Swim-up method is superior to density gradient centrifugation for preserving sperm DNA integrity during sperm processing.

Purpose: This study aimed to evaluate the effects of swim-up and density gradient centrifugation methods on sperm DNA fragmentation. Methods: Nineteen normozoospermic patient samples with ≥100 × 106 motile sperms were included in this study. Sperm DNA fragmentation, progressive motility, and progressive motile sperm number were measured before and after the swim-up method or density gradient centrifugation. Results: Sperm DNA fragmentation was not statistically different between swim-up-(14.4 ± 2.1%, p = 0.32) and density gradient centrifugation-processed (25.0 ± 3.0%, p = 0.20) and unprocessed semen samples (19.2 ± 1.9%). Sperm DNA fragmentation was significantly lower in swim-up-than in density gradient centrifugation-processed samples (p < 0.05). Sperm progressive motility was significantly higher (p < 0.05) in swim-up-(92.9 ± 1.0%) and density gradient centrifugation-processed (81.3 ± 2.0%) samples, with the former being higher, than in unprocessed semen samples (53.1 ± 3.7%). The recovery rate of progressive motile sperms was significantly lower in swim-up-(9.7 ± 1.4%) than in density gradient centrifugation-processed samples (17.2 ± 1.8%, p < 0.05). Conclusions: The swim-up method is superior to density gradient centrifugation, evidenced by less sperm DNA fragmentation and higher sperm progressive motility. The recovery rate of progressive motile sperms was better after density gradient centrifugation than after swim-up.

[A Case of Retroperitoneal Bronchogenic Cyst Diagnosed by Fluorescence in situ Hybridization].

A 50-year-old woman was referred to our hospital for consultation for a suspected left adrenal tumor detected by ultrasonography during a health check. Computed tomography and magnetic resonance imaging revealed a 4.7×3.4 cm tumor in the retroperitoneal space near the adrenal gland. The patient subsequently underwent laparoscopic tumor resection. Using fluorescence in situ hybridization (FISH), the resected tumor was diagnosed as a retroperitoneal bronchial cyst. Here we present a case of a definitive diagnosis of a retroperitoneal bronchial cyst using FISH, and review the cases of retroperitoneal bronchial cyst in the literature.

[A Case of Renal Anastomosing Hemangioma].

A 65-year-old man was found to have a 1.7 cm right renal mass by follow-up abdominal computed tomography for left total nephrectomy after a traffic accident. The renal mass progressed slowly to 2.2 cm in three years and enhanced magnetic resonance imaging revealed marked T2 weighting hyperintensity of the lesion. Although a radiologist (TK) suggested the diagnosis renal anastomosing hemangioma preoperatively, we could not deny the possibility of renal cell carcinoma completely. Therefore, the patient underwent robot-assisted laparoscopic partial nephrectomy. The tumor was successfully removed without any renal arterial clamping or parenchymal excision. Histopathologically, the lesion was composed of capillary-size blood vessels lined by a single layer of endothelial cells, and was diagnosed as a renal anastomosing hemangioma. There were no signs of postoperative recurrence during the 3 month follow-up.

Brain ventriculomegaly in Down syndrome mice is caused by Pcp4 dose-dependent cilia dysfunction.

Down syndrome is a leading cause of congenital intellectual disability caused by an additional copy of the chromosome 21. Patients display physiological and morphological changes affecting the brain and its function. Previously we showed that Ts1Cje and Ts2Cje, Down syndrome mouse models carrying overlapping trisomic segments of different length, show similar ventriculomegaly and neurogenesis dysfunction leading to the hypothesis of a cause-consequence relationship between these phenotypes. However, we here discovered that Ts1Rhr Down syndrome model, carrying an even shorter trisomic segment, was sufficient to trigger ventricular enlargement and ependymal cilia beating deficiency without affecting neurogenesis. We further found that Pcp4 gene on the Ts1Rhr trisomic segment is expressed in ependymal cells, and its resumption to two copies rescued both ventricular enlargement and cilia dysfunction in Ts1Rhr mice. This work underlines a Pcp4-dependent ciliopathy in Down syndrome brain affecting cerebrospinal fluid flow.

Potentiation of excitatory synaptic transmission ameliorates aggression in mice with Stxbp1 haploinsufficiency.

