Associations of prenatal antibiotic exposure and delivery mode on childhood asthma inception.

BACKGROUND: Prenatal antibiotic exposure and delivery mode may affect the gut microbiome in early life and influence the development of childhood asthma, but the combined effect of these 2 factors is unknown. OBJECTIVE: To identify the individual and combined effects of prenatal antibiotic exposure and delivery mode on the development of asthma in children and the potential mechanisms underlying these associations. METHODS: A total of 789 children from the Cohort for Childhood Origin of Asthma and Allergic Diseases birth cohort study were enrolled. Asthma was defined as a physician-confirmed diagnosis with asthma symptoms in the previous 12 months at age 7 years. Information on prenatal antibiotic exposure was obtained by mothers using a questionnaire. Logistic regression analysis was used. Gut microbiota analysis using 16S rRNA gene sequencing of fecal specimens obtained at 6 months was undertaken for 207 infants. RESULTS: Prenatal antibiotic exposure and cesarean section delivery (adjusted odds ratio [aOR], 95% confidence interval [CI], 5.70 [1.25-22.81] and 1.57 [1.36-6.14], respectively) were associated with childhood asthma, especially synergistically when compared with the vaginal delivery-prenatal antibiotic exposure reference group (aOR, 7.35; 95% CI, 3.46-39.61; Interaction P = .03). Prenatal antibiotic exposure was associated with childhood asthma with aORs 21.79 and 27.03 for 1 and 2 or more exposures, respectively. Considerable small-airway dysfunction (R5-R20 in impulse oscillometry) was observed with prenatal antibiotic exposure and cesarean section delivery, compared with those with spontaneous delivery without prenatal antibiotic exposure. There was no significant difference in the diversity of gut microbiota among the 4 groups. However, the relative abundance of Clostridium was significantly increased in infants with prenatal antibiotic exposure and delivered by means of cesarean section. CONCLUSION: Prenatal antibiotic exposure and delivery mode might modulate asthma development in children and small-airway dysfunction, potentially through early-life gut microbiota alterations.

Interaction of the TLR4 rs1927911 polymorphism with house dust mite sensitization in allergic rhinitis with its prognosis.

BACKGROUND: Sensitization to the house dust mite (HDM) plays important roles in the development of allergic rhinitis (AR). Toll-like receptor 4 (TLR4) is a key initiator of the innate immune system upon exposure to environmental factors. OBJECTIVE: The present study investigated the independent and interaction effects of HDM sensitization and TLR4 rs1927911 polymorphism on AR and its prognosis in children. METHODS: This study included 2,929 children (mean age, 7.8 yrs) from the Children's HEalth and Environmental Research study (CHEER), a prospective study with a 2-year-interval for 4 years. An ISAAC questionnaire was used with skin prick tests in all subjects. TaqMan genotyping was performed for TLR4 (rs1927911) polymorphism in 1,024 children. RESULTS: HDM sensitization increased risk of current AR (aOR, 2.50; 95% CI, 1.41-4.41; P for interaction = 0.005), current asthma at follow-up (aOR, 4.63; 95% CI, 2.41-8.88; P for interaction < 0.001) and allergic march (aOR, 2.57; 95% CI, 1.06-6.22; P for interaction = 0.002) by interacting with genotypes of TLR4 (rs1927911). HDM sensitization increased risk of persistence (aOR, 4.17; 95% CI, 1.77-9.83) and new diagnosis of AR (aOR, 2.48; 95% CI, 1.10-5.61), new sensitization to inhalant allergens (aOR, 10.67; 95% CI, 5.83-19.54), and new development of bronchial hyper-responsiveness (aOR, 5.29; 95% CI, 2.29-12.21) in children with CC genotype of TLR4 rs1927911. CONCLUSIONS: HDM sensitization affects AR and its prognosis by interacting with TLR4 rs1957911 polymorphism. The preventive and therapeutic strategies for AR in children need to be targeted in accordance with genetic susceptibility with HDM sensitization.

