.00 (235.00, 304.00)	277.00 (231.50, 308.50)	0.011	0.99
Hypersensitivity C-reactive protein, mg/L	0.28 (0.21, 0.36)	0.26 (0.22, 0.36)	0.590	0.56
25(OH)D, ng/mL	14.02 (9.55, 16.69)	16.71 (14.09, 20.98)	4.067	<0.001
Vitamin A, ng/mL	233.14 (196.55, 288.65)	280.54 (242.91, 330.38)	3.943	<0.001
25(OH)D			3.709	<0.001
Lack	66 (59.46)	12 (32.43)
Insufficient	36 (32.43)	11 (29.73)
Adequate	9 (8.11)	14 (37.84)
Vitamin A			3.772	<0.001
Lack	29 (26.13)	0
Insufficient	68 (61.26)	26 (70.27)
Adequate	14 (12.61)	11 (29.73)
Accept treatment with pulmonary surfactants within 5 minutes after birth			0.181	0.67
No	96 (86.49)	33 (89.19)
Yes	15 (13.51)	4 (10.81)

Data are presented as mean ± standard deviation, n (%) or median (interquartile range). 25(OH)D, 25-hydroxyvitamin D; NRDS, neonatal respiratory distress syndrome.

Table 2. Comparison of clinical data in both groups of mothers of premature infants.

Item	NRDS group (n=111)	Non-NRDS group (n=37)	t/Z/χ2	P
Age, years	30.96±4.53	30.84±4.23	0.149	0.88
Pregnancy, times	2.00 (1.00, 4.00)	2.00 (1.00, 3.00)	1.901	0.06
Production, times	2.00 (1.00, 2.00)	1.00 (1.00, 2.00)	1.354	0.18
Prepregnancy weight, kg	59.00 (52.00, 66.00)	58.00 (51.50, 65.00)	0.363	0.72
Prenatal weight, kg	69.00 (64.50, 80.00)	71.50 (65.00, 76.50)	0.598	0.55
Height, cm	162.00 (158.00, 164.00)	160.00 (157.50, 165.00)	0.040	0.97
Prenatal body mass index, kg/m2	27.04 (24.53, 30.11)	27.34 (24.74, 30.42)	0.221	0.83
Pregnancy related diseases
Pregnancy induced hypertension	18 (16.22)	5 (13.51)	0.154	0.69
Gestational diabetes	31 (27.93)	11 (29.73)	0.044	0.83
Hypothyroidism	21 (18.92)	5 (13.51)	0.560	0.45
Anemia	11 (9.91)	5 (13.51)	0.093	0.76
Prenatal completion of ≥1 course of glucocorticoids therapy			14.290	<0.001
Yes	54 (48.65)	5 (13.51)
No	57 (51.35)	32 (86.49)
Amniotic fluid properties			2.435	0.12
Clear	100 (90.09)	29 (78.38)
Pollution/bloodliness	11 (9.91)	8 (21.62)
Delivery method			2.927	0.09
Vaginal delivery	48 (43.24)	22 (59.46)
Cesarean section	63 (56.76)	15 (40.54)

Data are presented as mean ± standard deviation, n (%) or median (interquartile range). NRDS, neonatal respiratory distress syndrome.

Comparison of serum vitamin A and D deficiency in preterm infants whose mothers received prenatal and non-glucocorticoid therapy

Among the 148 preterm infants, 59 cases were treated with glucocorticoid, of which 17 cases were deficient (28.81%), 32 cases were insufficient (54.24%), and 10 cases were sufficient (16.95%). Serum vitamin D deficiency in 34 cases (57.63%), insufficient in 19 cases (32.20%), adequate in 7 cases (11.86%). Of the 89 patients who did not receive glucocorticoid therapy, 12 (13.48%) were deficient in serum vitamin A, 62 (69.66%) were insufficient, and 15 (16.85%) were adequate. Serum vitamin D deficiency in 44 cases (49.44%), insufficient in 28 cases (31.46%), adequate in 17 cases (19.10%). There was no statistically significant difference in serum vitamin A and D deficiency between preterm infants whose mothers received and did not receive glucocorticoid treatment (Z=1.523, 1.267; P=0.13, 0.21).

