Genetic Analysis of UGT1A1 Polymorphisms Using Preserved Dried Umbilical Cord for Assessing the Potential of Neonatal Jaundice as a Risk Factor for Autism Spectrum Disorder in Children.

Neonatal jaundice has been suggested as a perinatal risk factor for autism spectrum disorder (ASD). We examined UGT1A1 polymorphisms to assess the potential of neonatal jaundice as a risk factor for ASD in children by using DNA extracted from preserved umbilical cord. In total, 79 children with ASD were genotyped for UGT1A1*28 (c.-41-40dup), UGT1A1*6 (c.211 G > A), and UGT1A1*27 (c.686 C > A). The allele frequency of UGT1A1*6 (OR = 1.34, p = 0.26) and UGT1A1*28 (OR = 0.80, p = 0.54) and the prevalence of UGT1A1*28/*6 diplotypes did not differ significantly from those in the control population. No UGT1A1*27 allele was detected in the subjects. ASD symptom assessment scores were not associated with UGT1A1*28/*6/*27 genotypes or UGT1A1*28/*6 diplotypes. These results suggest that neonatal jaundice is not significantly associated with ASD.

A Novel Method for Measuring Serum Unbound Bilirubin Levels Using Glucose Oxidase-Peroxidase and Bilirubin-Inducible Fluorescent Protein (UnaG): No Influence of Direct Bilirubin.

The glucose oxidase-peroxidase (GOD-POD) method used to measure serum unbound bilirubin (UB) suffers from direct bilirubin (DB) interference. Using a bilirubin-inducible fluorescent protein from eel muscle (UnaG), a novel GOD-POD-UnaG method for measuring UB was developed. Newborn sera with an indirect bilirubin/albumin (iDB/A) molar ratio of <0.5 were classified into four groups of DB/total serum bilirubin (TB) ratios (<5%, 5-10%, 10-20%, and ≥20%), and the correlation between the UB levels and iDB/A ratio was examined. Linear regression analysis was performed to compare UB values from both methods with the iDB/A ratio from 38 sera samples with DB/TB ratio <5% and 11 samples with DB/TB ratio ≥5%. The correlation coefficient (r) between UB values and the iDB/A ratio for the GOD-POD method was 0.8096 (DB/TB ratio <5%, n = 239), 0.7265 (5-10%, n = 29), 0.7165 (10-20%, n = 17), and 0.4816 (≥20%, n = 16). UB values using the GOD-POD-UnaG method highly correlated with the iDB/A ratio in both <5% and ≥5% DB/TB ratio sera (r = 0.887 and 0.806, respectively), whereas a low correlation (r = 0.428) occurred for ≥5% DB/TB ratio sera using the GOD-POD method. Our GOD-POD-UnaG method can measure UB levels regardless of the presence of DB.

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants.

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Evaluation index for asymmetric ventricular size on brain magnetic resonance images in very low birth weight infants.

OBJECTIVE: Asymmetric ventriculomegaly is often evident on brain magnetic resonance imaging (MRI) in very low birth weight infants (VLBWI) and is interpreted as white matter injury. However, no evaluation index for asymmetric left-right and anterior-posterior ventricular sizes has been established. METHODS: In this retrospective multicenter cohort study, brain T2-weighted MRI was performed at term-equivalent ages in 294 VLBWI born between 2009 and 2011. The value of a lateral ventricular index (LVI) to evaluate asymmetric ventricular size, as well as the relationship between the LVI value and walking at a corrected age of 18 months was investigated. At the level of the foramen of Monro in a horizontal slice, asymmetry between the left and right sides and between the anterior and posterior horns was identified by the corrected width and was detected by a low concordance rate and κ statistic value. An LVI representing the sum of the widths of the four horns of the lateral ventricle corrected for cerebral diameter was devised. RESULTS: Asymmetric left-right and anterior-posterior ventricular sizes were confirmed. The LVI value was significantly higher in the non-walking VLBWI group (n = 39) than in the walking VLBWI group (n = 255; 18.2 vs. 15.8, p = 0.02). An LVI cut-off value of 21.5 was associated with non-walking. Multivariate analysis revealed that an LVI value >21.5 was an independent predictor of walking disability at the corrected age of 18 months (odds ratio 2.56, p = 0.008). CONCLUSIONS: The LVI value calculated via MRI may predict walking disability at a corrected age of 18 months in VLBWI.

