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Fragouli E.,University of Oxford | Alfarawati S.,University of Oxford | Daphnis D.D.,London Fertility Center | Goodall N.-N.,Reprogenetics LLC | And 4 more authors.
Human Reproduction | Year: 2011

BACKGROUNDRecent studies have suggested that biopsy of several trophectoderm (TE) cells from blastocysts followed by comparative genomic hybridization (CGH) analysis might represent an optimal strategy for aneuploidy detection, but few data on accuracy are available. The main question concerns the rate of mosaicism at the blastocyst stage, and to what extent this might cause misdiagnoses. We assessed blastocyst aneuploidy and mosaicism rates and evaluated the accuracy and efficiency of CGH and microarray-CGH (aCGH) for TE analysis.METHODSA total of 52 blastocysts, from 20 couples, were biopsied and their chromosomes examined by CGH. The remaining cells were spread and tested by fluorescent in situ hybridization (FISH). Of the 52 blastocysts, 20 underwent a second TE biopsy and were tested using aCGH.RESULTSCGH and aCGH produced results for 98 of TE samples. 42.3 of blastocysts were uniformly euploid, 30 were uniformly aneuploid and 32.4 were mosaic. Of the mosaic embryos, 15.4% were found to be composed of a mixture of different aneuploid cell lines, while 17 contained both normal and aneuploid cells. Mosaic diploidaneuploid blastocysts with >30 normal cells accounted for <6 of analysed embryos. CONCLUSIONSComprehensive chromosome screening and follow-up assessment of large numbers of cells provided a unique insight into the cytogenetics of human blastocysts. Meiotic and post-zygotic errors leading to mosaicism were common. However, most mosaic blastocysts contained no normal cells. Hence, CGH or aCGH TE analysis is an accurate aneuploidy detection tool and may assist in identifying viable euploid embryos with higher implantation potential. © 2010 The Author. Source


Alfarawati S.,University of Oxford | Alfarawati S.,Oxford Business Park | Fragouli E.,University of Oxford | Fragouli E.,Oxford Business Park | And 2 more authors.
Human Reproduction | Year: 2011

BACKGROUND: Balanced chromosomal rearrangements represent one of the most frequent indications for preimplantation genetic diagnosis (PGD). Although fluorescence in situ hybridization (FISH) has been successfully employed for diagnosis in such cases, this approach usually restricts assessment of the chromosomes involved in the rearrangement. Furthermore, with FISH-based strategies, it is sometimes necessary to create patient-specific protocols, increasing the waiting time and costs. In the current study, we explored the use of two comprehensive chromosome screening methods, conventional metaphase comparative genomic hybridization (CGH) and microarray-CGH (aCGH), as alternatives for PGD of chromosome rearrangements. Methods The study included 16 patients who underwent 20 cycles of PGD for a variety of chromosome rearrangements (reciprocal or Robertsonian translocations or inversions). Testing was performed at various embryonic stages using CGH (9 cases) or aCGH (11 cases). Results Results were obtained for 121 out of 132 samples (91.7%). Of the diagnosed samples, 48.8% were found to carry abnormalities associated with the rearrangement, either alone or in combination with other chromosomal abnormalities. A further 28.9% of samples were normal/balanced for the rearranged chromosomes, but affected by aneuploidy for other chromosomes. Only 22.3% of samples were chromosomally normal. Of the 15 patients who completed their treatment cycles, 5 became pregnant after one or two cycles resulting in four healthy births. The delivery rate per cycle was 21% (27% per embryo transfer). CONCLUSIONS This is the first study to describe the clinical application of comprehensive chromosome screening applied to polar bodies, blastomeres or trophectoderm cells from patients carrying inversions and translocations. Using these techniques, most patients requesting PGD for a chromosome rearrangement can be treated using a single protocol. Additionally, the detection of abnormalities affecting chromosomes unrelated to the rearrangement may assist in the selection of viable embryos for transfer. © The Author 2011. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. Source