The interaction between serum 25(OH)D and vitamin A on NRDS

The threshold value of serum 25(OH)D, vitamin A and NRDS dose response in premature infants was used as the cut-off value. Below or equal to the threshold value, it was low expression (+), and higher than the threshold value, it was high expression (−). After adjusting the confounding factors, the two indicators had an interaction effect on NRDS in premature infants. The risk of NRDS in premature infants with both low levels was 10.500 times higher than that with both high levels. The risk of NRDS caused by both low levels was 4.992 times that of other location factors (odds ratio =1, RERI =4.992), and the synergistic effect was 2.107 times that of the sum of the effects of the two alone (SI =2.107). In the risk of NRDS, 47.54% (AP =47.54%) was caused by the interaction between the two. See Table 3.

Table 3. Interaction of serum 25(OH)D and vitamin A on the occurrence of NRDS in premature infants.

25(OH)D/vitamin A	Occurrence (n=111)	No occurrence (n=37)	Odds ratio (95% CI)	RERI	AP	SI
+/+	42	4	1.000	4.992	47.54%	2.107
+/−	23	7	3.286 (1.115–9.681)
−/+	29	9	3.222 (1.179–8.808)
−/−	17	17	10.500 (3.081–35.79)

+, 25(OH)D, overall vitamin A levels ≤ average, −, 25(OH)D, overall vitamin A levels > average. 25(OH)D, 25-hydroxy vitamin D; AP, attribution percentage; CI, confidence interval; NRDS, neonatal respiratory distress syndrome; RERI, relative excess risk due to interaction; SI, synergy index.

Nomogram model

Based on the results of Tables 1,2, serum 25(OH)D, vitamin A levels and other main indicators of premature infants were included, and a nomogram model was drawn. The results showed that the C-index of the model for predicting the occurrence of NRDS in premature infants was 0.961 (95% CI: 0.931–0.990), which had a high predictive efficiency. See Figures 1,2.

Figure 1.

A nomogram model for predicting the risk of NRDS in premature infants using serum 25(OH)D and vitamin A as auxiliary indicators. 25(OH)D, 25-hydroxy vitamin D; NRDS, neonatal respiratory distress syndrome.

Figure 2.

A calibration plot of the nomogram model. NRDS, neonatal respiratory distress syndrome.

Discussion

Modern medical studies have confirmed that pulmonary surfactant, as a kind of phosphorus lipoprotein complex, is mainly synthesized in type II alveolar epithelial cells, which can reduce the surface tension of the lung and avoid end-expiratory alveolar atrophy. Once the secretion of pulmonary surfactant is insufficient, it can easily lead to alveolar collapse and induce NRDS (12). Premature infants have a higher risk of NRDS due to imperfect respiratory system development and less secretion of pulmonary surfactant, which seriously affects the health and long-term development of newborns (13). Studies have shown that birth weight, gestational age, maternal prenatal glucocorticoid application, birth Apgar score and other factors are related to the occurrence of NRDS in premature infants (14,15). The results of this study also found that compared with the non-NRDS group, the preterm infants in the NRDS group had smaller gestational age, lower birth weight, 1- and 5-min Apgar scores, which were similar to the above research reports. However, in this study, the proportion of pregnant women in the NRDS group who completed ≥1 glucocorticoid treatment before delivery was higher than that in the non-NRDS group, which was contrary to the results of Yin et al. (16). The reason may be related to the large difference in gestational age included in this study and Yin et al. However, most of the above factors are objective facts, which cannot be changed clinically after the birth of premature infants. Therefore, it is necessary to explore other indicators that are convenient for clinical intervention related to the occurrence of NRDS in premature infants, so as to provide new therapeutic targets and ideas for clinical practice.

