Invest Clin 67(1): 5 - 18, 2026 https://doi.org/10.54817/IC.v67n1a01

Corresponding author: Xiaohui Guo, No. 515, Huanghe Seventh Road, Bincheng District, Binzhou City 256610,

Shandong Province, China. Email: guoxiaohui3212919@163.com

Effects of Vitamin D deficiency

and supplementation on 25(OH)D3 levels

and neuropsychobehavioral development

in premature infants.

Xiaohui Guo1, Yanfeng Sun2, Yanhong Chen1, Feifei Xu1 and Yanfei Li1

1Department of Pediatrics, Binzhou People’s Hospital, Binzhou City, Shandong Province,

China.

2Department of Hematology, Binzhou People’s Hospital, Binzhou City, Shandong

Province, China.

Keywords: Vitamin D; Neuropsychobehavioral development; Premature infants;

Vitamin D; 25(OH)D3; Neurodevelopment; Psychomotor Performance;

Infant, Premature.

Abstract. This study systematically examined how vitamin D metabolic

imbalance impacts 25(OH)D3 levels and neuropsychological development in

premature infants and proposed a personalized supplementation approach. Pre-

mature infants were classified as adequate, insufficient, or deficient based on

umbilical cord blood 25(OH)D3 levels and then randomly assigned to either a

standard-dose group (800 IU/d) or an individualized supplementation group

(400-1000 IU/d) with vitamin D. In the vitamin D-deficient group, infants re-

ceiving personalized supplementation had significantly higher 25(OH)D3 levels

at three and nine months, adjusted for gestational age, than those receiving the

fixed dose, indicating that 1000 IU/d is more effective than 800 IU/d for cor-

recting deficiency (p<0.05). At nine and 18 months adjusted gestational age,

infants in the vitamin D-insufficient and deficient groups scored significantly

lower on the Gesell Developmental Scales across categories such as gross mo-

tor, fine motor, language, adaptive, and social skills compared to the adequate

group (p<0.05). Within the deficient group, those receiving personalized sup-

plementation scored higher in all five areas at both nine and 18 months ad-

justed gestational age compared to those on the fixed dose (p<0.05). The study

highlights notable differences in umbilical cord blood 25(OH)D3 levels among

premature infants, emphasizing that a customized vitamin D supplement pro-

tocol is more effective for correcting deficiencies.

6 Guo et al.

Investigación Clínica 67(1): 2026

Efectos de la deficiencia de vitamina D y la suplementación

sobre los niveles de 25(OH)D3 y el desarrollo neuropsicológico-

conductual en bebés prematuros.

Invest Clin 2026; 67 (1): 5 – 18

Palabras clave: Vitamina D; 25(OH)D3; Desarrollo Neuropsicológico/Desarrollo

Psicomotor; Recién Nacido; Prematuro.

Resumen. Este estudio exploró sistemáticamente el impacto del desequili-

brio metabólico de la vitamina D en los niveles de 25(OH)D3 y en el desarrollo

neuropsicológico de bebés prematuros, y propuso una estrategia de suplemen-

tación individualizada. Los bebés prematuros se categorizaron en grupos ade-

cuados, insuficientes y deficientes según los niveles de 25(OH)D3 en la sangre

del cordón umbilical, y luego se asignaron aleatoriamente a un grupo con dosis

estándar (800 UI/día) o a otro grupo con suplementación individualizada (400-

1000 UI/día) con vitamina D. Entre los bebés prematuros con deficiencia de

vitamina D, el grupo de suplementación individualizada presentó niveles signi-

ficativamente más altos de 25(OH)D3 a los 3 y 9 meses de edad gestacional co-

rregida, en comparación con el grupo de dosis estándar, lo que indica que una

dosis de 1000 UI/día fue más efectiva que 800 UI/día para corregir la deficien-

cia de vitamina D (p<0,05). A los 9 y 18 meses de edad gestacional corregida,

los bebés prematuros de los grupos con insuficiencia y deficiencia de vitamina

D obtuvieron puntuaciones significativamente más bajas en las Escalas de De-

sarrollo de Gesell para la habilidad motora gruesa, la habilidad motora fina, la

competencia lingüística, la capacidad adaptativa y la habilidad personal-social

en comparación con el grupo adecuado (p<0,05). Dentro del grupo deficiente,

el grupo de suplementación individualizada obtuvo puntuaciones más altas en

las cinco habilidades a los 9 y 18 meses de edad gestacional corregida en com-

paración con el grupo de dosis estándar (p<0,05). Existen diferencias signifi-

cativas en los niveles de 25(OH)D3 en la sangre del cordón umbilical entre los

bebés prematuros, y un protocolo de suplementación de vitamina D específico

para cada individuo es más eficaz para corregir la deficiencia de vitamina D.

