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Handyside A.H.,Gynaecology and Genetics Center | Handyside A.H.,University of Leeds
Biochimica et Biophysica Acta - Molecular Basis of Disease | Year: 2012

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis has shown that these are predominantly maternal in origin and trisomies most frequently occur through errors in the first meiotic division. Analysis of chromosome copy number in the three products of female meiosis, the first and second polar bodies and the corresponding zygote by microarray comparative genomic hybridisation (array CGH), in women of advanced maternal age undergoing IVF, has recently revealed a pattern of frequent multiple meiotic errors, caused by premature predivision of sister chromatids in meiosis I and a high incidence of errors in meiosis II. This pattern is similar to those observed in various mouse models which implicate the gradual depletion of cohesins, which are essential for cohesion of sister chromatids, as the primary cause of age related aneuploidy in female meiosis. However, defects in other aspects of meiosis including the formation and stabilisation of chiasmata and the spindle assembly checkpoint (SAC) may also contribute. The challenge remains to explain the molecular basis of 'physiological' rather than 'chronological' female ageing and the contribution of multifactorial causes from the fetal to adult ovary. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure. © 2012 Elsevier B.V.


Handyside A.H.,Gynaecology and Genetics Center | Handyside A.H.,University of Leeds | Harton G.L.,Genetics and IVF Institute | Mariani B.,Genetics and IVF Institute | And 5 more authors.
Journal of Medical Genetics | Year: 2010

The use of genome wide single nucleotide polymorphism (SNP) arrays for high resolution molecular cytogenetic analysis using a combination of quantitative and genotype analysis is well established. This study demonstrates that by Mendelian analysis of the SNP genotypes of the parents and a sibling or other appropriate family member to establish phase, it is possible to identify informative loci for each of the four parental haplotypes across each chromosome and map the inheritance of these haplotypes and the position of any crossovers in the proband. The resulting 'karyomap', unlike a karyotype, identifies the parental and grandparental origin of each chromosome and chromosome segment and is unique for every individual being defined by the independent segregation of parental chromosomes and the pattern of non-recombinant and recombinant chromosomes. Karyomapping, therefore, enables both genome wide linkage based analysis of inheritance and detection of chromosome imbalance where either both haplotypes from one parent are present (trisomy) or neither are present (monosomy/deletion). The study also demonstrates that karyomapping is possible at the single cell level following whole genome amplification and, without any prior patient or disease specific test development, provides a universal linkage based methodology for preimplantation genetic diagnosis readily available worldwide.


Handyside A.H.,Gynaecology and Genetics Center | Handyside A.H.,University of Leeds
Obstetrics, Gynaecology and Reproductive Medicine | Year: 2011

Preimplantation genetic diagnosis (PGD) of inherited conditions following in vitro fertilization (IVF) is now clinically well established worldwide and 7000 children have been born. The range of applications includes single gene defects, chromosome aneuploidy and structural abnormalities, and HLA matching, to identify histocompatible embryos for cord blood stem cell transplantation and treatment of existing children affected mainly by blood related conditions. Recently, the use of advanced technologies for genome-wide analysis, such as microarrays, has provided a universal approach for the diagnosis of both inherited genetic defects and chromosomal abnormalities affecting the viability of the embryo. Clinical pregnancy rates have steadily increased with improvements both in the accuracy of the diagnosis and in IVF. Clinical outcomes are similar to those in assisted conception generally, with an increase in prematurity mainly associated with multiple births and no significant increase in congenital abnormalities. Follow up of children born is ongoing but initial studies have been reassuring. © 2010.


Handyside A.H.,Gynaecology and Genetics Center | Handyside A.H.,University of Leeds | Montag M.,University of Heidelberg | Magli M.C.,SISMER | And 7 more authors.
European Journal of Human Genetics | Year: 2012

Chromosome aneuploidy is a major cause of pregnancy loss, abnormal pregnancy and live births following both natural conception and in vitro fertilisation (IVF) and increases exponentially with maternal age in the decade preceding the menopause. Molecular genetic analysis following natural conception and spontaneous miscarriage demonstrates that trisomies arise mainly in female meiosis and particularly in the first meiotic division. Here, we studied copy number gains and losses for all chromosomes in the two by-products of female meiosis, the first and second polar bodies, and the corresponding zygotes in women of advanced maternal age undergoing IVF, using microarray comparative genomic hybridisation (array CGH). Analysis of the segregation patterns underlying the copy number changes reveals that premature predivision of chromatids rather than non-disjunction of whole chromosomes causes almost all errors in the first meiotic division and unlike natural conception, over half of aneuploidies result from errors in the second meiotic division. Furthermore, most abnormal zygotes had multiple aneuploidies. These differences in the aetiology of aneuploidy in IVF compared with natural conception may indicate a role for ovarian stimulation in perturbing meiosis in ageing oocytes. © 2012 Macmillan Publishers Limited All rights reserved.


