Genetic analysis of embryo in a human case of spontaneous oocyte activation: a case report.

Parthenogenesis, a unique form of reproduction, is normally inhibited in mammals and a human embryo with parthenogenetic origin is not considered capable of producing offspring. The aim of this report is to analyze a parthenogenetic oocyte retrieved from a patient so as to have a better understanding on parthenogenesis and causes of infertility. A 38-year-old woman presented at our center with a history of primary infertility for 10 years and underwent an IVF-ICSI cycle. Three MII oocytes retrieved and one of which presented with 1 pronucleus before conducting ICSI and developed into an embryo 30 h post-retrieval. Blastomere biopsy, genome amplification, copy number variation (CNV) analysis and MultiSNPs analysis was performed on the embryo. The results showed that only one blastomere contains DNA and CNV analysis indicated a genotype of 48, XX, +17, +17 and the genetic contribution of biopsied embryo was of exclusively maternal origin. Such analysis might be beneficial for patients with a history of oocyte spontaneous activation in diagnosing case-specific aberrations and providing individualized therapeutic strategies such as preimplantation genetic diagnosis to choose a genetic normal embryo to transplant.

Single-cell RNA-sequencing reveals the existence of naive and primed pluripotency in pre-implantation rhesus monkey embryos.

Naive pluripotency exists in epiblast cells of mouse pre-implantation embryos. However, whether the naive pluripotency is transient or nonexistent in primate embryos remains unclear. Using RNA-seq in single blastomeres from 16-cell embryos through to hatched blastocysts of rhesus monkey, we constructed the lineage segregation roadmap in which the specification of trophectoderm, epiblast, and primitive endoderm is initiated simultaneously at the early blastocyst stage. Importantly, we uncovered the existence of distinct pluripotent states in monkey pre-implantation embryos. At the early- and middle-blastocyst stages, the epiblast cells have the transcriptome features of naive pluripotency, whereas they display a continuum of primed pluripotency characteristics at the late and hatched blastocyst stages. Moreover, we identified potential regulators that might play roles in the transition from naive to primed pluripotency. Thus, our study suggests the transient existence of naive pluripotency in primates and proposes an ideal time window for derivation of primate embryonic stem cells with naive pluripotency.

Long and small noncoding RNAs during oocyte-to-embryo transition in mammals.

Oocyte-to-embryo transition is a process during which an oocyte ovulates, is fertilized, and becomes a developing embryo. It involves the first major genome reprogramming event in life of an organism where gene expression, which gave rise to a differentiated oocyte, is remodeled in order to establish totipotency in blastomeres of an early embryo. This remodeling involves replacement of maternal RNAs with zygotic RNAs through maternal RNA degradation and zygotic genome activation. This review is focused on expression and function of long noncoding RNAs (lncRNAs) and small RNAs during oocyte-to-embryo transition in mammals. LncRNAs are an assorted rapidly evolving collection of RNAs, which have no apparent protein-coding capacity. Their biogenesis is similar to mRNAs including transcriptional control and post-transcriptional processing. Diverse molecular and biological roles were assigned to lncRNAs although most of them probably did not acquire a detectable biological role. Since some lncRNAs serve as precursors for small noncoding regulatory RNAs in RNA silencing pathways, both types of noncoding RNA are reviewed together.

Single Cell Restriction Enzyme-Based Analysis of Methylation at Genomic Imprinted Regions in Preimplantation Mouse Embryos.

The methylation of cytosines in DNA is a fundamental epigenetic regulatory mechanism. During preimplantation development, mammalian embryos undergo extensive epigenetic reprogramming, including the global erasure of germ cell-specific DNA methylation marks, to allow for the establishment of the pluripotent state of the epiblast. However, DNA methylation marks at specific regions, such as imprinted gene regions, escape this reprogramming process, as their inheritance from germline to soma is paramount for proper development. To study the dynamics of DNA methylation marks in single blastomeres of mouse preimplantation embryos, we devised a new approach-single cell restriction enzyme analysis of methylation (SCRAM). SCRAM allows for reliable, fast, and high-throughput analysis of DNA methylation states of multiple regions of interest from single cells. In the method described below, SCRAM is specifically used to address loss of DNA methylation at genomic imprints or other highly methylated regions of interest.

Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish.

Intracellular Ca2+ signaling regulates cellular activities during embryogenesis and in adult organisms. We generated stable Tg[ßactin2:GCaMP6s]stl351 and Tg[ubi:GCaMP6s]stl352 transgenic lines that combine the ubiquitously-expressed Ca2+ indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca2+ imaging. Using the Tg[ßactin2:GCaMP6s]stl351 line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca2+ transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca2+ signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca2+ signaling is diminished in ventralized ichabod/ß-catenin2 mutant embryos and ectopically induced in embryos dorsalized by excess ß-catenin. During gastrulation, we directly visualized Ca2+ signaling in the dorsal forerunner cells, which form in a Nodal signaling dependent manner and later give rise to the laterality organ. We found that excess Nodal increases the number and the duration of Ca2+ transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca2+ events from embryogenesis through adulthood, augmenting the zebrafish toolbox.

Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid?

OBJECTIVE: To investigate the blastocoelic fluid (BF) for the presence of DNA that could be amplified and analyzed; the extent to which its chromosomal status corresponds to that found in trophectoderm (TE) cells, polar bodies (PBs), or blastomeres; and the identification of segmental abnormalities. DESIGN: Longitudinal cohort study. SETTING: In vitro fertilization unit. PATIENT(S): Fifty-one couples undergoing preimplantation genetic screening or preimplantation genetic diagnosis for translocations by array-comparative genomic hybridization on PBs (n = 21) or blastomeres (n = 30). INTERVENTION(S): BFs and TE cells were retrieved from 116 blastocysts, whose chromosome status had already been established by PB or blastomere assessment. Separate chromosome analysis was performed in 70 BFs. MAIN OUTCOME MEASURE(S): Presence of DNA in BFs, evaluation of the chromosome condition, and comparison with the diagnosis made in TE cells and at earlier stage biopsies. RESULT(S): DNA detection was 82%, with a net improvement after refinement of the procedure. In 97.1% of BFs, the ploidy condition corresponded to that found in TE cells, with one false positive and one false negative. The rate of concordance per single chromosome was 98.4%. Ploidy and chromosome concordance with PBs were 94% and 97.9%, respectively; with blastomeres, the concordances were 95% and 97.7%, respectively. Segmental abnormalities, which were detected in PBs or blastomeres of 16 blastocysts, were also identified in the corresponding BFs. CONCLUSION(S): BF represents to a good extent the blastocyst ploidy condition and chromosome status when compared with TE cells. If the proportion of clinically useful BFs is improved, blastocentesis could become the preferred source of DNA for chromosomal testing.

Assessment of nuclear transfer techniques to prevent the transmission of heritable mitochondrial disorders without compromising embryonic development competence in mice.

To evaluate and compare mitochondrial DNA (mtDNA) carry-over and embryonic development potential between different nuclear transfer techniques we performed germinal vesicle nuclear transfer (GV NT), metaphase-II spindle-chromosome-complex (MII-SCC) transfer and pronuclear transfer (PNT) in mice. No detectable mtDNA carry-over was seen in most of the reconstructed oocytes and embryos. No significant differences were seen in mtDNA carry-over rate between GV NT (n=20), MII-SCC transfer (0.29 ± 0.63; n=21) and PNT (0.29 ± 0.75; n=25). Blastocyst formation was not compromised after either PNT (88%; n=18) or MII-SCC transfer (86%; n=27). Further analysis of blastomeres from cleaving embryos (n=8) demonstrated undetectable mtDNA carry-over in all but one blastomere. We show that NT in the germ line is potent to prevent transmission of heritable mtDNA disorders with the applicability for patients attempting reproduction.

Enhanced generation of human embryonic stem cells from single blastomeres of fair and poor-quality cleavage embryos via inhibition of glycogen synthase kinase ß and Rho-associated kinase signaling.