Received: 17-07-2025 Accepted: 07-09-2025

INTRODUCTION

Premature infants, defined as those

born before 37 weeks of gestation, encoun-

ter various health issues after birth because

their organ systems are not fully developed1.

In recent years, with the continued develop-

ment of perinatal medicine and significant

advances in neonatal intensive care and diag-

nostic techniques, the treatment of prema-

ture infants has been greatly enhanced, re-

sulting in a rising survival rate 2. Subsequent

to their survival, long-term issues such as

abnormal neurological development in pre-

mature infants have also received increas-

ing attention 3. The normal development of

the nervous system is directly related to the

future quality of life of premature infants,

including cognition, motor skills, language,

and other aspects 4. Therefore, identifying

Vitamin D deficiency and neuropsychobehavioral development 7

Vol. 67(1): 5 - 18, 2026

velopment 18. Research indicates that insuf-

ficient vitamin D levels in premature infants

may impair growth and development, includ-

ing intellectual and motor abilities, at one

year of age 19. Moreover, insufficient vitamin

D levels may affect the neurological and be-

havioral development of preterm infants,

particularly if they do not receive adequate

supplementation shortly after birth20. A study

demonstrated a positive association between

serum 25(OH)D3 levels in premature infants

and neuropsychological developmental quo-

tient scores, which were enhanced by vitamin

D supplementation administered both pre-

natally and postnatally 21. This suggests that

adequate vitamin D supplementation has

a positive effect on the neurodevelopment

and cognitive function of premature infants.

Timely vitamin D supplementation can alle-

viate vitamin D deficiency and mitigate its

adverse effects on neurodevelopment, mak-

ing it crucial for improving the long-term

prognosis of premature infants 22. Vitamin D

influences the morphogenesis and physiolog-

ical functions of the nervous system through

diverse mechanisms, including regulating

the expression of neurotrophic factors, in-

fluencing neurotransmitter synthesis, and

modulating calcium signaling pathways 23.

Vitamin D and its receptor (VDR) are ubiq-

uitously expressed across diverse tissues and

cell types in the human body, including neu-

rons and glial cells in the central nervous

system, and participate in neuronal prolif-

eration, differentiation, and apoptosis. Vita-

min D is essential for neurodevelopment via

epigenetic regulation mediated by VDR 24, 25.

1,25(OH)2D3 upregulates the expression of

synaptic proteins such as Synaptophysin and

PSD-95, promoting synaptic plasticity. Ad-

ditionally, vitamin D influences behavioral

control and cognitive function by regulating

the activity of the dopamine synthesis rate-

limiting enzyme (tyrosine hydroxylase) and

the glutamate transporter (EAAC-1) 26, 27.

Therefore, examining the impact of vitamin

D insufficiency and augmentation on serum

25[OH]D3 concentrations, alongside neuro-

factors that affect the neuropsychological

and behavioral development of premature

infants and implementing effective interven-

tions are of great significance for improving

their prognosis.

Vitamin D, a crucial fat-soluble nutrient,

holds a fundamental position in maintain-

ing human health 5. In recent years, in-depth

research on vitamin D and its roles in bone

health, immune function, and neurodevel-

opment has received increasing attention 6,

7. Especially among premature infants, pe-

diatric research has increasingly focused on

vitamin D status and corresponding supple-

mentation strategies 8. Premature infants of-

ten suffer from vitamin D deficiency 9, 10. On

the one hand, premature infants have inad-

equate vitamin D stores during pregnancy,

especially in the late gestational period, when

the amount of vitamin D obtained by the fe-

tus from the mother gradually increases; how-

ever, premature birth leads to relatively insuf-

ficient vitamin D stores 11. On the other hand,

premature infants have a limited capacity to

synthesize vitamin D through their skin after

birth, and breast milk, typically their prima-

ry source of nutrition, provides insufficient

vitamin D to meet their rapid growth and

developmental demands 12, 13. Furthermore,

premature infants may not receive adequate

sunlight exposure during their hospitaliza-

tion, thereby hindering vitamin D synthesis14.

