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Mechanisms of implantation such as determination of the attachment pole, fetal-maternal communication, and underlying causes of implantation failure are largely unexplored. Here, we performed single-cell RNA sequencing on peri-implantation embryos from both humans and mice to explore trophectoderm (TE) development and embryo-endometrium cross-talk. We found that the transcriptomes of polar and mural TE diverged after embryos hatched from the zona pellucida in both species, with polar TE being more mature than mural TE. The implantation poles show similarities in cell cycle activities, as well as in expression of genes critical for implantation and placentation. Embryos that either fail to attach in vitro or fail to implant in vivo show abnormalities in pathways related to energy production, protein metabolism, and 18S ribosomal RNA m6A methylation. These findings uncover the gene expression characteristics of humans and mice TE differentiation during the peri-implantation period and provide new insights into embryo implantation.
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Chromosomal mosaicism is common throughout human pre- and post-implantation development. However, the incidence and characteristics of mosaicism in human blastocyst remain unclear. Concerns and confusions still exist regarding the interpretation of chromosomal mosaicism on preimplantation genetic testing for aneuploidy (PGT-A) results and embryo development. Here, we aimed to estimate the genetic concordance between trophectoderm (TE), inner cell mass (ICM) and the corresponding human embryonic stem cells (hESCs), and to explore the characteristics of mosaicism in human blastocyst and hESCs on a single cell level. The single cell sequencing results of TE cells indicated that 65.71% of the blastocysts were mosaic (23 in 35 embryos), while the ICM sequencing results suggested that 60.00% of the blastocysts were mosaic (9 in 15 embryos). The incidence of mosaicism for the corresponding hESCs was 33.33% (2 in 6 embryos). No significant difference was observed between the mosaic rate of TE and that of ICM. However, the mosaic rate of the corresponding hESCs was significantly lower than that of TE and ICM cells, suggesting that the incidence of mosaicism may decline during embryonic development. Upon single cell sequencing, we found several "complementary" copy number variations (CNVs) that were usually not revealed in clinical PGT-A which used multi-cell DNA sequencing (or array analysis). This indicates the potential diagnostic risk of PGT-A based multi-cell analysis routinely in clinical practice. This study provided new insights into the characteristics, and considerable influences, of mosaicism on human embryo development, as well as the clinical risks of PGT-A based on multi-cell biopsies and bulk DNA assays.
Assuntos
Mosaicismo , Diagnóstico Pré-Implantação , Aneuploidia , Blastocisto , Variações do Número de Cópias de DNA/genética , Feminino , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Gravidez , Diagnóstico Pré-Implantação/métodosRESUMO
RESEARCH QUESTION: Can preimplantation genetic testing for structural rearrangement (PGT-SR) based on low-coverage next-generation sequencing (NGS) accurately discriminate between normal and carrier embryos of reciprocal translocation (RecT) and Robertsonian translocation (RobT)? DESIGN: A total of 109 couples with RecT or RobT were included in this study. The ages, bad obsteric histories (BOH), blood karyotype and IVF cycle information, including the number of cumulus-oocyte complexes, metaphase II oocytes, two pronuclei oocytes and blastocysts were recorded. 0.1â¯×â¯whole genome sequencing (WGS) of embryos followed by copy number variation (identifying unbalanced/balanced) and 2â¯×â¯WGS of parents and embryos followed by haplotype analysis (discriminating between normal and carrier) were carried out in PGT-SR cycles. The embryos without translocation were transferred and clinical outcomes evaluated. RESULTS: Among all the couples in this study, 67 patients had RecT and 42 had RobT. After unbalanced and balanced detection, 103 balanced embryos underwent a further normal and carrier discrimination procedure, and 53 normal embryos were identified. Finally, 32 normal embryos were transferred, with an ongoing pregnancy rate of 46.88% (15/32). All ongoing pregnancies underwent amniocentesis, and the amninocentesis karyotyping results showed 100% concordance with PGT-SR diagnosis. CONCLUSIONS: Our low-coverage NGS-based PGT-SR method can accurately discriminate between normal and carrier status of balanced embryos. The method is cost-effective and has broad clinical applicability.
