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1.
Aneuploidies as quantitative chromosome abnormalities are a main cause of failed development of morphologically normal embryos, implantation failures, and early reproductive losses. Preimplantation genetic screening (PGS) allows a preselection of embryos with a normal karyotype, thus increasing the implantation rate and reducing the frequency of early pregnancy loss after IVF. Modern PGS technologies are based on a genome-wide analysis of the embryo. The first pilot study in Russia was performed to assess the possibility of using semiconductor new-generation sequencing (NGS) as a PGS method. NGS data were collected for 38 biopsied embryos and compared with the data from array comparative genomic hybridization (array-CGH). The concordance between the NGS and array-CGH data was 94.8%. Two samples showed the karyotype 47,XXY by array-CGH and a normal karyotype by NGS. The discrepancies may be explained by loss of efficiency of array-CGH amplicon labeling. 相似文献
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Edith Coonen Joyce C. Harper Frans C. S. Ramaekers Joy D. A. Delhanty Anton H. N. Hopman Joep P. M. Geraedts Alan H. Handyside 《Human genetics》1994,94(6):609-615
The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation. 相似文献
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Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation. 总被引:26,自引:0,他引:26
Molecular genetic analysis of isolated single cells and other minute DNA samples is limited because there is insufficient DNA to perform more than one independent PCR amplification. One solution to this problem is to first amplify the entire genome, thus providing enough DNA for numerous subsequent PCRs. In this study we have investigated four different methods of whole genome amplification performed on single cells, and have identified a protocol that generates sufficient quantities of DNA for comparative genomic hybridisation (CGH) as well as more than 90 independent amplification reactions. Thus, numerous specific loci and the copy number of every chromosome can be assessed in a single cell. We report here the first reliable application of CGH to single cells from human preimplantation embryos (blastomeres) and to single fibroblasts, buccal cells and amniocytes. 相似文献
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Adeleh Taei Hamid Gourabi Ali Seifinejad Mehdi Totonchi Ebrahim Shahbazi Mojtaba Rezazadeh Valojerdi Poopak Eftekhari Leila Karimian Hossein Baharvand 《In vitro cellular & developmental biology. Animal》2010,46(3-4):395-402
In this study, we focused on the derivation of human embryonic stem cell (hESC) from preimplantation genetic screening (PGS)-analyzed and preimplantation genetic diagnosis (PGD)-analyzed embryos. Out of 62 fresh PGD/PGS-analyzed embryos, 22 embryos reached the blastocyst stage. From 12 outgrowth blastocysts, we derived four hESC lines onto a feeder layer. Surprisingly, karyotype analysis showed that hESC lines derived from aneuploid embryos had diploid female karyotype. One hESC line was found to carry a balanced Robertsonian translocation. All the cell lines showed hESC markers and had the pluripotent ability to differentiate into derivatives of the three embryonic germ layers. The established lines had clonal propagation with 22–31% efficiency in the presence of ROCK inhibitor. These results further indicate that hESC lines can be derived from PGD/PGS-analyzed embryos that are destined to be discarded and can serve as an alternative source for normal euploid lines. 相似文献
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LiYing Yan Yuan Wei Jin Huang XiaoHui Zhu XiaoDan Shi Xi Xia Jie Yan CuiLing Lu Ying Lian Rong Li Ping Liu Jie Qiao 《中国科学:生命科学英文版》2014,57(7):665-671
Preimplantation genetic diagnosis (PGD) gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection. Preimplantation genetic screening (PGS) is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage. However, how and to whom PGS should be provided is a controversial topic. The first successful case of PGD of a human being was reported in 1990, and there have been tremendous improvements in this technology since then. Both embryo biopsy and genetic technologies have been improved dramatically, which increase the accuracy and expand the indications of PGD/PGS. 相似文献
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Current status and future prospects of array-based comparative genomic hybridisation. 总被引:4,自引:0,他引:4