Genetic studies point to a major role of de novo mutations in neurodevelopmental disorders of intellectual disability, autism spectrum disorders, and epileptic encephalopathy. The STXBP1 gene encodes the syntaxin-binding protein 1 (Munc18-1) that critically controls synaptic vesicle exocytosis and synaptic transmission. This gene harbors a high frequency of de novo mutations, which may play roles in these neurodevelopmental disorders. However, the system and behavioral-level pathophysiological changes caused by these genetic defects remain poorly understood. Constitutional (Stxbp1+/-), dorsal-telencephalic excitatory (Stxbp1fl/+/Emx), or global inhibitory neuron-specific (Stxbp1fl/+/Vgat) mice were subjected to a behavioral test battery examining locomotor activity, anxiety, fear learning, and social interactions including aggression. Furthermore, measurements of local field potentials in multiple regions of the brain were performed. Stxbp1+/- male mice exhibited enhanced aggressiveness and impaired fear learning associated with elevated gamma activity in several regions of the brain including the prefrontal cortex. Stxbp1fl/+/Emx mice showed fear-learning deficits, but neither Stxbp1fl/+/Emx nor Stxbp1fl/+/Vgat mice showed increased aggressiveness. Pharmacological potentiation of the excitatory transmission at active synapses via the systemic administration of ampakine CX516, which enhances the excitatory postsynaptic function, ameliorated the aggressive phenotype of Stxbp1+/- mice. These findings suggest that synaptic impairments of the dorsal telencephalic and subcortical excitatory neurons cause learning deficits and enhanced aggression in Stxbp1+/- mice, respectively. Additionally, normalizing the excitatory synaptic transmission is a potential therapeutic option for managing aggressiveness in patients with STXBP1 mutations.

DYRK1A-haploinsufficiency in mice causes autistic-like features and febrile seizures.

Mutations and copy number variants affecting DYRK1A gene encoding the dual-specificity tyrosine phosphorylation-regulated kinase 1A are among the most frequent genetic causes of neurodevelopmental disorders including autism spectrum disorder (ASD) associated with microcephaly, febrile seizures and severe speech acquisition delay. Here we developed a mouse model harboring a frame-shift mutation in Dyrk1a resulting in a protein truncation and elimination of its kinase activity site. Dyrk1a+/- mice showed significant impairments in cognition and cognitive flexibility, communicative ultrasonic vocalizations, and social contacts. Susceptibility to hyperthermia-induced seizures was also significantly increased in these mice. The truncation leading to haploinsufficiency of DYRK1A in mice thus recapitulates the syndromic phenotypes observed in human patients and constitutes a useful model for further investigations of the mechanisms leading to ASD, speech delay and febrile seizures.

Effect of blastocyst shrinkage on assisted reproductive outcomes: a retrospective cohort study describing a new morphological evaluation of blastocyst pre-vitrification and post-warming.

BACKGROUND: The failure of frozen-thawed blastocysts to re-expand adequately within a few hours after warming has been reported to have a negative impact on assisted reproductive technology (ART) outcomes. However, the extent to which this failure truly affects ART outcomes has not yet been presented in a manner that is easily understandable to medical practitioners and patients. This study aimed to assess the effects of blastocyst shrinkage on ART outcomes and determine a more effective morphological evaluation approach for use in clinical settings. METHODS: This retrospective observational cohort study of frozen-thawed blastocyst transfer cycles was conducted from April 2017 to March 2022. Overall, 1,331 cycles were eligible for inclusion, of which 999 were good-quality blastocysts (GQB) and 332 were non-good-quality blastocysts (non-GQB). All frozen-thawed blastocyst transfer cycles performed during the specified study period were included in the study. Exclusion criteria were established to mitigate potential sources of bias as these cycles could impact implantations. We calculated rates and age-adjusted odds ratios of implantation, clinical pregnancy, ongoing pregnancy, and live birth of the re-expansion group, which showed sufficient expansion, and shrinkage group, which showed insufficient expansion. We also calculated the implantation, clinical pregnancy, ongoing pregnancy, and live birth rates of the re-expansion and shrinkage groups for each morphological scoring system parameter. RESULTS: A reduced ART outcome was observed with use of blastocysts with shrinkage after vitrification/warming. The age-adjusted odds ratios for implantation, clinical pregnancy, ongoing pregnancy, and live birth were lower in the shrinkage group than in the re-expansion group. CONCLUSIONS: This study examined the adverse effect of blastocyst shrinkage after warming and recovery culturing on reproductive outcomes in a clinically useful manner by retrospectively examining a substantial number of frozen-thawed embryo transfer cycles. The study findings can possibly reduce concerns regarding over- or under-estimation of blastocyst implantation by allowing providers and patients to refer to the data.