Ruminococcus gnavus ameliorates atopic dermatitis by enhancing Treg cell and metabolites in BALB/c mice.

BACKGROUND: Ruminococcus gnavus (R. gnavus) are mucin-degrading gut bacteria that play a key role in the early colonization of the gut by serving as endogenous sources of nutrients. They can also influence immune development. We had previously reported a lower abundance of R. gnavus in infants with atopic dermatitis (AD) compared with that in healthy subjects. However, the underlying mechanisms remain unclear. In this study, we investigated the effect of orally administered R. gnavus on antibiotic treatment-induced gut dysbiosis (and the underlying mechanism) in a mouse model of AD. METHODS: Four-week-old female BALB/C mice were administered antibiotic cocktails for 2 weeks. R. gnavus was orally administered throughout the study duration. At 6 weeks of age, AD was induced by epidermal sensitization with ovalbumin. AD phenotypes and systemic and gut immune responses were investigated. RESULTS: Orally administered R. gnavus significantly reduced AD-associated parameters (i.e., transepidermal water loss, clinical score, total serum immunoglobulin (Ig) E level, OVA-specific IgE level, and skin inflammation). R. gnavus treatment also resulted in significant downregulation of T helper 2-related cytokine mRNA and upregulation of interleukin (IL)-10 and Foxp3 in the skin. The population of CD4+ FOXP3+ T cells in mesenteric- and skin-draining lymph nodes and butyrate levels in the cecum increased in R. gnavus-administered AD mice. CONCLUSIONS: Immune modulation by orally administered R. gnavus may alleviate AD symptoms through the enhancement of regulatory T-cell counts and short-chain fatty acids production in AD mice.

Host-microbial interactions between PTGR2 and Bifidobacterium in the early life gut of atopic dermatitis children.

BACKGROUND: Gut microbiota dysbiosis is linked to the development and responses of the immune system and can play an important role in the onset of allergic diseases including atopic dermatitis (AD). This study investigated the association between host genetics and the gut microbiota in AD. METHODS: A global gene expression profiling of the gut epithelial colonocytes, genetic variations analysis, and the gut microbial composition analysis were performed. RESULTS: This study identified the upregulation of PTGR2 (p = .028), a gene involved in prostaglandin catalysis and inflammatory responses, as a potential risk factor for AD. In subsequent fine mapping analysis using 17 single nucleotide polymorphisms (SNPs) of PTGR2 in 864 Korean subjects (420 AD patients and 444 unaffected controls), several SNPs and haplotypes showed significant associations with AD and its SCORing AD (SCORAD) values (p = .002). To investigate host-microbial interactions, further gut microbiota data and genotypes were obtained from an independent cohort of 176 subjects (91 AD patients and 85 controls). From correlation analysis, a significantly negative association between SNP and Bifidobacterium abundance was observed in AD patients (p = .005). In additional observations of PTGR2-associated downstream molecules, NRF2 (p = .004) and several antioxidant genes (GSTT1, GCLC, GPX1; p < .05) showed significantly reduced expression in AD patients. CONCLUSIONS: Our current findings suggest that the interaction between PTGR2 dysregulated expression and a Bifidobacterium abundance affects a higher risk of AD and a more severe onset.

Food allergy in early childhood increases the risk of oral allergy syndrome in schoolchildren: A birth cohort study.

