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Wells D.,University of Oxford | Kaur K.,Oxford Genetics | Grifo J.,New York University | Glassner M.,Main Line Fertility Bryn Mawr | And 3 more authors.
Journal of Medical Genetics | Year: 2014

Background: The majority of human embryos created using in vitro fertilisation (IVF) techniques are aneuploid. Comprehensive chromosome screening methods, applicable to single cells biopsied from preimplantation embryos, allow reliable identification and transfer of euploid embryos. Recently, randomised trials using such methods have indicated that aneuploidy screening improves IVF success rates. However, the high cost of testing has restricted the availability of this potentially beneficial strategy. This study aimed to harness nextgeneration sequencing (NGS) technology, with the intention of lowering the costs of preimplantation aneuploidy screening. Methods: Embryo biopsy, whole genome amplification and semiconductor sequencing. Results: A rapid (<15 h) NGS protocol was developed, with consumable cost only two-thirds that of the most widely used method for embryo aneuploidy detection. Validation involved blinded analysis of 54 cells from cell lines or biopsies from human embryos. Sensitivity and specificity were 100%. The method was applied clinically, assisting in the selection of euploid embryos in two IVF cycles, producing healthy children in both cases. The NGS approach was also able to reveal specified mutations in the nuclear or mitochondrial genomes in parallel with chromosome assessment. Interestingly, elevated mitochondrial DNA content was associated with aneuploidy ( p<0.05), a finding suggestive of a link between mitochondria and chromosomal malsegregation. Conclusions: This study demonstrates that NGS provides highly accurate, low-cost diagnosis of aneuploidy in cells from human preimplantation embryos and is rapid enough to allow testing without embryo cryopreservation. The method described also has the potential to shed light on other aspects of embry genetics of relevance to health and viability. Source


Jaroudi S.,Institute of Reproductive science | Wells D.,University of Oxford
Methods in Molecular Biology | Year: 2013

The cytogenetic analysis of single cells, such as oocytes and polar bodies, is extremely challenging. The main problem is low probability of obtaining a metaphase preparation in which all of the chromosomes are sufficiently well spread to permit accurate analysis (no overlapping chromosomes, no chromosomes lost). As a result, a high proportion of the oocytes subjected to cytogenetic analysis are not suitable for traditional chromosome banding studies or for molecular cytogenetic methods such as spectral karyotyping (SKY) or multiplex fluorescence in situ hybridization (M-FISH). Fortunately, recent innovations in whole genome amplification and microarray technologies have provided a means to analyze the copy number of every chromosome in single cells with high accuracy. Here we describe the use of such methods for the investigation of chromosome and chromatid abnormalities in human oocytes and polar bodies. © 2013 Springer Science+Business Media, LLC. Source


Kashir J.,Womens Center | Jones C.,Womens Center | Mounce G.,Womens Center | Ramadan W.M.,Womens Center | And 8 more authors.
Fertility and Sterility | Year: 2013

Objective: To examine whether similar levels of phospholipase C zeta (PLC-ζ) protein are present in sperm from men whose ejaculates resulted in normal oocyte activation, and to examine whether a predominant pattern of PLC-ζ localization is linked to normal oocyte activation ability. Design: Laboratory study. Setting: University laboratory. Patient(s): Control subjects (men with proven oocyte activation capacity; n = 16) and men whose sperm resulted in recurrent intracytoplasmic sperm injection failure (oocyte activation deficient [OAD]; n = 5). Intervention(s): Quantitative immunofluorescent analysis of PLC-ζ protein in human sperm. Main Outcome Measure(s): Total levels of PLC-ζ fluorescence, proportions of sperm exhibiting PLC-ζ immunoreactivity, and proportions of PLC-ζ localization patterns in sperm from control and OAD men. Result(s): Sperm from control subjects presented a significantly higher proportion of sperm exhibiting PLC-ζ immunofluorescence compared with infertile men diagnosed with OAD (82.6% and 27.4%, respectively). Total levels of PLC-ζ in sperm from individual control and OAD patients exhibited significant variance, with sperm from 10 out of 16 (62.5%) exhibiting levels similar to OAD samples. Predominant PLC-ζ localization patterns varied between control and OAD samples with no predictable or consistent pattern. Conclusion(s): The results indicate that sperm from control men exhibited significant variance in total levels of PLC-ζ protein, as well as significant variance in the predominant localization pattern. Such variance may hinder the diagnostic application of quantitative PLC-ζ immunofluorescent analysis. Copyright © 2013 American Society for Reproductive Medicine, Published by Elsevier Inc. Source