Harton G.,Reprogenetics LLC | Braude P.,Center for Preimplantation Genetic Diagnosis | Lashwood A.,Center for Preimplantation Genetic Diagnosis | Schmutzler A.,University of Kiel | And 4 more authors.
Human Reproduction | Year: 2011

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. Subsequent years have seen the introduction of new technologies as well as the evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written/formulated, the Consortium believed it was timely to update the PGD guidelines. Rather than one document that covers all of PGD, the new guidelines are separated into four documents, including one relating to organization of the PGD centre and three relating to the methods used: DNA amplification, fluorescence in situ hybridization and biopsy/embryology. Here, we have updated the sections on organization of the PGD centre. One area that has continued to expand is Transport PGD, in which patients are treated at one IVF centre, whereas their gametes/embryos are tested elsewhere, at an independent PGD centre. Transport PGD/preimplantation genetic screening (PGS) has a unique set of challenges with respect to the nature of the sample and the rapid turn-around time required. PGS is currently controversial. Opinions of laboratory specialists and clinicians interested in PGD and PGS have been taken into account here. Current evidence suggests that PGS at cleavage stages is ineffective, but whether PGS at the blastocyst stage or on polar bodies might show improved delivery rates is still unclear. Thus, in this revision, PGS has been included. This document should assist everyone interested in PGD/PGS in developing the best laboratory and clinical practice possible. © 2010 The Author. Source


Harton G.L.,Reprogenetics LLC | Harton G.L.,Genetics and IVF Institute | Magli M.C.,Reproductive Medicine Unit | Lundin K.,Sahlgrenska University Hospital | And 4 more authors.
Human Reproduction | Year: 2011

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) Preimplantation Genetic Diagnosis (PGD) Consortium published a set of Guidelines for Best Practice to give information, support and guidance to potential, existing and fledgling PGD programmes (Thornhill AR, De Die-Smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, Moutou C, Robinson MD, Schmutzler AG, Scriven PN et al. ESHRE PGD Consortium best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2005;20:3548.). The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of ESHREs recent advice on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme; Organization of a PGD centre, fluorescence in situ hybridization-based testing, amplification-based testing and polar body and embryo biopsy for preimplantation genetic diagnosis/screening (PGD/PGS). Here we have updated the sections that pertain to embryology (including cryopreservation) and biopsy of embryos prior to PGD or PGS. Topics covered in this guideline include uses of embryo biopsy, laboratory issues relating to biopsy, timing of biopsy, biopsy procedure and cryopreserving biopsied embryos. © 2010 The Author. Source


Harton G.L.,Reprogenetics LLC | Harton G.L.,Genetics and IVF Institute | De Rycke M.,Universitair Ziekenhuis | Fiorentino F.,Genoma Laboratories | And 5 more authors.
Human Reproduction | Year: 2011

In 2005, the European Society for Human Reproduction and Embryology (ESHRE) PGD Consortium published a set of Guidelines for Best Practice PGD to give information, support and guidance to potential, existing and fledgling PGD programmes. The subsequent years have seen the introduction of a number of new technologies as well as the evolution of current techniques. Additionally, in light of recent advice from ESHRE on how practice guidelines should be written and formulated, the Consortium believed it was timely to revise and update the PGD guidelines. Rather than one document that covers all of PGD, as in the original publication, these guidelines are separated into four new documents that apply to different aspects of a PGD programme, i.e. Organization of a PGD centre, fluorescence in situ hybridization-based testing, Amplification-based testing and Polar Body and Embryo Biopsy for PGD/preimplantation genetic screening. Here, we have updated the sections that pertain to amplification-based PGD. Topics covered in this guideline include inclusion/exclusion criteria for amplification-based PGD testing, preclinical validation of tests, amplification-based testing methods, tubing of cells for analysis, set-up of local IVF centre and Transport PGD centres, quality control/quality assurance and diagnostic confirmation of untransferred embryos. © 2010 The Author. Source

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