STUDY QUESTION: Could selected pluripotency-enhancing small molecules (SMs) lead to efficient derivation of human embryonic stem cells (hESCs) from cleavage embryos-derived single blastomeres (SBs)? SUMMARY ANSWER: Inhibition of glycogen synthase kinase ß (GSK3ß) and Rho-associated kinase (ROCK) signaling can enhance the derivation of hESCs from cleavage embryo-derived SBs. WHAT IS KNOWN ALREADY: Parameters involved in sustaining the pluripotency of biopsied blastomeres for generating hESCs without causing injury to a viable embryo have remained obscure. This research seeks to improve the culture conditions for increasing the efficiency of deriving hESCs from SBs from cleavage-stage embryos by using SMs. STUDY DESIGN, SIZE, DURATION: In order to identify SMs which may enhance hESC generation from SBs, 11 pluripotency-enhancing SMs were screened and CHIR99021 (CH), a GSK3ß inhibitor, was selected. To optimize culture condition in hESC generation from SMs, we used ROCK inhibitor Y27632 (Y) and basic fibroblast growth factor in combination with CH or its alternative, Kenpaullone, in different time courses over 12 days. We also assessed a critical time point for CH + Y treatment of cleavage embryos from 4- to 8-cell embryo. In total, 224 embryos and 1607 SBs were used in the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Blastomeres of fair and poor-quality from 6- to 8-cell stage human embryos were mechanically dispersed and individually seeded into a 96-well plate that was precoated with mitotically inactivated feeder cells. Derivation of hESC line from each SB was carried out in hESC defined medium supplemented with SMs. Randomly selected hESC lines were evaluated by immunostaining for pluripotency markers, karyotype analysis and differentiation potential into the three embryonic germ layer derivatives. MAIN RESULTS AND THE ROLE OF CHANCE: We found that 3 µM CH was the only SM that was capable of directing SBs from fair and poor-quality 6-8-cell embryos into hESC lines. The application of hESC-conditioned medium had no additive effect on hESC establishment from SBs. Also, we indicated that CH combined with Y improved hESC generation efficiency by up to 31%. By using of Kenpaullone as an alternative to CH, we confirmed the involvement of GSK3 inhibition in hESC derivation from SBs. Interestingly, by treatment of 4-cell embryos, these SMs could enhance the derivation efficiency of SB-derived hESC lines up to 73% and the maximum number of hESC lines from SBs of one embryo was achieved in this state. LIMITATIONS, REASONS FOR CAUTION: The low quality of the embryos used in this study most likely had an effect on hESC generation. Furthermore, although we attempted to minimize any differences in inter-embryo quality, we cannot exclude the possibility that small differences in starting quality between embryos may have contributed to the differences observed, other than the addition of SMs. WIDER IMPLICATIONS OF THE FINDINGS: This approach would allow the establishment of autogeneic or allogeneic matched cells from embryos fertilized in vitro without destroying them. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the National Elite Foundation and the Royan Institute for Stem Cell Biology and Technology. The authors have no conflict of interest to declare.

Single-cell paired-end genome sequencing reveals structural variation per cell cycle.

The nature and pace of genome mutation is largely unknown. Because standard methods sequence DNA from populations of cells, the genetic composition of individual cells is lost, de novo mutations in cells are concealed within the bulk signal and per cell cycle mutation rates and mechanisms remain elusive. Although single-cell genome analyses could resolve these problems, such analyses are error-prone because of whole-genome amplification (WGA) artefacts and are limited in the types of DNA mutation that can be discerned. We developed methods for paired-end sequence analysis of single-cell WGA products that enable (i) detecting multiple classes of DNA mutation, (ii) distinguishing DNA copy number changes from allelic WGA-amplification artefacts by the discovery of matching aberrantly mapping read pairs among the surfeit of paired-end WGA and mapping artefacts and (iii) delineating the break points and architecture of structural variants. By applying the methods, we capture DNA copy number changes acquired over one cell cycle in breast cancer cells and in blastomeres derived from a human zygote after in vitro fertilization. Furthermore, we were able to discover and fine-map a heritable inter-chromosomal rearrangement t(1;16)(p36;p12) by sequencing a single blastomere. The methods will expedite applications in basic genome research and provide a stepping stone to novel approaches for clinical genetic diagnosis.