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Transferência Embrionária , Diagnóstico Pré-Implantação , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Transferência Embrionária/métodos , Feminino , Fertilização in vitro , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Gravidez , Diagnóstico Pré-Implantação/métodos , Translocação GenéticaRESUMO
Gonadal somatic cells are the main players in gonad development and are important for sex determination and germ cell development. Here, using a time-series single-cell RNA sequencing (scRNA-seq) strategy, we analyzed fetal germ cells (FGCs) and gonadal somatic cells in human embryos and fetuses. Clustering analysis of testes and ovaries revealed several novel cell subsets, including POU5F1+SPARC+ FGCs and KRT19+ somatic cells. Furthermore, our data indicated that the bone morphogenetic protein (BMP) signaling pathway plays cell type-specific and developmental stage-specific roles in testis development and promotes the gonocyte-to-spermatogonium transition (GST) in late-stage testicular mitotic arrest FGCs. Intriguingly, testosterone synthesis function transitioned from fetal Sertoli cells to adult Leydig cells in a stepwise manner. In our study, potential interactions between gonadal somatic cells were systematically explored and we identified cell type-specific developmental defects in both FGCs and gonadal somatic cells in a Turner syndrome embryo (45, XO). Our work provides a blueprint of the complex yet highly ordered development of and the interactions among human FGCs and gonadal somatic cells.
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Gônadas , Testículo , Humanos , Masculino , Linhagem da Célula/genética , RNA-Seq , Células Intersticiais do Testículo/metabolismoRESUMO
Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease characterized by the development of renal cysts and progression to renal failure. Preimplantation genetic testing-monogenic disease (PGT-M) is an alternative option to obtain healthy babies. However, de novo PKD1 mutation of one of the spouses or the absence of a positive family history poses a serious challenge to PGT-M. Here, we described a comprehensive strategy which includes preimplantation genetic testing for aneuploidies (PGT-A) study and monogenic diagnosis study for ADPKD patients bearing de novo mutations. The innovation of our strategy is to use the gamete (polar body or single sperm) as proband for single-nucleotide polymorphism (SNP) linkage analysis to detect an embryo's carrier status. Nine ADPKD couples with either de novo mutation or without a positive family history were recruited and a total of 34 embryos from 13 PGT-M cycles were examined. Within these nine couples, two successfully delivered healthy babies had their genetic status confirmed by amniocentesis. This study provides a creative approach for embryo diagnosis of patients with de novo mutations or patients who lack essential family members for linkage analysis.
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Aneuploidia , Testes Genéticos/métodos , Nascido Vivo/epidemiologia , Mutação , Rim Policístico Autossômico Dominante/diagnóstico , Diagnóstico Pré-Implantação/métodos , Canais de Cátion TRPP/genética , Adulto , Transferência Embrionária , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/patologia , Feminino , Fertilização in vitro , Ligação Genética , Células Germinativas/metabolismo , Células Germinativas/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Rim Policístico Autossômico Dominante/epidemiologia , Rim Policístico Autossômico Dominante/genética , Gravidez , Adulto JovemRESUMO
PURPOSE: To determine whether next-generation sequencing (NGS) could be used to directly detect different mutations of Duchenne muscular dystrophy (DMD) during preimplantation genetic testing (PGT). METHODS: From Sep. 2016 to Aug. 2018, a total of six couples participated in this study. Four cases carried DMD exon deletions and two carried exon duplications. Trophectoderm cells were biopsied at day 5 or 6 and NGS was used in the genetic testing of the biopsied cells after whole-genome amplification. We developed a new method-DIRected Embryonic Cell Testing of Exon Deletion/Duplication (DIRECTED) to directly detect the single-gene mutation by NGS. Linage analysis based on single-nucleotide polymorphism (SNP) was used to validate the results from DIRECTED. RESULTS: In the four deletion cases, DIRECTED was used to detect DMD exon deletion in 16 biopsied embryos. All DIRECTED results were consistent with linkage analysis, indicating this method was reliable in detecting deletions around 1 Mb. In the two cases carrying exon duplications, no blastocyst was obtained for biopsy. Nonetheless, preliminary experiment results suggested that DIRECTED could also be used for direct detection of exon duplications in embryos. CONCLUSIONS: Exon deletions or duplications in DMD of preimplantation embryos could be detected directly by NGS-based methods during PGT.