BACKGROUND: The level of pollen in Korea has increased over recent decades. Research suggests that oral allergy syndrome (OAS) may be more frequent in childhood than previously recognized. We aimed to investigate the prevalence and characteristics of OAS in children aged 6-10 years from a general-population-based birth cohort. METHODS: We analyzed 930 children from the cohort for childhood origin of asthma and allergic diseases (COCOA). Allergic diseases were diagnosed annually by pediatric allergists. The skin prick tests were performed with 14 common inhalant allergens and four food allergens for the general population of children aged 3 and 7 years. RESULTS: Of the 930 eligible children, 44 (4.7%) aged 6-10 years were diagnosed with OAS. The mean age at onset was 6.74 years. OAS prevalence was 7.2% among children with allergic rhinitis (AR) and 19.1% among those with pollinosis, depending on comorbidity. OAS was more prevalent in schoolchildren with atopic dermatitis, food allergy, and sensitization to food allergens and grass pollen in early childhood. In schoolchildren with AR, only a history of food allergy until the age of 3 years increased the risk of OAS (aOR 2.971, 95% CI: 1.159-7.615). CONCLUSION: Food allergy and food sensitization in early childhood were associated with OAS in schoolchildren with AR. Further study is required to elucidate the mechanism by which food allergy in early childhood affects the development of OAS.

Dog Ownership in Early Life Increased the Risk of Nonatopic Asthma in Children.

BACKGROUND: It is still debatable whether dog ownership during early childhood is a risk factor for the development of allergic diseases. OBJECTIVE: We investigated the association of dog ownership in early life with sensitization and asthma in childhood. METHODS: Data from the Cohort for Childhood Origin of Asthma and Allergic diseases were used to investigate the association between dog ownership at any time from pregnancy to 1 year of age and sensitization to aeroallergens at 3 and 7 years old, bronchial hyperresponsiveness (BHR), and asthma at 7 years old. We analyzed the cytokine levels in cord blood (CB) and indoor environmental measurement concentrations in the mother's residence obtained at 36 weeks of pregnancy. RESULTS: Sensitization to dogs at age 3 and 7 did not differ between dog ownership and nonownership, but dog ownership during early life decreased the risk of sensitization to aeroallergens at age 7 (aOR = 0.44, 95% CI 0.21-0.90). Dog ownership significantly increased the risk of nonatopic BHR (aOR = 2.86; 95% CI 1.32-6.21). In addition, dog ownership was associated with asthma, especially nonatopic asthma at 7 years old (aOR = 2.73, 95% CI 1.02-7.32; aOR = 7.05, 95% CI 1.85-26.90, respectively). There were no significant differences in the concentrations of IL-13 or interferon-γ in CB or indoor environmental measurements according to dog ownership during pregnancy. CONCLUSION: Early-life dog exposure in this birth cohort has been shown to reduce atopy but increase the risk of nonatopic BHR and nonatopic asthma at 7 years old.

Effect of early-life antibiotic exposure and IL-13 polymorphism on atopic dermatitis phenotype.

BACKGROUND: Although atopic dermatitis (AD) is associated with certain gene variants, the rapidly increasing incidence of AD suggests that environmental factors contribute to disease development. In this study, we investigated the association of AD incidence and phenotype with antibiotic exposure within 6 months of age, considering the dose administered and genetic risk. METHODS: This study included 1637 children from the COCOA cohort. Pediatric allergists assessed the presence of AD at each visit and obtained information about antibiotic exposure for more than 3 days. IL-13 (rs20541) polymorphism was genotyped by the TaqMan method. We stratified the AD phenotypes into four groups and used multinomial logistic regression models for analysis. RESULTS: Antibiotic exposure within 6 months of age was found to increase the risk of AD within 3 years of life (aOR = 1.40; 95% CI, 1.09-1.81) in dose-dependent manner. Antibiotic exposure more than twice increased the risk of the early-persistent AD phenotype (aOR = 2.50; 95% CI, 1.35-4.63). There was a weak interaction between genetic polymorphisms and environmental factors on the development of AD (p for interaction = 0.06). Children with the IL-13 (rs20541) GA + AA genotype have a higher risk of the early-persistent AD phenotype when exposed to antibiotics more than twice than those with the IL-13 (rs20541) GG genotype and without exposure to antibiotics (aOR = 4.73; 95% CI, 2.01-11.14). CONCLUSION: Antibiotic exposure within 6 months was related to the incidence of early-persistent AD and a dose-dependent increase in the incidence of AD in childhood, whose effect was modified by the IL-13 (rs20541) genotype.

