This study protocol describes a randomized, placebo-controlled, double-blinded, and multicenter trial to be conducted at seven medical centers (Guangdong Women and Children Hospital, Dongguan Maternal and Child Health Hospital, Zhongshan Boai Hospital, Shunde Hospital-Southern Medical University, Dongguan Hospital-Southern Medical University, Nanfang Hospital-Southern Medical University, and Affiliated Maternal and Child Hospital of Foshan-Southern Medical University) in tertiary hospitals. The participating NICUs are selected by an expert committee based on the distance to the Guangdong Cord Blood Bank (GDCBB) and the level of intensive care that these NICUs could provide. The Guangdong Cord Blood Bank is a public provincial blood bank affiliated with the Guangdong Women and Children Hospital and collects cord blood routinely at these enrolled hospitals. To ensure that the cord blood is processed and infused to infants within 24 h after birth, the distance between these centers should be short. Furthermore, to reduce the differences in therapeutic treatment between the selected centers, a uniform guideline regarding the treatment of patients has been devised in all clinical centers. These centers hold quality control meetings every 3 months to ensure research consistency and report and analyze the possible side effects caused by MNC infusion. In this study, a total of 60 pairs of monozygotic twin premature infants fulfilling the eligibility criteria will be enrolled. For each pair, one random number will be assigned to the first baby of the twins; if the last digit of this random number is odd, the first baby will be assigned to the intervention group, and the second baby will be automatically assigned to the control group; in contrast, if the last digit of this random number is even, the first baby will be assigned to the control group, and the second will be automatically assigned to the intervention group. The protocol for this study has been developed based on the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) checklist (Supplementary material S1, S2). We have followed the Consolidated Standards of Reporting Trials (CONSORT) guidelines and the study design is illustrated in Figure 1.

The primary objective of this trial is to evaluate the efficacy of ACBMNC infusion for the prevention of BPD in very preterm monozygotic twins of less than 32 GW.

The secondary objectives of this trial are as follows: (1) to compare the infant mortality rate at 36 weeks of postmenstrual age; (2) to compare the rates of other common preterm complications such as intraventricular hemorrhage (IVH), necrotizing enterocolitis (NEC), retinopathy of prematurity (ROP), respiratory distress syndrome (RDS), ventilation-associated pneumonia (VAP), hypoxic ischemic encephalopathy (HIE), late-onset sepsis (LOS), periventricular leukomalacia (PVL), and anemia; (3) to compare the duration of mechanical ventilation and oxygen therapy in the two groups; (4) to determine the re-intubation rate and time of return to birth weight (BW); (5) to compare the duration of antibiotic usage; (6) to determine the long-term outcomes during a 2-year follow-up, including anthropometric characteristics, respiratory outcomes, and neurodevelopmental outcomes via a standardized neurological examination; (7) to compare the results of single-cell RNA sequencing for cord blood cells and blood cells 3–10 days after intervention and detect whether reactive oxygen species and inflammatory cytokines are present in serum.

Infants fulfilling all the following inclusion criteria will be enrolled in this trial: (1) infants born at study hospitals; (2) monozygotic twins (this will be confirmed by ultrasonographic diagnosis before birth and confirmed again with obstetricians during and after delivery, as monochorionic diamniotic twins and monochorionic monoamniotic twins are both monozygotic twins; therefore, both will be eligible) (24); (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) those enrolled within the first 24 postnatal hours; (5) those free of severe perinatal asphyxia (defined as an Apgar score of 0–3 for more than 5 min, a cord blood gas pH < 7.00, or both); (6) those free of severe congenital anomalies or genetic syndromes; (7) those free of mother infections such as chorioamnionitis, TORCH syndrome (chorioamnionitis, TORCH syndrome, were diagnosed as previously described (24, 25); (8) written informed consent is obtained from the parents or guardians of the infants; (9) either of the twins has available UCB, and the cell number should be at least 5 × 10⁷ cells per kilogram.

Infants will be excluded from the study if (1) they exhibit severe congenital abnormalities (detected via prenatal ultrasound); (2) their condition is critical and are expected to die within the first 24 h; (3) they are diagnosed with twin-to-twin transfusion syndrome confirmed by prenatal ultrasonography (24).

