Professor Darren Griffin

Professor of Genetics
Director, Centre for Interdisciplinary Studies of Reproduction

About

Professor Darren Griffin joined the school in 2004 from Brunel University. His main interests are in the study of chromosomes, principally in humans (from spermatogenesis to preimplantation development) and birds. Other interests include allelic variation and its relationship to fatness and studies relating to eLearning. In 2007 he became a BBSRC Career Development Fellow with a remit to exploit microarray technology for studies of copy number variation in birds and humans.
Darren is a member of the Centre for Interdisciplinary Studies of Reproduction (CISoR)
He also regularly coordinates the International Chromosome Conferences.
Research Career 

  • 2008 Doctor of Science, University of Manchester 
  •  2007 Fellow of the Royal College of Pathologists 
  • 2007 BBSRC Career Development Fellow 
  • 2004 Vice President of the International Chromosome and Genome Society 
  • 2002 Postgraduate Certificate, Teaching and Learning in HE, Brunel University. 
  • 2002 Fellow of the Institute of Biology. 
  • 2001 Editorial Board 'Prenatal Diagnosis'. 
  • 1992 Doctor of Philosophy, Human Genetics, University College London. 
  • 1988 Bachelor of Science (with honours), Genetics and Cell Biology, University of Manchester. 

Research interests

Chromosome Segregation in Human Sperm and Preimplantation Embryos 

It is now well established that men with severely compromised semen parameters can have increased levels of aneuploidy in their sperm. We are interested in fundamental investigations into this phenomenon, in particular, the role of genetic recombination and changes in genome organisation. 

Through deeper understanding this we aim to better comprehend the causes of male fertility and the mechanisms of chromosome non-disjunction. We have evidence of the efficacy of Chinese Herbal Medicine in decreasing high aneuploidy rates in infertile men and for anti-oestrogenic properties in the medicinal herbs. Work has recently re-visited examination of the human preimplanatation embryo with research into the degree to which aneuploidy is transmitted from sperm to embryo and the role of genome organization in preimplantation development. We have close links with the London Bridge Fertility Clinic and Bridge Genoma.

Current projects:

Comparative Genomics of Avian Species 
The ability to distinguish each chicken chromosome has a number of applications in comparative genomics, developmental biology, molecular ecology, genome organization, and agriculture. We have developed resources that enable us to identify all chicken chromosomes individually. Work has now progressed to generating cytogenetic maps in a range of avian species and studying the role of chromosome evolution in birds.

Computer based learning
Our newest research interest is in the generation, evaluation and dissemination of computer-based learning tools in genetics, cytogenetics and human reproduction. In 2002 we launched "Learning Interactive" a University spin off activity specializing in production, dissemination and sale of these computer-based learning materials.

Publications

Article

  • Turner, K. et al. (2019). Karyomapping for simultaneous genomic evaluation and aneuploidy screening of preimplantation bovine embryos: The first live-born calves. Theriogenology [Online] 125:249-258. Available at: https://doi.org/10.1016/j.theriogenology.2018.11.014.
    In cattle breeding, the development of genomic selection strategies based on single nucleotide polymorphism (SNP) interrogation has led to improved rates of genetic gain. Additionally, the application of genomic selection to in-vitro produced (IVP) embryos is expected to bring further benefits thanks to the ability to test a greater number of individuals before establishing a pregnancy and to ensure only carriers of desirable traits are born. However, aneuploidy, a leading cause of developmental arrest, is known to be common in IVP embryos. Karyomapping is a comprehensive screening test based on SNP typing that can be used for simultaneous genomic selection and aneuploidy detection, offering the potential to maximize pregnancy rates. Moreover, Karyomapping can be used to characterize the frequency and parental origin of aneuploidy in bovine IVP embryos, which have remained underexplored to date. Here, we report the use of Karyomapping to characterize the frequency and parental origin of aneuploidy in IVP bovine embryos in order to establish an estimate of total aneuploidy rates in each parental germline. We report an estimate of genome wide recombination rate in cattle and demonstrate, for the first time, a proof of principle for the application of Karyomapping to cattle breeding, with the birth of five calves after screening. This combined genomic selection and aneuploidy screening approach was highly reliable, with calves showing 98% concordance with their respective embryo biopsies for SNP typing and 100% concordance with their respective biopsies for aneuploidy screening. This approach has the potential to simultaneously improve pregnancy rates following embryo transfer and the rate of genetic gain in cattle breeding, and is applicable to basic research to investigate meiosis and aneuploidy.
  • Joseph, S. et al. (2018). Chromosome Level Genome Assembly and Comparative Genomics between Three Falcon Species Reveals an Unusual Pattern of Genome Organisation. Diversity [Online] 10:113. Available at: https://doi.org/10.3390/d10040113.
    Whole genome assemblies are crucial for understanding a wide range of aspects of falcon biology, including morphology, ecology, and physiology, and are thus essential for their care and conservation. A key aspect of the genome of any species is its karyotype, which can then be linked to the whole genome sequence to generate a so-called chromosome-level assembly. Chromosome-level assemblies are essential for marker assisted selection and genotype-phenotype correlations in breeding regimes, as well as determining patterns of gross genomic evolution. To date, only two falcon species have been sequenced and neither initially were assembled to the chromosome level. Falcons have atypical avian karyotypes with fewer chromosomes than other birds, presumably brought about by wholesale fusion. To date, however, published chromosome preparations are of poor quality, few chromosomes have been distinguished and standard ideograms have not been made. The purposes of this study were to generate analyzable karyotypes and ideograms of peregrine, saker, and gyr falcons, report on our recent generation of chromosome level sequence assemblies of peregrine and saker falcons, and for the first time, sequence the gyr falcon genome. Finally, we aimed to generate comparative genomic data between all three species and the reference chicken genome. Results revealed a diploid number of 2n = 50 for peregrine falcon and 2n = 52 for saker and gyr through high quality banded chromosomes. Standard ideograms that are generated here helped to map predicted chromosomal fragments (PCFs) from the genome sequences directly to chromosomes and thus generate chromosome level sequence assemblies for peregrine and saker falcons. Whole genome sequencing was successful in gyr falcon, but read depth and coverage was not sufficient to generate a chromosome level assembly. Nonetheless, comparative genomics revealed no differences in genome organization between gyr and saker falcons. When compared to peregrine falcon, saker/gyr differed by one interchromosomal and seven intrachromosomal rearrangements (a fusion plus seven inversions), whereas peregrine and saker/gyr differ from the reference chicken genome by 14/13 fusions (11 microchromosomal) and six fissions. The chromosomal differences between the species could potentially provide the basis of a screening test for hybrid animals.
  • Griffin, D. et al. (2018). Jurassic spark: Mapping the genomes of birds and other dinosaurs Galkina, S. and Vishnevskaya, M. eds. Comparative Cytogenetics [Online] 12:322-323, Abstract L13. Available at: https://doi.org/10.3897/CompCytogen.v12i3.27748.
    The ultimate aim of a genome assembly is to create a contiguous length of sequence from the p- to q- terminus of each chromosome. Most assemblies are however highly fragmented, limiting their use in studies of gene mapping, phylogenomics and genomic organisation. To overcome these limitations, we developed a novel scaffold-to-chromosome anchoring method combining reference-assisted chromosome assembly (RACA) and fluorescence in situ hybridisation (FISH) to position scaffolds from de novo genomes onto chromosomes. Using RACA, scaffolds were ordered and orientated into ‘predicted chromosome fragments’ (PCFs) against a reference and outgroup genome. PCFs were verified using PCR prior to FISH mapping. A universal set of FISH probes developed through the selection of conserved regions were then used to map PCFs of peregrine falcon (Falco peregrinus Tunstall, 1771), pigeon (Columba livia Gmelin, 1789), ostrich (Struthio camelus Linnaeus, 1758), saker falcon (Falco cherrug Gray, 1834) the budgerigar (Melopsittacus undulatus Shaw, 1805). Using this approach, we were able to improve the N50 of genomes seven-fold. Results revealed that Interchromosomal breakpoint regions are limited to regions with low sequence conservation, shedding light on why most avian species have very stable karyotypes.