Congenital Cytomegalovirus Infection in Children with Autism Spectrum Disorder: Systematic Review and Meta-Analysis.

Association of congenital cytomegalovirus (CMV) infection with autism spectral disorder (ASD) has been suggested since 1980s. Despite the observed association, its role as a risk factor for ASD remains to be defined. In the present review, we systematically evaluated the available evidence associating congenital CMV infection with ASD using PubMed, Web of Science, Cochrane Library, and Embase databases. Any studies on children with CMV infection and ASD were evaluated for eligibility and three observational studies were included in meta-analysis. Although a high prevalence of congenital CMV infection in ASD cases (OR 11.31, 95% CI 3.07-41.66) was indicated, too few events (0-2 events) in all included studies imposed serious limitations. There is urgent need for further studies to clarify this issue.

Involvement of WNT Signaling in the Regulation of Gestational Age-Dependent Umbilical Cord-Derived Mesenchymal Stem Cell Proliferation.

Mesenchymal stem cells (MSCs) are a heterogeneous cell population that is isolated initially from the bone marrow (BM) and subsequently almost all tissues including umbilical cord (UC). UC-derived MSCs (UC-MSCs) have attracted an increasing attention as a source for cell therapy against various degenerative diseases due to their vigorous proliferation and differentiation. Although the cell proliferation and differentiation of BM-derived MSCs is known to decline with age, the functional difference between preterm and term UC-MSCs is poorly characterized. In the present study, we isolated UC-MSCs from 23 infants delivered at 22-40 weeks of gestation and analyzed their gene expression and cell proliferation. Microarray analysis revealed that global gene expression in preterm UC-MSCs was distinct from term UC-MSCs. WNT signaling impacts on a variety of tissue stem cell proliferation and differentiation, and its pathway genes were enriched in differentially expressed genes between preterm and term UC-MSCs. Cell proliferation of preterm UC-MSCs was significantly enhanced compared to term UC-MSCs and counteracted by WNT signaling inhibitor XAV939. Furthermore, WNT2B expression in UC-MSCs showed a significant negative correlation with gestational age (GA). These results suggest that WNT signaling is involved in the regulation of GA-dependent UC-MSC proliferation.

Gestational Age-Dependent Increase of Survival Motor Neuron Protein in Umbilical Cord-Derived Mesenchymal Stem Cells.

BACKGROUND: Spinal muscular atrophy (SMA) is the most common genetic neurological disease leading to infant death. It is caused by loss of survival motor neuron (SMN) 1 gene and subsequent reduction of SMN protein in motor neurons. Because SMN is ubiquitously expressed and functionally linked to general RNA metabolism pathway, fibroblasts (FBs) are most widely used for the assessment of SMN expression in SMA patients but usually isolated from skin biopsy samples after the onset of overt symptoms. Although recent translational studies of SMN-targeted therapies have revealed the very limited time window for effective SMA therapies during perinatal period, the exact time point when SMN shortage became evident is unknown in human samples. In this study, we analyzed SMN mRNA and protein expression during perinatal period by using umbilical cord-derived mesenchymal stem cells (UC-MSCs) obtained from preterm and term infants. METHODS: UC-MSCs were isolated from 16 control infants delivered at 22-40 weeks of gestation and SMA fetus aborted at 19 weeks of gestation (UC-MSC-Control and UC-MSC-SMA). FBs were isolated from control volunteer and SMA patient (FB-Control and FB-SMA). SMN mRNA and protein expression in UC-MSCs and FBs was determined by RT-qPCR and Western blot. RESULTS: UC-MSC-Control and UC-MSC-SMA expressed the comparable level of MSC markers on their cell surface and were able to differentiate into adipocytes, osteocytes, and chondrocytes. At steady state, SMN mRNA and protein expression was decreased in UC-MSC-SMA compared to UC-MSC-Control, as observed in FB-SMA and FB-Control. In response to histone deacetylase inhibitor valproic acid, SMN mRNA and protein expression in UC-MSC-SMA and FB-SMA was increased. During perinatal development from 22 to 40 weeks of gestation, SMN mRNA and protein expression in UC-MSC-Control was positively correlated with gestational age. CONCLUSION: UC-MSCs isolated from 17 fetus/infant of 19-40 weeks of gestation are expressed functional SMN mRNA and protein. SMN mRNA and protein expression in UC-MSCs is increased with gestational age during perinatal development.