Antoine M Snijders Daniel Pinkel Donna G Albertson 《Briefings in Functional Genomics and Prot》2003,2(1):37-45
The majority of human cancers as well as many developmental abnormalities harbour chromosomal imbalances, many of which result in the gain and/or loss of genomic material. Conventional comparative genomic hybridisation (CGH) has been used extensively to map DNA copy number changes to chromosomal positions. The introduction of microarray CGH provided a powerful tool to precisely detect and quantify genomic aberrations and map these directly onto the sequence of the human genome. In the past several years, a number of different approaches towards array-based CGH have been undertaken. This paper reviews these approaches and presents some of the recently-developed applications of this new technology in both research and clinical settings. 相似文献
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Background
Malignant fibrous histiocytomas (MFHs), or undifferentiated pleomorphic sarcomas, are in general high-grade tumours with extensive chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, as well as novel gene targets of potential importance for MFH development and/or progression, we have analysed DNA copy number changes in 33 MFHs using microarray-based comparative genomic hybridisation (array CGH).Principal findings
In general, the tumours showed numerous gains and losses of large chromosomal regions. The most frequent minimal recurrent regions of gain were 1p33-p32.3, 1p31.3-p31.2 and 1p21.3 (all gained in 58% of the samples), as well as 1q21.2-q21.3 and 20q13.2 (both 55%). The most frequent minimal recurrent regions of loss were 10q25.3-q26.11, 13q13.3-q14.2 and 13q14.3-q21.1 (all lost in 64% of the samples), as well as 2q36.3-q37.2 (61%), 1q41 (55%) and 16q12.1-q12.2 (52%). Statistical analyses revealed that gain of 1p33-p32.3 and 1p21.3 was significantly associated with better patient survival (P = 0.021 and 0.046, respectively). Comparison with similar array CGH data from 44 leiomyosarcomas identified seven chromosomal regions; 1p36.32-p35.2, 1p21.3-p21.1, 1q32.1-q42.13, 2q14.1-q22.2, 4q33-q34.3, 6p25.1-p21.32 and 7p22.3-p13, which were significantly different in copy number between the MFHs and leiomyosarcomas.Conclusions
A number of recurrent regions of gain and loss have been identified, some of which were associated with better patient survival. Several specific chromosomal regions with significant differences in copy number between MFHs and leiomyosarcomas were identified, and these aberrations may be used as additional tools for the differential diagnosis of MFHs and leiomyosarcomas. 相似文献11.
Ercelen N Turtar E Gultomruk M Comert H Coskun H Mercan R Nuhoglu A 《Genetics and molecular research : GMR》2011,10(4):4093-4103
Preimplantation genetic diagnosis is a preventive approach for identifying genetic abnormalities in early stages of reproduction. We used preimplantation genetic aneuploidy screening in 230 cycles of patients with indications of advanced maternal age, recurrent implantation failure, recurrent spontaneous abortions, or severe male factor. Biopsied blastomeres from embryos with six to eight blastomeres on day 3 were fixed and fluorescence in situ hybridization was utilized on chromosomes 13, 16, 18, 21, 22, X, and Y. Among 945 morphologically normal embryos, 314 were diagnosed as chromosomally normal. Trisomy and monosomy were observed in 36% of the cases (18% each). Embryo transfer was used in 144 cycles, resulting in 41 pregnancies. Thirty-seven healthy babies were delivered, with a take-home baby rate of 24.2% and an implantation rate of 22%. We recommend preimplantation genetic aneuploidy screening as a valuable technique to select normal chromosome embryos in order to avoid multiple pregnancies due to the multiple embryo transfers that are normally necessary to ensure pregnancy in poor prognosis in vitro fertilization patients. 相似文献
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Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization 总被引:26,自引:0,他引:26