    Our combined FISH and bioinformatics approach represents a step-change in the mapping of genome assemblies, allowing comparative genomic research at a higher resolution than was previously possible. The universal probe set facilitates research into avian karyotype evolution and the role of chromosome rearrangements in adaptation and phenotypic diversity in birds. Indeed, they have been used on over 20 avian species plus non-avian reptiles (including turtles), shedding light into the evolution of dinosaur species. Non-avian dinosaurs remain subjects of intense biological enquiry while pervading popular culture and the creative arts. While organismal studies focus primarily on their morphology, relationships, likely behaviour, and ecology there have been few academic studies that have made extensive extrapolations about the nature of non-avian dinosaur genome structure prior to the emergence of modern birds. We have used multiple avian whole genome sequences assembled at a chromosomal level, to reconstruct the most likely gross genome organization of the overall genome structure of the diapsid ancestor and reconstruct the sequence of inter and intrachromosomal events that most likely occurred along the Archosauromorpha-Archosauria-Avemetatarsalia-Dinosauria-Theropoda-Maniraptora-Avialae lineage from the lepidosauromorph-archosauromorph divergence ~275 million years ago through to extant neornithine birds.
  • O'Connor, R. et al. (2018). Reconstruction of the diapsid ancestral genome permits chromosome evolution tracing in avian and non-avian dinosaurs. Nature Communications [Online] 9. Available at: http://dx.doi.org/10.1038/s41467-018-04267-9.
    Genomic organisation of extinct lineages can be inferred from extant chromosome-level
    genome assemblies. Here, we apply bioinformatic and molecular cytogenetic approaches to
    determine the genomic structure of the diapsid common ancestor. We then infer the events
    that likely occurred along this lineage from theropod dinosaurs through to modern birds. Our
    results suggest that most elements of a typical ‘avian-like’ karyotype (40 chromosome pairs,
    including 30 microchromosomes) were in place before the divergence of turtles from birds
    ~255 mya. This genome organisation therefore predates the emergence of early dinosaurs
    and pterosaurs and the evolution of flight. Remaining largely unchanged interchromosomally
    through the dinosaur–theropod route that led to modern birds, intrachromosomal changes
    nonetheless reveal evolutionary breakpoint regions enriched for genes with ontology terms
    related to chromatin organisation and transcription. This genomic structure therefore appears
    highly stable yet contributes to a large degree of phenotypic diversity, as well as underpinning
    adaptive responses to major environmental disruptions via intrachromosomal repatterning.
  • Parks, J. et al. (2018). The impact of infertility diagnosis on embryo-endometrial dialogue. Reproduction [Online] 155:543-552. Available at: https://doi.org/10.1530/REP-17-0566.
    Initial stages of implantation involve bi-directional molecular crosstalk between the blastocyst and endometrium. This study investigated an association between infertility etiologies, specifically advanced maternal age (AMA) and endometriosis, on the embryo-endometrial molecular dialogue prior to implantation. Co-culture experiments were performed with endometrial epithelial cells (EEC) and cryopreserved day 5 blastocysts (n = 41 ≥ Grade 3BB) donated from patients presenting with AMA or endometriosis, compared to fertile donor oocyte controls. Extracellular vesicles isolated from co-culture supernatant were analyzed for miRNA expression and revealed significant alterations correlating to AMA or endometriosis. Specifically, AMA resulted in 16 miRNAs with increased expression (P ≤ 0.05) and strong evidence for negative regulation toward 206 target genes. VEGFA, a known activator of cell adhesion, displayed decreased expression (P ≤ 0.05), validating negative regulation by 4 of these increased miRNAs: miR-126; 150; 29a; 29b (P ≤ 0.05). In endometriosis patients, a total of 10 significantly altered miRNAs displayed increased expression compared to controls (miR-7b; 9; 24; 34b; 106a; 191; 200b; 200c; 342-3p; 484) (P ≤ 0.05), targeting 1014 strong evidence-based genes. Three target genes of miR-106a (CDKN1A, E2F1 and RUNX1) were independently validated. Functional annotation analysis of miRNA-target genes revealed enriched pathways for both infertility etiologies, including disrupted cell cycle regulation and proliferation (P ≤ 0.05). These extracellular vesicle-bound secreted miRNAs are key transcriptional regulators in embryo-endometrial dialogue and may be prospective biomarkers of implantation success. One of the limitations of this study is that it was a stimulated, in vitro model and therefore may not accurately reflect the in-vivo environment.
  • O’Connor, R. et al. (2018). Chromosome-level assembly reveals extensive rearrangement in saker falcon and budgerigar, but not ostrich, genomes. Genome Biology [Online] 19. Available at: https://doi.org/10.1186/s13059-018-1550-x.
    Background: The number of de novo genome sequence assemblies is increasing exponentially; however, relatively
    few contain one scaffold/contig per chromosome. Such assemblies are essential for studies of genotype-to-phenotype
    association, gross genomic evolution, and speciation. Inter-species differences can arise from chromosomal changes
    fixed during evolution, and we previously hypothesized that a higher fraction of elements under negative selection
    contributed to avian-specific phenotypes and avian genome organization stability. The objective of this study is to
    generate chromosome-level assemblies of three avian species (saker falcon, budgerigar, and ostrich) previously
    reported as karyotypically rearranged compared to most birds. We also test the hypothesis that the density of
    conserved non-coding elements is associated with the positions of evolutionary breakpoint regions.

    Results: We used reference-assisted chromosome assembly, PCR, and lab-based molecular approaches, to generate
    chromosome-level assemblies of the three species. We mapped inter- and intrachromosomal changes from the avian
    ancestor, finding no interchromosomal rearrangements in the ostrich genome, despite it being previously described as
    chromosomally rearranged. We found that the average density of conserved non-coding elements in evolutionary
    breakpoint regions is significantly reduced. Fission evolutionary breakpoint regions have the lowest conserved
    non-coding element density, and intrachromomosomal evolutionary breakpoint regions have the highest.