Enlarged brain ventricles and impaired neurogenesis in the Ts1Cje and Ts2Cje mouse models of Down syndrome.

Down syndrome (DS) is the most common cause of mental retardation. Although structural and neurogenic abnormalities have been shown in the brains of DS patients, the molecular etiology is still unknown. To define it, we have performed structural and histological examinations of the brains of Ts1Cje and Ts2Cje, 2 mouse models for DS. These mice carry different length of trisomic segments of mouse chromosome 16 that are orthologous to human chromosome 21. At 3 months of age, ventricular enlargements were observed in both Ts1Cje and Ts2Cje brains at a similar degree. Both mice also showed decreases of the number of doublecortin-positive neuroblasts and thymidine-analog BrdU-labeled proliferating cells in the subventricular zone of the lateral ventricles (LVs) and in the hippocampal dentate gyrus at a similar degree, suggesting impaired adult neurogenesis. Additionally, at embryonic day 14.5, both strains of mice, when compared with diploid littermates, had smaller brains and decreased cortical neurogenesis that could possibly contribute to the ventricular enlargements observed in adulthood. Our findings suggest that the trisomic segment of the Ts1Cje mouse, which is shared with Ts2Cje, contains the genes that are responsible for these abnormal phenotypes and could be relevant to the mental retardation associated with DS.

DSCAM deficiency causes loss of pre-inspiratory neuron synchroneity and perinatal death.

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam(-/-)) died within 24 h after birth. Whole-body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Furthermore, a medulla-spinal cord preparation of Dscam(-/-) mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory neurons located in the rostral ventrolateral medulla and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam(-/-) mice. Dscam(+/-) mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam(-/-) mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner.

Central respiratory failure in a mouse model depends on the genetic background of the host.

To define the mechanisms of human congenital central respiratory failure, we are examining gene-deficient mice with central respiratory failure. However, the influence of the genetic background of the mice may play an important role in the phenotype of the mice. Therefore, we examined developmental respiratory adaptation in several mouse strains. Neonatal mice from P0 to P3 were examined by whole-body plethysmography and the electro- physiological analysis using brainstem-spinal cord preparations. Our results show that respiratory maturation becomes progressively fixed after birth and that the rate of progression depends on the genetic background of the mice. In particular, the progression of C57BL/6 mice was delayed compared to that of BALB/c mice.

Loss of pre-inspiratory neuron synchroneity in mice with DSCAM deficiency.

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam (-/-)) died within 24 hours after birth. Whole body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Further, a medulla-spinal cord preparation of Dscam (-/-) mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory (Pre-I) neurons located in the rostral ventrolateral medulla (RVLM) and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam (-/-) mice. Dscam (+/-) mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam (-/-) mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner. These results have been published (Amano et al. 2009).

Impairment of spatial memory accuracy improved by Cbr1 copy number resumption and GABAB receptor-dependent enhancement of synaptic inhibition in Down syndrome model mice.

Down syndrome is a complex genetic disorder caused by the presence of three copies of the chromosome 21 in humans. The most common models, carrying extra-copies of overlapping fragments of mouse chromosome 16 that is syntenic to human chromosome 21, are Ts2Cje, Ts1Cje and Ts1Rhr mice. In electrophysiological analyses using hippocampal slices, we found that the later phase of the depolarization during tetanic stimulation, which was regulated by GABAB receptors, was significantly smaller in Ts1Cje and Ts2Cje mice than that in WT controls but not in Ts1Rhr mice. Furthermore, isolated GABAB receptor-mediated inhibitory synaptic responses were larger in Ts1Cje mice. To our knowledge, this is the first report that directly shows the enhancement of GABAB receptor-mediated synaptic currents in Ts1Cje mice. These results suggest that GABAB receptor-mediated synaptic inhibition was enhanced in Ts1Cje and Ts2Cje mice but not in Ts1Rhr mice. The Cbr1 gene, which is present in three copies in Ts1Cje and Ts2Cje but not in Ts1Rhr, encodes carbonyl reductase that may facilitate GABAB-receptor activity through a reduction of prostaglandin E2 (PGE2). Interestingly, we found that a reduction of PGE2 and an memory impairment in Ts1Cje mice were alleviated when only Cbr1 was set back to two copies (Ts1Cje;Cbr1+/+/-). However, the GABAB receptor-dependent enhancement of synaptic inhibition in Ts1Cje was unaltered in Ts1Cje;Cbr1+/+/- mice. These results indicate that Cbr1 is one of the genes responsible for DS cognitive impairments and the gene(s) other than Cbr1, which is included in Ts1Cje but not in Ts1Rhr, is responsible for the GABAB receptor-dependent over-inhibition.