Humans obtain vitamin D primarily from two

sources: skin synthesis of 7-dehydrocholes-

terol under UV-B light to form vitamin D3,

and dietary intake of vitamin D2 15. Both vi-

tamin D forms undergo hepatic hydroxylation

to yield 25(OH)D3, which then undergoes

renal metabolism to form its biologically ac-

tive form, 1,25(OH)2D3 16. This metabolic

pathway requires the collaboration of various

organs, with 25(OH)D3 being the main circu-

lating form and the key indicator for evaluat-

ing vitamin D levels 17.

Vitamin D deficiency not only affects

the bone development of premature infants

but may also have far-reaching impacts on

their neuropsychological and behavioral de-

8 Guo et al.

Investigación Clínica 67(1): 2026

cognitive and behavioral progress in preterm

infants, possesses considerable theoretical

and practical significance.

At present, there is no universally

agreed standard for the dose and schedule of

vitamin D supplements for premature babies,

with variations in recommendations among

countries and organizations 28, 29. Generally

speaking, the range of vitamin D supple-

mentation for premature infants is 400-

1000 IU/day, and the specific dosage needs

to be adjusted according to factors such as

gestational age, birth weight, and feeding

mode 30. Numerous studies have explored

the effects of vitamin D supplementation on

25(OH)D3 levels in premature infants, re-

vealing that adequate supplementation can

significantly elevate serum 25(OH)D3 levels;

nonetheless, it is crucial to recognize that

the connection between dosage and 25(OH)

D3 levels is non-linear, and excessively high

doses may pose a risk of vitamin D excess or

toxicity 31, 32. Secondly, the duration of sup-

plementation also affects the outcome, with

studies showing that long-term supplemen-

tation maintains stable 25(OH)D3 levels

better than short-term supplementation33.

In addition, factors such as gestational age,

birth weight, and liver and kidney function

status in premature infants also affect the

metabolism and utilization efficiency of vi-

tamin D 34. One of the research hotspots is

the comparison of the effects of different

supplementation regimens. Drawing on the

aforementioned research background and

evidence, this study aims to determine the

optimal dose and schedule for administer-

ing vitamin D supplements to premature

infants, while comprehensively assessing the

potential long-term effects of such supple-

mentation on their 25(OH)D3 levels and

neuropsychobehavioral development.

Patients and Methods

This is a clinical study conducted in the

neonatal unit at our hospital’s Children’s

Medical Center. The designed operational

procedure is shown below.

Clinical data

A total of 175 premature infants admit-

ted between January 2020 and December

2021 who underwent umbilical cord blood

25(OH)D3 testing within 24 hours after birth

were selected as study subjects. Inclusion

criteria included: gestational age less than

37 weeks; no severe complications; stable vi-

tal signs; mothers without severe pregnancy-

related complications; and participants free

from conditions or medications that could

affect calcium and vitamin D metabolism.

Exclusion criteria included: twin or multiple

pregnancies; congenital malformations af-

fecting normal body structure and function;

genetic metabolic diseases; brain hypopla-

sia with obvious neurological abnormalities;

and mothers with hypertension, diabetes,

metabolic diseases, or other conditions dur-

ing pregnancy that could negatively impact

the fetus.

Grouping and intervention

All premature infants underwent umbil-

ical cord blood 25(OH)D3 testing within 24

hours of birth and were classified into three

groups: adequate (>30 ng/mL), insufficient

(20-30 ng/mL), and deficient (<20 ng/mL).

