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Munne S.,Reprogenetics | Fragouli E.,Reprogenetics UK | Colls P.,Reprogenetics | Katz-Jaffe M.G.,Colorado Center for Reproductive Medicine | And 3 more authors.
Reproductive BioMedicine Online | Year: 2010

Fluorescence in-situ hybridization (FISH) has been the principal method used for the identification and preferential transfer of chromosomally normal embryos, in the context of both preimplantation genetic diagnosis (PGD) and screening (PGS). Generally, the probe combinations used during PGS have focused on chromosomes frequently identified as abnormal in prenatal samples or material derived from first-trimester spontaneous abortions. Recent data, however, obtained with the use of comparative genomic hybridization (CGH), have suggested that commonly used PGS strategies may fail to detect a large number of aneuploidies affecting preimplantation embryos. Some chromosomes, which have been relatively neglected in PGS protocols thus far, display a disproportionate contribution to embryo aneuploidy and should be prioritized for screening. Using CGH data, it is possible to design new probe combinations that examine between 10 and 12 chromosomes and are capable of accurately diagnosing 89-91% of anomalies seen in embryos. At present, 24-chromosome tests, such as CGH, array CGH or single nucleotide polymorphism arrays, remain relatively costly and, in some cases, are yet to be fully validated. For these reasons, a cost-effective method, capable of accurately detecting almost all aneuploid embryos, represents an attractive alternative to comprehensive chromosome screening approaches. © 2009, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Fragouli E.,Reprogenetics UK | Wells D.,Reprogenetics UK | Wells D.,University of Oxford
Seminars in Reproductive Medicine | Year: 2012

Chromosome abnormalities are extremely common in human oocytes and embryos and are associated with a variety of negative outcomes for both natural cycles and those using assisted conception techniques. Embryos containing the wrong number of chromosomes (aneuploidy) may fail to implant in the uterus, miscarry, or lead to children with serious medical problems (e.g., Down syndrome). Preimplantation genetic screening (PGS) is a method that seeks to improve the outcomes of assisted reproductive treatments, such as in vitro fertilization (IVF), by ensuring that the embryos chosen for transfer to the uterus are chromosomally normal. Here we summarize published and novel data concerning the frequency and variety of chromosomal abnormalities seen in oocytes and embryos at the cleavage and blastocyst stages of development. Clinical outcomes of studies using PGS are presented, and the controversy over the use of chromosome screening as a tool for embryo selection is discussed. We describe validation and preliminary clinical data from the new generation of methods being used for PGS, including comparative genomic hybridization (CGH), microarrays (aCGH and single nucleotide polymorphism arrays), and quantitative polymerase chain reaction. These methodologies allow comprehensive chromosomal analysis, provide high accuracy, and have yielded encouraging preliminary clinical data. The combination of advances in genetics and embryology seems poised to usher in a new era in the treatment of infertility. Copyright © 2012 by Thieme Medical Publishers, Inc.

Munne S.,Reprogenetics | Held K.R.,Reprogenetics Germany | Magli C.M.,S.I.S.Me.R | Ata B.,Uludag University | And 5 more authors.
Fertility and Sterility | Year: 2012

Objective: To determine the extent of intra-age and intercycle variations in the frequency of first polar body aneuploidy in two consecutive cycles of oocyte retrieval undertaken by the same patient within 1 year. Design: Retrospective study. Setting: Fertility centers. Patient(s): Infertile couples undergoing IVF. Intervention(s): Patients underwent two consecutive cycles of preimplantation genetic screening through first polar body biopsy within 1 year. Main Outcome Measure(s): Meiosis I aneuploidy. Result(s): A total of 226 patients underwent 452 cycles of preimplantation genetic screening. Differences within age groups were wide, with 0-100% of oocytes being chromosomally normal in all age groups. Euploidy rates between centers were significantly different (48% vs. 25%). Intercycle differences for the same patient were also wide (0-100%), but with 68.5% of patients having less than ±2 euploid eggs of difference between cycles. Conclusion(s): Although euploidy rate decreased on average with advancing maternal age, the high intra-age and intercenter variation in oocyte chromosome abnormalities emphasize the difficulty in estimating how many euploid oocytes a specific woman will have. This may have repercussions for fertility preservation where a defined number of eggs are currently frozen just based on maternal age. © 2012 by American Society for Reproductive Medicine.

