Long-term morbidities among very low birth weight infants remain a significant challenge. Oxidative stress is a key factor in the pathogenesis of 'free radical (FR) diseases of prematurity,' including retinopathy of prematurity, bronchopulmonary dysplasia, necrotizing enterocolitis, and intraventricular hemorrhage. Red blood cell (RBC) transfusions are recognized as a contributing factor to FR-related diseases. RBCs contain adult hemoglobin (HbA), which has a lower affinity for oxygen. This characteristic increases oxygen delivery and tissue uptake, leading to a potentially harmful state of hyperoxia and over-generation of FRs. The strategy employs a multidisciplinary approach to evaluate the impact of cord blood transfusions in anemic newborns. Results will be assessed in relation to short- and long-term neonatal outcomes to determine the effectiveness of this new preventive strategy. Improving the current data are critical for setting action priorities for and monitoring progress

Specific AIM 1 To assess whether CB-RBC transfusions can offer a protective advantage of CB-RBC transfusions in preterm newborns by preserving HbF, reducing the risk of oxidative stress-related complications, and potentially lowering the incidence of 'free radical diseases of prematurity' .

Biomarkers of oxidative stress will be correlated with short term outcome of the newborns (diagnosis at discharge). Moreover with the aim to evaluate the social, economic, organizational implications of this intervention the investigators will perform an Health Impact Assessment (HIA).

The goal is to provide policymakers, healthcare providers, and stakeholders with evidence-based insights on the effectiveness, cost-effectiveness, safety, and potential impact of CB-RBC transfusions.

Specific AIM 2 To evaluate pathways involved in inflammatory and hypoxic-hyperoxic processes through microRNAs expression. OS cascade comprises the activation of microglia, infiltration of macrophages and release of pro-inflammatory mediators such as cytokines, chemokines, nitric oxide, free radicals. MicroRNAs, (miRs), play an important role in the regulation of OS modulated genes, with the potentiality to induce protective protein synthesis such as neuroglobin and sirtuin. Specifically, miRNA 107 e 17-5p, involved in angiogenesis and arterial remodeling are upregulated; miRNA 223-3p associated with chronic kidney disease is downregulated.

Specific AIM 3 To assess the protective role CB-RBC transfusions in newborns, by performing neurodevelopmental outcome of enrolled babies through clinical and neurobehavioral evaluations at 3-6-9-12-month of age.

Experimental Design Aim 1 This study is designed as a multicenter, randomized, controlled, double-blind trial.

Preterm neonates born between 24+0 and 31+6 weeks of gestational age who require at least one red blood cell transfusion during hospitalization will be randomized in a 1:1 ratio to receive either standard A-RBCs (control group) or CB-RBCs (intervention group). Randomization will be stratified by gestational age and study center and conducted using a centralized computer generated system. Treating physicians, caregivers, and outcome assessors including clinical, laboratory, and imaging personnel will be blinded to treatment allocation.

--Interventions: All transfusions will follow uniform clinical guidelines and thresholds for transfusion, aligned with current Italian neonatal transfusion standards. The experimental intervention involves transfusion with CB-RBC concentrates prepared from cord blood units donated to public cord blood banks. These units are leukodepleted using BioR Flex filters, fractionated via Compomat G5 cell separators, suspended in SAG-M additive solution, and stored in DEHP-free pediatric bags. All units will be gamma-irradiated before administration and transfused within 24 hours of irradiation. Safety screening will include infectious disease testing (HIV, HBV, HCV, syphilis, bacterial and fungal cultures), identical to the procedures for adult donor blood.

--Sample Size: Based on an expected 50% incidence of the composite endpoint in the control group and a hypothesized reduction to 30% in the CB-RBC group (absolute risk reduction = 20%), a one-sided Chi-square test with alpha = 0.05 and 80% power requires 74 infants per group. To account for an interim analysis at 50% of enrollment and for up to 10% attrition rate, a total of 200 infants (100 per group) will be enrolled.

--Data Collection and Quality Assurance: Data will be captured using the REDCap electronic data capture system, with predefined validation rules and role-specific access rights. Clinical and perinatal data, transfusion details, and outcomes will be recorded prospectively.

Experimental Design Aim 2

--Biological Sampling and Time Points:

Peripheral blood samples will be collected from each enrolled neonate at three defined time points:

• B1 - Before the first transfusion (baseline);

• B2 - 48 hours (±6h) after the transfusion;

• B3 - 14 days after the transfusion. At each time point, 1 ml of whole blood will be obtained and processed for molecular and biochemical analyses. Samples will be aliquoted and stored at -80°C until batch analysis. Pre-analytical procedures, storage, and shipping conditions will follow standardized SOPs across centers. All laboratory staff performing analyses will be blinded to treatment allocation, ensuring unbiased biomarker evaluation.

  • Oxidative Stress and Antioxidant Biomarkers:

The oxidative stress (OS) profile will be assessed using a panel of protein and lipid biomarkers, including:

• Protein oxidation: Advanced Oxidation Protein Products (AOPP);
• Lipid peroxidation: Isoprostanes (IsoPs), malondialdehyde (MDA);
• Non-enzymatic antioxidants: Vitamin E, glutathione (GSH), and ascorbic acid (AA);
• Enzymatic antioxidants: Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx).