Very Low Birth Weight Monochorionic Diamniotic Twins as a Risk Factor for Symptomatic Patent Ductus Arteriosus.

BACKGROUND: Some prior studies have shown that symptomatic patent ductus arteriosus (sPDA) is highly familial. Although it is estimated that both genetic and environmental factors may contribute to sPDA, evidence is still lacking. OBJECTIVE: The aim of this study was to determine the risk factors for sPDA, focusing on the genetic and in utero environment by analyzing very low birth weight (VLBW) singletons and twins. METHODS: This retrospective case-control study reviewed the medical records of 445 VLBW infants (25 weeks ≤ gestational age <32 weeks, 600 g ≤ birth weight <1,500 g) and compared the incidence of sPDA among monochorionic diamniotic (MD) twins (n = 65), dichorionic diamniotic (DD) twins (n = 66), and singletons (n = 314). RESULTS: Stepwise multiple regression analysis showed that twin siblings (p = 0.001), gestational week (p < 0.001), antenatal steroid use (p = 0.021), and premature rupture of membranes (p = 0.002) were independent predictors of sPDA. Incidence of sPDA in MD twin siblings was significantly higher than that in singletons (p < 0.01), whereas no significant difference was found between singletons and DD twins or between MD and DD twins. CONCLUSIONS: The current results show that being a VLBW MD twin is an independent risk factor for sPDA, and that both genetic and in utero environmental factors may contribute to its development.

Familial Hemophagocytic Lymphohistiocytosis Presenting as Hydrops Fetalis.

Background Familial hemophagocytic lymphohistiocytosis (FLH) is an autosomal recessive disorder of immune regulation that leads to a hyperinflammatory syndrome. Fetal onset FHL is extremely rare and is considered to be the most severe form of FHL. Case We report a preterm case of FHL that presented as hydrops fetalis. The infant was treated with a chemotherapy regimen based on the HLH-2004 protocol from the third day of life. However, he had persistent cytopenia and died on the 18th day of life due to bacteremia. The detection of defective perforin expression in the patient's natural killer cells and mutations in the PRF1 gene resulted in a molecular diagnosis of FHL. Conclusion We suggest that early diagnosis and the development of an appropriate immunosuppressive strategy that can induce and maintain remission until hematopoietic stem cell transplantation can be performed are required to improve the outcomes of fetal onset FHL.

Successful management of an extremely premature infant with congenital candidiasis.

Congenital candidiasis, which presents with a variety of clinical symptoms, is very rare in both term and preterm infants, and less than 100 neonatal cases have been reported in the medical literature. We describe the case of an extremely premature infant with congenital candidiasis, who was successfully treated and survived without major sequelae. A male infant was born at 25 weeks' gestation (weight, 834 g). He exhibited diffuse erythematous papules. Samples of his skin, pharyngeal mucus, gastric fluid, and tracheal aspirate were found to be Candida albicans-positive while blood cultures were negative. Further histopathological examinations revealed that Candida albicans mycelia had invaded the umbilical cord. After prompt antifungal therapy, the patient's skin lesions improved markedly, and he was discharged from hospital without any major complications. This report highlights the importance of characteristic skin lesions for the early diagnosis of Candida infections, especially in extremely premature infants.