    Conclusions: The tools used here can generate inexpensive, efficient chromosome-level assemblies, with > 80%
    assigned to chromosomes, which is comparable to genomes assembled using high-density physical or genetic
    mapping. Moreover, conserved non-coding elements are important factors in defining where rearrangements,
    especially interchromosomal, are fixed during evolution without deleterious effects.
  • Damas, J. et al. (2018). Reconstruction of avian ancestral karyotypes reveals differences in the evolutionary history of macro- and microchromosomes. Genome Biology [Online] 19. Available at: https://doi.org/10.1186/s13059-018-1544-8.
    Background
    Reconstruction of ancestral karyotypes is critical for our understanding of genome evolution, allowing for the identification of the gross changes that shaped extant genomes. The identification of such changes and their time of occurrence can shed light on the biology of each species, clade and their evolutionary history. However, this is impeded by both the fragmented nature of the majority of genome assemblies and the limitations of the available software to work with them. These limitations are particularly apparent in birds, with only 10 chromosome-level assemblies reported thus far. Algorithmic approaches applied to fragmented genome assemblies can nonetheless help define patterns of chromosomal change in defined taxonomic groups.

    Results
    Here, we make use of the DESCHRAMBLER algorithm to perform the first large-scale study of ancestral chromosome structure and evolution in birds. This algorithm allows us to reconstruct the overall genome structure of 14 key nodes of avian evolution from the Avian ancestor to the ancestor of the Estrildidae, Thraupidae and Fringillidae families.

    Conclusions
    Analysis of these reconstructions provides important insights into the variability of rearrangement rates during avian evolution and allows the detection of patterns related to the chromosome distribution of evolutionary breakpoint regions. Moreover, the inclusion of microchromosomes in our reconstructions allows us to provide novel insights into the evolution of these avian chromosomes, specifically.
  • Griffin, D. and Ogur, C. (2018). Chromosomal analysis in IVF: just how useful is it? Reproduction [Online] 156:F29-F50. Available at: https://doi.org/10.1530/REP-17-0683.
    Designed to minimize chances of transferring genetically abnormal embryos, preimplantation genetic diagnosis (PGD) involves
    in vitro fertilization (IVF), embryo biopsy, diagnosis and selective embryo transfer. Preimplantation genetic testing for aneuploidy
    (PGT-A) aims to avoid miscarriage and live born trisomic offspring and to improve IVF success. Diagnostic approaches include
    fluorescence in situ hybridization (FISH) and more contemporary comprehensive chromosome screening (CCS) including array
    comparative genomic hybridization (aCGH), quantitative polymerase chain reaction (PCR), next-generation sequencing (NGS) and
    karyomapping. NGS has an improved dynamic range, and karyomapping can detect chromosomal and monogenic disorders
    simultaneously. Mosaicism (commonplace in human embryos) can arise by several mechanisms; those arising initially meiotically (but
    with a subsequent post-zygotic ‘trisomy rescue’ event) usually lead to adverse outcomes, whereas the extent to which mosaics that
    are initially chromosomally normal (but then arise purely post-zygotically) can lead to unaffected live births is uncertain. Polar body
    (PB) biopsy is the least common sampling method, having drawbacks including cost and inability to detect any paternal contribution.
    Historically, cleavage-stage (blastomere) biopsy has been the most popular; however, higher abnormality levels, mosaicism and
    potential for embryo damage have led to it being superseded by blastocyst (trophectoderm – TE) biopsy, which provides more cells for
    analysis. Improved biopsy, diagnosis and freeze-all strategies collectively have the potential to revolutionize PGT-A, and there is
    increasing evidence of their combined efficacy. Nonetheless, PGT-A continues to attract criticism, prompting questions of when we
    consider the evidence base sufficient to justify routine PGT-A? Basic biological research is essential to address unanswered questions
    concerning the chromosome complement of human embryos, and we thus entreat companies, governments and charities to fund
    more. This will benefit both IVF patients and prospective parents at risk of aneuploid offspring following natural conception. The aim
    of this review is to appraise the ‘state of the art’ in terms of PGT-A, including the controversial areas, and to suggest a practical ‘way
    forward’ in terms of future diagnosis and applied research.
  • Fowler, K. et al. (2018). The production of pig preimplantation embryos in vitro: Current progress and future prospects. Reproductive Biology [Online] 18:203-211. Available at: https://doi.org/10.1016/j.repbio.2018.07.001.
    Human assisted reproductive technology procedures are routinely performed in clinics globally, and some of these approaches are now common in other mammals such as cattle. This is currently not the case in pigs. Given that the global population is expected to increase by over two billion people between now and 2050, the demand for meat will also undoubtedly increase. With this in mind, a more sustainable way to produce livestock; increasing productivity and implementing methods that will lead to faster genetic selection, is imperative. The establishment of routine and production scale pig embryo in vitro production could be a solution to this problem. Producers would be able to increase the overall number of offspring born, animal transportation would be more straightforward and in vitro produced embryos could be produced from the gametes of selected elite. Here we review the most recent developments in pig embryology, outline the current barriers and key challenges that exist, and outline research priorities to surmount these difficulties.
  • McCallie, B. et al. (2017). Infertility diagnosis has a significant impact on the transcriptome of developing blastocysts. Molecular Human Reproduction [Online] 23:549-556. Available at: https://doi.org/10.1093/molehr/gax034.
    STUDY QUESTION: Is the human blastocyst transcriptome associated with infertility diagnosis, specifically: polycystic ovaries (PCO), male factor (MF) and unexplained (UE)?
    SUMMARY ANSWER: The global blastocyst transcriptome was significantly altered in association with a PCO, MF and UE infertility diagnosis.
    WHAT IS KNOWN ALREADY: Infertility diagnosis has an impact on the probability for a successful outcome following an IVF cycle. Limited information is known regarding the relationship between a specific infertility diagnosis and blastocyst transcription during preimplantation development.
    STUDY DESIGN, SIZE, DURATION: Blastocysts created during infertility treatment from patients with specific infertility diagnoses (PCO, MF and UE) were analyzed for global transcriptome compared to fertile donor oocyte blastocysts (control).
    PARTICIPANTS/MATERIALS, SETTING, METHODS: Surplus cryopreserved blastocysts were donated with patient consent and institutional review board approval. Female patients were <38 years old with male patients <40 years old. Blastocysts were grouped according to infertility diagnosis: PCO (n = 50), MF (n = 50), UE (n = 50) and fertile donor oocyte controls (n = 50). Pooled blastocysts were lysed for RNA isolation followed by microarray analysis using the SurePrint G3 Human Gene Expression Microarray. Validation was performed on significant genes of interest using real-time quantitative PCR (RT-qPCR).
    MAIN RESULTS AND THE ROLE OF CHANCE: Transcription alterations were observed for all infertility etiologies compared to controls, resulting in differentially expressed genes: PCO = 869, MF = 348 and UE = 473 (P < 0.05; >2-fold). Functional annotation of biological and molecular processes revealed both similarities, as well as differences, across the infertility groups. All infertility etiologies displayed transcriptome alterations in signal transducer activity, receptor binding, reproduction, cell adhesion and response to stimulus. Blastocysts from PCO patients were also enriched for apoptotic genes while MF blastocysts displayed enrichment for genes involved in cancer processes. Blastocysts from couples with unexplained infertility displayed transcription alterations related to various disease states, which included mechanistic target of rapamycin (mTOR) and adipocytokine signaling. RT-qPCR validation confirmed differential gene expression for the following genes: BCL2 like 10 (BCL2L10), heat shock protein family A member 1A (HSPA1A), heat shock protein family A member 1B (HSPA1B), activating transcription factor 3 (ATF3), fibroblast growth factor 9 (FGF9), left-right determination factor 1 (LEFTY1), left-right determination factor 2 (LEFTY2), growth differentiation factor 15 (GDF15), inhibin beta A subunit (INHBA), adherins junctions associated protein 1 (AJAP1), cadherin 9 (CDH9) and laminin subunit alpha 4 (LAMA4) (P < 0.05; >2-fold).
  • Gould, R. and Griffin, D. (2017). Karyomapping and how is it improving preimplantation genetics? Expert Review of Molecular Diagnostics [Online]. Available at: https://doi.org/10.1080/14737159.2017.1325736.
    Introduction: Preimplantation genetic diagnosis and screening (PGD/PGS) has been applied clinically for >25 years however inherent drawbacks include the necessity to tailor each case to the trait in question, and that technology to detect monogenic and chromosomal disorders respectively is fundamentally different.