Intracellular protein and nucleic acid measured in eight cell types using deep-ultraviolet mass mapping.

We present measurements by deep-ultraviolet mass mapping of nucleic acid (NA) and protein for five commonly cultured and three primary cell types. The dry mass distribution at submicron resolution was determined on a single-cell basis for 250-500 cells from each of these types. Since the method carries a direct reference to a spectrophotometric standard (molar extinction coefficient), we are able to calibrate the absolute weight distributions both on a cell-to-cell basis within each type and across types. We also provide a calibration in absolute mass units for fluorescence-based measurements (flow cytometry and fluorescence microscopy). As might be expected the cultured cell lines show a high concentration of nucleic acids in the nuclear compartment, much larger than the genomic 2C number even in the G1 stage. The whole-cell nucleic-acid/protein ratio was found to be a characteristic of cell lines that persists independent of cell cycle and, as a result, this ratio has some value for phenotyping. Primary chicken red blood cells (cRBC), often used as a cytometry standard, were determined to have a nuclear-isolated nucleic acid content much closer to the genomic number than the cultured cell lines (cRBC: 3.00 pg total NA, 2.30 pg DNA, and 0.70 pg RNA). The individual blastomeres (n = 54) from mouse embryos at eight-cell stage were measured and found to vary by more than a factor or two in total protein and nucleic acid content (0.8-2.3 ng total protein, 70-150 pg total NA). The ratio of nucleic acid to protein was more nearly constant for each blastomere from a particular embryo and this ratio was found to be an identifying characteristic that varies from embryo to embryo obtained from a single flushing of a mouse.

Multinucleation of a sibling blastomere on day 2 suggests unsuitability for embryo transfer in IVF-preimplantation genetic screening cycles.

OBJECTIVE: To evaluate the impact of multinucleation of a sibling blastomere of day 2 embryos on the rate of aneuploidy detected by day 3 preimplantation genetic screening (PGS) biopsy and the effect on subsequent implantation and pregnancy rates. DESIGN: Retrospective cohort study. SETTING: University-based IVF center. PATIENT(S): A total of 141 couples undergoing their first IVF-PGS cycle for idiopathic recurrent pregnancy loss (RPL) or multiple failed IVF implantations. INTERVENTION(S): Biopsy of single-nucleated blastomeres for PGS analysis of chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22 by fluorescence in situ hybridization. MAIN OUTCOME MEASURE(S): Aneuploidy, implantation, and pregnancy rates. RESULT(S): PGS revealed an increased incidence of aneuploidy when comparing multinucleated day 2 embryos with single-nucleated embryos (85% vs. 78%; relative risk 0.92 (95% confidence interval 0.84-1.00). Transfer of single-nucleated euploid embryos resulted in clinical pregnancy and implantation rates of 28% and 24%. Transfer of multinucleated euploid embryos resulted in no implantations. CONCLUSION(S): The presence of multinucleated blastomeres on day 2 of embryo development, 1 day before biopsy, predicts an increase of aneuploidy and poor pregnancy outcomes in IVF-PGS cycles.

Birth of piglets from in vitro-produced, zona-intact porcine embryos vitrified in a closed system.

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN(2)). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a "closed" system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.

Transcript profiling of individual twin blastomeres derived by splitting two-cell stage murine embryos.

