Chromosomal copy number analysis of products of conception by conventional karyotyping and next-generation sequencing.

PURPOSE: Chromosomal abnormalities are a major cause of spontaneous abortion, and conventional G-banded karyotyping (G-banding) is mainly utilized for chromosomal analysis. Recently, next-generation sequencing (NGS) has been introduced for chromosomal analysis. Here, we aimed to investigate the applicability and utility of NGS-based chromosomal analysis of products of conception (POC) on chorionic villus samples from spontaneous abortion. METHODS: The results of chromosomal analysis of 7 chorionic villus samples from spontaneous abortion were compared between conventional G-banding and NGS-based chromosomal copy number analysis. Age dependency and frequency of each chromosomal aneuploidy were evaluated for 279 cases analyzed by NGS. RESULTS: Excluding two cases (culture failure and maternal cell contamination), the results were consistent between G-banding and NGS. For cases analyzed by NGS, the rate of chromosomal abnormality increased in a maternal age-dependent manner. The frequency of each chromosomal aneuploidy detected by NGS was almost the same as that previously reported. Finally, NGS analysis was possible for difficult cases by G-banding analysis, such as culture failure, maternal cell contamination, long-term storage cases, and low cell number. CONCLUSIONS: Chromosome analysis using NGS not only obtains comparable results to conventional G-banding, but also can analyze POC more accurately and efficiently.

Evaluation of Neutrophil Activation Status According to the Phenotypes of Adult Asthma.

PURPOSE: Neutrophils are considered key effector cells in the pathogenic mechanisms of airway inflammation in asthma. This study assessed the activation status of neutrophils in adult asthmatics, and the therapeutic potential of FTY720, a synthetic sphingosine-1-phosphate analog, on activated neutrophils using an in vitro stimulation model. METHODS: We isolated peripheral blood neutrophils (PBNs) from 59 asthmatic patients (including 20 aspirin-exacerbated respiratory disease [AERD] and 39 aspirin-tolerant asthma [ATA] groups). PBNs were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharide (LPS) and their activation status was determined based on reactive oxygen species (ROS) production, cell surface expression of CD11b, interleukin (IL)-8 and matrix metallopeptidase (MMP)-9 release. PBNs were primed with FTY720 to evaluate its anti-inflammatory action. RESULTS: In vitro PBN stimulation with fMLP or LPS induced a significant increase in ROS/CD11b/IL-8/MMP-9 levels (P < 0.05 for all). In asthmatics, fMLP-induced ROS level was significantly correlated with values of forced expiratory volume in 1 second/forced vital capacity (r = -0.278; P = 0.036), maximal mid-expiratory flow (r = -0.309; P = 0.019) and PC20 methacholine (r = -0.302; P = 0.029). In addition, ROS levels were significantly higher in patients with AERD and in those with severe asthma than in those with ATA or non-severe asthma (P < 0.05 for all). FTY720 treatment could suppress ROS/CD11b levels, and LPS-induced IL-8 and MMP-9 levels (P < 0.05 for all). Responders to FTY720 treatment had significantly higher neutrophil counts in sputum (P = 0.004). CONCLUSIONS: Our findings suggest a useful in vitro PBN stimulation model for evaluating the neutrophil functional status and the therapeutic potentials of neutrophil-targeting candidates in asthmatics.

Prediction of Postnatal Growth Failure among Very Low Birth Weight Infants.

Postnatal growth failure (PGF) in preterm infants remains an important clinical issue. In this study, we analysed the incidence of PGF among very low birth weight (VLBW) infants and evaluated the risk factors for PGF based on the data of 2799 VLBW infants obtained from the Korean Neonatal Network database from 2013 to 2014. PGF was defined as a decrease in weight Z score between birth and discharge of more than -1.28 using the Fenton growth charts. Risk factors were evaluated in relation to birth weight for gestational age, namely small (SGA) or appropriate (AGA) for gestational age, using propensity score matching used for between-group differences. The overall incidence of PGF was 45.5%, with a rate of 68.9% in the SGA group and 36.2% in the AGA group. PGF was negatively correlated with gestation and birth weight; additionally, PGF was associated with a higher incidence of co-morbidities. Predictors of PGF in the SGA group were respiratory distress syndrome and days to attain 100 mL/kg of enteral feeding. The only predictor of PGF in the AGA group was days to attain 100 mL/kg of enteral feeding. Early initiation and aggressive progression of enteral nutrition may decrease the incidence of PGF.

Analysis of National Pharmacovigilance Data Associated with Statin Use in Korea.

