A Retrospective Analysis Of Clinical Characteristics Of Very Preterm Infants And An Experimental Study On Isolation Of Mesenchymal Stem Cell From Human Umbilical Cord Blood

PART Ⅰ A Retrospective Analysis of Clinical Characteristics of Very Preterm InfantsObjective:Internationally, the neonatal outcomes of very preterm (VPT) infants (born at<32weeks of GA) has became the highest priority of clinical investigation. However, information regarding this topic is still scant in Chinese tertiary NICU. In the past two decades, the driving forces for major tertiary care centers with NICUs have emerged dramatically with an increase in the preterm birth rate in China. This change in the pattern of provided services has improved the morbidity and mortality of low gestational age (GA) high-risk infants, who are born at level Ⅰ and Ⅱ nurseries in community hospitals and transferred to a level Ⅲ medical center NICU for additional care. We have established a tertiary pediatric hospital (BaYi Children’s Hospital, affiliated to the General Hospital of Beijing Military Region) with a specialized neonatal care center that serves as a referral centre for the city of Beijing in2007. The neonatal care center has developed with450infant incubators, more than8000admissions in2011and four subspecialty NICUs up to2012. As the largest tertiary care center, it serves a largely outborn population and cares for all regional neonates requiring complex medical and surgical subspecialty care in Beijing. Over the past years, active treatment of VPT infants has been a major topic of discussion in China, but information regarding clinical outcomes of VPT infants in tertiary NICU setting is scant. The aim of this study was to evaluate clinical practice, mortality and morbidity of VPT infants during hospitalization in this largest NICU from October2010to September2011. We paid special attention to the outcomes of outborn infants trasnported from lower level hospitals.Methods:Research personnel retrospectively analyzed clinical data collected by a standardized data collection sheet through medical record review. The study group consisted of infants born at<32weeks of GA and admitted within24hours of postnatal life to the VPT-NICU of BaYi Children’s Hospital, between October1st,2010and September30th,2011. The admitted infants aged over24hours often with incomplete prenatal data or medical records were not included in the present study.SPSS (SPSS for Windows, version20.0, IBM-SPSS, Chicago, IL, USA) was used for statistical analysis. The primary goal of this study was to provide descriptive statistics of the patient population. Continuous variables were presented as means and SD or medians and range or interquartile range (IQR,25th to75th percentile), depending on whether their distributions were or were not highly skewed; categorical variables as counts or rates, and odds ratios (ORs) with95%confidence intervals (CI). Comparison between continuous variables was made by using a Mann-Whitney U test. Univariate analyses on categorical data were performed by using a2-tailed Pearson χ2or Fisher’s exact test wherever appropriate. Logistic regression models were used to analyze the risk factors for death in VPT infants. Multivariate logistic regression analysis included GA, BW categories, gender, caesarean section, small for gestational age (SGA), premature rupture of membrane (PROM)>24h and admission age>2h as independent factors. Values of P<0.05were considered to be signifcant.Results:1. Study PopulationA total of1262case records were retrospectively reviewed. Of these records,729infants born before32weeks’GA were subsequently admitted to the NICU within24hours of postnatal life; that is, they met the inclusion criteria for this study.Of these VPT infants,22.5%were extremely preterm (EPT) infants (GA≤28wk, n=164),65.6%very low birth weight (VLBW,<1500g, n=478) infants and9.3%extremely low birth weight (ELBW,<1000g, n=68) infants.The mean GA and BW of all infants was29.8±1.4(range23.9-31.9) weeks,1399±285(range610-2415) g, respectively. BW increased with every week of gestation (P<0.001). The male/female ratio was1.4:1. Boys weighed more than girls (1430±290vs1356±272g; P=0.001), whereas there was no significant difference in GA (29.7±1.4vs29.9±1.4weeks; P=0.054).76(10.4%)VPT infants were small for gestational age infants,199(27.3%) multiple births. The mean admission age was2.2±2.3(range0.3-24) h.2. Perinatal Characteristics and Delivery Room InterventionsOf all VPT infants(n=729),74.6%VPT infants received regular prenatal care,19.3%prenatal care more than once or no prenatal care.27.3%infants were treated with prenatal steroids. Cesarean sections were done in362(49.7%) deliveries and the cesarean section delivery rate increased with GA (OR1.607;95%CI1.425-1.812; P <0.001), with the steepest increase between GA of≤26and31weeks (17.9%at≤26weeks and70.1%at24weeks). There were more girls than boys (55.5%vs45.6%; P=.008) among infants born by cesarean section and also more SGA compared to those by vaginal route (17.7%vs3.3%; P<0.001).21.0%infants were born with PROM>24h,8.8%received antibiotics. Diagnosed gestational diseases were occurred in63.3%VPT infants, including21.9%gestational hypertension,33.6% PROM,9.9%both placenta praevia or abruption and gestational diabetes mellitus.29.2%(n=213) VPT infants were treated with oxygen for resuscitation in the delivery room, and of whom48.3%received by nasal cannula or mask, the remaining51.7%received by tracheal intubation;3.4%received chest compression and1.9received resuscitation drugs.3. Primary diagnosis in NICUInfants at the lowest GA and BW were at the greatest risk for morbidities of prematurity. Diseases of respiratory system were the most common diagnosis in VPT infants. Overall,68.4%of infants experienced neonatal respiratory distress syndrome (RDS),48.4%pneumonia of newborn,8.4%pneumorrhagia,7.5%pulmonary hypertension,3.0%bronchopulmonary dysplasia (BPD),2.2%pneumothorax and so on. The other main diagnosis included68.4%patent ductus arteriosus (PDA),39.4%apnea,12.6%sepsis,21.5%intraventricular hemorrhage (IVH),1.5%periventricular leukomalacia (PVL),3.0%necrotizing enterocolitis (NEC),1.7%retinopathy of prematurity (ROP) more than stage3and so on.4. Characteristics of Diseases of Respiratory System and Clinical PracticesOf all RDS infants (n=499),89.6%received pulmonary surfactant (PS), and13.2%received PS more than once. Campared with no PS treated infants with RDS, the survival rate of PS treated infants was insignificant, while lower mean GA, higher rates of morbidities, longer mean length of mechanical ventilation (MV) and oxygen supply was found in PS treated infants. The survival rate, mean GA, rates of morbidities, mean length of MV and oxygen supply was no significant difference between infants received PS in the delivery room and infants received PS in the NICU. In addition, the effect of different surfactants (poractant alpha, Curosurf and calfactant, Calfsurf) has not shown any significant difference in the survial rate (Curosurf vs Calfsurf:86.6%vs92.7%; P=0.105), rates of main severe morbidities, mean length of MV and oxygen supply. However, infants who received Curosurf were noted to have indications that they were more critically ill than Calfsurf infants, with lower mean GA and BW, longer length of hospital stay (P<0.05).All of infants with RDS were treated with MV (n=499), and of whom15.8% only received nasal continuous positive airway pressure (nCPAP),71.5%received only conventional mechanical ventilation (CMV) and/or nCPAP, and12.6%received high frequency oscillatory ventilation (HFOV). The survival rate of infants who received HFOV was the lowest, the mean length of MV was the longest, the rates of main complications and severe morbidities (including pneumorrhagia, pneumothorax, IVH grade3or4) were the highest. For the infants treated with only CMV and/or nCPAP, the survival rate and rates of main complications and severe morbidities were insignificant except a higher rate of pneumorrhagia (11.2%vs0%; P<0.01) compared with infants received only nCPAP.The positive rate of tracheal aspirates culture in pneumonia of newborns was16.1%(n=57). The main microorganisms recovered from tracheal aspirates culture were Acinetobacter baumannii (49.1%), Klebsiella pneumoniae (31.6%), Escherichia coli (10.5%) and Pseudomonas aeruginosa (7.0%).Of all infants with pulmonary hypertension (n=55),43.6%were treated with HFOV,80.0%treated with iNO. Infants with pulmonary hypertension often diagnosed with PDA. The proportion of infants with PDA who were treated surgically were higher in infants with pulmonary hypertension compared with no pulmonary hypertension infants (16.4%vs1.9%; P<0.001). For the infants who were treated with iNO (n=48),16.7%received PDA ligation, which accounded for36.4%of infants with PDA treated surgiallly.As one of the severe pulmonary morbidities,22(3.6%) of606infants needed oxygen therapy at36weeks and developed BPD. Postnatal steroids was given to7(31.8%) infants by intravenous injection after BPD diagnosed.3(13.6%) infants of BPD discharged home on oxygen and the remaining19(86.4%) infants were survived until discharge without oxygen.5. Other Primary Diagnosis and Clinical PracticesAll infants underwent≥1cranial ultrasound evaluation after admission;77.9%(n=572) of these results were normal. The lowest GA of infants were experienced the highest risk of brain injury. The proportion of EPT infants with FVH was42.1%, and24.3%in VLBW infants. Of92infants diagnosed with sepsis,76.1%blood culture of them were positive.The main microorganisms recovered from blood culture were Klebsiella pneumoniae (37.1%), Staphylococcus (30.0%), Fungus(12.9%), Escherichia coli (7.1%) Acinetobacter baumannii (5.7%) and so on.22(3.6%) infants developed for NEC and of whom36.4%died, which included62.5%withdrawn and only37.5%received surgical treatment.Among729cases,87.5%(n=638) underwent at least an ophthalmologic examination before hospital discharge, death, or transfer.16.6%of these infants were diagnosed with ROP any stage, with47.2%in127EPT infants