It has been reported that the synthesis of pulmonary surfactant is regulated by a variety of glucocorticoids, including steroids (17). Both 25(OH)D and vitamin A have steroid glucocorticoid effects, which can affect cell differentiation, growth and repair, and maintain the integrity of epithelial tissue structure. Zhu et al. found that the incidence of serum 25(OH)D and vitamin A deficiency in preterm infants with NRDS was higher than that in those without NRDS (18). Lin et al. also pointed out that the level of 1,25(OH)₂D₃ in newborns with NRDS was significantly lower than that in healthy newborns (19). This study found that compared with the non NRDS group, the serum 25(OH)D and vitamin A levels of preterm infants in the NRDS group were lower, which was also consistent with the above research results. 25(OH)D can promote the proliferation and differentiation of fibroblasts and alveolar type II epithelial cells, mediate the production of choline and phosphatidylglycerol, induce the formation of pulmonary surfactant, promote lung development and alveolar maturation, regulate the body’s immune response, reduce the inflammatory injury of lung tissue, and promote the repair of damaged lung tissue (20). Dogan et al. also pointed out that 25(OH)D deficiency can not only inhibit the proliferation of fibroblasts in alveolar type II cells (21), affect the secretion of pulmonary surfactant, but also lead to the consumption of lung volume and alveolar number, affect lung development and increase the risk of NRDS, which also supports the conclusions of this study indirectly. In addition, Elfarargy et al. found that pulmonary surfactant protein-B is an important protein in pulmonary surfactant (22). Vitamin A can form “Rheumatoid Arthritis reaction element” in cells, up-regulate the expression of pulmonary surfactant protein-B gene, promote the secretion of pulmonary surfactant, accelerate alveolar development and promote lung maturation. In addition, vitamin A can also promote lung vascularization and lung maturation by promoting vascular endothelial growth factor and nitric oxide production, increasing alveolar surface tension and lung compliance, improving ventilation function, and reducing the risk of NRDS (23). Therefore, the lower the serum vitamin A level, the higher the risk of NRDS in preterm infants.

Further analysis of this study found that serum 25(OH)D and vitamin A levels had an interaction effect on the occurrence of NRDS. The risk of NRDS at low levels was 10.50 times that at high levels. Studies have shown that vitamin A can not only increase the synthesis of 25(OH)D, but also promote the expression of vitamin D receptor and improve the utilization of 25(OH)D (24,25). Therefore, when the level of serum vitamin A decreases, it may affect the synthesis of 25(OH)D, resulting in a decrease in its level, and ultimately synergistically increase the risk of NRDS in premature infants. In addition, this study also analyzed the value of the above two indicators to assist other major clinical indicators in predicting the occurrence of NRDS in premature infants through a nomogram model. The results showed that the C-index of nomogram model in predicting the risk of NRDS in premature infants was 0.961, suggesting that the model had high predictive value. In this regard, it is recommended that clinical monitoring of serum 25(OH)D and vitamin A levels in premature infants can be used to assist other risk factors to guide the early screening of high-risk premature infants with NRDS. For those with abnormal reduction of the two levels, exogenous 25(OH)D and vitamin A should be supplemented in time, which may play a positive role in promoting lung development and reducing the occurrence of NRDS in premature infants.

However, this study is a retrospective analysis with a relatively small number of subjects included, and the relationship between maternal use of vitamin supplements during pregnancy and serum levels of 25(OH)D and vitamin A, as well as the occurrence of NRDS in premature infants, was not analyzed, and only analyzed the serum 25(OH)D and vitamin A levels at a single time point after delivery of premature infants. Dynamic monitoring of these levels before and after treatment was not conducted. Future prospective studies with large sample sizes are needed to include information on maternal use of vitamin supplements during pregnancy and other related nutritional indicators, and to observe the dynamic changes in serum 25(OH)D and vitamin A levels before and after treatment in premature infants with NRDS, in order to further clarify their significance in disease prevention and treatment.

In summary, serum 25(OH)D and vitamin A levels may be related to the risk of NRDS in premature infants.

Conclusions

This study demonstrates that premature infants with NRDS have significantly lower levels of serum 25(OH)D and vitamin A compared to those without NRDS. The interaction between these two vitamins significantly increases the risk of NRDS, with a combined deficiency resulting in a 10.5-fold higher risk. The development of a nomogram model incorporating these serum markers shows high predictive accuracy for NRDS risk. These findings highlight the potential clinical benefit of monitoring and addressing deficiencies in 25(OH)D and vitamin A as part of early intervention strategies to reduce NRDS incidence in premature infants.

Supplementary

The article’s supplementary files as

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Third Affiliated Hospital of Nanjing Medical University (No. [2024] YLJSC010). Informed consent was obtained from the parents or legal guardians of all participants under the age of 16 years.

Data Sharing Statement

Available at https://tp.amegroups.com/article/view/10.21037/tp-2025-388/dss

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DOI: 10.21037/tp-2025-388