The aim of this study was to evaluate clinical manifestations, age distribution and risk factors of adverse drug reactions (ADRs) of statins. Korean Adverse Event Reporting System (KAERS) database records (July 2009-June 2014) on statin-treated adults were used. ADRs classified as 'certain', 'probable' and 'possible' based on the WHO-Uppsala Monitoring Centre criteria were analysed. The frequency of ADRs was compared between adults (18-64 years) and older people (age ≥65 years) groups. In total, 2161 ADRs from 1690 patients (579, 34.3% older people) were included for analysis. Mean patient age and ADRs per patient were 60.46 ± 12.72 years and 1.28, respectively. ADRs were reported with atorvastatin (48.4%), rosuvastatin (23.0%), pitavastatin (10.4%) and simvastatin (9.1%). The frequent ADRs were gastrointestinal (421 events, 19.5%), musculoskeletal (331, 15.3%), skin (312, 14.4%) and hepatobiliary disorders (286, 13.2%). Skin disorders were significantly more frequent in adults compared to those in older patients (16.3% versus 12.4%, p = 0.021). Common clinical symptoms were myalgia (263 events, 12.2%), dyspepsia (133, 6.2%) and pruritus (103, 4.8%). Myalgia was more frequently reported in adults (12.7% versus 9.5%, p = 0.039) and dizziness was more frequent in older people (3.4% versus 5.8%, p = 0.015). According to KAERS data, leading statin ADRs were gastrointestinal and musculoskeletal disorders. Myalgia and dyspepsia were the common clinical symptoms.

Long-chain fatty acid triglyceride (TG) metabolism disorder impairs male fertility: a study using adipose triglyceride lipase deficient mice.

STUDY QUESTION: Does the deletion of adipose triglyceride lipase (Atgl) gene impair male fertility? SUMMARY ANSWER: The deletion of Atgl gene impaired male fertility but the effect was partially reversed by a low long-chain triglyceride (TG) diet. WHAT IS KNOWN ALREADY: ATGL specifically hydrolyses long-chain fatty acid TG to diacylglycerol and a high level of expression of ATGL in testes has been reported. However, the role of ATGL in male fertility is unknown. STUDY DESIGN, SIZE, DURATION: To investigate the effect of deletion of Atgl gene on male fertility, cauda epididymides and testes were collected from wild-type, heterozygous and homozygous Atgl-deficient mice at 10 weeks of age and epididymal sperm analysis and histological analysis of the testes were performed. To investigate whether a medium-chain triglycerides (MCTs) replacement diet mitigated the impaired male fertility by deletion of Atgl gene, homozygous Atgl-deficient mice were fed a MCT replacement diet, or a standard diet including long-chain triglycerides (LCTs) in a control group, for 6 weeks from 5 weeks of age (n = 22). The systematic and local effects of the MCT replacement diet on spermatogenesis and sperm maturation in the epididymis were analyzed at 10 weeks of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Hematoxylin and eosin staining in paraffin-embedded sections of testes and Oil Red O staining in frozen sections of testes were performed. The epididymal sperm concentrations were analyzed. Statistical analyses were performed using the Student's t-test or Mann-Whitney U test with Shapiro-Wilk Normality test. MAIN RESULTS AND THE ROLE OF CHANCE: Although heterozygous mice were fertile and showed a similar number of epididymal total and motile sperm concentrations to wild-type mice, the deletion of Atgl gene in homozygous mice led to accumulation of TG deposits in testes and impaired spermatogenesis. The deletion of Atgl gene also impaired the sperm maturation process required for sperm to acquire the ability to move forward in the epididymis. The MCT replacement diet for 6 weeks increased the plasma level of non-esterified fatty acid (NEFA) (1.5-fold, P = 0.005), but not the plasma total cholesterol (T-Cho) and TG levels. In testes, the MCT replacement diet decreased the number of Oil Red O stain positive vacuoles (-40%, P < 0.001) and increased testis tissue weight (1.1-fold, P = 0.012), total sperm concentration (1.5-fold, P = 0.011) and motile sperm concentration (2.1-fold, P < 0.001) compared to the control group. However, there was no significant change in the sperm survival rate between the two groups. LARGE SCALE DATA: None. LIMITATIONS REASONS FOR CAUTION: One previous study reported that Atgl-deficient male mice were fertile. In most studies heterozygous Atgl(+/-) mice were used to generate homozygous Atgl-deficient Atgl(-/-) mice. Although the same gene targeting mice were used in this study and the formation of vaginal plugs were observed after mating with Atgl(-/-) male mice, there were no pregnant wild-type mice observed after mating with Atgl(-/-) male mice. WIDER IMPLICATIONS OF THE FINDINGS: Local TG metabolism in the male reproductive system could affect spermatogenesis and sperm motility in men. The MCT replacement diet could be an effective therapy for idiopathic non-obstructive oligozoospermia or asthenozoospermia in men with low levels of serum NEFA. STUDY FUNDING AND COMPETING INTEREST(S): This study was supported in part by the Japan Society for the Promotion of Science JSPS KAKENHI Grant (Nos. JP24249080, JP25462557, JP16K11086). The authors declare no conflict of interest.

Effect of lipid metabolism on male fertility.