Each group was further divided into a stan-

dard vitamin D dosage group or an individu-

alized supplementation group. All premature

infants received vitamin D3 supplementa-

tion starting on the first day after birth. The

standard-dose group received a routine sup-

plementation of 800 IU/day, while the indi-

vidualized group received 400, 800, or 1000

Premature infants

Umbilical cord blood 25(OH)D testing

Adequate group

(>30 ng/mL)

Insufficient group

(20-30 ng/mL)

Deficient group

(<20 ng/mL)

Standard-dose group

Individualized supplementation group

Standard-dose group

Individualized supplementation group

Standard-dose group

Individualized supplementation group

Follow up for 3 years

Detection of serum 25(OH)D3 level Analysis Gesell Developmental Scale to

assess preterm infant development

Vitamin D deficiency and neuropsychobehavioral development 9

Vol. 67(1): 5 - 18, 2026

IU/day for three months, depending on their

25(OH)D3 levels (adequate, insufficient, or

deficient, respectively). After three months,

the dose was increased to 400 IU/day for all

infants and maintained at this level until age

3 years. Follow-up visits occurred in the out-

patient clinic over three years, and cases lost

to follow-up were excluded. Ultimately, 175

infants completed the follow-up: 51 in the

adequate group (25 in the standard dosage

group and 26 in the individualized group),

54 in the insufficient group (26 in the stan-

dard dosage group and 28 in the individual-

ized group), and 70 in the deficient group

(35 in each group). All premature infants re-

ceived oral vitamin D3 drops in capsule form

[400 IU/capsule, Sinopharm Xingsha Phar-

maceutical (Xiamen) Co., Ltd.].

Observation indicators

(1) Serum 25(OH)D3 levels served as

a crucial indicator for assessing vitamin D

nutritional status. Regular monitoring of

serum 25(OH)D3 levels at 24 hours after

birth, at 3 months, at 9 months’ corrected

gestational age (CGA), and at 18 months’

CGA can provide insights into the dynamic

changes in vitamin D status in premature

infants and evaluate the effectiveness of vita-

min D supplementation.

(2) The Gesell Developmental Scales

were used to evaluate the developmental

status of premature infants. This scale cov-

ers adaptive ability, gross motor skills, fine

motor skills, language ability, and personal-

social skills, offering a comprehensive view

of the children’s intellectual development.

Assessments with the Gesell Developmental

Scales were performed at 9 months CGA and

18 months CGA. A pediatric nurse trained

to administer the scales conducted these

assessments at each time point. The scale

includes adaptive ability, gross motor skills,

fine motor skills, linguistic ability, and per-

sonal-social ability, fully reflecting children’s

intellectual growth. For children aged 0-3,

the scale contains 514 items, all completed

within 60 minutes. Results are expressed

as a Developmental Quotient (DQ). DQ is

calculated as Measured Developmental Age

divided by Chronological Age, multiplied by

100. A DQ of 130 or higher indicates supe-

rior development; 110 to 129, good develop-

ment; 80 to 109, average development; 70 to

79, borderline low development; and below

70, indicates intellectual developmental de-

lay.

Statistical analysis

Statistical analysis was done with SPSS

25.0. Normally distributed data were report-

ed as mean ± SD and analyzed using a t-test

(for two groups) or ANOVA with Bonferroni

correction (for multiple groups). Categori-

cal data were presented as the number of in-

dividuals and the composition ratio (%), and

the chi-square test was employed for analy-

sis. A p-value < 0.05 was considered statisti-

cally significant.

RESULTS

Comparison of Baseline characteristics

Baseline demographic features showed

no significant differences among the ade-

quate, insufficient, and deficient groups (p

> 0.05), ensuring comparability of subse-

quent intervention effects (Table 1).

Comparison of 25(OH)D3 levels among

the three groups of infants at 24 hours

after birth, 3 months after birth,

9 months CGA, and 18 months CGA

At 24 hours after birth, 3 months after

birth, and 9 months CGA, the 25(OH)D3 lev-

els in both the vitamin D insufficient group

and the deficient group were lower than

those in the adequate group (p<0.05). At

18 months CGA, there were no statistically

significant differences in 25(OH)D3 levels

among the three groups of infants (p>0.05

(Table 2).

10 Guo et al.

Investigación Clínica 67(1): 2026

Comparison of 25(OH)D3 levels between

the standard dosage group and the indi-

vidualized supplementation group within

the deficient group

Within the deficient group, there were

no statistically significant differences in

25(OH)D3 levels between the standard-dose

and individualized-supplementation groups

at 24 hours after birth (p>0.05). However,

at 3 months after birth, 9 months CGA, and

18 months CGA, the 25(OH)D3 levels in the

individualized supplementation group were

significantly higher than those in the stan-

dard dose group (p<0.05) (Table 3).