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Distrofina/genética , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Distrofia Muscular de Duchenne/diagnóstico , Diagnóstico Pré-Implantação , Adulto , Biópsia , Blastocisto/metabolismo , Blastocisto/patologia , Éxons/genética , Feminino , Deleção de Genes , Triagem de Portadores Genéticos , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mutação/genética , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
STUDY QUESTION: Is it possible to evaluate the methylome of individual oocytes to investigate the DNA methylome alterations in metaphase II (MII) oocytes with reduced embryo developmental potential? SUMMARY ANSWER: The DNA methylome of each human first polar body (PB1) closely mirrored that of its sibling MII oocyte; hypermethylated long interspersed nuclear element (LINE) and long terminal repeats (LTRs) and methylation aberrations in PB1 promoter regions may indicate poor embryo development. WHAT IS KNOWN ALREADY: The developmental potential of an embryo is determined by the oocyte's developmental competence, and the PB1 is a good substitute to examine the chromosomal status of the corresponding oocyte. However, DNA methylation, a key epigenetic modification, also regulates gene expression and embryo development. STUDY DESIGN, SIZE, DURATION: Twelve pairs of PB1s and sibling MII oocytes were biopsied and sequenced to compare their methylomes. To further investigate the methylome of PB1s and the potential epigenetic factors that may affect oocyte quality, MII oocytes (n = 74) were fertilized through ICSI, while PB1s were biopsied and profiled to measure DNA methylation. The corresponding embryos were further cultured to track their development potential. The oocytes and sperm samples used in this study were donated by healthy volunteers with signed informed consent. PARTICIPANTS/MATERIALS, SETTING, METHODS: Single-cell methylome sequencing was applied to obtain the DNA methylation profiles of PB1s and oocytes. The DNA methylome of PB1s was compared between the respective group of oocytes that progressed to blastocysts and the group of oocytes that failed to develop. DNA methylation levels of corresponding regions and differentially methylated regions were calculated using customized Perl and R scripts. RNA-seq data were downloaded from a previously published paper and reanalysed. MAIN RESULTS AND THE ROLE OF CHANCE: The results from PB1-MII oocyte pair validated that PB1 contains nearly the same methylome (average Pearson correlation is 0.92) with sibling MII oocyte. LINE and LTR expression increased markedly after fertilization. Moreover, the DNA methylation levels in LINE (including LINE1 and LINE2) and LTR were significantly higher in the PB1s of embryos that could not reach the blastocyst stage (Wilcoxon-Matt-Whitney test, P < 0.05). DNA methylation in PB1 promoters correlated negatively with gene expression of MII oocyte. Regarding the methylation status of the promoter regions, 66 genes were hypermethylated in the developmental arrested group, with their related functions (significantly enriched in several Gene Ontology terms) including transcription, positive regulation of adenylate cyclase activity, mitogen-activated protein kinase (MAPK) cascade and intracellular oestrogen receptor signalling pathway. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Data analysis performed in this study focused on the competence of human oocytes and compared them with maternal genetic and epigenetic profiles. Therefore, data regarding the potential regulatory roles of paternal genomes in embryo development are lacking. WIDER IMPLICATIONS OF THE FINDINGS: The results from PB1-oocyte pairs demonstrated that PB1s shared similar methylomes with their sibling oocytes. The selection of the good embryos for transfer should not only rely on morphology but also consider the DNA methylation of the corresponding PB1 and therefore MII oocyte. The application of early-stage analysis of PB1 offers an option for high-quality oocyte and embryo selection, which provides an additional tool for elective single embryo transfer in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key Research and Development Program of China (2018YFC1004003, 2017YFA0103801), the National Natural Science Foundation of China (81730038, 3187144, 81521002) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16020703). The authors have no conflicts of interest to declare.
