Study design

A retrospective study was conducted on EPIs born in the obstetrics department of a tertiary hospital in Wenzhou from 1 January 2021 to 31 March 2023 and admitted to NICU. Inclusion criteria were: ① Gestational age < 28 weeks; ② Apgar scores at one minute, five minutes and ten minutes after birth were 8–10 points(muscle tone not deducted [10]); ③ Transferred directly to the NICU after birth; ④ Early enteral nutrition (EN) and education for family members on breast milk feeding within 12 h after birth [11];⑤ Oxygen saturation is maintained above 88%. Exclusion criteria comprised: ① Mothers with contraindications to breastfeeding; ② Infant born with complications such as (congenital malformations or genetic metabolic diseases; cystic periventricular leukomalacia); ③ Children who were re-admitted to hospital (those who were readmitted within one month after being discharged). Further exclusion criteria were: ① Infant with fasting due to illness; ② treatment interruption, or transfer to another hospital for various reasons.

This study was approved by the Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University· Yuying Children ‘s Hospital (2023-K-29-01).

Calculation of sample size

According to the retrospective design, this study adopts G.Power to calculate the sample size, the effect size is set to 0.3, the test efficiency is set to 0.9, and α is set to 0.05. The total sample size of the three groups is calculated as n = 144 cases, allowing for a 20% drop out rate for the sample. In the end, at least three groups of research subjects with a total of 172 cases will be needed.

Breast milk feeding

According to the early EN of premature infants and the classification of breast milk feeding [11], breast milk feeding was divided into three groups: high proportion, medium proportion and low proportion of breast milk feeding. Within the groups, the proportion of breast milk feeding comprising the total feeding amount was ≥ 80% for the high proportion of breast milk feeding (including exclusive breast milk feeding) group; 20% ~ < 80% of breast milk feeding for the medium proportion group; and, < 20% for the low proportion of breast milk feeding group. In the NICU, the breast milk volume for each EPIs is documented using the electronic medical record system. The proportion of breast milk volume is defined as the percentage of breast milk intake in the total EN consumed up to 37 weeks of corrected age.

Nutrition plan

Breast milk is provided immediately once available. When the mother delivers breast milk to the NICU, it is the preferred source of infant nutrition. Infants with inadequate breast milk intake are supplemented with preterm infant formula, with a preference for breastfeeding if breast milk is available. When the total breast milk feeding volume reaches 50–80 mL / kg / d, breast milk fortifier is added. Breast milk education, acceptance, storage, and feeding are conducted in accordance with the evidence-based guidelines for breast milk feeding of hospitalized neonates in China [12].

EN: ① Initiation time: preterm infants without intrauterine distress and relatively stable conditions start feeding within 12 h after birth. Those with intrauterine distress or unstable conditions start feeding 12–24 h after birth, or it may be appropriately extended to 24–48 h. ② Initial feeding volume: very low birth weight infants (VLBWI) start with 1–2 ml, every 2 h; extremely low birth weight infants (ELBWI) start with 0.5–1 ml, every 2 h. ③ Milk volume increase plan: micro-feeding is carried out on days 1–4 after birth. If tolerated, the rate of milk increase for VLBW preterm infants is 20–30mL / (kg·d), and for ELBW preterm infants, it’s 15–25 ml / (kg·d), until the milk volume reaches 150–180 ml / (kg·d) or the calorie intake reaches 110–130 kcal / (kg·d) (1 kcal = 4.184 kJ).

Parenteral Nutrition (PN): Start PN support within 24 h after birth. Implement PN based on gestational age, weight, day of life, and clinical conditions. Adjust the PN dosage according to the volume of enteral feeding. Discontinue PN when the milk volume reaches 120mL/ (kg·d) or the calorie intake from EN reaches 90 kcal / (kg·d). Use the “all-in-one” parenteral nutrition solution uniformly configured by the hospital’s intravenous configuration center.

Start nasogastric tube feeding after birth and gradually transition to oral feeding at a corrected gestational age of 32–34 weeks.