This study is approved by the ethics committee of the Guangdong Women and Children Hospital. The ethics number is 202101265.

Our previous study showed that ACBMNC infusion was effective in reducing respiratory support duration in preterm infants (18). A randomized study in Korea by Won Soon Park showed that severe BPD incidence significantly reduced from 53% to 19% with mesenchymal stem cell intervention in the 23–24 GW group (n = 33) (20). The rate of BPD among very preterm infants at our NICU was 30% (pA), which is similar to the incidence rate reported previously (26). The sample size of this study was determined for assessing the efficacy of ACBMNCs compared with placebo with respect to the primary outcome in order to decrease the incidence of BPD or death with a two-sided α level of 0.05% and 80% power. We expect the intended (or at least acceptable) effect of ACBMNC infusion to be at a rate of 20% decrease in the frequency of BPD. To achieve this rate, at least 60 patients per group will be required based on the following formula:n=(pA(1−pA)/κ+pB(1−pB))((z1−α/2+z1−β)/(pA−pB))2)

κ = nA/nB is the matching ratio; α is type I error; β is type II error, meaning 1 − β is power.

Around 20 pairs of monozygotic twin births of less than 32 gestational weeks occur annually in Guangdong Women and Children Hospital and Dongguan Maternal and Child Health Hospital. Around 6 pairs of monozygotic twin births of less than 32 gestational weeks take place annually in Zhongshan Boai Hospital, Shunde Hospital, Southern Medical University, Dongguan Hospital, Southern Medical University, Nanfang Hospital, Southern Medical University, and Affiliated Maternal and Child Hospital of Foshan, Southern Medical University. Therefore, we assume that 60 pairs of monozygotic twins will be enrolled annually and the trial could be completed within 1 year.

The randomization sequence will be generated electronically using SPSS (version 21). Following enrollment, the subjects will be assigned to treatment levels or groups after the verification of their eligibility and consent status. A random number will be assigned by computer for each pair of enrolled twins. For each pair, one random number will be assigned to the first baby of the twins; if the last number of this random number is odd, the first baby will be assigned to the intervention group, and the second baby of the twins will be automatically assigned to the control group; in contrast, if the last number of this random number is even, the first baby of the twins will be assigned to the control group, and the second will be automatically assigned to the intervention group. Those enrolled in the ACBMNC group will receive an infusion of ACBMNC within 24 h after birth. Those in the placebo group will receive an infusion of a placebo solution consisting of normal saline with similar volume based on the cell number results in their cord blood after processing. The ell dose for all patients will be 5 × 10⁷ cells per kilogram (18, 27).

The study data will be reviewed by the hospital ethics committee during the trial. Nobody will be made aware of the treatment-group assignments of the infants. After the enrollment is confirmed, the research assistants of the respective project leaders will confirm the random number. There will be at least one research nurse and physician in each center. Research nurses and physicians who are aware of the allocation of treatment will not be included in the babies’ care team and they will not participate in making care decisions for the babies in the trial. They will be asked to prepare the processed cord blood MNC or normal saline. Infusion bags will be covered with shading bags and MNCs will be infused by using a light-blocking infusion tube after preparation, so that the nurses who carry out the infusion and all physicians who treat the baby are not aware of the treatment assignment (Figures 2B,C). The infusion is then done by the supervisor nurse of the patients. The staff who collect and analyze the patients’ data and who follow up the patients are also blinded with the assignment. What is more, the infants’ parents are also not aware of the assignment. Therefore, this study is a double-blinded one.

Written informed consent is obtained from the parents or guardians of all infants before delivery. For each monozygotic twin, neonatologists will be summoned at least 1 h before delivery, and their brief is to study patient data and confer with the obstetrician to verify whether the patients fulfill the enrollment criteria. An intrahospital transport team will be organized, which includes three neonatologists (one of them will be a senior physician). A transport incubator and ventilator with an air oxygen mixer and heater humidifier that can provide intermittent positive-pressure ventilation will be equipped for intrahospital transport from the delivery suite to the NICU in the same hospital (Figure 2C). Two blood-collection bags (WEGO, China) containing 28 ml of citrate-phosphate-dextrose anticoagulant and several blood-collection tubes are prepared.