Mid-pregnancy PM2.5 exposure affects sex-specific growth trajectories via ARRDC3 methylation.

Prenatal particulate matter <2.5 µm (PM2.5) is associated with adverse birth growth. However, the longitudinal growth impacts have been little studied, and no mechanistic relationships have been described. We investigated the association between prenatal PM2.5 exposure and growth trajectories, and the possible role of epigenetics. We enrolled 1313 neonates with PM2.5 data measured by ordinary kriging from the COhort for Childhood Origin of Asthma and allergic diseases, followed up at 1, 3, and 5 years to evaluate growth. Differential DNA methylation and pyrosequencing of cord blood leukocytes was evaluated according to the prenatal PM2.5 levels and birth weight (BW). PM2.5 exposure during the second trimester (T2) caused the lowest BW in both sexes, further adjusted for indoor PM2.5 levels [female, aOR 1.39 (95% CI 1.05-1.83); male, aOR 1.36 (95% CI 1.04-1.79)]. Bayesian distributed lag models with indoor PM2.5 adjustments revealed a sensitive window for BW effects at 10-26 weeks gestation, but only in females. Latent class mixture models indicated that a persistently low weight-for-height percentile trajectory was more prevalent in the highest PM2.5 exposure quartile at T2 in females, compared to a persistently high trajectory (36.5% vs. 20.3%, P = 0.022). Also, in the females only, the high PM2.5 and low BW neonates showed significantly greater ARRDC3 methylation changes. ARRDC3 methylation was also higher only in females with low weight at 5 years of age. Higher fetal PM2.5 exposure during T2 may cause a decreased growth trajectory, especially in females, mediated by ARRDC3 hyper-methylation-associated energy metabolism.

TNF-α (rs1800629) polymorphism modifies the effect of sensitization to house dust mite on asthma and bronchial hyperresponsiveness in children.

Asthma is a complex disease, with various genetic and environmental factors implicated in its development. Sensitization to the house dust mite (HDM) is closely linked with the development of respiratory allergies, including asthma. However, some children sensitized to HDM do not complain of any symptoms of respiratory allergies, even though HDM is correlated with an increased risk for developing asthma, suggesting the involvement of other factors. Tumor necrosis factor (TNF)-α is associated with the pathophysiologies of asthma in combination with its genetic polymorphism. The aim of the present study was to elucidate the associations between sensitization to HDM, polymorphism of TNF-α rs1800629, and asthma/bronchial hyperresponsiveness (BHR). Our results revealed that sensitization to HDM is associated with asthma diagnosis in lifetime, current asthma, and BHR in Korean children. Furthermore, the genetic polymorphism of TNF-a rs1800629 was found to modify and interact with these associations. This study suggests that prevention strategies for childhood asthma need to be targeted according to genetic susceptibility.

Association of IL13 genetic polymorphisms with atopic dermatitis: Fine mapping and haplotype analysis.

BACKGROUND: Although previous studies had reported an important role of interleukin 13 (IL13) and its genetic polymorphisms in atopic dermatitis (AD), many of these previous reports focused on the missense variant rs20541 (Gln144Arg) without fine mapping of the gene region. OBJECTIVE: To analyze the potential associations of other IL13 variants and their haplotypes with AD and assess total serum immunoglobulin E (IgE) levels. METHODS: We performed fine mapping of single-nucleotide polymorphisms (SNPs) within the IL13 gene in a pilot study of 495 children with AD and 444 healthy controls. Then, we conducted a replication study of 757 children with AD and 1620 healthy controls to evaluate the association between the rs20541 variant of IL13 and AD. RESULTS: In the pilot study, the rs20541 and rs1295685 SNPs in the 3'-untranslated region of IL13 had significant associations with AD (P < .001 and .01, respectively). In addition, 2 haplotypes (BL2_ht1 and BL2_ht2), which harbored the significant rs20541 and rs1295685 SNPs, had an association with AD (minimum P = .006). BL2_ht1 and BL2_ht2 had nominal signals associated with the total serum IgE levels (P < .05) but not with the severity of AD (P > .05). In the replication study, rs20541 was associated with the total serum IgE levels but not with the severity of AD. CONCLUSION: An additional IL13 gene SNP, rs1295685, has a strong linkage disequilibrium with rs20541, and its haplotypes are associated with AD and the total serum IgE levels.