Increased levels of interleukin-6 in tracheal aspirate fluid are indicative of fetal inflammation in ventilated extremely low gestational age newborns.

OBJECTIVE: To determine whether increased serum and/or tracheal aspirate fluid (TAF) levels of IL-6 at birth are associated with fetal inflammation in ventilated extremely low gestational age newborns (ELGAN). METHOD: A total of 36 ELGAN who required mechanical ventilation were enrolled in this study. The patients were classified into two groups: 19 infants who displayed histological evidence of funisitis, which is a marker of fetal inflammation, (funisitis group) and 17 infants without funisitis (comparison group). TAF samples were obtained during routine endotracheal suctioning performed within 2 hours of birth. RESULTS: The funisitis group exhibited significantly higher TAF IL-6 levels than the comparison group (2245 vs. 113 pg/mg total protein; p<0.001). The serum IL-6 levels of the funisitis group were also significantly elevated compared with those of the comparison group (median: 737 vs. 136 pg/mL, p=0.017). Receiver operating characteristic curve analysis of the association between IL-6 levels and the presence of funisitis revealed that the TAF IL-6 concentration had a higher area under the curve (0.947) than the serum IL-6 concentration (0.719). At a cut-off value of 216 pg/mg total protein, the TAF IL-6 level exhibited sensitivity and specificity values of 94.7% and 86.7%, respectively, for detecting funisitis. CONCLUSION: Elevated TAF IL-6 levels at birth are strongly associated with funisitis. The TAF IL-6 concentration is a useful marker for detecting fetal inflammation in ventilated ELGAN.

Patterns of increases in interleukin-6 and C-reactive protein as predictors for white matter injury in preterm infants.

BACKGROUND: The aim of this study was to determine whether patterns of increases in serum interleukin-6 (IL-6) and C-reactive protein (CRP) levels at birth were associated with the development of white matter injury (WMI) in preterm infants with a fetal inflammatory response (FIR). METHODS: One hundred infants who were born at <32 weeks gestation and had funisitis, as determined by histological evidence of FIR, were studied. Infants were divided into four groups according to IL-6 and CRP levels at birth, with cut-off values of 200 pg/mL and 0.4 mg/dL, respectively. We compared the incidence of WMI, determined by MRI at term-equivalent age, among these groups. RESULTS: The number of infants in each group was 12, 43, 0, and 45 in the high IL-6 and high CRP (HH) group, high IL-6 and low CRP (HL) group, low IL-6 and high CRP (LH) group, and low IL-6 and low CRP (LL) group, respectively. The incidence of WMI was significantly higher in the HH group than in the HL group and LL group (83%, 40%, and 34%, respectively). Multiple logistic regression analysis revealed that a combined elevation in IL-6 and CRP levels was an independent predictor for the development of WMI (odds ratio, 8.3). CONCLUSION: A combined elevation in serum IL-6 and CRP levels at birth was associated with the development of WMI in preterm infants with FIR.

Neonatal leukemoid reaction associated with Candida albicans chorioamnionitis.

Chorioamnionitis due to Candida species is relatively rare, despite the high prevalence (20-25%) of Candida vulvovaginitis during pregnancy. We describe a case of neonatal leukemoid reaction (NLR) associated with Candida albicans chorioamnionitis. A male infant was born at 31 weeks' gestation and weighed 1864 g. Laboratory tests at birth indicated marked leukocytosis (i.e. total leukocyte count 89.8 × 10(9) /L including 66% polymorphonuclear leukocytes and 15% band forms). Samples of the infant's pharyngeal mucus and tracheal aspirate were positive for Candida albicans. On further histopathology of the placenta, C. albicans mycelia had invaded the placenta, chorioamniotic membrane, and umbilical cord. Although it is not very common, C. albicans chorioamnionitis should be considered in preterm infants with NLR.

N-Terminal pro-brain natriuretic peptide levels in dichorionic diamniotic twins with selective intrauterine growth restriction.