General information questionnaire

Databases such as CNKI, Wanfang database, VIP database and PubMed were searched, literatures related to breast milk feeding were read, variables were screened in combination with clinical practice and expert opinions, and a general data questionnaire was formulated, including the following two parts. ① Demographic data: infant sex, gestational age, birth weight, mode of delivery, Score for Neonatal Acute Physiology Perinatal Extension II (SNAPPE-II) [13], length of hospital stay, maternal education level, and paternal education level of the preterm infants. ② Growth and development assessment: length, weight, and head circumference at a corrected gestational age of 37 weeks.

Test of infant motor performance(TIMP)

TIMP was established by American scholar Girolamil et al. [14] in 1983, and it is suitable for premature infants with a corrected gestational age of 34 weeks to infants with a corrected age of 4 months. The purpose is to evaluate the motor control, postural coordination and functional activity-related motor ability of premature infants and young infants, which has a predictive effect on the later motor development of infants. There are 42 items in the TIMP assessment, of which items 1 to 13 are observation items, including selective postural control, midline alignment, and movement quality. Items 14–42 were elicited items: including sitting position (items 14–18), supine position (items 19––27), turning over (items 28–31), lateral position (items 32–34), prone position (items 35–39), standing position (items 40–42) in terms of infant head, response to visual and auditory stimuli, defensive movements, trunk movement, limb movement, etc. In the first 13 observation items, if the baby achieves the corresponding performance, they will score 1 point; if there is no corresponding performance, the score will be 0 points. The last 29 induced items are graded from 0 to 6 points. The original score is obtained by adding the scores of 42 items (13 observation items + 29 eliciting items). The original score ranges from 0 to 142 points and takes 20 to 40 min. EPIs were assessed at 37 weeks corrected gestational age.

Neonatal neurobehavioral assessment

Neonatal neurobehavioral assessment (NBNA), a method for assessing the neurobehavior of newborns in China, was developed by Professor Bao Xiulan, an early childhood education expert and a professional at Beijing Union Medical College Hospital. This method integrates the strengths of the NBAS, theNeonatal Behavioral Assessment Scale, by Professor Brazelton from Boston University, USA, and the neonatal neuromotor assessment method by Amiel-Tison from France, combined with her own experience. The NBNA comprises 20 items and provides a comprehensive reflection of the functional state of the brain in newborns [15]. The NBNA comprises five parts: ① Behavioral ability (six items, including response to sound and light stimuli, startle response, facial response, response to red ball, comfort response); ② Passive muscle tension ( four items, including scarf sign, forearm rebound, popliteal angle, lower limb rebound ); ③ Active muscle tension ( four items, including neck flexor and extensor active contraction, hand grip, traction response and support response upright); ④ Primitive reflexes (three items, including sucking action, hugging reflex, step or place response); ⑤ General assessment (three items, including wakefulness, crying, and activity). The score for each item has three partitions: 0 points, 1 point and 2 points. The total score is 40 points, and the abnormal NBNA is defined as the total score ≤ 35 points. EPIs were assessed at 37 weeks corrected gestational age.

Bedside cranial ultrasound examination

Using the portable color Doppler ultrasound diagnostic device, the M-turbo, manufactured by Sonosite with a probe frequency range of 4 to 8 MHz, specialized neonatal neurosonologists conduct regular bedside cranial examinations on EPIs during the first, second, third, and fourth weeks after birth, aiming to compile the cumulative incidence within the first four weeks.

Data quality control

EPIs were evaluated by professional nurses with TIMP and NBNA operating qualifications in the neonatology department. Data receipts were collected by two systematically trained graduate students from January to March 2023 in strict accordance with the inclusion and exclusion criteria. General information on premature infants was collected by reviewing the electronic medical record system. The electronic medical record system was reviewed to ascertain the proportion of breast milk feeding; data were entered into ab Excel form after being checked by two people. Guidance from statistical experts was sought for any problems found during data analysis to ensure the accuracy of the data. A complete set of data were kept by a designated research team member.

Statistical analyses

Statistical analysis was performed using SPSS 27.0 software. The measurement data conforming to the normal distribution is described by mean ± standard deviation, and the comparison between groups is carried out by one-way analysis of variance. Enumeration data were expressed as cases and percentages, and χ2 test was used for comparison between groups. For dimensions with statistical significance, further post-hoc tests were performed, and p < 0.05 was considered statistically significant.