Harper J.C.,University College London | Sengupta S.,University College London | Vesela K.,Center for Reproductive Medicine and Preimplantation Diagnosis | Thornhill A.,Gynaecology and Genetics Center | And 4 more authors.
Human Reproduction | Year: 2010

Accreditation according to an internationally recognized standard is increasingly acknowledged as the single most effective route to comprehensive laboratory quality assurance, and many countries are progressively moving towards compulsory accreditation of medical testing laboratories. The ESHRE PGD Consortium and some regulatory bodies recommend that all PGD laboratories should be accredited or working actively towards accreditation, according to the internationally recognized standard ISO 15189, 'Medical laboratories-Particular requirements for quality and competence'. ISO 15189 requires comprehensive quality assurance. Detailed management and technical requirements are defined in the two major chapters. The management requirements address quality management including the quality policy and manual, document control, non-conformities and corrective actions, continual improvement, auditing, management review, contracts, referrals and resolution of complaints. Technical requirements include personnel competence (both technical and medical), equipment, accommodation and environment, and pre-analytical, analytical and post-analytical processes. Emphasis is placed on the particular requirements of patient care: notably sample identification and traceability, test validation and interpretation and reporting of Result s. Quality indicators must be developed to monitor contributions to patient care and continual improvement. We discuss the implementation of ISO 15189 with a specific emphasis on the PGD laboratory, highlight elements of particular importance or difficulty and provide suggestions of effective and efficient ways to obtain accreditation. The focus is on the European environment although the principles are globally applicable.


Magli M.C.,SISMER | Montag M.,University of Bonn | Kster M.,University of Bonn | Muzi L.,SISMER | And 11 more authors.
Human Reproduction | Year: 2011

Background The purpose of this study was to assess the technical aspects related to polar body (PB) biopsy, which might have an influence on the results of the microarray comparative genomic hybridization analysis. Furthermore, a comparison was made between two biopsy methods (mechanical and laser). Methods Biopsy of the first and second PB (PB1 and PB2) was performed by mechanical-or laser-assisted biopsy in two different IVF centres. PBs were separately amplified by whole genome amplification. Results The method of biopsy, mechanical or laser had no influence on the proportion of successfully biopsied oocytes. Especially, for the PB2, the timing of biopsy after ICSI was directly correlated to amplification efficiency. Conclusions Special care has to be taken with respect to the timing of biopsy of the PB2. Mechanical-and laser-assisted biopsy give the same performance in terms of diagnostic efficiency. © 2011 The Author.


Handyside A.H.,Gynaecology and Genetics Center | Handyside A.H.,University of Leeds
Reproductive BioMedicine Online | Year: 2011

Bisignano et al. (2011) argue that, for preimplantation genetic diagnosis (PGD) of aneuploidy for all 24 chromosomes, microarray-based comparative genomic hybridization (array CGH) is superior to the use of single-nucleotide polymorphism (SNP) genotyping arrays. Published studies indicate that both technologies accurately detect aneuploidy of whole chromosomes or chromosome segments. However, given the extra theoretical resolution and parent-of-origin information provided by SNP-based approaches, these may be particularly suited to certain applications such as PGD of single-gene defects or translocation chromosome imbalance combined with comprehensive detection of aneuploidy. A consensus on how to validate aneuploidy testing and all other clinically relevant information resulting from genome-wide analysis is needed urgently. © 2011 Elsevier Inc. All rights reserved.