Comparison of Gesell Developmental Scales

scores among the three groups of infants at

9 months CGA and 18 months CGA

At both the 9- and 18-month CGAs, sig-

nificant differences were observed in scores

across the five abilities among the three in-

fant groups (p<0.05). Pairwise comparisons

revealed that the scores for the five abilities

in both the insufficient and deficient groups

were lower than those in the adequate group

(p<0.05). Nonetheless, the scores of the five

abilities did not show significant statistical dif-

ferences when comparing the insufficient and

deficient groups (p>0.05) (Tables 4 and 5).

Table 1. Comparison of baseline characteristics.

Variables Adequate group

(n=51)

Insufficient group

(n=54)

Deficient group

(n=70) F/χ2p

Gender (n, %)

male 23 (45.10) 25 (46.30) 33 (47.14) 0.050 0.975

female 28 (54.90) 29 (53.70) 37 (52.86)

Mode of delivery (n, %)

spontaneous labor 25 (49.02) 26 (48.15) 31 (44.29) 0.318 0.853

cesarean section 26 (50.98) 28 (51.85) 39 (55.71)

Gestational age (weeks) 35.33±0.74 35.43±0.63 35.40±0.71 0.248 0.781

Birth weight (g) 2462.82±211.96 2489.46±212.43 2459.87±205.66 0.344 0.709

Mother age (years) 28.96±5.38 28.76±4.59 28.94±5.33 0.026 0.974

1 min Apgar score (scores) 8.33±0.62 8.30±0.71 8.30±0.73 0.046 0.955

5 min Apgar score (scores) 9.04±0.34 9.11±0.32 9.04±0.27 0.971 0.381

Baseline characteristics are presented as mean ± SD or n (%). Between-group differences were assessed using one-

way ANOVA for continuous variables and the χ² test for categorical variables.

Table 2. Comparison of 25(OH)D3 levels at different time points in the 3 groups.

Groups n24 h after birth 3 months after birth 9 months CGA 18 months CGA

Adequate group 51 35.74±4.27 34.95±4.38 35.82±3.88 35.18±4.83

Insufficient group 54 24.15±2.14a 29.48±3.44a 31.07±4.38a34.53±4.61

Deficient group 70 13.18±2.31ab 23.54±4.24ab 29.93±5.00a34.65±4.10

F853.143 118.449 26.8893 0.327

p0.000 0.000 0.000 0.722

Data are presented as mean ± SD in ng/mL. Between-group comparisons at each time point were performed using

one-way ANOVA, followed by Bonferroni post hoc tests. ap<0.05 vs. Adequate group; bp<0.05 vs. Insufficient group.

CGA: corrected gestational age.

Vitamin D deficiency and neuropsychobehavioral development 11

Vol. 67(1): 5 - 18, 2026

Comparison of Gesell Developmental

Scales scores between the standard dosage

group and the individualized supplemen-

tation group within the deficient group at

9 months CGA and 18 months CGA

Within the deficient group, at both

9-month and 18-month CGA, infants in

the individualized supplementation group

scored higher on gross motor ability, fine

motor ability, linguistic competence, adap-

tive capacity, and personal-social ability

than those in the standard dosage group

(p<0.05). See Tables 6 and 7.

Table 3. Comparison of 25(OH)D3 levels between the standard dosage group

and the individualized supplementation group within the deficient group.

Groups n24 h after birth 3 months after birth 9 months CGA 18 months CGA

Standard dose group 35 13.15±2.46 21.34±3.55 27.29±4.18 32.82±3.81

Individualized

supplementation group 35 13.22±2.18 25.74±3.73 32.56±4.37 36.47±3.55

t0.126 5.049 5.146 4.152

p0.900 0.000 0.000 0.000

Data are presented as mean ± SD in ng/mL. Between-group differences were evaluated using two-sample t-tests for

independent samples. CGA: corrected gestational age.

Table 4. Comparison of Gesell Developmental Scales scores among the three groups

of infants at 9 months CGA.