and23.4%in410VLBW infants.8.5%of122EPT infants with VLBW were performed retina photocoagulation for ROP stage≥3while infants born at higher GAs or BW≥1500g were not detected for development of severe ROP. The median length of oxygen supply was significantly longer in infants with ROP than those without ROP (26vs7days; P<0.001). It was also significantly longer in EPT infants treated with ROP photocoagulation than those ROP infants without surgery (44vs29days; P<0.001).The rate of infants with PDA was inversely related to GAs.82.3%(n=380) of infants with PDA received oral ibuprofen administration, and of whom,34.2%received ibuprofen more than one course. The lower GAs infants had, with the more courses of ibuprofen they were treated, the lower proportion of PDA closed and the higher rate of PDA surgery after ibuprofen administration.56.2%of infants with PDA were closed after the first course of ibuprofen administered,22.7%closed with more courses of ibuprofen administered, and4.8%received PDA surgery. Only17.7%of infants with PDA were not treated with ibuprofen, and of whom,90.2%closed spontaneously, which was significantly higher than the rate (78.9%) in the infants treated with ibuprofen (P<0.05). Infants who received ibuprofen were found to have indications that they were more critically ill than no ibuprofen treated infants, with lower GA and BW, higher rate of pneumorrhagia, longer length of MV and oxygen supply, and more money spended in the NICU, while no significant difference in the survival rate (P=0.623). Similarly, infants who received more courses of ibuprofen often had lower GA, higher rate of pulmonary hypertension, iNO treated and PDA ligation, longer length of MV, oxygen supply and hospital stay, and more money spended in the NICU, whereas no significant difference in the survival rate (P=0.495) compared with infants treated with one course of ibuprofen.6. Neonatal Transport and Outcome-based ComparisonAn extremely high proportion (90.0%) of admissions were born at lower level centers and subsequently transported, the remaining10.0%were inborns. The median age of NICU admission was2.0(IQR:1.5-3) hours for the outborns, which was significantly longer than0.5(IQR:0.5-1) hours for the inborns (P<0.001). No difference was found in both the mean GA and BW for the inborn and outborn infants. The rates of cesarean section and PROM>24h were higher in inborns than the rates in outborns, while more multiple births in outborns than inborns (P<0.05). Outborns experienced higher but insignificant need for intensive delivery room and NICU interventions compared with inborns (P=0.084).566(78.6%) infants were treated with supplemental oxygen therapy in transport. The main types of oxygen supply were oxyhood (55.7%) and endotracheal intubation (41.9%) in the transportation. The rate of infants treated with oxygen supply was conversely related to GA and BW (P<0.001). However, rates of oxygen use and MV were similar for the inborn and outborn infants.Rates of diseases of respiratory system, main clinical practices and IVH were found no significant difference in the inborn and outborn infants (P>0.05). A little but not so high mortality was seen in the outborns when compared to the inborns (8.7%vs5.5%; P=0.348). Rates of main complications, length of MV, oxygen supply and hospital stay were also found insignificant difference among the inborn and outborn survivors (P>0.05).Compared with different admission age, the mean GA and BW of infants with admission age≤2h were no significant difference with those of infants with admission age>2h. For the perinatal characteristics, tube baby, gestational hypertension diseases, regular prenatal care, prenatal steroids and cesarean section were prevalent in infants with admission age≤2h, while asphyxia and intensive delivery room interventions were more common in infants with admission age>2h. For the outcomes of VPT infants in the NICU, higher survival rate (91.2%vs84.8%; P<0.05) and lower proportion of withdrawal of care (11.2%vs21.7%; P<0.01) were found in infants with admission age<2h compared with infants with admission age>2h. There was no difference in occurrence of main severe morbidities, length of MV, oxygen use and hospital stay, and cost of hospital (P>0.05).7. Mortality Rates and Risk Factors of DeathThe overall mortality rate of VPT infants until discharge from the hospital was8.4%(n=61), which consisted of hospital deaths either in the process of intensive care (1.8%) or after the withdrawn/withhold of support (6.6%). The death rate of VLBW and ELBW infants was10.5%and26.5%, respectively, which was higher compared to4.4%of infants with BW≥1500g (P<0.001). In the lower ranges of GA and BW (both P<0.001), a striking stepwise reductions in mortality rate occur significantly with each additional week of gestation and250g increase in BW. Most of deaths occurred within neonatal period:44.3%occurred in the early neonatal period (0-6days postpartum),49.2%during the late neonatal period (7-28days postpartum), while only6.6%of deaths after