Karyotypic studies of aborted fetuses have been used to draw the inference that the proportion of conceptuses with chromosome abnormalities is very high. Fluorescent in situ hybridization (FISH) studies of blastomeres from early cleavage embryos have provided some support for this inference but they are limited to the study of a few chromosomes. We describe the novel application of comparative genomic hybridization (CGH) to the study of numerical and structural abnormalities of single blastomeres from disaggregated 3-day-old human embryos. CGH results were obtained for 63 blastomeres from 12 embryos. Identification of all chromosomes with the exception of chromosomes 17, 19, 20 and 22 was possible. The embryos divided into four groups: (1) embryos with a normal CGH karyotype seen in all blastomeres; (2) embryos with consistent aneuploidy suggesting meiotic non-disjunction had occurred; (3) embryos that were mosaic generally with one or more cells showing aneuploidy for one or two chromosomes but some with cells showing extensive aneuploidy; and (4) one embryo with extensive aneuploidy in all blastomeres. The extensive aneuploidy in group 4 is interpreted as corresponding to the random aneuploidy seen in "chaotic" embryos reported by using interphase FISH. Partial chromosome loss and gain following chromosome breakage was observed in one embryo. Our analysis provides basic biological information on the occurrence of constitutional and post-zygotic chromosome abnormalities in early human embryos. Used in conjunction with embryo biopsy, diagnostic CGH should allow the exclusion of a proportion of embryos that appear normal but that have a poor probability of survival and, therefore, may improve the implantation rate after in vitro fertilization. 相似文献
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Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene 总被引:1,自引:0,他引:1
Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro. The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques. Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous singlecopy gene or an integrated foreign gene. The endogenous single-copy gene encoding the beta chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells. To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance were bred to nontransgenic females, and the resulting blastocysts were analyzed. The neo gene was detected in approximately half of the embryos. The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunity to study genetics in early development. 相似文献
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Glucose metabolism by preimplantation pig embryos 总被引:2,自引:0,他引:2
Pig embryos were collected, 2-7 days after oestrus, in modified BMOC-2 containing glucose as the only energy source. Embryos were incubated individually in medium containing [5-(3)H]-, [1-(14)C]- or [6-(14)C]glucose. Total glucose metabolism, as measured by [5-(3)H]glucose use, increased steadily from the 1-cell to the 8-cell stage. Total glucose use increased (P less than 0.05) at the compacted morula stage and was highest (P less than 0.05) at the blastocyst stage. Production of 14CO2 from embryos metabolizing [1-(14)C]glucose increased steadily from the unfertilized ovum to the 8-cell stage. Metabolism of [1-(14)C]glucose increased at the compacted morula stage (P less than 0.05) and continued to increase (P less than 0.05) to the blastocyst stage. Metabolism of [6-(14)C]glucose increased steadily from the unfertilized ovum to the compacted morula stage. Metabolism of [6-(14)C]glucose was highest (P less than 0.05) for the blastocyst stage. Percentage pentose phosphate pathway activity of total glucose metabolism before the 4-cell stage was higher (greater than 5%) than that of 8-cell to blastocyst stage embryos (approximately 1%). When embryo metabolism was determined on a per cell basis for each isotope, the compacted morulae stage (16 cells) had a higher total glucose metabolism than all other embryo stages (P less than 0.05), while early blastocyst (32 cells) and blastocyst (64 cells) stage embryos metabolized more [5-(3)H]glucose than all stages except compacted morulae (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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María -Dolores Rey Pilar Prieto 《Molecular breeding : new strategies in plant improvement》2017,37(3):32
Simple, reliable methods for the identification of alien genetic introgressions are required in plant breeding programmes. The use of genomic dot-blot hybridisation allows the detection of small Hordeum chilense genomic introgressions in the descendants of genetic crosses between wheat and H. chilense addition or substitution lines in wheat when molecular markers are difficult to use. Based on genomic in situ hybridisation, DNA samples from wheat lines carrying putatively H. chilense introgressions were immobilised on a membrane, blocked with wheat genomic DNA and hybridised with biotin-labelled H. chilense genomic DNA as a probe. This dot-blot screening reduced the number of plants necessary to be analysed by molecular markers or in situ hybridisation, saving time and money. The technique was sensitive enough to detect a minimum of 5 ng of total genomic DNA immobilised on the membrane or about 1/420 dilution of H. chilense genomic DNA in the wheat background. The robustness of the technique was verified by in situ hybridisation. In addition, the detection of other wheat relative species such as Hordeum vulgare, Secale cereale and Agropyron cristatum in the wheat background was also reported. 相似文献