Groups ngross motor

ability

fine motor

ability

linguistic

competence

adaptive

capacity

personal-social

ability

Adequate group 51 96.31±8.61 97.45±9.36 96.49±7.79 97.22±10.24 96.22±9.88

Insufficient group 54 88.63±6.22a 88.78±8.51a 90.46±7.35a91.56±8.21a 90.11±10.19a

Deficient group 70 87.57±5.65a 87.34±9.62a 89.70±9.23a90.50±9.57a88.64±9.60a

F16.216 19.494 11.165 8.224 9.258

p0.000 0.000 0.000 0.000 0.000

Data are presented as mean ± SD. Between-group comparisons at each time point were performed using one-way

ANOVA, followed by Bonferroni post hoc tests. ap<0.05 vs. Adequate group. CGA: corrected gestational age.

Table 5. Comparison of Gesell Developmental Scales scores among the three groups

of infants at 18 months of CGA.

Groups ngross motor

ability

fine motor

ability

linguistic

competence

adaptive

capacity

personal-social

ability

Adequate group 51 96.45±9.16 97.78±10.22 97.65±7.84 97.98±10.21 97.35±10.21

Insufficient group 54 92.61±7.98a91.35±8.77a 92.33±8.64a93.44±8.16a92.65±8.54a

Deficient group 70 90.06±7.92a89.67±8.64a 90.71±7.97a92.84±7.33a91.90±7.33a

F8.721 12.223 11.164 6.012 6.503

p0.000 0.000 0.000 0.003 0.002

Data are presented as mean ± SD. Between-group comparisons at each time point were performed using one-way

ANOVA, followed by Bonferroni post hoc tests. ap<0.05 vs. Adequate group. CGA: corrected gestational age.

12 Guo et al.

Investigación Clínica 67(1): 2026

DISCUSSION

Vitamin D plays numerous physio-

logical roles in the human body, including

maintaining bone health, modulating the

immune system, influencing cell differen-

tiation, and contributing to neurodevelop-

ment, among other functions 35. Vitamin D

can affect the development of the normal

fetal brain by regulating the expression of

neurotrophic factors, modulating cytokines

activity, synthesizing neurotransmitters,

modulating intracellular calcium signaling,

and controlling the activity of genes and

proteins responsible for neuronal differen-

tiation and metabolic processes 36. During

the final stage of pregnancy, the fetus’s need

for vitamin D increases significantly to sup-

port rapid bone growth and calcification 37.

The mother transfers vitamin D to the fetus

through the placenta, helping the fetus es-

tablish sufficient vitamin D stores to meet

early postnatal growth demands 38. Prema-

ture infants, due to their shorter gestational

age at birth, have relatively inadequate vi-

tamin D stores, and their rapid growth and

development further increase their need for

vitamin D 39. Additionally, premature infants

have thinner skin and less subcutaneous fat,

which decreases their ability to synthesize

vitamin D, making them more vulnerable

to deficiency 40. In our study, we examined

25(OH)D3 levels in the umbilical cord blood

of preterm infants, revealing a high rate of

vitamin D deficiency in this population.

The results of this research indicate

that preterm infants with insufficient vi-

tamin D had significantly lower 25(OH)D3

concentrations at 24 hours, 3 months, and

9 months CGA than those with normal vita-

Table 6. Comparison of Gesell Developmental Scales scores between the standard dosage group

and the individualized supplementation group within the deficient group at 9 months CGA.

Groups ngross motor

ability

fine motor

ability

linguistic

competence

adaptive

capacity

personal-social

ability

Standard dosage group 35 85.20±9.57 84.37±9.66 86.97±8.01 87.31±9.77 85.29±9.51

Individualized

supplementation group 35 91.29±8.53 90.31±8.74 92.43±9.66 93.69±8.35 92.00±8.57

F2.809 2.698 2.573 2.933 3.103

p0.007 0.009 0.012 0.005 0.003

Note: Data are presented as mean ± SD. Between-group differences were evaluated using two-sample t-tests for

independent samples. CGA: corrected gestational age.

Table 7. Comparison of Gesell Developmental Scales scores between the standard dosage group

and the individualized supplementation group within the deficient group at 18 months CGA.