    Areas Covered: The area of preimplantation genetics has evolved over the last 25 years, adapting to changes in technology and the need for more efficient, streamlined diagnoses. Karyomapping allows the determination of inheritance from the (grand)parental haplobocks through assembly of inherited chromosomal segments. The output displays homologous chromosomes, crossovers and the genetic status of the embryos by linkage comparison, as well as chromosomal disorders. It also allows for determination of heterozygous SNP calls, avoiding the risks of allele dropout, a common problem with other PGD techniques. Manuscripts documenting the evolution of preimplantation genetics, especially those investigating technologies that would simultaneously detect monogenic and chromosomal disorders, were selected for review.

    Expert Commentary: Karyomapping is currently available for detection of single gene disorders; ~1000 clinics worldwide offer it (via ~20 diagnostic laboratories) and ~2500 cases have been performed. Due an inability to detect post-zygotic trisomy reliably however and confounding problems of embryo mosaicism, karyomapping has yet to be applied clinically for detection of chromosome disorders.
  • Sanders, K. and Griffin, D. (2017). Chromosomal Preimplantation Genetic Diagnosis: 25 Years and Counting. Journal of Fetal Medicine [Online] 4:51-56. Available at: http://dx.doi.org/10.1007/s40556-017-0123-5.
    Preimplantation genetic diagnosis (PGD), first successfully carried out in humans in the early 1990s, initially involved the PCR sexing of embryos by Y- (and later also X-) chromosome specific detection. Because of the problems relating to misdiagnosis and contamination of this technology however the PCR based test was superseded by a FISH-based approach involving X and Y specific probes. Sexing by FISH heralded translocation screening, which was shortly followed by preimplantation genetic screening (PGS) for Aneuploidy. Aneuploidy is widely accepted to be the leading cause of implantation failure in assisted reproductive technology (ART) and a major contributor to miscarriage, especially in women of advanced maternal age. PGS (AKA PGD for aneuploidy PGD-A) has had a chequered history, with conflicting lines of evidence for and against its use. The current practice of trophectoderm biopsy followed by array CGH or next generation sequencing is gaining in popularity however as evidence for its efficacy grows. PGS has the potential to identify viable embryos that can be transferred thereby reducing the chances of traumatic failed IVF cycles, miscarriage or congenital abnormalities and facilitating the quickest time to live birth of chromosomally normal offspring. In parallel to chromosomal diagnoses, technology for PGD has allowed for improvements in accuracy and efficiency of the genetic screening of embryos for monogenic disorders. The number of genetic conditions available for screening has increased since the early days of PGD, with the human fertilization and embryology authority currently licensing 419 conditions in the UK [1]. A novel technique known as karyomapping that involves SNP chip screening and tracing inherited chromosomal haploblocks is now licensed for the PGD detection of monogenic disorders. Its potential for the universal detection of chromosomal and monogenic disorders simultaneously however, has yet to be realized.
  • Griffin, D. (2017). What is Karyomapping and where does it fit in the world of preimplantation genetic diagnosis (PGD)? Medical Research Archives [Online] 5. Available at: http://journals.ke-i.org/index.php/mra/article/view/1256.
    The first application of preimplantation genetic diagnosis (PGD) recently celebrated its 25th birthday. Aside from the very early days when chromosomal diagnoses were used (by sexing) for the selective implantation of embryos unaffected by sex linked disorders, the paths of chromosomal and monogenic PGD have diverged. For monogenic disorders, progress has been impeded by the need to tailor each diagnosis to the mutation in question. For chromosomal diagnoses, fluorescent in situ hybridization (FISH) technology was replaced by array comparative genomic hybridization (aCGH), and then next generation sequencing (NGS). Karyomapping is a novel approach that allows the detection of the inheritance of (grand) parental haploblocks through the identification of inherited chromosomal segments. It involves genome-wide single nucleotide polymorphism (SNP) analysis of parental DNA, a reference from a related individual of known disease status (typically an affected child) and amplified DNA form biopsied cells of the (usually lastocyst) embryos in question. Identification of informative loci for each of four parental haplotypes is followed by direct comparison to the reference, ultimately creating a Karyomap. The Karyomapping programme (Illumina) displays homologous chromosomes, points of crossing over and the haplotype of each of the embryos. It also detects meiotic trisomy, monosomy, triploidy and uniparental heterodisomy (some of which NGS and aCGH will not). Inherent in the design is the analysis of “key SNPs” (heterozygous informative calls) thereby avoiding the risk of misdiagnoses caused by the phenomenon of allele drop out (ADO). Karyomapping is currently in use for the detection of monogenic disorders and around 1000 clinics offer it worldwide making use of about 20 diagnostic laboratories. At the time of writing, over two and a half thousand clinical cases have been performed. Because of the limited detection of some post-zygotic errors such as post-zygotic trisomy which can also lead to mosaicism, Karyomapping has not yet been fully applied clinically for aneuploidy screening. The diagnostic potential of the technique will be fully recognised with the application of this technology on clinical cases.
  • O'Connor, R. et al. (2017). Isolation of subtelomeric sequences of porcine chromosomes for translocation screening reveals errors in the pig genome assembly. Animal Genetics [Online] 48:395-403. Available at: http://dx.doi.org/10.1111/age.12548.
    Balanced chromosomal aberrations have been shown to affect fertility in most species studied, often leading to hypoprolificacy (reduced litter size) in domestic animals such as pigs. With an increasing emphasis in modern food production on the use of a small population of high quality males for artificial insemination, the potential economic and environmental costs of hypoprolific boars, bulls, rams etc. are considerable. There is therefore a need for novel tools to facilitate rapid, cost-effective chromosome translocation screening. This has previously been achieved by standard karyotype analysis; however, this approach relies on a significant level of expertise and is limited in its ability to identify subtle, cryptic translocations. To address this problem, we developed a novel device and protocol for translocation screening using subtelomeric probes and fluorescence in situ hybridisation. Probes were designed using BACs (bacterial artificial chromosomes) from the subtelomeric region of the short (p-arm) and long (q-arm) of each porcine chromosome. They were directly labelled with FITC or Texas Red (p-arm and q-arm respectively) prior to application of a ‘Multiprobe’ device, thereby enabling simultaneous detection of each individual porcine chromosome on a single slide. Initial experiments designed to isolate BACs in subtelomeric regions led to the discovery of a series of incorrectly mapped regions in the porcine genome assembly (from a total of 82 BACs, only 45 BACs mapped correctly). Our work therefore highlights the importance of accurate physical mapping of newly sequenced genomes. The system herein described allows for robust and comprehensive analysis of the porcine karyotype, an adjunct to classical cytogenetics that provides a valuable tool to expedite efficient, cost effective food production.
  • Griffin, D. et al. (2017). Continuing to deliver: the evidence base for pre-implantation genetic screening. BMJ [Online] 356:j752. Available at: https://doi.org/10.1136/bmj.j752.
  • Saretzki, G. et al. (2017). Preterm infants have significantly longer telomeres than their term born counterparts. PLOS ONE [Online] 12:e0180082. Available at: https://doi.org/10.1371/journal.pone.0180082.
    There are well-established morbidities associated with preterm birth including respiratory, neurocognitive and developmental disorders. However several others have recently emerged
    that characterise an `aged' phenotype in the preterm infant by term-equivalent age. These include hypertension, insulin resistance and altered body fat distribution. Evidence
    shows that these morbidities persist into adult life, posing a significant public health concern. In this study, we measured relative telomere length in leukocytes as an indicator of biological
    ageing in 25 preterm infants at term equivalent age. Comparing our measurements with those from 22 preterm infants sampled at birth and from 31 term-born infants, we tested the
    hypothesis that by term equivalent age, preterm infants have significantly shorter telomeres (thus suggesting that they are prematurely aged). Our results demonstrate that relative telomere
    length is highly variable in newborn infants and is significantly negatively correlated with gestational age and birth weight in preterm infants. Further, longitudinal assessment in
    preterm infants who had telomere length measurements available at both birth and term age (n = 5) suggests that telomere attrition rate is negatively correlated with increasing gestational
    age. Contrary to our initial hypothesis however, relative telomere length was significantly shortest in the term born control group compared to both preterm groups and longest
    in the preterm at birth group. In addition, telomere lengths were not significantly different between preterm infants sampled at birth and those sampled at term equivalent age. These
    results indicate that other, as yet undetermined, factors may influence telomere length in the preterm born infant and raise the intriguing hypothesis that as preterm gestation declines,
    telomere attrition rate increases.
  • Caujolle, S. et al. (2017). Speckle variance OCT for depth resolved assessment of the viability of bovine embryos. Biomedical Optics Express [Online] 8:5139-5150. Available at: http://dx.doi.org/10.1364/BOE.8.005139.
    The morphology of embryos produced by in vitro fertilization (IVF) is commonly used to estimate their viability. However, imaging by standard microscopy is subjective and unable to assess the embryo on a cellular scale after compaction. Optical coherence tomography is an imaging technique that can produce a depth-resolved profile of a sample and can be coupled with speckle variance (SV) to detect motion on a micron scale. In this study, day 7 post-IVF bovine embryos were observed either short-term (10 minutes) or longterm (over 18 hours) and analyzed by swept source OCT and SV to resolve their depth profile and characterize micron-scale movements potentially associated with viability. The percentage of en face images showing movement at any given time was calculated as a method to detect the vital status of the embryo. This method could be used to measure the levels of damage sustained by an embryo, for example after cryopreservation, in a rapid and non-invasive way.
  • Saintas, E. et al. (2017). Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS. PLoS ONE [Online] 12:e0172140. Available at: http://dx.doi.org/10.1371/journal.pone.0172140.
    The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
  • Capalbo, A. et al. (2016). Artificial oocyte activation with calcium ionophore does not cause a widespread increase in chromosome segregation errors in the second meiotic division of the oocyte. Fertility and Sterility [Online] 105:807-814.e2. Available at: http://doi.org/10.1016/j.fertnstert.2015.11.017.
    Objective