Perturbation of the immune cells and prenatal neurogenesis by the triplication of the Erg gene in mouse models of Down syndrome.

Some mouse models of Down syndrome (DS), including Ts1Cje mice, exhibit impaired prenatal neurogenesis with yet unknown molecular mechanism. To gain insights into the impaired neurogenesis, a transcriptomic and flow cytometry analysis of E14.5 Ts1Cje embryo brain was performed. Our analysis revealed that the neutrophil and monocyte ratios in the CD45-positive hematopoietic cells were relatively increased, in agreement with the altered expression of inflammation/immune-related genes, in Ts1Cje embryonic brain, whereas the relative number of brain macrophages was decreased in comparison to wild-type mice. Similar upregulation of inflammation-associated mRNAs was observed in other DS mouse models, with variable trisomic region lengths. We used genetic manipulation to assess the contribution of Erg, a trisomic gene in these DS models, known to regulation hemato-immune cells. The perturbed proportions of immune cells in Ts1Cje mouse brain were restored in Ts1Cje-Erg+/+/Mld2 mice, which are disomic for functional Erg but otherwise trisomic on a Ts1Cje background. Moreover, the embryonic neurogenesis defects observed in Ts1Cje cortex were reduced in Ts1Cje-Erg+/+/Mld2 embryos. Our findings suggest that Erg gene triplication contributes to the dysregulation of the homeostatic proportion of the populations of immune cells in the embryonic brain and decreased prenatal cortical neurogenesis in the prenatal brain with DS.

Increased lipid peroxidation in Down's syndrome mouse models.

Elevated oxidative stress has been suggested to be associated with the features of Down's syndrome (DS). We previously reported increased oxidative stress in cultured cells from the embryonic brain of Ts1Cje, a mouse genetic DS model. However, since in vivo evidence for increased oxidative stress is lacking, we here examined lipid peroxidation, a typical marker of oxidative stress, in the brains of Ts1Cje and another DS mouse model Ts2Cje with an overlapping but larger trisomic segment. Accumulations of proteins modified with the lipid peroxidation-derived products, 13-hydroperoxy-9Z,11E-octadecadienoic acid and 4-hydroxy-2-nonenal were markedly increased in Ts1Cje and Ts2Cje brains. Analysis with oxidation-sensitive fluorescent probe also showed that reactive oxygen species themselves were increased in Ts1Cje brain. However, electron spin resonance analysis of microdialysate from the hippocampus of Ts1Cje showed that antioxidant activity remained unaffected, suggesting that the reactive oxygen species production was accelerated in Ts1Cje. Proteomics approaches with mass spectrometry identified the proteins modified with 13-hydroperoxy-9Z,11E-octadecadienoic acid and/or 4-hydroxy-2-nonenal to be involved in either ATP generation, the neuronal cytoskeleton or antioxidant activity. Structural or functional impairments of these proteins by such modifications may contribute to the DS features such as cognitive impairment that are present in the Ts1Cje mouse.

Impaired cortico-striatal excitatory transmission triggers epilepsy.

STXBP1 and SCN2A gene mutations are observed in patients with epilepsies, although the circuit basis remains elusive. Here, we show that mice with haplodeficiency for these genes exhibit absence seizures with spike-and-wave discharges (SWDs) initiated by reduced cortical excitatory transmission into the striatum. Mice deficient for Stxbp1 or Scn2a in cortico-striatal but not cortico-thalamic neurons reproduce SWDs. In Stxbp1 haplodeficient mice, there is a reduction in excitatory transmission from the neocortex to striatal fast-spiking interneurons (FSIs). FSI activity transiently decreases at SWD onset, and pharmacological potentiation of AMPA receptors in the striatum but not in the thalamus suppresses SWDs. Furthermore, in wild-type mice, pharmacological inhibition of cortico-striatal FSI excitatory transmission triggers absence and convulsive seizures in a dose-dependent manner. These findings suggest that impaired cortico-striatal excitatory transmission is a plausible mechanism that triggers epilepsy in Stxbp1 and Scn2a haplodeficient mice.

Association study between the Down syndrome cell adhesion molecule (DSCAM) gene and bipolar disorder.