Groups ngross motor

ability

fine motor

ability

linguistic

competence

adaptive

capacity

personal-

social ability

Standard dosage group 35 86.43±7.18 86.49±8.03 86.77±7.53 88.91±6.29 87.57±5.65

Individualized

supplementation group 35 93.69±6.98 92.86±8.14 94.66±6.34 96.77±6.15 96.23±6.21

F4.288 3.297 4.738 5.284 6.100

p0.000 0.002 0.000 0.000 0.000

Data are presented as mean ± SD. Between-group differences were evaluated using two-sample t-tests for indepen-

dent samples. CGA: corrected gestational age.

Vitamin D deficiency and neuropsychobehavioral development 13

Vol. 67(1): 5 - 18, 2026

min D levels. This suggests that variations

in umbilical cord blood vitamin D levels at

birth significantly influence 25(OH)D3 lev-

els in premature infants during the early

postnatal period. Despite postnatal vitamin

D3 supplementation, premature infants in

the deficient group did not achieve 25(OH)

D3 levels comparable to those in the ade-

quate group within a relatively short time-

frame. This may relate to the physiological

characteristics of premature infants, whose

livers and kidneys are not fully developed,

limiting their ability to metabolize and con-

vert vitamin D, resulting in a slower increase

in 25(OH)D3 levels after supplementation

in the deficient group 41. It may also be af-

fected by the dosage of supplementation and

individual differences. Although a standard

vitamin D3 supplement was given, absorp-

tion and utilization efficiency vary among in-

dividuals, making it difficult for premature

infants in the deficient group to quickly cor-

rect their deficiency 42. By 18 months CGA,

there were no statistically significant differ-

ences in 25(OH)D3 levels among the three

infant groups. This indicates that, after a pe-

riod of supplementation, premature infants

in the deficient group had sufficient time to

increase their levels, gradually closing the

gap with the adequate group and eventually

reaching comparable levels at 18 months

CGA. This improvement may be due to the

gradual maturation of liver and kidney func-

tion as infants grow, enhancing their ability

to metabolize vitamin D, thereby allowing

better utilization of supplemental vitamin

D3 and a subsequent rise in 25(OH)D3 levels

43. It may also be related to the cumulative

effect of the dose and duration of supple-

mentation. After a longer period, prema-

ture infants in the deficient group gradually

compensated for their intrauterine vitamin

D deficiency, resulting in 25(OH)D3 levels

comparable to those in the other groups.

At 3, 9, and 18 months of CGA, the group

receiving individualized supplementation ex-

hibited significantly higher 25(OH)D3 levels

than the standard-dosage group. In the early

postnatal period, infants with vitamin D de-

ficiency should undergo closer monitoring

of 25(OH)D3 levels and dosage and route

of supplementation adjusted to individual

circumstances to promote a rapid increase

in 25 (OH) D3 levels. By approximately 18

months CGA, monitoring frequency can

be adjusted, as vitamin D levels among the

groups have converged. Additionally, these

findings support the development of more

scientifically grounded and rational vitamin

D supplementation protocols for premature

infants, such as stratifying infants by umbili-

cal cord blood vitamin D levels and applying

targeted strategies at different stages to en-

sure adequate vitamin D for healthy growth.

This study investigates how vitamin

D deficiency affects the neuropsychologi-

cal and behavioral development of preterm

infants. At 9 and 18 months CGA, preterm

infants with insufficient or deficient vita-

min D levels scored significantly lower on

the Gesell Developmental Scales in areas

such as gross motor, fine motor, language,

adaptive behavior, and personal-social skills

compared to those with adequate vitamin

D levels. These findings indicate that vita-

min D deficiency may adversely affect the

neuropsychological and behavioral develop-

ment of preterm infants. Vitamin D plays

several roles in the nervous system, includ-

ing supporting neuronal development and

differentiation and regulating the produc-

tion and release of neurotransmitters 44, 45.

