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(searched for: doi:10.1093/humrep/des366)
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Silvia Mateo, , Lluc Coll, , Montserrat Boada
Published: 1 January 2017
JBRA Assisted Reproduction, Volume 21, pp 203-207; https://doi.org/10.5935/1518-0557.20170039

Abstract:
The combination of FISH and aCGH has been shown to be a suitable approach to increase the knowledge about monopronucleated ICSI-derived embryos. FISH analysis of blastocysts derived from monopronucleated ICSI zygotes enabled us to conclude that aCGH underestimates haploidy. Some diploid embryos diagnosed by aCGH are in fact mosaic. In cases where these embryos would be used for reproductive purposes, extra analysis of parental genome origin is recommended.
, L. Guzman, A. Mertzanidou, Kurt Jacobs, C. Ortega-Hrepich, , , , ,
Published: 4 December 2014
Human Reproduction, Volume 30, pp 653-663; https://doi.org/10.1093/humrep/deu329

Abstract:
Do cleavage-stage embryos obtained from oocytes matured in vitro after pre-incubation with a phosphodiesterase inhibitor (IBMX) carry more chromosomal abnormalities than those generated from oocytes matured in vivo? The rate and type of chromosomal abnormalities in normally developing cleavage-stage embryos generated with an in vitro maturation (IVM) system including pre-incubation with IBMX are not different from those observed in supernumerary embryos obtained from oocytes matured in vivo. Very limited information is available about the chromosomal constitution of IVM embryos. Previous studies were carried out using FISH on single biopsied blastomeres or arrested whole embryos and only provided fragmentary information on chromosomal abnormalities in IVM embryos. There is no systematic study of chromosomal abnormalities in all blastomeres of human Day 3 embryos with good morphology. Between July 2012 and December 2012, 16 young (age <35 years old) egg donors underwent 18 IVM cycles for the generation of research embryos. Eighteen embryos developed to Day 3 and were analysed using array comparative genomic hybridization (aCGH). Immature oocytes were retrieved from 2 to 10 mm follicles after mild ovarian stimulation with gonadotrophins but without hCG ovulation trigger. At collection, oocytes were pre-incubated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and matured in vitro. After IVM culture, mature oocytes were microinjected with sperm from a single donor. Embryos were cultured to Day 3 after ICSI and all blastomeres of 18 good-morphology embryos were collected individually for aCGH. Oocyte maturation rate in vitro was 50.2% (120/239). The mean fertilization rate was 68.3% (82/120) and 30.5% (25/82) of fertilized oocytes developed into a morphologically good quality embryo on Day 3 after ICSI. Of these, 18 embryos that developed well up to Day 3 were analysed using aCGH. Eighty of the 123 blastomeres analysed showed at least one chromosomal abnormality. Three out of eighteen embryos had completely normal cells. A single embryo carried a meiotic abnormality, 11 embryos were mosaic and three were chaotic. Although the aneuploidy data of this study are too limited to allow statistical analysis, these data are comparable to our own published data on the chromosome constitution of whole day 3 and day 4 embryos after conventional ART. Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. Although the limited number of embryos analysed here precludes firm conclusions, they provide valuable data on possible causes of the reduced potential of IVM embryos. This is the first study to describe the complete chromosome complement of all single blastomeres of good-morphology day 3 embryos obtained with IVM (including the presence of IBMX in a pre-incubation medium). The results demonstrate that a high proportion of good-morphology embryos are aneuploid and that there is no obvious increase in aneuploidies as a result of IVM which seems to suggest that the reduced efficiency of IVM technology compared with standard IVF may be accounted for by factors other than aneuploidy, such as cytoplasmic defects or reduced endometrial receptivity. This study was funded by the TBM (Applied Biomedical Research with Societal Finality) programme of the IWT (Agency for Innovation through Science and Technology – Flanders, 110680) and by a Methusalem grant of the Vrije Universiteit Brussel. C.S. is a post-doctoral fellow of the Fund for Scientific Research Flanders (FWO – Vlaanderen). K.J. is a PhD student funded by the FWO. The University of Adelaide owns a patent family associated with IVM technologies that is licensed to Cook Medical. R.B.G. and J.G.T. are inventors. The remaining authors have no conflict of interest to declare.
A. Mertzanidou, , , H. Van de Velde,
Human Reproduction, Volume 28, pp 1716-1724; https://doi.org/10.1093/humrep/det079

Abstract:
What is the incidence of aneuploidy and mosaicism in all cells of top-quality Day-4 embryos analysed by array-based comparative genomic hybridization (array CGH)? Our data show extensive abnormalities in Day-4 embryos. Numerous studies on human embryos at Day 3 and Day 5 of development show that they frequently contain aneuploid cells and are mosaic, although Day-5 embryos contain proportionally more normal cells than at Day 3. In contrast, only limited data exist on Day 4 of preimplantation development, despite the fact that it is the suggested stage for the initiation of the process of self-correction. Thirteen embryos were analysed: four fresh good-quality preimplantation genetic diagnosis (PGD) embryos and nine good-quality surplus embryos cryopreserved on Day 3 and donated for research. On Day 4, following removal of the zona pellucida, all blastomeres were disaggregated and collected. The genomic DNA of 283 single blastomeres from disaggregated embryos was amplified. Array CGH was carried out using 24SureTM Cytochip microarrays. After scanning of the microarray slides, the images were analysed using BlueFuse Software (BlueGnome). Combined with selective microsatellite analysis, hypothetical reconstructions of embryo chromosome complements were made following each of the first four cleavage divisions. No chromosome imbalance was detected for one PGD embryo, the other three were mosaic containing between 16 and 75% abnormal cells. All nine frozen–thawed embryos were abnormal. Six were mosaic with between 30 and 100% abnormal cells; three had abnormalities of meiotic origin, two of which displayed mitotic abnormalities. Evidence was also found of mitotic unbalanced structural chromosome rearrangements. The higher rate of abnormality of frozen–thawed embryos is based on a small number of embryos and cannot be tested statistically. The aneuploidy can mostly be explained by anaphase lag and non-disjunction. In some cases, we hypothesize endoreduplication followed by a cellular division with multipolar spindles to explain the results. Array CGH technology determines relative quantification of chromosomal domains but does not allow for the visualization of chromosomal rearrangements, assessment of ploidy or detection of uniparental isodisomy. Conclusions drawn on segmental abnormalities should be treated with caution. The division trees presented are hypothetical models projecting back in time that try to explain observations in single blastomeres of Day 4 embryos. The limited number of embryos analysed does not allow drawing firm conclusions, but nevertheless provides valuable data on the origin of aneuploidy in human embryos. Our data show extensive abnormalities in Day-4 embryos. We found no evidence of self-correction at this stage of development, suggesting that this process may start at a later stage of development. This research was supported by the Instituut voor de aanmoediging van innovatie door Wetenschap en Technologie in Vlaanderen (IWT-Vlaanderen). C.S. is a postdoctoral fellow at the Flemish Fund for Scientific Research [Fonds voor Wetenschappelijk Onderzoek (FWO) Vlaanderen]. There are no competing interests.
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