neonatal period.The results of the multivariate logistic regression analysis showed that lower GA, invasive MV, admission age>2h, air leak, pulmonary hemorrhage, no PS therapy and presence of a major anomaly were the independent risk factors for death.8. Length and Costs of HospitalizationThe median LOS among survivors was43days, and decreased with increasing GA, from92days at26weeks to37days at31weeks (P<0.001). PMA at discharge decreased from39.2weeks for surviving infants born at GAs of26weeks to35.7weeks or36.3weeks for those born at30or31weeks, respectively. The median cost of surviving infants was1.4-fold to the entire annual income of a Beijing urban resident in2011(32903CNY). The median cost of hospital in infants with GA≤26wk and31wk was132771and38142CNY, respectively.9. Withdrawal or Withholding of Care18.0%(n=131) of VPT infants support were withdrawn from the treatment, including27.4%in EPT infants. The withdrawal rate of support was20.5%(n=98) among VLBW infants and25.0%among ELBW infants. Of all infants withdrawn from the treatment,51%were withdrawn due to the parents’inability to afford the high cost of continued medical treatment.36.6%of infants died after withdrawal of support in the care unit. No distinction of survival was detected in the gender discrepancy (males vs females:90.9%vs92.6%; P=0.412) and the treatment withdrawn happened almost equally to female and male infants (18.4%vs17.7%; P=0.803).10. Outcome-based Comparison with Developed CountriesThe rate of provide active obstetric care and initiate neonatal intensive care for the most-premature infats was obviously lower in Chinese NICU than the rate in developed countries. An extremely high rate of VPT infants transported was found in the present study, while a much lower rate was found in developed countries. More antenatal transferred VPT infants were found in developed NICU. The overall survival rate of the VPT infants was similar with the rate in developed countries; specifically, a relative lower proportion and survival rate of EPT was found in this study.Conclusions:1. This study is the first report to summarize the management, mortality and morbidity among very preterm infants born at less than32weeks of gestation at the very preterm-NICU of the largest neonatal intensive care center in China.89.5%of VLBW infants survived to discharge, this nearly achieves the rate of90%of infants born with VLBW in subspecialty perinatal centers in the United States in2010.2. The rate of provide active obstetric care (including prenatal steriods, cesarean section) and initiate neonatal intensive care for the most-premature infants (timely fetus or infant transferred to a higher level hospital) was obviously lower in the present data than the rate in developed countries, which would result in delaying the time to rescue the VPT infants and partially explain the worse outcomes of EPT infants. As we know, prenatal steroids treatment has been applied in clinical practice decades and recent reports have affirmed that antenatal exposure to steroids was associated with a lower rate of severe morbidity, death or neurodevelopment impairment among VPT infants in developed countries.3. This VPT-NICU has been the largest VPT infants referral centre in China.90%of infants were born at lower level centers and subsequently transported to the NICU. As many studies reported, outborn VPT infants may have poor outcomes with regards to survival and morbidity when compared to inborn infatns. An unexpected finding was that no significant difference was found in the rates of mortality and servere morbidity between the inborn and outborn infants; the results may suggest that both short-distance within about2hours neonatal ground transport by well-developed hospital referral/infant transport systems and high-volume NICU may minimize the adverse effects of transport.4. Of concern, the cost of hospital stay, especially in EPT infants with severe morbidity, were very high in the present study. The direct costs incurred during neonatal intensive care unit hospitalization for such infants approximated the annual income of an urban resident family. In addition, it should be noted that death after withdrawal or withholding form intensive care, contributed to the high mortality which accounted for79%of the total death in this article. The high cost of continued medical treatment and the high risk of poor outcomes, often withdraw or withhold the parents’decisions to provide active intensive care for VPT infants in China. In fact, socioeconomic and culture status has been a substantial impact on the outcomes of VPT infants.PART II A Lab Study on Isolation of Mesenchymal Stem Cell from Human Umbilical Cord BloodObjective:Mesenchymal stem cells (MSCs) consist of a rare population of multipotent progenitors having the capacity for self-renewal and differentiation into various lineages of mesenchymal tissues. This ability makes MSCs an attractive tool in the field of therapeutic use. These cells can be isolated from different tissues such as bone marrow, umbilical