Prenatal PM2.5 exposure and vitamin D-associated early persistent atopic dermatitis via placental methylation.

BACKGROUND: The effects of prenatal particulate matter with an aerodynamic diameter ranging from 0.1 µm to 2.5 µm (PM2.5) and vitamin D on atopic dermatitis (AD) phenotypes have not been evaluated. DNA methylation and cord blood (CB) vitamin D could represent a plausible link between prenatal PM2.5 exposure and AD in an offspring. OBJECTIVE: To determine the critical windows of prenatal PM2.5 exposure on the AD phenotypes, if vitamin D modulated these effects, and if placental DNA methylation mediated these effects on AD in offspring. METHODS: Mother-child pairs were enrolled from the birth cohort of the Cohort for Childhood Origin of Asthma and allergic diseases (COCOA) study. PM2.5 was estimated by land-use regression models, and CB vitamin D was measured by chemiluminescence immunoassay. AD was identified by the parental report of a physician's diagnosis. We defined the following 4 AD phenotypes according to onset age (by the age of 2 years) and persistence (by the age of 3 years): early-onset transient and persistent, late onset, and never. Logistic regression analysis and Bayesian distributed lag interaction model were used. DNA methylation microarray was analyzed using an Infinium Human Methylation EPIC BeadChip (Illumina, San Diego, California) in placenta. RESULTS: PM2.5 exposure during the first trimester of pregnancy, especially during 6 to 7 weeks of gestation, was associated with early-onset persistent AD. This effect increased in children with low CB vitamin D, especially in those with PM2.5 exposure during 3 to 7 weeks of gestation. AHRR (cg16371648), DPP10 (cg19211931), and HLADRB1 (cg10632894) were hypomethylated in children with AD with high PM2.5 and low CB vitamin D. CONCLUSION: Higher PM2.5 during the first trimester of pregnancy and low CB vitamin D affected early-onset persistent AD, and the most sensitive window was 6 to 7 weeks of gestation. Placental DNA methylation mediated this effect.

Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine ß-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine ß-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the ß-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine ß-casein gene.

Rapamycin rescues the poor developmental capacity of aged porcine oocytes.

Unfertilized oocytes age inevitably after ovulation, which limits their fertilizable life span and embryonic development. Rapamycin affects mammalian target of rapamycin (mTOR) expression and cytoskeleton reorganization during oocyte meiotic maturation. The goal of this study was to examine the effects of rapamycin treatment on aged porcine oocytes and their in vitro development. Rapamycin treatment of aged oocytes for 24 h (68 h in vitro maturation [IVM]; 44 h+10 µM rapamycin/24 h, 47.52±5.68) or control oocytes (44 h IVM; 42.14±4.40) significantly increased the development rate and total cell number compared with untreated aged oocytes (68 h IVM, 22.04±5.68) (p<0.05). Rapamycin treatment of aged IVM oocytes for 24 h also rescued aberrant spindle organization and chromosomal misalignment, blocked the decrease in the level of phosphorylated-p44/42 mitogen-activated protein kinase (MAPK), and increased the mRNA expression of cytoplasmic maturation factor genes (MOS, BMP15, GDF9, and CCNB1) compared with untreated, 24 h-aged IVM oocytes (p<0.05). Furthermore, rapamycin treatment of aged oocytes decreased reactive oxygen species (ROS) activity and DNA fragmentation (p<0.05), and downregulated the mRNA expression of mTOR compared with control or untreated aged oocytes. By contrast, rapamycin treatment of aged oocytes increased mitochondrial localization (p<0.05) and upregulated the mRNA expression of autophagy (BECN1, ATG7, MAP1LC3B, ATG12, GABARAP, and GABARAPL1), anti-apoptosis (BCL2L1 and BIRC5; p<0.05), and development (NANOG and SOX2; p<0.05) genes, but it did not affect the mRNA expression of pro-apoptosis genes (FAS and CASP3) compared with the control. This study demonstrates that rapamycin treatment can rescue the poor developmental capacity of aged porcine oocytes.

