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Chatzimeletiou K.,Section of Reproductive Medicine | Morrison E.E.,University of Leeds | Panagiotidis Y.,Iakentro Advanced Medical Center | Vanderzwalmen P.,Ivf Center Prof Zech | And 4 more authors.
Human Reproduction | Year: 2012

BACKGROUND: Vitrification of human blastocysts is being used increasingly to cryopreserve supernumerary embryos following IVF. In this study, we investigate the effects of aseptic vitrification on the cytoskeleton and development of human blastocysts, by analysing survival rates and spindle and chromosome configurations by fluorescence and confocal laser scanning microscopy. METHODS: A total of 55 fresh blastocysts and 55 day 5 dimethylsulphoxide/ethylene glycol vitrified blastocysts, which were allowed to remain in culture for 24 h post-warming, were rapidly fixed in ice cold methanol, and immunostained with an a-tubulin antibody to visualize microtubules in combination with antibodies against acetylated tubulin (to visualize spindles, poles and mid bodies), gamma tubulin (to identify spindle poles) and 4(6-diamidino-2-phenylindole) to visualize DNA. RESULTS: In total, 213 spindles were analysed in the control (fresh) group of which 183/213 (85.9) were normal, 20/213 (9.4) were abnormally shaped, 9/213 (4.2) were multipolar and 1/213 (0.5) was monopolar. A total of 175 spindles were analysed in the vitrified group, of which 120/175 (68.6) were normal, 39/175 (22.3) were abnormally shaped, 10/175 (5.7) were multipolar and 6/175 (3.4) were monopolar. The incidence of multipolar spindles was similar in the two groups, but the level of abnormally shaped spindles, often associated with chromosome lagging, or congression failure, was significantly higher in the vitrified group compared with the fresh group (P< 0.05). CONCLUSIONS: The high survival rate following thawing and the large proportion of normal spindle/chromosome configurations suggests that vitrification at the blastocyst stage on Day 5 does not adversely affect the development of human embryos and the ability of spindles to form and continue normal cell divisions. However, there was a significantly higher incidence of abnormal spindles in the vitrified group compared with the fresh group, notably of spindles with a focused and an unfocused pole as well as chromosome bridging and disorganized middle spindle fibres at telophase. Further investigation is warranted to elucidate the mitotic stages that are more vulnerable to damage during vitrification, the fate of the abnormal spindles and any potential effects that may be reflected on the chromosomal constitution of the developing blastocysts. © The Author 2011. Source


Ioannou D.,University of Kent | Ioannou D.,London Bridge Fertility Gynaecology and Genetics Center | Meershoek E.J.,Kreatech Diagnostics | Christopikou D.,Embryogenesis IVF Unit | And 4 more authors.
Chromosome Research | Year: 2011

Organisation of chromosome territories in interphase nuclei has been studied in many systems and positional alterations have been associated with disease phenotypes (e.g. laminopathies, cancer) in somatic cells. Altered nuclear organisation is also reported in developmental processes such as mammalian spermatogenesis where a "chromocentre" model is proposed with the centromeres and sex chromosomes repositioning to the nuclear centre. The purpose of this study was to test the hypothesis that alterations in nuclear organisation of human spermatozoa are associated with defects upstream in spermatogenesis (as manifest in certain infertility phenotypes). The nuclear address of (peri-) centromeric loci for 18 chromosomes (1-4, 6-12, 15-18, 20, X and Y) was assayed in 20 males using established algorithms for 3D extrapolations of 2D data. The control group comprised 10 fertile sperm donors while the test group was 10 patients with severely compromised semen parameters including high sperm aneuploidy. All loci examined in the control group adopted defined, interior positions thus providing supporting evidence for the presence of a chromocentre and interior sex chromosome territories. In the test group however there were subtle alterations in the nuclear address for certain centromeres in individual patients and, when all patient results were pooled, some different nuclear addresses were observed for chromosomes 3, 6, 12 and 18. Considering the extensive impairment of spermatogenesis in the test group (evidenced by compromised semen parameters and increased chromosome abnormalities), the observed differences in nuclear organisation for centromeric loci compared to the controls were modest. A defined pattern of nuclear reorganisation of centromeric loci in sperm heads therefore appears to be a remarkably robust process, even if spermatogenesis is severely compromised. © 2011 Springer Science+Business Media B.V. Source


Natesan S.A.,Illumina | Handyside A.H.,Illumina | Handyside A.H.,London Bridge Fertility Gynaecology and Genetics Center | Handyside A.H.,University of Kent | And 11 more authors.
Reproductive BioMedicine Online | Year: 2014

Preimplantation genetic diagnosis (PGD) for monogenic disorders has the drawback of time and cost associated with tailoring a specific test for each couple, disorder, or both. The inability of any single assay to detect the monogenic disorder in question and simultaneously the chromosomal complement of the embryo also limits its application as separate tests may need to be carried out on the amplified material. The first clinical use of a novel approach ('karyomapping') was designed to circumvent this problem. In this example, karyomapping was used to confirm the results of an existing PGD case detecting both chromosomal abnormalities and a monogenic disorder (Smith-Lemli-Opitz [SLO] syndrome) simultaneously. The family underwent IVF, ICSI and PGD, and both polar body and cleavage stage biopsy were carried out. Following whole genome amplification, array comparative genomic hybridisation of the polar bodies and minisequencing and STR analysis of single blastomeres were used to diagnose maternal aneuploidies and SLO status, respectively. This was confirmed, by karyomapping. Unlike standard PGD, karyomapping required no a-priori test development. A singleton pregnancy and live birth, unaffected with SLO syndrome and with no chromosome abnormality, ensued. Karyomapping is potentially capable of detecting a wide spectrum of monogenic and chromosome disorders and, in this context, can be considered a comprehensive approach to PGD. © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. Source

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