cord, adipose tissue, dental pulp, and umbilical cord blood (UCB). UCB is an interesting source of these cells because the collection process is painless and non-invasive, it causes no harm to the mother or infant, and it is a material usually discarded. Unfortunately, reliable procedures for efficient expansion and differentiation of UCB-MSCs remain to be established. The isolation, characterisation, in vitro expansion and differentiation of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were among the current aims of this study, and achieving these goals is a pre-requisite for extensive use of this novel approach for the treatment of a number of human diseases. Additionally, the factors that influence the rate of success of MSCs isolation culture and the yields of MSCs from UCB of different gestational age deliveries were investigated.Methods:(1) UCB units from full-term deliveries were collected with informed consent of the mothers. We separated cells on Ficoll density gradient, and the monocuclear cells (MNCs) fraction was collected and washed in PBS. MNCs were seeded in culture at a density of1×10/L into a25cm2culture flask, precoated with5%fetal bovine serum (FBS), in DMEM/F12+10%FBS medium in the presence of100units/ml penicillin and100U/ml streptomycin, at37℃, in5%CO2. Every3to4days, we changed the medium until subconfluency was obtained, and cells were harvested with a trypsin-EDTA solution and replated at at a density of1×108/L. The morphology of MSCs derived from the UCM was taken. Cumulative population doubling levels were calculated for each subcultivation. Immunophenotyping of MSCs was detected using flow cytometry. The adipogenic and chondrogenetic differentiation studies were induced.(2) UCB was collected at birth in neonates of three different gestational groups, cultured with the same culture conditions. The relationship of the yields of MSCs derived from UCB with several factors such as GA, the collected volume of UCB and the MNCs count of UCB and the relationship among these factors were investigated. Results:(1) After plating the MNCs, a few cells attached to the plastic culture dishes and formed adherent cells within4to5d. Most of those cells were monocytes, which fused toform osteoclast-like cells. The onset of colony formation could be observed at first after2weeks. These appeared in80%to90%of the flask coated with FBS and reached a subconfluent condition within4to5weeks. The expression of cell-surface antigens by flow cytometry was evaluated at passage4. The isolated cells significantly expressed MSCs markers CD105、CD90、CD73and HLA-ABC, while they lacked expression of the hematopoietic markers CD14, CD34and CD45. Differentiation capacity of MSC derived from HUCB was cultured in differentiation medium to induce adipogenic and chondrogenetic differentiation respectively. The MSCs demonstrated a multilineage capacity of adipogenic and chondrogenetic differentiation. The cumulative population doubling levels increased sharply for the the passage3to passage7and apparent slowly after the passage9. The bioactivities of MSCs had no changes after cell culture from thawing of frozen cells.(2) There were significant correlations between the success rate and such factors as the gestaional age (GA), the volume and the MNCs count of UCB.①The success rate of generating MSCs cells from UCB was up to52.8%. The GA of UCB deliveries with succesful isolating MSCs (the successful UCB) was significantly lower than that of UCB deliveries without isolating MSCs (the unsuccessful UCB;34.0±4.2vs36.8±3.3wk;t=2.184, P=0.036). The trend but insignificantly toward the success rate declined with the increasing GA (χ2=3.769, P=0.152).②The volume of the unsuccessful UCB was significantly more than that of the unsuccessful UCB (43.2±13.6vs32.1±11.5ml; t=2.635, P=0.013). There was a positive correlation between the volume and the GA in the successful UCB (F=4.003, P=0.039).③The density of MNCs in the unsuccessful UCB was significantly higher than that of the unsuccessful UCB (32.9±11.7vs23.7±6.5×106/ml;t=2.863, P=0.008). In average, the yield of the MNCs was comparable between the three GA groups (F=1.087, P=0.361).④The MSCs count as percentage of MNCs was in a significant inverse correlation with GA; that is, the density of MSCs and GA, which reduces as the maturity increased (F=13.309, P=0.000).Conclusions:(1) MSCs can be isolated and cultivated successfully in vitro from UCB with the optimal culture conditions,1×1011/L mononuclear cells seeded in DMEM/F12culture medium containing10%FBS and a culture flask precoated with FBS. The bioactivities of these MSCs were identical with that of the MSCs reported.(2) The success rate of generating MSCs cells from UCB was low. Crucial points to isolate MSCs cells from UCB were the gestaional age (GA), the volume and the MNCs count of UCB. Lower GA, more volume and higher density of MNCs of UCB seems like with higher success rate isolating MSCs cells. The UCB from preterm infants is a better source of MSCs than that from full term neonates.