    To study the effect of artificial oocyte activation (AOA) on chromosome segregation errors in the meiotic divisions.

    Design

    Prospective cohort study with historical control.

    Setting

    Private/academic IVF centers.

    Patient(s)

    Fifty-six metaphase II oocytes were donated from 12 patients who had undergone IVF between June 2008 and May 2009.

    Intervention(s)

    Oocytes were activated by 40 minutes' exposure to 100 μM calcium-ionophore. The activated oocyte was tubed and analyzed by array comparative genomic hybridization and/or single-nucleotide polymorphism genotyping and maternal haplotyping (meiomapping). A control sample of embryos derived from normally fertilized oocytes was included for comparison.

    Main Outcome Measure(s)

    Incidence of chromosome segregation errors in artificially activated and normally fertilized oocytes in relation to pronuclear evaluation.

    Result(s)

    Of 49 oocytes that survived the warming procedure, thirty-nine (79.6%) activated. Most activated normally, resulting in extrusion of the second polar body and formation of a single or no pronucleus (2PB1PN: 30 of 39, 76.9%; or 2PB0PN: 5 of 39, 12.8%). Twenty-seven of these were analyzed, and 16 (59.3%) were euploid, showing no effect of AOA on meiotic segregation. Single-nucleotide polymorphism analysis of normally activated oocytes confirmed normal segregation of maternal chromosomes. No difference in the proportion of meiosis II type errors was observed between artificially activated oocytes (28.6%; 95% confidence interval 3.7%–71.0%) compared with embryos obtained from normally fertilized oocytes (44.4%; 95% confidence interval 13.7%–78.8%). The abnormally activated oocytes, with ≥2PN (4 of 39, 10.3%) were diploid, indicating a failure to coordinate telophase of meiosis II with polar body extrusion.

    Conclusion(s)

    From this preliminary dataset, there is no evidence that AOA causes a widespread increase in chromosome segregation errors in meiosis II. However, we recommend that it be applied selectively to patients with specific indications.
  • O'Connor, R. et al. (2016). Gross genome evolution in the Dinosauria Griffin, D. K. et al. eds. Chromosome Research [Online] 24:S36-S37, Abstract O19. Available at: http://dx.doi.org/10.1007/s10577-016-9532-x.
    The Dinosaurs dominated the terrestrial environment for around 170 million years and are probably the most successful land vertebrate group to have existed. They survived several mass extinction events before finally all non-avian species were wiped out 66 million years ago in the Cretaceous-Paleogene extinction event. The neornithes (modern birds) are their living descendants. Despite the huge phenotypic diversity seen in birds, they, and some non-avian reptiles (e.g. some turtle species) display remarkably similar karyotypes with a characteristic pattern of macro and micro chromosomes, small genome size and few repetitive elements. This suggests that these were features present early in their evolution.