In invertebrates and amphibians, informational macromolecules in egg cytoplasm are organized to provide direction to the formation of embryonic lineages, but it is unclear whether vestiges of such prepatterning exist in mammals. Here we examined whether twin blastomeres from 2-cell stage mouse embryos differ in mRNA content. mRNA from 26 blastomeres derived from 13 embryos approximately mid-way through their second cell cycle was subjected to amplification. Twenty amplified samples were hybridized to arrays. Of those samples that hybridized successfully, 12 samples in six pairs were used in the final analysis. Probes displaying normalized values >0.25 (n = 4573) were examined for consistent bias in expression within blastomere pairs. Although transcript content varied between both individual embryos and twin blastomeres, no consistent asymmetries were observed for the majority of genes, with only 178 genes displaying a >1.4-fold difference in expression across all six pairs. Although class discovery clustering showed that blastomere pairs separated into two distinct groups in terms of their differentially expressed genes, when the data were tested for significance of asymmetrical expression, only 39 genes with >1.4-fold change ratios in six of six blastomere pairs passed the two-sample t-test (P < 0.05). Transcripts encoding proteins implicated in RNA processing and cytoskeletal organization were among the most abundant, differentially distributed mRNA, suggesting that a stochastically based lack of synchrony in cell cycle progression between the two cells might explain at least some and possibly all of the asymmetries in transcript composition.

Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease.

Mutations in mitochondrial DNA (mtDNA) are a common cause of genetic disease. Pathogenic mutations in mtDNA are detected in approximately 1 in 250 live births and at least 1 in 10,000 adults in the UK are affected by mtDNA disease. Treatment options for patients with mtDNA disease are extremely limited and are predominantly supportive in nature. Mitochondrial DNA is transmitted maternally and it has been proposed that nuclear transfer techniques may be an approach for the prevention of transmission of human mtDNA disease. Here we show that transfer of pronuclei between abnormally fertilized human zygotes results in minimal carry-over of donor zygote mtDNA and is compatible with onward development to the blastocyst stage in vitro. By optimizing the procedure we found the average level of carry-over after transfer of two pronuclei is less than 2.0%, with many of the embryos containing no detectable donor mtDNA. We believe that pronuclear transfer between zygotes, as well as the recently described metaphase II spindle transfer, has the potential to prevent the transmission of mtDNA disease in humans.

The possibility of a false positive arising from sperm DNA in genetic diagnosis of bovine embryos.

The present study was conducted to evaluate the effect of accessory sperm cells that adhered to the zona pellucida or blastomeres on the accuracy of genetic diagnosis of preimplantation embryos. The properties of sperm cells as a template for DNA amplification were examined using bovine sperm cells frozen-thawed or incubated in PBS after thawing for 7 days at 39 C. Sexing by loop-mediated isothermal amplification (LAMP) and claudin-16 genotyping by polymerase chain reaction (PCR) were performed using 10, 50 and 100 sperm cells. When sexing based on LAMP was performed, no amplified DNA was detected in 10 sperm-derived samples, whereas male-specific (10-60%) and gender-natural DNA (30-100%) sequences were detected in 50 and 100 sperm-derived samples. The detection rates for gender-natural DNA sequences were higher in incubated sperm samples than in sperm samples immediately after freeze-thawing. The detection rates for claudin-16 were low (7-13%) regardless of the concentration of sperm cells and the period of incubation after thawing. The present results showed that male-specific DNA, gender-natural DNA and claudin-16 sequences were not usually amplified from a small number of sperm cells (< or =10 cells). However, when a large number of sperm cells (> or =50 cells) were present, male-specific and gender-natural DNA sequences were amplified at a high rate, and claudin-16 DNA sequences were also occasionally detected. These results raise the possibility that accessory sperm cells may reduce the accuracy of the genetic diagnosis of bovine embryos. Therefore, steps to prevent the contamination of sperm cells, such as removal of the zona pellucida and washing of sample blastomeres, are necessary to obtain an accurate result.

A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes.

Mammalian mitochondrial DNA (mtDNA) is inherited principally down the maternal line, but the mechanisms involved are not fully understood. Females harboring a mixture of mutant and wild-type mtDNA (heteroplasmy) transmit a varying proportion of mutant mtDNA to their offspring. In humans with mtDNA disorders, the proportion of mutated mtDNA inherited from the mother correlates with disease severity. Rapid changes in allele frequency can occur in a single generation. This could be due to a marked reduction in the number of mtDNA molecules being transmitted from mother to offspring (the mitochondrial genetic bottleneck), to the partitioning of mtDNA into homoplasmic segregating units, or to the selection of a group of mtDNA molecules to re-populate the next generation. Here we show that the partitioning of mtDNA molecules into different cells before and after implantation, followed by the segregation of replicating mtDNA between proliferating primordial germ cells, is responsible for the different levels of heteroplasmy seen in the offspring of heteroplasmic female mice.