After the neonates are delivered, delayed cord clamping is done for 30–60 s if the babies do not show severe asphyxia and do not need immediate resuscitation. After the twins are born and before the placenta is delivered, both umbilical veins are sterilized and then punctured with a 17-gauge needle. A part of the cord blood will be collected to the blood-collection tubes for clinical blood tests and single-cell RNA sequencing within 24 h after collection, and then cord blood is collected using a blood-collection bag (WEGO, China) by trained medical staff (Figures 2A,B).

Enrollment will be evaluated again by the neonatologists according to the inclusion and exclusion criteria. The blood-collection bag is closed and sealed after the collection is completed. The cord blood bag is labeled with the full name of the neonate, the hospital name, and the birth time and then placed in a cord blood storage container at 4°C storage and sent to the GDCBB within 2 hours. The GDCBB is a provincial, public cord blood bank with the clinical certification of the American Association of Blood Bank (AABB) and authentication of the Chinese Ministry of Health. The GDCBB has been equipped with a thermostat that is specifically used for cord blood transportation; it is able to maintain a temperature of 4°C during transportation. The transportation time duration (both from and back to the hospital) is within 2 hours depending on the traffic condition.

Procedures for cord blood processing will be performed in accordance with cord blood bank guidelines as described previously (28).

Briefly, after arriving at the GDCBB, the blood bags are transported to a 10000-grade aseptic operational workshop via a transmission window. Two milliliter of cord blood is taken to detect viruses [human immunodeficiency virus, hepatitis B virus, hepatitis C virus, cytomegalovirus by polymerase chain reaction (PCR)] and bacterial infections (including Treponema pallidum-antibody detection or blood smear). The results are obtained shortly before the transfusion is started. The blood bags are then centrifuged at 4°C with a gravity of 50 g for 8 min in a low-temperature centrifuge (RT, Beckman, United States). Then, red blood cells are removed. After that, the blood bags are turned straight and centrifuged for 10 min in the same condition as before. Then, MNCs are obtained after the plasma is separated by using the plasma separation clip (Figure 2D). Finally, a 1 ml MNC sample is taken to determine the following parameters: MNC count (Sysmex XE-5000 automated flow cytometer, Japan), CD34 count (BD calibur flow cytometry, BD Bioscience, United States), colony-forming unit-granulocyte and macrophage (CFU-GM) count under an inverted microscope after being cultured in methyl cellulose medium (SIGMA-ALDRICH, United States), sterility detection by an automatic bacteria detector (Thermo Fisher 6240, MA, United States), and cell viability by using the 7-aminoactinomycin D detection kit via flow cytometry analysis (Figure 2E). After processing, if the cell number reaches at least 5 × 10⁷ cells per kilogram, the cells will be titrated according to the cell concentration in 5% albumin to ensure that the final infusion volume ranges from 5 to 20 ml/kg and stored in the transparent separation bag in a 4°C refrigerator and then transported in the thermostat used for cord blood transportation. The cord blood cells will be sent back to the hospitals where they are collected. The patients are finally enrolled in the allocation. Even if only one of the twins has enough cord blood for the purposes of study participation, both will be enrolled as part of the randomization process.

All infusions will be administered in the NICU. Infusion and subject identities will be double-checked by research nursing and physician staff. Infusions will be monitored by research and clinical staff. ACBMNCs or placebo are administered intravenously via a central catheter or a peripheral catheter if a central catheter is not available. ACBMNC infusion should be performed within 4 h of patients arriving in hospital. The maximum volume will be less than 20 ml/kg. An infusion pump is used to control the infusion rate at less than 10 ml/kg/h. A blood filter will be used to filter the cell settings or blood clots before infusion. Prior to infusion, the intravascular (IV) line is prestored with 2 ml MNCs. We will infuse the MNCs by positioning the syringe and IV line vertically (29). During infusion, the nurses will shake the cells gently every half an hour. After infusion, 2-ml normal saline will be used to flush the intravascular catheter (Figure 2F).

