Multi-omics (analysis of peripheral blood immune cells subset, peripheral blood MNCs transcriptome, soluble inflammatory cytokine profile in blood and airway secretion, lung and gut microbiota, and the interaction) analysis was used to profile immune alternation of infants with intravenous ACBMNC infusion in very preterm monozygotic twins

This was a randomized, placebo-controlled, double-blinded trial involving eight pairs of VPMTs who were admitted to NICU to receive respiratory support right after birth. The infants were assigned (1:1) to receiving at least 2×107 ACB-MNCs/kg or normal saline, intravenously, within 24-h post-enrollment within each pair. Multi-omics (analysis of peripheral blood immune cells subset, peripheral blood MNCs transcriptome, soluble inflammatory cytokine profile in blood and airway secretion, lung and gut microbiota, and the interaction) analysis was used to profile immune alternation of infants with ACB-MNCs infusion, along with paired controls. Feasibility, safety and clinical outcomes improvement of the ACB-MNCs infusion in both short and long term were also assessed.

Inclusion criteria

1. born at study hospitals;
2. monozygotic twins (confirmed by ultrasonographic diagnosis before birth and confirm again with obstetricians during and after delivery, as monochorionic diamniotic twins and monochorionic monoamniotic Twins twins were both monozygotic twins, therefore they will be both eligible);
3. gestational age (GA) <32 weeks (GA was calculated based on the date of the last menstrual period of the mother and an ultrasonographic screening performed during the first trimester of pregnancy);
4. enrolled within the first 24 postnatal hours;
5. free of severe perinatal asphyxia (defined as an Apgar score of 0-3 for more than 5 minutes, a cord blood gas pH <7.00, or both);
6. free of severe congenital anomalies or genetic syndromes;
7. free of maternal sepsis24,25;
8. Written informed consent is obtained from the parents or guardians of the infants;
9. Either of the twins has available umbilical cord blood (UCB) , and the cell number was no more than 10×107 cells per kilogram, but should not be less than 2×107 cells per kilogram.

Exclusion criteria

(1) they exhibit severe congenital abnormalities (detected via prenatal ultrasound); (2) expected to die within the first 24 hours; (3) diagnosed with severe twin-to-twin transfusion syndrome confirmed by prenatal ultrasonography24.

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Once the enrollment was confirmed, the research physician contacted the research nurse. They were informed with the assignment and prepared the processed cord blood MNC or normal saline. After preparation, infusion bag was covered with shading bag and infused by light-blocking infusion tube, so that the nurses who conducted the infusion and all physicians who treated the baby were not aware of the treatment assignment. We used the code name of ACBMNC number 1 or number 2 to name the infusion fluid in the computerized physician order entry. The staff who collected and analyzed the patients' data and who followed up the patients were also blinded with the assignment. The research nurse and physician that knew the allocation of treatment did not make decisions in baby's clinical care. The parents or guardians were also not aware of the assignment. Therefore, this study was double-blinded.