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Epigenoma , Corpos Polares , China , Desenvolvimento Embrionário/genética , Humanos , Masculino , OócitosRESUMO
Epigenetic dynamics, such as DNA methylation and chromatin accessibility, have been extensively explored in human preimplantation embryos. However, the active demethylation process during this crucial period remains largely unexplored. In this study, we use single-cell chemical-labeling-enabled C-to-T conversion sequencing (CLEVER-seq) to quantify the DNA 5-formylcytosine (5fC) levels of human preimplantation embryos. We find that 5-formylcytosine phosphate guanine (5fCpG) exhibits genomic element-specific distribution features and is enriched in L1 and endogenous retrovirus-K (ERVK), the subfamilies of repeat elements long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs), respectively. Unlike in mice, paired pronuclei in the same zygote present variable difference of 5fCpG levels, although the male pronuclei experience stronger global demethylation. The nucleosome-occupied regions show a higher 5fCpG level compared with nucleosome-depleted ones, suggesting the role of 5fC in organizing nucleosome position. Collectively, our work offers a valuable resource for ten-eleven translocation protein family (TET)-dependent active demethylation-related study during human early embryonic development.
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Blastocisto/metabolismo , Citosina/análogos & derivados , Desmetilação do DNA , Citosina/metabolismo , Desenvolvimento Embrionário , Genoma Humano , Humanos , Elementos Reguladores de Transcrição , Análise de Célula ÚnicaRESUMO
Characterizing the video quality seen by an end-user is a critical component of any video transmission system. In packet-based communication systems, such as wireless channels or the Internet, packet delivery is not guaranteed. Therefore, from the point-of-view of the transmitter, the distortion at the receiver is a random variable. Traditional approaches have primarily focused on minimizing the expected value of the end-to-end distortion. This paper explores the benefits of accounting for not only the mean, but also the variance of the end-to-end distortion when allocating limited source and channel resources. By accounting for the variance of the distortion, the proposed approach increases the reliability of the system by making it more likely that what the end-user sees, closely resembles the mean end-to-end distortion calculated at the transmitter. Experimental results demonstrate that variance-aware resource allocation can help limit error propagation and is more robust to channel-mismatch than approaches whose goal is to strictly minimize the expected distortion.
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Algoritmos , Compressão de Dados/métodos , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Internet , Multimídia , Processamento de Sinais Assistido por Computador , Gravação em Vídeo/métodos , Gráficos por Computador , TelecomunicaçõesRESUMO
The problem of application-layer error control for real-time video transmission over packet lossy networks is commonly addressed via joint source-channel coding (JSCC), where source coding and forward error correction (FEC) are jointly designed to compensate for packet losses. In this paper, we consider hybrid application-layer error correction consisting of FEC and retransmissions. The study is carried out in an integrated joint source-channel coding (IJSCC) framework, where error resilient source coding, channel coding, and error concealment are jointly considered in order to achieve the best video delivery quality. We first show the advantage of the proposed IJSCC framework as compared to a sequential JSCC approach, where error resilient source coding and channel coding are not fully integrated. In the USCC framework, we also study the performance of different error control scenarios, such as pure FEC, pure retransmission, and their combination. Pure FEC and application layer retransmissions are shown to each achieve optimal results depending on the packet loss rates and the round-trip time. A hybrid of FEC and retransmissions is shown to outperform each component individually due to its greater flexibility.