Monochorionic diamniotic (MD) twins with selective intrauterine growth restriction (sIUGR) have known associations with cardiac complications. However, the cardiac load of dichorionic diamniotic (DD) twins with sIUGR (DD-sIUGR) remains unclear. N-terminal pro-brain natriuretic peptide (NT-pro BNP) is a convenient marker of cardiac dysfunction in neonates, and is elevated in MD twins with sIUGR (MD-sIUGR). However, there are no reports assessing serum NT-pro BNP levels in DD-sIUGR. Here, we aimed to clarify serum NT-pro BNP levels at birth in DD-sIUGR, and to compare them with those of MD-sIUGR. Forty-one DD twin pairs admitted to our center between October 2007 and January 2013 were enrolled in this study and separated into two groups: nine twins with sIUGR (DD-sIUGR group) and 32 twins without sIUGR (DD without sIUGR group). Sixteen MD twins with sIUGR (MD-sIUGR group) served as positive controls. Serum NT-pro BNP levels at birth in DD-sIUGR [median 2,115 pg/ml (range, 443-6,590 pg/ml)] were significantly higher than in DD without sIUGR [1,080 pg/ml (range, 313-3,470 pg/ml); p=0.001], and significantly lower than in MD twins with sIUGR [4,520 pg/ml (range, 529-62,400 pg/ml); p=0.04]. Serum NT-pro BNP levels between larger and smaller DD co-twins were significantly correlated (r = 0.582; p<0.0001). In conclusion, serum NT-pro BNP levels at birth in DD twins with sIUGR were higher than those without, and lower than in MD twins with sIUGR.

N-terminal pro-brain natriuretic peptide levels in monochorionic diamniotic twins with twin-to-twin transfusion syndrome treated by fetoscopic laser photocoagulation.

Twin-to-twin transfusion syndrome (TTTS) affects 15% of monochorionic diamniotic (MD) twin pregnancies, and is associated with adverse perinatal outcome. Recently, fetoscopic laser photocoagulation (FLP) has been widely accepted as the most definitive therapy to treat TTTS. N-terminal pro-brain natriuretic peptide (NT-proBNP) is a powerful diagnostic marker of cardiac dysfunction in neonates, and is elevated in MD twins with TTTS. However, there are no reports assessing the effect of FLP on neonatal cardiac overload in TTTS by measuring the serum NT-proBNP levels at birth. Here, we aimed to compare serum NT-proBNP levels at birth in MD twins with TTTS treated with FLP or not. Twelve MD twin pairs with TTTS admitted to our center between October 2007 and September 2012 were enrolled in this study. The MD twin pairs were separated into two groups: seven twins (12 newborn infants) with FLP (FLP group) and five twins (nine newborn infants) without FLP (non-FLP group). Gestational age, birthweight, and Apgar scores were significantly higher in the FLP group than that in the non-FLP group. Serum NT-proBNP levels at birth were significantly lower in the FLP group than in the non-FLP group [1425 pg/ml (range, 466-9560) vs. 29900 pg/ml (range, 7300-77900), respectively; p=0.0003]. The serum NT-proBNP levels of larger and smaller co-twins were significantly correlated with each other (r=0.750; p=0.026). In conclusion, serum NT-proBNP levels at birth are lower in MD twins with TTTS after FLP treatment than in those without FLP.

Increased volume of tracheal aspirate fluid predicts the development of bronchopulmonary dysplasia.