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A protein isolated from serum is required in the cell suspension for the chemotactic response of normal mouse peritoneal macrophages to complement-derived C5a. Macrophage stimulating protein (MSP) has a molecular weight of 100000 and an isoelectric point of 7.0. It is resistant to changes in pH over a range of 1.3–11, is heat labile especially after partial purification and does not survive proteolytic enzyme attack. Binding to ConA Sepharose suggests that it contains a carbohydrate moiety. Its concentration in normal serum is very low and it is detectable only by virtue of a sensitive bioassay. An upper limit has been estimated at 75 ng/ml; the actual concentration may be considerably lower. 相似文献
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Currently, the stage of embryo development has been proposed as one of many criteria for identifying healthy embryos in infertility clinics with the fastest embryos being highlighted as the healthiest. However the validity of this as an accurate criterion with respect to genomic imprinting is unknown. Given that embryo development in culture generally requires an extra day compared to in vivo development, we hypothesized that loss of imprinting correlates with slower rates of embryonic development. To evaluate this, embryos were recovered at the 2-cell stage, separated into four groups based on morphological stage at two predetermined time points, and cultured to blastocysts. We examined cell number, embryo volume, embryo sex, imprinted Snrpn and H19 methylation, imprinted Snrpn, H19, and Cdkn1c expression, and expression of genes involved in embryo metabolism-Atp1a1, Slc2a1, and Mapk14-all within the same individual embryo. Contrary to our hypothesis, we observed that faster developing embryos exhibited greater cell numbers and embryo volumes as well as greater perturbations in genomic imprinting and metabolic marker expression. Embryos with slower rates of preimplantation development were most similar to in vivo derived embryos, displaying similar cell numbers, embryo volumes, Snrpn and H19 imprinted methylation, H19 imprinted expression, and Atp1a1 and Slc2a1 expression. We conclude that faster development rates in vitro are correlated with loss of genomic imprinting and aberrant metabolic marker expression. Importantly, we identified a subset of in vitro cultured embryos that, according to the parameters evaluated, are very similar to in vivo derived embryos and thus are likely most suitable for embryo transfer. 相似文献
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J C Strefford N J Foot T Chaplin M J Neat R T Oliver B D Young L K Jones 《Cytogenetics and cell genetics》2001,94(1-2):9-14
The cell line U937, which has been used extensively for studies of myeloid differentiation, bears the t(10;11)(p13;q14) translocation which results in a fusion between the MLLT10 (myeloid/lymphoid or mixed-lineage leukemia [trithorax, Drosophila, homolog]; translocated to 10; alias AF10) gene and the Ap-3-like clathrin assembly protein, PICALM (Clathrin assembly lymphoid myeloid leukaemia). Apart from this translocation, very little is known about the other genetic alterations in this cell line that may represent significant events in disease progression. In this study, conventional G-banding, CGH and M-FISH have been used to characterise fully all of the cytogenetic alterations present in the U937 cell line. M-FISH analysis confirmed the presence of the t(10;11) and an apparently normal copy of both chromosomes 10 and 11. A t(1;5) translocation was observed as well as several unbalanced rearrangements. CGH detected amplifications resulting from duplications of 2q, 6p and 13q. These changes could result in fusion gene products involved in carcinogenesis or the positions of putative oncogenes and tumour suppressor genes. A good correlation between conventional G-banding, CGH and M-FISH was observed. 相似文献
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