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Algoritmos , Redes de Comunicação de Computadores , Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Armazenamento e Recuperação da Informação/métodos , Processamento de Sinais Assistido por Computador , Gravação em Vídeo/métodos , Gráficos por Computador , Sistemas Computacionais , Análise Numérica Assistida por ComputadorRESUMO
AIM: To study the therapeutic mechanism of Ginkgo biloba exocarp polysaccharides (GBEP) on gastric cancer. METHODS: Thirty patients with gastric cancer were treated with oral GBEP capsules. The area of tumors was measured by electron gastroscope before and after treatment, then the inhibitory and effective rates were calculated. The ultrastructures of tumor cells were examined by transmissional electron microscope. Cell culture, MTT, flow cytometry were performed to observe proliferation, apoptosis and changes of relevant gene expression of human gastric cancer SGC-7901 cells. RESULTS: Compared with the statement before treatment, GBEP capsules could reduce the area of tumors, and the effective rate was 73.4%. Ultrastructural changes of the cells indicated that GBEP could induce apoptosis and differentiation in tumor cells of patients with gastric cancer. GBEP could inhibit the growth of human gastric cancer SGC-7901 cells following 24-72 h treatment in vitro at 10-320 mg/L, which was dose- and time-dependent. GBEP was able to elevate the apoptosis rate and expression of c-fos gene, but reduce the expression of c-myc and bcl-2 genes also in a dose-dependent manner. CONCLUSION: The therapeutic mechanism of GBEP on human gastric cancer may relate to its effects on the expression of c-myc, bcl-2 and c-fos genes, which can inhibit proliferation and induce apoptosis and differentiation of tumor cells.
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Ginkgo biloba , Fitoterapia , Extratos Vegetais/uso terapêutico , Polissacarídeos/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Administração Oral , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologiaRESUMO
OBJECTIVE: To observe the clinical efficacy of Ginkgo biloba exocarp polysaccharides (GBEP) capsule preparation in treating upper digestive tract malignant tumors of middle and late stage. METHODS: Eighty-six patients of the upper digestive tract malignant tumors were treated with GBEP capsule preparation taken orally. The clinical symptoms and the qualities of life of the patients with single GBEP and combined with operation, radiotherapy or intervention chemotherapy were observed. The tumor size was measured by electronic gastroscope before and after treatment with single GBEP. Objective response rate (RR) of the tumor was calculated. The survival period of patient was observed. The changes of blood routine examination in the patients treated with radiotherapy were observed. RESULTS: GBEP preparation could markedly improve the patients'clinical symptoms. Karnofsky scoring of the patients markedly increased after treatment. There were 2 CR (complete response, 6.3%), 22 PR (partial response, 68.8%)and 5 SD (stable disease, 15.6%) of 32 cases with single GBEP preparation. The survival periods of the 32 cases were markedly prolonged. The preparation could relieve the inhibited hematopietic function and the weight loss due to radiotherapy. CONCLUSION: GBEP capsule preparation has some definite therapeutic effects on upper digestive tract malignant tumors of middle and late stage.
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Neoplasias Esofágicas/tratamento farmacológico , Ginkgo biloba , Preparações de Plantas/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Trato Gastrointestinal Superior/patologia , Administração Oral , Adulto , Idoso , Cápsulas , Terapia Combinada , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/radioterapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fitoterapia , Preparações de Plantas/administração & dosagem , Polissacarídeos/administração & dosagem , Polissacarídeos/uso terapêutico , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/radioterapia , Taxa de Sobrevida , Resultado do Tratamento , Trato Gastrointestinal Superior/efeitos dos fármacos , Trato Gastrointestinal Superior/efeitos da radiaçãoRESUMO
OBJECTIVE: To investigate the effects of ambient conditions on the antibacterial activity of inorganic antibacterial agent based on sodium titanium phosphate. METHODS: The number of live E. Coli ATCC 44113 was counted after the suspension was shaken and incubated in flask with antibacterial agent under ambient conditions. RESULT: The number of live bacteria increased when the concentration of bacteria increased in the range of 1.2 x 10(2) to 1.2 x 10(8) cfu/ml. The antibacterial activity increased with the increase of ambient temperature in the range of 16 approximately 46 degrees C, showing good antibacterial activity at human body temperature. With the increase of the concentrations of co-existing NaCl and protein, the antibacterial activity was found to decrease at first and level off at 0.01 g/ml and 0.0005 g/ml respectively. CONCLUSION: The antibacterial agent has to be used together with organic antibacterial agent in order to achieve good antibacterial effect when the ambient bacterial concentration is high. The antibacterial agent is promising to be used in the preparation of antibacterial cloth because it is highly effective at body temperature and expected to remain antibacterial in perspiration.