Ellagic acid treatment during in vitro maturation of porcine oocytes improves development competence after parthenogenetic activation and somatic cell nuclear transfer.

Ellagic acid (EA) is a natural polyphenol and a free radical scavenger with antioxidant properties. This study investigated the protective effects of EA during in vitro maturation (IVM) of porcine oocytes. To determine the optimal concentration, IVM medium was supplemented with various concentrations of EA. Treatment with 10 µM EA (10 EA) resulted in the highest cleavage rate, blastocyst formation rate, and total cell number per blastocyst and the lowest percentage of apoptotic cell in parthenogenetic blastocysts. In the 10 EA group, abnormal spindle and chromosome misalignment were rescued and the ratio of phosphorylated p44/42 to total p44/42 was increased. Furthermore, the reactive oxygen species and glutathione levels were significantly decreased and increased, respectively, and antioxidant genes (Nrf2, HO-1, CAT, and SOD1) were significantly upregulated in the 10 EA group. mRNA expression of developmental-related (CDX2, POU5F1, and SOX2) and anti-apoptotic (BCL2L1) genes was significantly upregulated in the 10 EA group, while mRNA expression of pro-apoptotic genes (BAK, FAS, and CASP3) was significantly downregulated. Ultimately, following somatic cell nuclear transfer, the blastocyst formation rate was significantly increased and the percentage of apoptotic cell in blastocysts was significantly decreased in the 10 EA group. In conclusion, addition of 10 EA to IVM medium improved oocyte maturation and the subsequent embryo development capacity through antioxidant mechanisms. These findings suggest that EA can enhance the efficiencies of assisted reproductive technologies.

Piperine improves the quality of porcine oocytes by reducing oxidative stress.

Oxidative stress caused by light and high temperature arises during in vitro maturation (IVM), resulting in low-quality embryos compared with those obtained in vivo. To overcome this problem, we investigated the influence of piperine (PIP) treatment during maturation of porcine oocytes on subsequent embryo development in vitro. Porcine oocytes were cultured in IVM medium supplemented with 0, 50, 100, 200, or 400 µM PIP. After parthenogenetic activation, the blastocyst (BL) formation was significantly higher and the apoptosis rate was significantly lower using 200 µM PIP-treated oocytes (200 PIP). In the 200 PIP group, the level of reactive oxygen species at the metaphase II stage was decreased, accompanied by an increased level of glutathione and increased expression of antioxidant processes (Nrf2, CAT, HO-1, SOD1, and SOD2). Consistently, chromosome misalignment and aberrant spindle organization were alleviated and phosphorylated p44/42 mitogen-activated protein kinase activity was increased in the 200 PIP group. Expression of development-related (CDX2, NANOG, POU5F1, and SOX2), anti-apoptotic (BCL2L1 and BIRC5), and pro-apoptotic (BAK, FAS, and CASP3) processes was altered in the 200 PIP group. Ultimately, embryo development was improved in the 200 PIP group following somatic cell nuclear transfer. These findings suggest that PIP improves the quality of porcine oocytes by reducing oxidative stress, which inevitably arises via IVM. In-depth mechanistic studies of porcine oocytes will improve the efficiencies of assisted reproductive technologies.