Fragouli E.,University of Oxford | Alfarawati S.,Reprogenetics UK | Spath K.,University of Oxford | Wells D.,University of Oxford
Molecular Human Reproduction | Year: 2014

Morphological assessments are the main way in which fertility clinics select in vitro generated embryo(s) for transfer to the uterus. However, it is widely acknowledged that the microscopic appearance of an embryo is only weakly correlated with its viability. Furthermore, the extent to which morphology is affected by aneuploidy, a genetic defectcommon in human preimplantation embryos, remains unclear. Aneuploidy is of great relevance to embryo selection as it represents one of the most important causes of implantation failure and miscarriage. The current studyaimed to examine whether morphological appearance can assist in identifying embryos at risk of aneuploidy. Additionally, the data produced sheds light on how chromosomal anomalies impact development from the cleavage to the blastocyst stage. A total of 1213 embryos were examined. Comprehensive chromosome analysis was combined with well-established criteria for the assessment of embryo morphology. At the cleavage stage, chromosome abnormalities were common even amongst embryos assigned the best morphological scores, indicating that aneuploidy has little effect on microscopic appearance at fixed time points up until Day 3 of development.However, at the blastocyst stage aneuploidies were found to be significantly lesscommonamong embryos of optimal morphological quality, while such abnormalities were overrepresented amongst embryos considered to be of poor morphology. Despite the link between aneuploidy and blastocyst appearance, many chromosomally abnormal embryos were able to achieve the highest morphological scores. In particular, blastocysts affected by forms of aneuploidy with the greatest capacity to produce clinical pregnancies (e.g. trisomy 21) were indistinguishable fromeuploid embryos. The sex ratiowas seen to be equal throughout preimplantation development. Interestingly, however, females were overrepresented amongst the fastest growing cleavage-stage embryos, whereas a sex-related skew in the opposite direction was noted for the most rapidly developing blastocysts. In summary, this study confirms that, at the cleavage stage, chromosome abnormalities have little if any effect on morphological scores assigned using traditional criteria. At the blastocyst stage some forms of aneuploidy begin to affect microscopic appearance, but in most instances the impact is subtle. In the case of the most clinically relevant aneuploidies (those capable of forming a pregnancy) there was no detectable effect on morphology at any preimplantation stage. © The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Fragouli E.,Reprogenetics UK | Spath K.,University of Oxford | Alfarawati S.,Reprogenetics UK | Kaper F.,Illumina | And 7 more authors.
PLoS Genetics | Year: 2015

Mitochondria play a vital role in embryo development. They are the principal site of energy production and have various other critical cellular functions. Despite the importance of this organelle, little is known about the extent of variation in mitochondrial DNA (mtDNA) between individual human embryos prior to implantation. This study investigated the biological and clinical relevance of the quantity of mtDNA in 379 embryos. These were examined via a combination of microarray comparative genomic hybridisation (aCGH), quantitative PCR and next generation sequencing (NGS), providing information on chromosomal status, amount of mtDNA, and presence of mutations in the mitochondrial genome. The quantity of mtDNA was significantly higher in embryos from older women (P=0.003). Additionally, mtDNA levels were elevated in aneuploid embryos, independent of age (P=0.025). Assessment of clinical outcomes after transfer of euploid embryos to the uterus revealed that blastocysts that successfully implanted tended to contain lower mtDNA quantities than those failing to implant (P=0.007). Importantly, an mtDNA quantity threshold was established, above which implantation was never observed. Subsequently, the predictive value of this threshold was confirmed in an independent blinded prospective study, indicating that abnormal mtDNA levels are present in 30% of non-implanting euploid embryos, but are not seen in embryos forming a viable pregnancy. NGS did not reveal any increase in mutation in blastocysts with elevated mtDNA levels. The results of this study suggest that increased mtDNA may be related to elevated metabolism and are associated with reduced viability, a possibility consistent with the ‘quiet embryo’ hypothesis. Importantly, the findings suggest a potential role for mitochondria in female reproductive aging and the genesis of aneuploidy. Of clinical significance, we propose that mtDNA content represents a novel biomarker with potential value for in vitro fertilisation (IVF) treatment, revealing chromosomally normal blastocysts incapable of producing a viable pregnancy. © 2015 Fragouli et al.

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