Cholesterol and lipid homeostasis is important for male fecundity. However, the plasma total cholesterol level does not reflect sperm concentration and motility. Adipose tissue in mammals is the main tissue contributing to the dynamic equilibrium of lipid synthesis and catabolism. However, recent studies suggested that local lipolysis has an important role in male fertility. If so, which plasma lipid metabolism parameter reflects sperm concentration and motility? Leptin receptor-deficient db/db mice show hyperphagia, hyperglycemia, hypercholesterolemia, hypertriglyceridemia, morbid obesity and are reported to be infertile. Impairment of spermatogenesis and sperm motility in db/db mice was observed in our experiments. Medium-chain triglycerides (MCTs) are more quickly metabolized as fuel compared to long-chain triglycerides (LCTs). If the LCTs are replaced with MCTs in the diet, even in hyperphagia, does it affect spermatogenesis and sperm motility? In this study, we investigated the effect of a MCT replacement diet on sperm parameters using db/db mice. Six weeks of MCT replacement diet improved not only spermatogenesis but also the maturation processes in the epididymis for sperm to acquire the ability to move forward. Plasma chylomicron and large VLDL levels showed positive correlation with total and motile sperm concentrations. The MCT replacement diet could be an effective treatment for idiopathic non-obstructive oligozoospermia or asthenozoospermia men with low levels of chylomicron and large VLDL.

The protein phosphatase 2C clade A protein OsPP2C51 positively regulates seed germination by directly inactivating OsbZIP10.

Protein phosphatase 2C clade A members are major signaling components in the ABA-dependent signaling cascade that regulates seed germination. To elucidate the role of PP2CA genes in germination of rice seed, we selected OsPP2C51, which shows highly specific expression in the embryo compared with other protein phosphatases based on microarray data. GUS histochemical assay confirmed that OsPP2C51 is expressed in the seed embryo and that this expression pattern is unique compared with those of other OsPP2CA genes. Data obtained from germination assays and alpha-amylase assays of OsPP2C51 knockout and overexpression lines suggest that OsPP2C51 positively regulates seed germination in rice. The expression of alpha-amylase synthesizing genes was high in OsPP2C51 overexpressing plants, suggesting that elevated levels of OsPP2C51 might enhance gene expression related to higher rates of seed germination. Analysis of protein interactions between ABA signaling components showed that OsPP2C51 interacts with OsPYL/RCAR5 in an ABA-dependent manner. Furthermore, interactions were observed between OsPP2C51 and SAPK2, and between OsPP2C51 and OsbZIP10 and we found out that OsPP2C51 can dephosphorylates OsbZIP10. These findings suggest the existence of a new branch in ABA signaling pathway consisting of OsPYL/RCAR-OsPP2C-bZIP apart from the previously reported OsPYL/RCAR-OsPP2C-SAPK-bZIP. Overall, our result suggests that OsPP2C51 is a positive regulator of seed germination by directly suppressing active phosphorylated OsbZIP10.

Functional characterization and reconstitution of ABA signaling components using transient gene expression in rice protoplasts.

The core components of ABA-dependent gene expression signaling have been identified in Arabidopsis and rice. This signaling pathway consists of four major components; group A OsbZIPs, SAPKs, subclass A OsPP2Cs and OsPYL/RCARs in rice. These might be able to make thousands of combinations through interaction networks resulting in diverse signaling responses. We tried to characterize those gene functions using transient gene expression for rice protoplasts (TGERP) because it is instantaneous and convenient system. Firstly, in order to monitor the ABA signaling output, we developed reporter system named pRab16A-fLUC which consists of Rab16A promoter of rice and luciferase gene. It responses more rapidly and sensitively to ABA than pABRC3-fLUC that consists of ABRC3 of HVA1 promoter in TGERP. We screened the reporter responses for over-expression of each signaling components from group A OsbZIPs to OsPYL/RCARs with or without ABA in TGERP. OsbZIP46 induced reporter most strongly among OsbZIPs tested in the presence of ABA. SAPKs could activate the OsbZIP46 even in the ABA independence. Subclass A OsPP2C6 and -8 almost completely inhibited the OsbZIP46 activity in the different degree through the SAPK9. Lastly, OsPYL/RCAR2 and -5 rescued the OsbZIP46 activity in the presence of SAPK9 and OsPP2C6 dependent on ABA concentration and expression level. By using TGERP, we could characterize successfully the effects of ABA dependent gene expression signaling components in rice. In conclusion, TGERP represents very useful technology to study systemic functional genomics in rice or other monocots.

Insulin pump therapy in transient neonatal diabetes mellitus.

Neonatal diabetes mellitus (NDM) is a rare disease requiring insulin treatment. Its treatment is primarily focused on maintaining adequate glycemic control and avoiding hypoglycemia. Although insulin pump therapy is frequently administered to adults and children, there is no consensus on the use of insulin pumps in NDM. A 10 day-old female infant was referred to us with intrauterine growth retardation and poor weight gain. Hyperglycemia was noted, and continuous intravenous insulin infusion was initiated. However, the patient's serum glucose levels fluctuated widely, and maintaining the intravenous route became difficult within the following weeks. Continuous subcutaneous insulin infusion with an insulin pump was introduced on the twenty-fifth day of life, and good glycemic control was achieved without any notable adverse effects including hypoglycemia. We suggest that the insulin pump is a safe and effective mode for treating NDM and its early adoption may shorten the length of hospital stays in patients with NDM.