Natesan S.A.,Illumina | Bladon A.J.,Illumina | Coskun S.,King Faisal Specialist Hospital And Research Center | Qubbaj W.,King Faisal Specialist Hospital And Research Center | And 13 more authors.
Genetics in Medicine | Year: 2014

Purpose:Our aim was to compare the accuracy of family- or disease-specific targeted haplotyping and direct mutation-detection strategies with the accuracy of genome-wide mapping of the parental origin of each chromosome, or karyomapping, by single-nucleotide polymorphism genotyping of the parents, a close relative of known disease status, and the embryo cell(s) used for preimplantation genetic diagnosis of single-gene defects in a single cell or small numbers of cells biopsied from human embryos following in vitro fertilization.Methods:Genomic DNA and whole-genome amplification products from embryo samples, which were previously diagnosed by targeted haplotyping, were genotyped for single-nucleotide polymorphisms genome-wide detection and retrospectively analyzed blind by karyomapping.Results:Single-nucleotide polymorphism genotyping and karyomapping were successful in 213/218 (97.7%) samples from 44 preimplantation genetic diagnosis cycles for 25 single-gene defects with various modes of inheritance distributed widely across the genome. Karyomapping was concordant with targeted haplotyping in 208 (97.7%) samples, and the five nonconcordant samples were all in consanguineous regions with limited or inconsistent haplotyping results.Conclusion:Genome-wide karyomapping is highly accurate and facilitates analysis of the inheritance of almost any single-gene defect, or any combination of loci, at the single-cell level, greatly expanding the range of conditions for which preimplantation genetic diagnosis can be offered clinically without the need for customized test development.Genet Med 16 11, 838-845. Source


Alfarawati S.,University of Oxford | Alfarawati S.,Institute of Reproductive science | Fragouli E.,University of Oxford | Fragouli E.,Institute of Reproductive science | And 3 more authors.
PLoS Genetics | Year: 2012

Balanced chromosomal rearrangements represent one of the most common forms of genetic abnormality affecting approximately 1 in every 500 (0.2%) individuals. Difficulties processing the abnormal chromosomes during meiosis lead to an elevated risk of chromosomally abnormal gametes, resulting in high rates of miscarriage and/or children with congenital abnormalities. It has also been suggested that the presence of chromosome rearrangements may also cause an increase in aneuploidy affecting structurally normal chromosomes, due to disruption of chromosome alignment on the spindle or disturbance of other factors related to meiotic chromosome segregation. The existence of such a phenomenon (an inter-chromosomal effect-ICE) remains controversial, with different studies presenting contradictory data. The current investigation aimed to demonstrate conclusively whether an ICE truly exists. For this purpose a comprehensive chromosome screening technique, optimized for analysis of minute amounts of tissue, was applied to a unique collection of samples consisting of 283 oocytes and early embryos derived from 44 patients carrying chromosome rearrangements. A further 5,078 oocytes and embryos, derived from chromosomally normal individuals of identical age, provided a robust control group for comparative analysis. A highly significant (P = 0.0002) increase in the rate of malsegregation affecting structurally normal chromosomes was observed in association with Robertsonian translocations. Surprisingly, the ICE was clearly detected in early embryos from female carriers, but not in oocytes, indicating the possibility of mitotic rather than the previously suggested meiotic origin. These findings have implications for our understanding of genetic stability during preimplantation development and are of clinical relevance for patients carrying a Robertsonian translocation. The results are also pertinent to other situations when cellular mechanisms for maintaining genetic fidelity are relaxed and chromosome rearrangements are present (e.g. in tumors displaying chromosomal instability). © 2012 Alfarawati et al. Source

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