OBJECTIVES: Defects of neurodevelopmental processes are suggested to underlie the pathogenesis of bipolar disorder. Down syndrome cell adhesion molecule (DSCAM), a member of neural immunoglobulin superfamily playing a diverse role for neural development, is mapped to chromosome 21q22, a linkage locus for bipolar disorder, and is, therefore, an interesting candidate for the disease. METHODS: We performed a variation screening of the gene and association studies in 22 multiplex bipolar pedigrees of Caucasian descent and 119 Japanese patients with bipolar disorder and 140 controls. Expression levels of DSCAM were also examined in postmortem brains from the Stanley Medical Research Institute. RESULTS: We found 27 single nucleotide polymorphisms in DSCAM. Possible associations of SNP DC141 (IVS27-15A>G; P=0.042) and DC142 (IVS29+328C>A; P=0.036) were observed in pedigree samples, and G allele of DC141 was correlated with increased expression levels of DSCAM (P=0.038) in postmortem brains. Possible association of DC136 (4749C>T), which is in the same haplotype block with DC141 and DC142, was detected in Japanese populations (P=0.049). CONCLUSIONS: These results suggest the possible contribution of DSCAM gene in bipolar disorder, and warrant further investigations.

Alterations of in vivo CA1 network activity in Dp(16)1Yey Down syndrome model mice.

Down syndrome, the leading genetic cause of intellectual disability, results from an extra-copy of chromosome 21. Mice engineered to model this aneuploidy exhibit Down syndrome-like memory deficits in spatial and contextual tasks. While abnormal neuronal function has been identified in these models, most studies have relied on in vitro measures. Here, using in vivo recording in the Dp(16)1Yey model, we find alterations in the organization of spiking of hippocampal CA1 pyramidal neurons, including deficits in the generation of complex spikes. These changes lead to poorer spatial coding during exploration and less coordinated activity during sharp-wave ripples, events involved in memory consolidation. Further, the density of CA1 inhibitory neurons expressing neuropeptide Y, a population key for the generation of pyramidal cell bursts, were significantly increased in Dp(16)1Yey mice. Our data refine the 'over-suppression' theory of Down syndrome pathophysiology and suggest specific neuronal subtypes involved in hippocampal dysfunction in these model mice.

Ts1Cje Down syndrome model mice exhibit environmental stimuli-triggered locomotor hyperactivity and sociability concurrent with increased flux through central dopamine and serotonin metabolism.

Ts1Cje mice have a segmental trisomy of chromosome 16 that is orthologous to human chromosome 21 and display Down syndrome-like cognitive impairments. Despite the occurrence of affective and emotional impairments in patients with Down syndrome, these parameters are poorly documented in Down syndrome mouse models, including Ts1Cje mice. Here, we conducted comprehensive behavioral analyses, including anxiety-, sociability-, and depression-related tasks, and biochemical analyses of monoamines and their metabolites in Ts1Cje mice. Ts1Cje mice showed enhanced locomotor activity in novel environments and increased social contact with unfamiliar partners when compared with wild-type littermates, but a significantly lower activity in familiar environments. Ts1Cje mice also exhibited some signs of decreased depression like-behavior. Furthermore, Ts1Cje mice showed monoamine abnormalities, including increased extracellular dopamine and serotonin, and enhanced catabolism in the striatum and ventral forebrain. This study constitutes the first report of deviated monoamine metabolism that may help explain the basis for abnormal behaviors, including the environmental stimuli-triggered hyperactivity, increased sociability and decreased depression-like behavior in Ts1Cje mice.

Transcriptional profiling of a mouse model for Lafora disease reveals dysregulation of genes involved in the expression and modification of proteins.

Lafora's progressive myoclonus epilepsy (Lafora disease: LD) is caused by mutations in the EPM2A or NHLRC1 gene, but cellular mechanisms of the pathogenesis remain unclear. In an attempt to understand and elucidate the disease pathway, we have investigated the global gene expression profile in a mouse model for LD that developed a phenotype similar to that observed in human patients, including presence of Lafora bodies, neurodegeneration and profound neurological disturbances. We found 62 differentially expressed genes in the Epm2a knockout mice brains. These genes encode factors involved in protein catabolism, phosphatase, transcription factors, and molecules involved in protein translation, and homeostasis. The two largest functional groups of mRNAs that showed altered expression were predicted to be involved in post-translational modification of proteins and transcriptional regulation, suggesting that defects in protein activity and/or turnover may be the key trigger in the pathophysiology of LD. Furthermore we show that changes in gene expression are not limited to brain and are seen in other organs that develop Lafora bodies. Our study may provide valuable insights into the pathophysiology of LD and may aid in developing potential therapeutic targets.