Lipid mediators related to inflammation and oxidative balance, such as Resolvin D1, will also be quantified, alongside neuroglobin and sirtuin-1, which are involved in cellular protection mechanisms.

• MicroRNA Profiling:

Total RNA will be extracted from 400 μl of serum using the miRVana PARIS kit (Invitrogen). Reverse transcription will be performed using the TaqMan Advanced miRNA cDNA Synthesis Kit, followed by quantification using TaqMan miRNA assays on a 7900HT Fast PCR system.

MicroRNAs of interest include miR-107, miR-17-5p, and miR-223-3p, selected for their involvement in oxidative stress, angiogenesis, and vascular remodeling. Expression will be normalized using hsa-miR-16-5p as the endogenous control.

The 2^deltadeltaCt method will be used to quantify expression changes, and miRNAs with Ct > 35 will be excluded from analysis. Total RNA will be isolated from 400μl of serum using the miRVana PARIS kit (Invitrogen, AM1556). Samples will be stored at -80°C. Reverse transcription will be performed using the TaqMan Advanced miRNA cDNA Synthesis Kit (Applied Biosystems, A28007). Candidate miRNAs will be quantified using TaqMan Advanced miRNA assays (Applied Biosystems) on a 7900HT Fast PCR System, with hsa-miR-16-5p (Assay ID: 477860_mir) as an endogenous control. Expression levels will be analyzed using the 2^deltadeltaCt method; miRNAs with Ct > 35 will be considered not expressed and excluded.

--Comparative Analyses: Between-group comparisons (CB-RBC vs A-RBC) will be conducted at each time point and across time points (B1-B3), allowing evaluation of both absolute differences and temporal changes in biomarker levels. Analyses will be adjusted for baseline clinical variables, transfusion volume, and number of transfusions received.

This aim will allow to link the transfusion source to molecular signatures of oxidative stress and inflammation, providing mechanistic insight into the observed clinical outcomes

Experimental Design Aim 3 --Follow-up Time Points--

• Neurodevelopmental assessments will be conducted at:

  • 1 month corrected age;
  • 3 months corrected age;
  • 6 months corrected age;
  • 12 months corrected age. These time points will allow for early detection of neurological delays and tracking of developmental trajectories over the first year of life.

• Neurodevelopmental Assessment Tools:

Standardized and validated instruments will be used to ensure comparability and reliability across study sites:

• The Bayley Scales of Infant and Toddler Development - Third Edition (BSID-III) will be used at 12 months to assess cognitive, motor, and language development;

• Structured neurological examinations will be performed at each follow-up visit to detect signs of abnormal motor tone, reflexes, or neurosensory deficits;

• Information on growth, oxygen dependence, feeding difficulties, and the presence of multi-organ sequelae will be collected longitudinally.

  --Neuroimaging: Magnetic Resonance Imaging (MRI) of the brain will be conducted according to routine clinical practice and scored retrospectively using the Woodward scoring system, which quantifies white and gray matter injury, ventricular dilation, and global structural anomalies. MRI reviewers will be blinded to treatment allocation and clinical outcomes to ensure unbiased evaluation.

• Analysis Plan The primary neurodevelopmental outcome will be the proportion of infants with BSID-III scores below 85 (i.e., more than one standard deviation below the normative mean) in at least one domain (cognitive, motor, or language) at 12 months.

Group comparisons will be performed using Chi-square tests, and multivariable logistic regression models will be used to adjust for potential confounders, including gestational age, IVH grade, BPD, and birth weight.

Secondary analyses will explore continuous BSID-III scores, composite neurodevelopmental indices, and MRI findings.

Correlations between biological markers (from Aim 2) and neurodevelopmental outcomes will be examined to identify predictive biomarkers and potential mechanistic pathways

Inclusion Criteria:

• Preterm neonates born between 24+0 and 31+6 weeks of gestational age;
• Requirement for at least one red blood cell transfusion during hospitalization, according to current Italian transfusion thresholds;
• Written informed consent obtained from parents or legal guardians prior to any study procedure.

Exclusion Criteria:

• Gestational age > 32+0 weeks;
• Pregnancy complicated by maternal-fetal alloimmunization (e.g., hemolytic disease of the newborn);
• Pregnancy complicated by fetal hydrops;
• Major congenital anomalies or genetic syndromes;
• Previous red blood cell transfusions (prior to enrollment);
• Perinatal hemorrhage at delivery;
• Documented congenital infections (TORCH).

De-identified individual participant data underlying the reported results will be made available upon reasonable request to qualified researchers, following publication of the primary results and subject to institutional and ethical approval

At the end of the sudy

Access to de-identified individual participant data will be provided to qualified researchers upon reasonable request, subject to approval by the study investigators and institutional ethics requirements. Data sharing will be permitted only for scientifically sound research purposes and after execution of a data access agreement