    The availability whole genome sequences and the recent sequencing of around 50 avian genomes, 6 of which were assembled at sufficient read depth and coverage to permit visualization at the chromosomal level, has facilitated the reconstruction of the overall genome structure (karyotype) of both Saurian (bird-reptile) and Avian ancestors. Subsequent use of bioinformatic tools permitted the retracing of the gross evolutionary changes that have occurred along the Dinosaur (and various avian) lineages. Gene ontology analysis of homologous synteny blocks (HSBs) and evolutionary breakpoint regions (EBRs) of chromosomes has allowed us to search for enrichment for genes involved in chromosome rearrangement (consistent with the formation of the signature fragmented karyotype of birds (and probably dinosaurs)). Preliminary analyses of EBRs suggest that they appear to be enriched for genes involved in body size, consistent with the overall gross reduction in body size as dinosaurs evolved into birds. Our results also suggest a period of inter-and intra-chromosomal rearrangements up until around the divergence of turtles (approximately 210 MYA) with a relatively “fixed” pattern thereafter where intra-chromosomal rearrangement plus a few identifiable fissions predominated. It is reasonable therefore to speculate that this ‘avianstyle’ genome may be one of the key factors in the success of this extraordinarily diverse animal group, allowing rapid speciation through increased propensity for random segregation and genetic recombination.
  • Farré, M. et al. (2016). Novel Insights into Chromosome Evolution in Birds, Archosaurs, and Reptiles. Genome Biology and Evolution [Online] 8:2442-2451. Available at: http://doi.org/10.1093/gbe/evw166.
    Homologous synteny blocks (HSBs) and evolutionary breakpoint regions (EBRs) in mammalian chromosomes are enriched for distinct DNA features, contributing to distinct phenotypes. To reveal HSB and EBR roles in avian evolution, we performed a sequence-based comparison of 21 avian and 5 outgroup species using recently sequenced genomes across the avian family tree and a newly-developed algorithm. We identified EBRs and HSBs in ancestral bird, archosaurian (bird, crocodile, and dinosaur), and reptile chromosomes. Genes involved in the regulation of gene expression and biosynthetic processes were preferably located in HSBs, including for example, avian-specific HSBs enriched for genes involved in limb development. Within birds, some lineage-specific EBRs rearranged genes were related to distinct phenotypes, such as forebrain development in parrots. Our findings provide novel evolutionary insights into genome evolution in birds, particularly on how chromosome rearrangements likely contributed to the formation of novel phenotypes.
  • Turner, K. et al. (2016). Multicolor detection of every chromosome as a means of detecting mosaicism and nuclear organization in human embryonic nuclei. Panminerva medica [Online] 58:175-90. Available at: https://www.minervamedica.it/it/riviste/panminerva-medica/articolo.php?cod=R41Y2016N02A0175.
    Fluorescence in-situ hybridization (FISH) revolutionized cytogenetics using fluorescently labelled probes with high affinity with target (nuclear) DNA. By the early 1990s FISH was adopted as a means of preimplantation genetic diagnosis (PGD) sexing for couples at risk of transmitting X-linked disorders and later for detection of unbalanced translocations. Following a rise in popularity of PGD by FISH for sexing and the availability of multicolor probes (5-8 colors), the use of FISH was expanded to the detection of aneuploidy and selective implantation of embryos more likely to be euploid, the rationale being to increase pregnancy rates (referral categories were typically advanced maternal age, repeated IVF failure, repeated miscarriage or severe male factor infertility). Despite initial reports of an increase in implantation rates, reduction in trisomic offspring and spontaneous abortions criticism centered around experimental design (including lack of randomization), inadequate control groups and lack of report on live births. Eleven randomized control trials (RCTs) (2004-2010) showed that preimplantation genetic screening (PGS) with FISH did not increase delivery rates with some demonstrating adverse outcomes. These RCTs, parallel improvements in culturing and cryopreservation and a shift to blastocyst biopsy essentially outdated FISH as a tool for PGS and it has now been replaced by newer technologies (array CGH, SNP arrays, qRT-PCR and NGS). Cell-by-cell follow up analysis of individual blastomeres in non-transferred embryos is however usually prohibitively expensive by these new approaches and thus FISH remains an invaluable resource for the study of mosaicism and nuclear organization. We thus developed the approach described herein for the FISH detection of chromosome copy number of all 24 human chromosomes. This approach involves 4 sequential layers of hybridization, each with 6 spectrally distinct fluorochromes and a bespoke capturing system. Here we report previously published studies and hitherto unreported data indicating that 24 chromosome FISH is a useful tool for studying chromosome mosaicism, one of the most hotly debated topics currently in preimplantation genetics. Our results suggest that mosaic embryo aneuploidy is not highly significantly correlated to maternal age, probably due, in part, to the large preponderance of post-zygotic (mitotic) errors. Chromosome loss (anaphase lag) appears to be the most common mechanism, followed by chromosome gain (endoreduplication), however 3:1 mitotic non-disjunction of chromosomes appears to be rare. Nuclear organization (i.e. the spatial and temporal topology of chromosomes or sub-chromosomal compartments) studies indicate that human morula or blastocyst embryos (days 4-5) appear to adopt a "chromocentric" pattern (i.e. almost all centromeric signals reside in the innermost regions of the nuclear volume). By the blastocyst stage however, a more ordered organization with spatial and temporal cues important for embryo development appears. We have however found no association between aneuploidy and nuclear organization using this approach despite our earlier studies. In conclusion, while FISH is mostly "dead and buried" for mainstream PGS, it still has a place for basic biology studies; the development of a 24 chromosome protocol extends the power of this analysis.
  • Victor, A. et al. (2016). Accurate quantitation of mitochondrial DNA reveals uniform levels in human blastocysts irrespective of ploidy, age, or implantation potential. Fertility and Sterility [Online] 107:34-42. Available at: http://doi.org/10.1016/j.fertnstert.2016.09.028.
  • Al Dibouni, Z. et al. (2016). Incidence, Sex Ratio and Perinatal Outcomes of IVF and ICSI Monozygotic Twin Pregnancies Following either Cleavage or Blastocyst Stage Embryo Transfer. Human Genetics & Embryology [Online] 6. Available at: http://www.dx.doi.org/10.4172/2161-0436.1000132.
    To determine if prolonged time in embryo culture has an effect on the rate, sex ratio, and perinatal outcomes of monozygotic twins (MZT) following either cleavage stage or blastocyst embryo transfer after assisted conception. This is a retrospective study of 2,316 consecutive clinical pregnancies resulting from cleavage stage transfer (CT) and blastocyst transfer (BT). Criteria examined included (i) incidences (ii) sex ratios (iii) gestational age and birth weight; (iv) perinatal outcomes of these pregnancies from cleavage stage and blastocyst transfer procedures. Monozygotic twin pregnancies were identified by (i) presence of a gestational sac containing more than one fetal pole with cardiac activity, (ii) the number of gestational sacs or fetal hearts exceeds the number of embryos transferred and (iii) twin pregnancies following a single embryo transfer. Overall the incidence of twinning was 1.64% (38 out of 2,316 pregnancies). The frequency of twinning was 2.3 × higher following BT (18 out of 649) compared to CT (20 out of 1,667). IVF techniques skewed the sex ratio in favour of males while ICSI significantly favoured females. There was no statistically significant difference between transfer type and gestational age, birth weight and perinatal outcome. All pregnancies resulted in the birth of 86 infants. In our experience, BT more than doubles the chances of conceiving a monozygotic twin pregnancy, however IVF techniques lead to a greater likelihood of male birth(s) if twins are conceived. Appropriate pre-conception counselling should be given to advise the potential risks associated with both types of transfer as well as using alternative methods such as single embryo transfer to reduce the risk of multiple gestations.