A deficiency of vitamin D may slow nervous

system development, impairing cognitive

functions, motor coordination, and social

skills in premature infants 46, 47. Moreover,

within the deficiency group, at both 9 and

18 months CGA, infants receiving individu-

alized vitamin D supplementation scored

higher across all five skill areas than those

given a standard dose. This suggests that an

individualized vitamin D supplementation

approach not only more effectively corrects

deficiency but also enhances the neuropsy-

chological and behavioral development of

preterm infants. Such tailored supplementa-

14 Guo et al.

Investigación Clínica 67(1): 2026

tion more effectively addresses the specific

needs of premature infants for vitamin D,

thereby maintaining healthy serum levels

and supporting nervous system develop-

ment. Adequate vitamin D levels are crucial

for the proper development and function of

nerve cells, the formation and connectivity

of neural synapses, and overall neuropsycho-

logical and behavioral health in preemies.

Additionally, personalized supplementation

may also modulate immune function, lower

infection rates, and indirectly support ner-

vous system development 48. These findings

underscore the importance of customized

vitamin D supplementation strategies in the

care of preterm infants.

The results of this study carry impor-

tant implications for clinical practice. First-

ly, it reminds healthcare professionals to

routinely test umbilical cord blood 25(OH)

D3 levels in premature infants at birth to

promptly identify those with vitamin D de-

ficiency. Secondly, a personalized approach

to vitamin D supplementation offers an effi-

cient way to address vitamin D deficiency in

premature infants and cater to their unique

requirements. In clinical settings, health-

care providers can develop customized vita-

min D supplementation plans for premature

infants based on individual circumstances to

enhance overall health and well-being.

Limitations of the Study

Although this study provides valuable

insights, it is important to acknowledge its

limitations. First, the relatively small sample

size may introduce bias into the results. Sec-

ond, the observation period of the study is

relatively short, extending only to 18 months

CGA, and the long-term effects on the neu-

ropsychological and behavioral development

of premature infants remain unclear. Future

research should increase the sample size and

conduct multicenter, large-sample studies

to enhance the reliability and generalizabil-

ity of the findings. Additionally, extending

the observation period to follow premature

infants into school age or even adulthood

would allow for a more comprehensive as-

sessment of the long-term impact of vitamin

D deficiency and supplementation on their

neuropsychological and behavioral develop-

ment. Further research could also explore

the specific molecular mechanisms by which

vitamin D influences neuropsychological

and behavioral development in premature

infants, providing a theoretical foundation

for developing more targeted treatment

strategies. Moreover, combining other nutri-

ents and treatment approaches could help

evaluate their overall effect on the growth

and development of premature babies, offer-

ing more comprehensive support for their

healthy development.

This study systematically examined

how vitamin D metabolic imbalance affects

25(OH)D3 levels and neuropsychological

and behavioral development in premature

infants, while proposing an individualized

supplementation strategy. The results show

significant differences in umbilical cord

blood 25(OH)D3 levels among premature

babies, and that a tailored vitamin D supple-

ment plan is more effective at correcting de-

ficiency. This personalized approach not only

resolves vitamin D deficiency in premature

infants but also positively influences their

neuropsychological and behavioral growth.

Acknowledgment

None.

Funding

Special Fund for Inspection and Test-

ing Science and Technology of China Inter-

national Scientific Exchange Foundation

(Z2021LSD009).

Consent to publish

The manuscript has neither been previ-

ously published nor is under consideration

by any other journal. The authors have all ap-

proved the content of the paper.

Vitamin D deficiency and neuropsychobehavioral development 15

Vol. 67(1): 5 - 18, 2026

Consent to Participate

We obtained a signed informed consent

form from each participant’s representative.

Ethic Approval

This study was approved by the Ethics

Committee of the Binzhou People’s Hospital.

Data Availability Statement

The data that support the findings of

this study are available from the correspond-

ing author upon reasonable request.

ORCID number of authors

• Xiaohui Guo (XG):

0009-0001-7638-8078

• Yanfeng Sun (YS):

0009-0006-2636-6846

• Yanhong Chen (YC):

0009-0005-0502-5287

• Feifei Xu (FX):

0009-0007-5341-7992

• Yanfei Li (YL):

0009-0001-1945-3347

Author contribution

XG: Edited and refined the manuscript

with a focus on critical intellectual contri-

butions. YC, FX, YL: Participated in collect-

ing, assessing, and interpreting the data.

Made significant contributions to date in-

terpretation and manuscript preparation.

XG, YS: Provided substantial intellectual in-

put during the drafting and revision of the

manuscript.

Conflicts of interest

The authors declare that they have no

financial conflicts of interest.

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