Assessment of safety is conducted before, during, and 12 and 24 h after infusion. The heart rate, systolic, diastolic, and mean arterial blood pressure, and arterial blood oxygen saturation level are monitored continually and documented; these are also monitored during the period of hospitalization and return visits. The levels of arterial blood gas and blood glucose in peripheral blood are monitored and kept stable during the whole treatment period. Infusion reactions (fever, hemolysis, hyperbilirubinemia, and urine color changes) and signs of circulatory overload (tachycardia, dyspnea, edema, and hepatomegaly) are observed and checked during infusion and 48 h after infusion.

The trial will be strictly monitored by the members of a safety monitoring board comprising an ethics committee member, project principal, good clinical practice member, and statisticians, and they will be notified of any severe adverse events (including death, LOS, NEC, severe IVH, and cystic periventricular leukomalacia). Other adverse or unexpected events will be reviewed immediately after they are discovered. After each interim analysis (every 3 months), the data safety monitoring board will make a decision on whether to stop or continue the trial based on safety monitoring and sequential analysis of the primary outcome.

All patients in the study will be given intensive care therapy in accordance with uniform guidelines, which include positive-pressure mechanical ventilation, oxygen therapy, and exogenous pulmonary surfactant (PS) (Curosurf, Chiesi, Parma, Italy) replacement. Oxygen saturation targets (90%–95%), ventilatory support, postnatal steroids, and transfusion guidelines are uniformly given and applied to all enrolled patients. Inhaled nitric oxide is given if a patient with severe hypoxemic respiratory failure presents with severe persistent pulmonary hypertension of newborn (PPHN) confirmed by cardiac ultrasound with an estimated peak systolic pulmonary artery pressure that is higher than 35 mm Hg or more than two-thirds of the systemic systolic pressure. Ibuprofen is given to neonates who have significant patent ductus arteriosus (PDA) (28). Postnatal systemic dexamethasone is reserved only for infants who have severe underlying lung disease and intractable respiratory failure. In such infants, a short course of dexamethasone (three to five doses of 0.25 mg/kg every 12 h) is given at the discretion of the attending physician. Chest radiography is performed after the umbilical venous catheter is placed. Arterial blood gas is monitored at least every 24 h until weaning from ventilation. All clinical diagnoses are defined according to a standard reference (29).

The following data will be collected and entered into an online collaborative database: (1)

Basic clinical characteristics: hospital name, patient name, the recorder name, birth date and time, sex, GA, birth weight, height, head circumference, delivery mode, delayed cord clamping time, and Apgar score at 1, 5, and 10 min. Maternal information includes maternal age, race, pregnancy complications, and prenatal glucocorticoid use.

Primary outcome:

– Surviving without BPD.

All infants will be followed up three times (corrected GA of 40 weeks and corrected age of 12 and 24 months), and the following parameters will be recorded.

Follow-up content at corrected GA of 40 weeks:

– Main caregiver and the education level.

Mean and standard deviation are reported for continuous variables, and the number and percentage are reported for categorical variables. Group comparisons of categorical variables for outcomes and co-interventions including death, BPD, and BPD severity are performed using nonparametric tests, the Fisher's exact test, or chi-square test, as appropriate. The relative risk (RR) is calculated as the ratio of the cumulative incidence rates of an event occurring in the intervention infusion group to the control group, and the number needed for treatment as the inverse of these two cumulative incidence rates. The results are shown as RR with 95% confidence interval (CI). For continuous variables, the mean values at each time point in each group of the variables are derived from a generalized linear model and the differences in the values over time are compared using an unpaired Student's t-test between the two groups. Kaplan–Meier cumulative incidence plots are generated to show time-to-event end points, namely, extubation and weaning to room air. All statistical tests are two-tailed, and p-values < 0.05 are deemed statistically significant. The Breslow–Day test and Cochran–Mantel–Haenszel (CMH) test will be used to adjust the data derived from different participating centers. The results will be reported as odds ratio (OR) with 95% CI. All statistical tests will be two-tailed and a p-value < 0.05 will be considered statistically significant. All statistical analysis will be performed using SPSS 21.0 (IBM).