  • Coates, A. et al. (2016). Differences in pregnancy outcomes in donor egg frozen embryo transfer (FET) cycles following preimplantation genetic screening (PGS): a single center retrospective study. Journal of Assisted Reproduction and Genetics [Online] 34:71-78. Available at: http://doi.org/10.1007/s10815-016-0832-z.
  • Damas, J. et al. (2016). Upgrading short read animal genome assemblies to chromosome level using comparative genomics and a universal probe set. Genome Research [Online] 27:875-884. Available at: http://dx.doi.org/10.1101/gr.213660.116.
    Most recent initiatives to sequence and assemble new species' genomes de-novo fail to achieve the ultimate endpoint to produce contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of sub-chromosomal sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, PCR-based scaffold verification and physical mapping to chromosomes. Multi-genome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffolds to chromosomes on all avian genomes. As proof of principle, we assembled genomes of the pigeon (Columbia livia) and peregrine falcon (Falco peregrinus) to chromosome level comparable, in continuity, to avian reference genomes. Both species are of interest for breeding, cultural, food and/or environmental reasons. Pigeon has a typical avian karyotype (2n=80) while falcon (2n=50) is highly rearranged compared to the avian ancestor. Using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved non-coding elements (CNEs) and that the chromosomal fission sites are further limited to long CNE 'deserts.' This corresponds with fission being the rarest type of rearrangement in avian genome evolution. High-throughput multiple hybridization and rapid capture strategies using the current BAC set provide the basis for assembling numerous avian (and possibly other reptilian) species while the overall strategy for scaffold assembly and mapping provides the basis for an approach that (provided metaphases can be generated) could be applied to any animal genome.
  • Ioannou, D. et al. (2016). Impact of sperm DNA chromatin in the clinic. Journal of Assisted Reproduction and Genetics [Online] 33:157-166. Available at: http://doi.org/10.1007/s10815-015-0624-x.
  • O'Connor, R. et al. (2016). Upgrading molecular cytogenetics to study reproduction and reproductive isolation in mammals, birds, and dinosaurs. Cytogenetic and Genome Research 148:151-152, Abstract VII.13.
    The past 10–15 years have seen a revolution in the field of genomics, first with the human genome project, followed by those of key model and agricultural species (chicken, pig, cattle, sheep) and, most recently, ∼ 60 de novo avian genome assemblies. The ultimate aim of a genome assembly is to create a contiguous unbroken length of sequence from p- to q-terminus to facilitate studies of gene mapping, trait linkage, phylogenomics, and gross genomic organization/change. Chromosome rearrangements are biologically relevant both in the context of reduction in reproductive capability of individual animals and in the establishment in reproductive isolation as species evolve and diverge. Moreover, a karyotype effectively represents a low-resolution map of the genome of any species. In investigating all these aspects, FISH remains the tool of choice, and this study describes a step change in its use.
  • Wrenzycki, C. et al. (2016). Hypomethylation and Genetic Instability in Monosomy Blastocysts May Contribute to Decreased Implantation Potential. PLOS ONE [Online] 11:e0159507. Available at: http://doi.org/10.1371/journal.pone.0159507.
    DNA methylation is a key epigenetic mechanism responsible for gene regulation, chromatin remodeling, and genome stability, playing a fundamental role during embryonic development. The aim of this study was to determine if these epigenetic marks are associated with chromosomal aneuploidy in human blastocysts. Surplus, cryopreserved blastocysts that were donated to research with IRB consent were chosen with varying chromosomal aneuploidies and respective implantation potential: monosomies and trisomies 7, 11, 15, 21, and 22. DNA methylation analysis was performed using the Illumina Infinium HumanMethylation450 BeadChip (~485,000 CpG sites). The methylation profiles of these human blastocysts were found to be similar across all samples, independent of chromosome constitution; however, more detailed examination identified significant hypomethylation in the chromosome involved in the monosomy. Real-time PCR was also performed to determine if downstream messenger RNA (mRNA) was affected for genes on the monosomy chromosome. Gene dysregulation was observed for monosomy blastocysts within significant regions of hypo-methylation (AVEN, CYFIP1, FAM189A1, MYO9A, ADM2, PACSIN2, PARVB, and PIWIL3) (P < 0.05). Additional analysis was performed to examine the gene expression profiles of associated methylation regulators including: DNA methyltransferases (DNMT1, DNMT3A, DNMT3B, DNMT3L), chromatin modifying regulators (CSNK1E, KDM1, PRKCA), and a post-translational modifier (PRMT5). Decreased RNA transcription was confirmed for each DNMT, and the regulators that impact DNMT activity, for only monosomy blastocysts (P < 0.05). In summary, monosomy blastocysts displayed hypomethylation for the chromosome involved in the error, as well as transcription alterations of associated developmental genes. Together, these modifications may be contributing to genetic instability and therefore be responsible for the limited implantation potential observed for full monosomy blastocysts.
  • Hornak, M. et al. (2016). Aneuploidy Detection and mtDNA Quantification in Bovine Embryos with Different Cleavage Onset Using a Next-Generation Sequencing-Based Protocol. Cytogenetic and Genome Research [Online] 150:60-67. Available at: http://doi.org/10.1159/000452923.
    Bovine embryos are now routinely used in agricultural systems as a means of disseminating superior genetics worldwide, ultimately with the aim of feeding an ever-growing population. Further investigations, common for human IVF embryos, thus have priority to improve cattle IVF, as has screening for aneuploidy (abnormal chromosome number). Although the incidence and consequences of aneuploidy are well documented in human preimplantation embryos, they are less well known for the embryos of other animals. To address this, we assessed aneuploidy levels in thirty-one 2-cell bovine embryos derived from early- and late-cleaving zygotes. Contemporary approaches ( Whole Genome Amplification and next-generation sequencing) allowed aneuploidy assessment for all chromosomes in oocytes from donors aged 4-7 years. We also quantified mitochondrial DNA (mtDNA) levels in all blastomeres assessed, thereby testing the hypothesis that they are related to levels of aneuploidy. The overall incidence of aneuploidy in this cohort of bovine embryos was 41.1% and correlated significantly with the timing of cleavage (77.8% in late-cleaving vs. 31.8% in early-cleaving embryos). Moreover, based on mtDNA sequence read counts, we observed that the median mtDNA quantity is significantly lower in late-cleaving embryos. These findings further reinforce the use of the bovine system as a model for human IVF studies.