BACKGROUND: Elevated cytokine concentrations were observed in tracheal aspirate fluid (TAF) of infants on mechanical ventilation who subsequently developed bronchopulmonary dysplasia (BPD). However, there are few reports that systematically evaluate the amount of TAF as an indicator of BPD development. AIM: To clarify whether TAF volume during the first week of life predicts BPD development in extremely low gestational age newborns (ELGANs). STUDY DESIGN: We analyzed 51 infants, born at gestational age of <28 weeks and ventilated for more than 7 days after birth, among whom, 26 were diagnosed with BPD based on the clinical definition of oxygen dependence at 36 weeks postmenstrual age (BPD group) and 25 were included in the non-BPD group. Sum of TAF scores (STS) was calculated by semi-quantification of TAF volume at each suctioning and the suctioning frequency during the first week of life. RESULTS: STS was significantly higher in the BPD group than in the non-BPD group (median (interquartile range): 77 (29-126) vs. 28 (22-59), p<0.001). STS (cut-off, 60) with area under the curve in receiver operating analysis of 0.75 was significantly predictive of BPD development. Multivariate logistic regression analysis adjusted for perinatal characteristics showed that STS≥60 was a significant risk factor for BPD development (odds ratio, 7.50; confidence interval, 1.16-48.40, p=0.034). CONCLUSION: Increased TAF volume during the first week of life was an independent predictor for BPD development in ventilated ELGANs, indicating that increased pulmonary capillary permeability may influence the pathogenesis of BPD.

Effect of hydrocortisone therapy on severe leaky lung syndrome in ventilated preterm infants.

BACKGROUND: The aim of this study was (i) to determine the incidence and risk factors of severe leaky lung syndrome (sLLS), persistent pulmonary edema characterized by massive tracheal secretions and resistance to surfactant therapy, in extremely low gestational age newborns requiring ventilatory support; and (ii) to evaluate the effects of hydrocortisone (HC) therapy for sLLS on tracheal aspirate fluid (TAF) volume and ß2-microglobulin levels in TAF. METHODS: Infants born at <28 weeks gestation requiring ventilation beyond day of life (DOL) 7 were included. Daily TAF volume changes were assessed using a TAF scoring system. Levels of TAF ß2-microglobulin, an indicator of capillary leakage, were measured at DOL0, 7, before, and 4 days after starting HC therapy (started at 4 mg/kg/day; tapered for 1-3 weeks). RESULTS: Of the 54 infants enrolled, 24 (44%) were diagnosed with sLLS. Lower gestational age, lower birthweight, and higher TAF ß2-microglobulin levels at DOL7 were independent risk factors for sLLS. Seventeen infants with sLLS received HC therapy starting at DOL17 (median), with subsequent decreases in TAF volume and ß2-microglobulin levels. CONCLUSIONS: The incidence of sLLS, as defined in this study, was 44% in extremely low gestational age newborns requiring ventilator support beyond a week. HC therapy effectively reduced TAF volume and ß2-microglobulin levels, suggesting suppression of increased permeability of pulmonary capillaries in infants with sLLS.

Time-course effect of a single dose of hydrocortisone for refractory hypotension in preterm infants.

BACKGROUND: The aim of this study was to determine the time-course effect of a single dose of hydrocortisone (HC) on arterial blood pressure in extremely low gestational age newborns (ELGAN) with refractory hypotension during the first 3 days of life. METHODS: We carried out a matched case-control study of a cohort of 116 infants born between 23 and 27 weeks' gestation at a tertiary center. Twelve infants (10%) were treated with HC for refractory hypotension (HC group). HC was administered at a dose of 2 mg/kg when mean arterial pressure (MAP) was below 30 mmHg (25 mmHg for infants <25 weeks of gestational age) despite the use of inotropes and volume expanders. Changes in the MAP after the HC administration were compared with those in the infants who were not treated with HC and matched for gestational age (Control group). RESULTS: The mean MAP before administration of HC was significantly lower in the HC group than that in the control group (24.0 ± 3.2 vs 33.3 ± 4.8 mmHg; P < 0.01). The mean MAP in the HC group increased significantly at 2 h after HC treatment, and then reached levels comparable to those in the Control group at 5 h (31.3 ± 4.0 vs 33.9 ± 4.7 mmHg; P= 0.18), and remained at normal levels until 12 h after HC treatment. CONCLUSION: A single dose of HC treatment induces a rapid and sustained improvement in blood pressure in ELGAN with refractory hypotension.