Hens K.,Maastricht University | Hens K.,Center for Society and the Life science | Dondorp W.,Maastricht University | Handyside A.H.,Gynaecology and Genetics Center | And 8 more authors.
Human Reproduction Update | Year: 2013

Background: Genetic testing of preimplantation embryos has been used for preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Microarray technology is being introduced in both these contexts, and whole genome sequencing of blastomeres is also expeted to become possible soon. The amount of extra information such tests will yield may prove to be beneficial for embryo selection, will also raise various ethical issues. We present an overview of the developments and an agenda-setting exploration of the ethical issues. Methods: The paper is a joint endeavour by the presenters at an explorative 'campus meeting' organized by the European Society of Human Reproduction and Embryology in cooperation with the department of Health, Ethics & Society of the Maastricht University (The Netherlands). Results: The increasing amount and detail of information that new screening techniques such as microarrays and whole genome sequencing offer does not automatically coincide with an increasing understanding of the prospects of an embryo. From a technical point of view, the future of comprehensive embryo testing may go together with developments in preconception carrier screening. From an ethical point of view, the increasing complexity and amount of information yielded by comprehensive testing techniques will lead to challenges to the principle of reproductive autonomy and the right of the child to an open future, and may imply a possible larger responsibility of the clinician towards the welfare of the future child. Combinations of preconception carrier testing and embryo testing may solve some of these ethical questions but could introduce others. Conclusions: As comprehensive testing techniques are entering the IVF clinic, there is a need for a thorough rethinking of traditional ethical paradigms regarding medically assisted reproduction. The Author 2013. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.


Ioannou D.,University of Kent | Meershoek E.J.,Kreatech Diagnostics | Thornhill A.R.,University of Kent | Thornhill A.R.,Gynaecology and Genetics Center | And 2 more authors.
Molecular and Cellular Probes | Year: 2011

From the late 1980s onwards, the use of DNA probes to visualise sequences on individual chromosomes (fluorescent in-situ hybridisation - FISH) revolutionised the study of cytogenetics. Following single colour experiments, more fluorochromes were added, culminating in a 24 colour assay that could distinguish all human chromosomes. Interphase cytogenetics (the detection of chromosome copy number in interphase nuclei) soon followed, however 24 colour experiments are hampered for this application as mixing fluorochromes to produce secondary colours produces images that are not easily distinguishable from overlapping signals. This study reports the development and use of a novel protocol, new fast hybridising FISH probes, and a bespoke image capture system for the assessment of chromosome copy number in interphase nuclei. The multicolour probe sets can be used individually or in sequential hybridisation layers to assess ploidy of all 24 human chromosomes in the same nucleus. Applications of this technique are in the investigation of chromosome copy number and the assessment of nuclear organisation for a range of different cell types including human sperm, cancer cells and preimplantation embryos. © 2011 Elsevier Ltd.


Gabriel A.S.,University of Kent | Hassold T.J.,Washington State University | Thornhill A.R.,University of Kent | Thornhill A.R.,Gynaecology and Genetics Center | And 4 more authors.
Chromosome Research | Year: 2011

Trisomy causes mental retardation, pregnancy loss, IVF failure, uniparental disomy and several other pathologies, and its accurate detection is thus clinically essential. Most trisomies arise at meiosis I and are associated with increasing maternal age and reduction or alteration in recombination patterns. Investigations into the relationship between trisomy and meiotic recombination have used short tandem repeat markers; however, this approach is limited by the resolution with which the position of crossovers can identified. As cytogenetics enters the post-genomic era, recent work has used array comparative genomic hybridisation (aCGH) to screen for trisomy of all 24 chromosomes, determining chromosome copy number by dosage analysis. However, aCGH has a fundamental drawback for studying the aetiology of trisomy since neither the parent and phase of origin nor uniparental disomy can be ascertained. The development of SNP microarrays has made it possible to analyse multiple loci for sequence variation, and the proprietary software provided can determine the presence of aneuploidy by algorithms based on fluorescence intensity. To the best of our knowledge, however, such software is not equipped to determine the phase of origin of the error or the position of any chiasmata. In this study, therefore, we present an algorithm to determine the parent of origin, the phase of origin and the location of chiasmata in a series of nine "trisomy triplets" (i.e. samples derived from father, mother and their trisomic foetus). Novel adaptations of well-established principles are applied along with a simple algorithm written in Microsoft Excel for visualisation of the results. Such analysis has a range of applications in preimplantation and prenatal diagnosis. © 2011 Springer Science+Business Media B.V.

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