Effect of human adipose tissue-derived mesenchymal-stem-cell bioactive materials on porcine embryo development.

Human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) secrete bioactive materials that are beneficial for tissue repair and regeneration. In this study, we characterized human hAT-MSC bioactive material (hAT-MSC-BM), and examined the effect of hAT-MSC-BM on porcine embryo development. hAT-MSC-BM was enriched with several growth factors and cytokines, including fibroblast growth factor 2 (FGF2), vascular endothelial growth factor A (VEGFA), and interleukin 6 (IL6). Among the various concentrations and days of treatment tested, 10% hAT-MSC-BM treatment beginning on culture Day 4 provided the best environment for the in vitro growth of parthenogenetic porcine embryos. While the addition of 10% fetal bovine serum (FBS) increased the hatching rate and the total cell number of parthenogenetic porcine embryos compared with the control and hAT-MSC culture medium group, the best results were from the group cultured with 10% hAT-MSC-BM. Mitochondrial activity was also higher in the 10% hAT-MSC-BM-treated group. Moreover, the relative mRNA expression levels of development and anti-apoptosis genes were significantly higher in the 10% hAT-MSC-BM-treated group than in control, hAT-MSC culture medium, or 10% FBS groups, whereas the transcript abundance of an apoptosis gene was slightly lower. Treatment with 10% hAT-MSC-BM starting on Day 4 also improved the development rate and the total cell number of in vitro-fertilized embryos. This is the first report on the benefits of hAT-MSC-BM in a porcine embryo in vitro culture system. We conclude that hAT-MSC-BM is a new, alternative supplement that can improve the development of porcine embryos during both parthenogenesis and fertilization in vitro.

Post-death cloning of endangered Jeju black cattle (Korean native cattle): fertility and serum chemistry in a cloned bull and cow and their offspring.

To preserve Jeju black cattle (JBC; endangered native Korean cattle), a pair of cattle, namely a post-death cloned JBC bull and cow, were produced by somatic cell nuclear transfer (SCNT) in a previous study. In the present study, we examined the in vitro fertilization and reproductive potentials of these post-death cloned animals. Sperm motility, in vitro fertilization and developmental capacity were examined in a post-death cloned bull (Heuk Oll Dolee) and an extinct nuclear donor bull (BK94-13). We assessed reproductive ability in another post-death cloned cow (Heuk Woo Sunee) using cloned sperm for artificial insemination (AI). There were no differences in sperm motility or developmental potential of in vitro fertilized embryos between the post-death cloned bull and its extinct nuclear donor bull; however, the embryo development ratio was slightly higher in the cloned sperm group than in the nuclear donor sperm group. After one attempt at AI, the post-death cloned JBC cow became pregnant, and gestation proceeded normally until day 287. From this post-death cloned sire and dam, a JBC male calf (Heuk Woo Dolee) was delivered naturally (weight, 25 kg). The genetic paternity/maternity of the cloned JBC bull and cow with regard to their offspring was confirmed using International Society for Animal Genetics standard microsatellite markers. Presently, Heuk Woo Dolee is 5 months of age and growing normally. In addition, there were no significant differences in blood chemistry among the post-death cloned JBC bull, the cow, their offspring and cattle bred by AI. This is the first report showing that a pair of cattle, namely, a post-death cloned JBC bull and cow, had normal fertility. Therefore, SCNT can be used effectively to increase the population of endangered JBC.

Effect of Glycosaminoglycans on In vitro Fertilizing Ability and In vitro Developmental Potential of Bovine Embryos.

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 µg/ml of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

Effect of Supplementation of Cryoprotectant Solution with Hydroxypropyl Cellulose for Vitrification of Bovine Oocytes.