[Distribution of foreign mitochondrial DNA during the first splittings of the transmitochondrial mouse embryos].

Distribution of human mitochondrial DNA (mtDNA) among separate murine blastomeres was analyzed during the splitting of embryos in which the suspension of human mitochondria had been injected at the one- or two-cell stage. Human mtDNA was detected by PCR with species specific primers. The total amount of the two- and four-cell murine embryos analyzed in the study was 339. In all embryos examined the copies of human mitochondrial genome were revealed along with murine mtDNA, which indicated the phenomenon of an artificially modeled heteroplasmy. The foreign mtDNA was not ubiquitous among the blastomeres of transmitochondrial embryos. Mathematical analysis of the results showed that in the period between the injection of human mitochondria and the subsequent splitting no equal distribution of the human mtDNA occurred in the cytoplasm. These results also point at the presence of more than 2-3 segregation units of mtDNA in the entire pool of mitochondria (about 5 x 10(2)) introduced into an embryo by microinjection.

Fluorescence in situ hybridization on single cells. (Sex determination and chromosome rearrangements).

Fluorescence in situ hybridization (FISH) is the technique of choice for preimplantation genetic diagnosis (PGD) selection of female embryos in families with X-linked disease, for which there is no mutation-specific test. FISH with target-specific DNA probes is also the primary technique used for PGD detection of chromosome imbalance associated with Robertsonian translocations, reciprocal translocations, inversions, and other chromosome rearrangements, because the DNA probes, labeled with different fluorochromes or haptens, detect the copy number of their target loci. The methods described outline strategies for PGD for sex determination and chromosome rearrangements. These methods are assessment of reproductive risks, the selection of suitable probes for interphase FISH, spreading techniques for blastomere nuclei, and in situ hybridization and signal scoring using directly labeled and indirectly labeled probes.

Single cell polymerase chain reaction for preimplantation genetic diagnosis: methods, strategies, and limitations.

We provide an overview of the methodology involved in single cell polymerase chain reaction (PCR), especially for single lymphocytes or cultured lymphoblasts and blastomeres. The first step toward single cell PCR is isolation of single cells; the protocols given can be carried out using basic instruments such as a stereomicroscope. We also describe the alkaline lysis method for cell lysis as well as the design and execution of single cell PCR, either in simplex or in multiplex. Finally, we briefly discuss the different methods for analyzing PCR products and obtaining an accurate diagnosis. Special attention is given to measures that avoid contamination with extraneous DNA and reduce allele dropout.

Single-cell chromosomal imbalances detection by array CGH.

Genomic imbalances are a major cause of constitutional and acquired disorders. Therefore, aneuploidy screening has become the cornerstone of preimplantation, prenatal and postnatal genetic diagnosis, as well as a routine aspect of the diagnostic workup of many acquired disorders. Recently, array comparative genomic hybridization (array CGH) has been introduced as a rapid and high-resolution method for the detection of both benign and disease-causing genomic copy-number variations. Until now, array CGH has been performed using a significant quantity of DNA derived from a pool of cells. Here, we present an array CGH method that accurately detects chromosomal imbalances from a single lymphoblast, fibroblast and blastomere within a single day. Trisomy 13, 18, 21 and monosomy X, as well as normal ploidy levels of all other chromosomes, were accurately determined from single fibroblasts. Moreover, we showed that a segmental deletion as small as 34 Mb could be detected. Finally, we demonstrated the possibility to detect aneuploidies in single blastomeres derived from preimplantation embryos. This technique offers new possibilities for genetic analysis of single cells in general and opens the route towards aneuploidy screening and detection of unbalanced translocations in preimplantation embryos in particular.