The technology of successful cryopreservation is a very important factor in research and commercial applications. However, the survival and development of the vitrified-thawed (VT) oocytes are lower than those of non-vitrified-thawed (non-VT) oocytes. This study investigated the effect of the addition of hydroxypropyl cellulose (HPC) to a vitrification solution of bovine oocytes. For the vitrification, bovine metaphase II oocytes were pretreated with a solution containing 10% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 5 min, then exposed to a solution containing 30% ethylene glycol supplemented with 0, 10, 50, or 100 µg/mL HPC for 30 sec, and then directly plunged into liquid nitrogen. Oocytes exposed to 0, 10, 50, and 100 µg/mL HPC were named the 0, 10, 50, and 100 HPC groups, respectively. Samples were thawed via sequential incubation in Dulbecco's phosphate-buffered saline (D-BPS) supplemented with 10% fetal bovine serum and decreasing concentrations of sucrose (1, 0.5, 0.25, and 0.125 M) for 1 min each time. After thawing, VT oocytes were treated at 0.05% hyaluronidase, and cumulus cells were removed by mechanical pipetting. The oocytes were washed with HEPES-buffered Tyrode's medium and incubated in a droplet of previously cultured in vitro maturation medium for 1 h to recover. The survival rate of the oocytes was significantly higher in the 50 HPC group (84.2%) than in the 0 (75.4%), 10 (80.4%), and 100 (75.5%) HPC groups. The reactive oxygen species (ROS) levels of the non-VT and 50 HPC groups were lower than the 0, 10, and 100 HPC groups. The mRNA levels of proapoptotic genes (Bax) were lower in the non-VT, 0, and 50 HPC groups than in the other groups. The mRNA expression levels of antiapoptotic genes (BCl2) was higher in the non-VT than in the other groups. The mRNA level of a stress-related gene (Hsp70) was lower in the 50 HPC than in the other groups. At day 8, the developmental capacity of embryos obtained via parthenogenetic activation (PA) was determined in the non-VT, 0 HPC, and 50 HPC groups. The cleavage rate of the non-VT group was significantly higher, but the blastocyst development rate and total cell number per blastocyst did not significantly differ between the non-VT and 50 HPC groups. The mRNA levels of proapoptotic genes (Bax and Caspase-3) and a stress-related gene (Hsp70) were higher in the 0 HPC group than in the non-VT and 50 HPC groups. In conclusion, supplementation of vitrification solution with HPC improves the survival rate of VT bovine oocytes and the development capacity of embryos derived from these oocytes via PA.

Atopic Dermatitis With Coexisting Food Allergy in Early Life Is Associated With Childhood Asthma.

PURPOSE: Atopic dermatitis (AD) and food allergy (FA) are associated with respiratory comorbidities, in the concept of 'atopic march.' However, children with AD and a coexisting FA have various disease courses, and the mechanism of atopic march remains unclear. In this study, we investigated whether the phenotype of AD with coexisting FA in early life affected asthma or allergic rhinitis (AR) in school children. METHODS: A total of 1,579 children from the Panel Study on Korean Children (PSKC) cohort were followed-up in 2013. The participants diagnosed with AD in this cohort were classified by the age of AD onset and persistence as well as FA history. We compared the presence of comorbidities-asthma and rhinitis-among different AD phenotypes. RESULTS: Asthma and AR with current symptoms within 12 months at age 6-8 years were associated with early-onset persistent AD phenotype, regardless of coexisting FA. AD with FA conferred a higher risk of recent wheezing at 8 years of age than AD without FA (adjusted odds ratio, 8.09; 95% confidence interval, 2.54-25.76). Children with early-onset persistent AD with FA manifested a distinctive trajectory with a higher prevalence of wheezing and AR at age 5-8 years than those without AD. CONCLUSIONS: AD with FA in early life is strongly associated with asthma and AR in school children, and the early-onset persistent AD with FA had a strong additive effect on the risk of asthma at school age. Classifying AD phenotypes regarding FA in early life will help predict and prevent asthma and AR in school children.