  • Parks, J. et al. (2016). Corona cell RNA sequencing from individual oocytes revealed transcripts and pathways linked to euploid oocyte competence and live birth. Reproductive BioMedicine Online [Online] 32:518-526. Available at: http://doi.org/10.1016/j.rbmo.2016.02.002.
    Corona cells surround the oocyte and maintain a close relationship through transzonal processes and gap junctions, and may be used to assess oocyte competence. In this study, the corona cell transcriptome of individual cumulus oocyte complexes (COCs) was investigated. Isolated corona cells were collected from COCs that developed into euploid blastocysts and were transferred in a subsequent frozen embryo transfer. Ten corona cell samples underwent RNA-sequencing to generate unique gene expression profiles. Live birth was compared with negative implantation after the transfer of a euploid blastocyst using bioinformatics and statistical analysis. Individual corona cell samples produced a mean of 21.2 million sequence reads, and 307 differentially expressed transcrpits (P < 0.05; fold change ≥2). Enriched pathway analysis showed Wnt signalling, mitogen-activated protein kinases signalling, focal adhesion and tricarboxylic acid cycle to be affected by implantation outcome. The Wnt/beta-catenin signalling pathway, including genes APC, AXIN and GSK3B, were independently validated by real-time quantitative reverse transcription. Individual, corona cell transcriptome was successfully generated using RNA-sequencing. Key genes and signalling pathways were identified in association with implantation outcome after the transfer of a euploid blastocyst in a frozen embryo transfer. These data could provide novel biomarkers for the non-invasive assessment of embryo viability.
  • Taylor, T. et al. (2016). Technique to ‘Map' Chromosomal Mosaicism at the Blastocyst Stage. Cytogenetic and Genome Research [Online] 149:262-266. Available at: http://doi.org/10.1159/000449051.
    The purpose of this study was to identify a technique that allows for comprehensive chromosome screening (CCS) of individual cells within human blastocysts along with the approximation of their location in the trophectoderm relative to the inner cell mass (ICM). This proof-of-concept study will allow for a greater understanding of chromosomal mosaicism at the blastocyst stage and the mechanisms by which mosaicism arises. One blastocyst was held by a holding pipette and the ICM was removed. While still being held, the blastocyst was further biopsied into quadrants. To separate the individual cells from the biopsied sections, the sections were placed in calcium/magnesium-free medium with serum for 20 min. A holding pipette was used to aspirate the sections until individual cells were isolated. Individual cells from each section were placed into PCR tubes and prepped for aCGH. A total of 18 cells were used for analysis, of which 15 (83.3%) amplified and provided a result and 3 (16.7%) did not. Fifteen cells were isolated from the trophectoderm; 13 (86.7%) provided an aCGH result, while 2 (13.3%) did not amplify. Twelve cells were euploid (46,XY), while 1 was complex abnormal (44,XY), presenting with monosomy 7, 10, 11, 13, and 19, and trisomy 14, 15, and 21. A total of 3 cells were isolated from the ICM; 2 were euploid (46,XY) and 1 did not amplify. Here, we expand on a previously published technique which disassociates biopsied sections of the blastocyst into individual cells. Since the blastocyst sections were biopsied in regard to the position of the ICM, it was possible to reconstruct a virtual image of the blastocyst while presenting each cell's individual CCS results.
  • Ottolini, C. et al. (2015). Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates. Nature Genetics [Online] 47:727-735. Available at: http://doi.org/10.1038/ng.3306.
    Crossover recombination reshuffles genes and prevents errors in segregation that lead to extra or missing chromosomes (aneuploidy) in human eggs, a major cause of pregnancy failure and congenital disorders. Here we generate genome-wide maps of crossovers and chromosome segregation patterns by recovering all three products of single female meioses. Genotyping >4 million informative SNPs from 23 complete meioses allowed us to map 2,032 maternal and 1,342 paternal crossovers and to infer the segregation patterns of 529 chromosome pairs. We uncover a new reverse chromosome segregation pattern in which both homologs separate their sister chromatids at meiosis I; detect selection for higher recombination rates in the female germ line by the elimination of aneuploid embryos; and report chromosomal drive against non-recombinant chromatids at meiosis II. Collectively, our findings show that recombination not only affects homolog segregation at meiosis I but also the fate of sister chromatids at meiosis II.
  • Thornhill, A. et al. (2015). Karyomapping—a comprehensive means of simultaneous monogenic and cytogenetic PGD: comparison with standard approaches in real time for Marfan syndrome. Journal of Assisted Reproduction and Genetics [Online] 32:347-356. Available at: http://dx.doi.org/10.1007/s10815-014-0405-y.
  • Ottolini, C. et al. (2015). Karyomapping identifies second polar body DNA persisting to the blastocyst stage: implications for embryo biopsy. Reproductive BioMedicine Online [Online] 31:776-782. Available at: http://doi.org/10.1016/j.rbmo.2015.07.005.
    Blastocyst biopsy is now widely used for both preimplantation genetic screening (PGS) and preimplantation genetic diagnosis (PGD). Although this approach yields good results, variable embryo quality and rates of development remain a challenge. Here, a case is reported in which a blastocyst was biopsied for PGS by array comparative genomic hybridization on day 6 after insemination, having hatched completely. In addition to a small trophectoderm sample, excluded cell fragments from the subzonal space from this embryo were also sampled. Unexpectedly, the array comparative genomic hybridization results from the fragments and trophectoderm sample were non-concordant: 47,XX,+19 and 46,XY, respectively. DNA fingerprinting by short tandem repeat and amelogenin analysis confirmed the sex chromosome difference but seemed to show that the two samples were related but non-identical. Genome-wide single nucleotide polymorphism genotyping and karyomapping identified that the origin of the DNA amplified from the fragments was that of the second polar body corresponding to the oocyte from which the biopsied embryo developed. The fact that polar body DNA can persist to the blastocyst stage provides evidence that excluded cell fragments should not be used for diagnostic purposes and should be avoided when performing embryo biopsies as there is a risk of diagnostic errors.
  • Schmid, M. et al. (2015). Third Report on Chicken Genes and Chromosomes 2015 Schmid, M., Smith, J. and Burt, D. W. eds. Cytogenetic and Genome Research [Online] 145:78-179. Available at: http://dx.doi.org/10.1159/000430927.
    Opening insights into new technologies in avian genomics

    The chicken has long been a model organism for genetic and developmental studies. It is now beginning to take its place as a model genome, opening up the fields of phylogenetics and comparative genomics like never before. This report comes at a time of huge technological advances (particularly in sequencing methodologies) and summarizes the current efforts to complete the gaps in the genome. It describes the progress that has been made in genomic annotation, particularly with respect to noncoding RNAs and genetic variants. Reviews of comparative genomics, avian evolution and sex determination are included as well as transcriptomic case studies and developments in epigenetic studies. The Third Report on Chicken Genes and Chromosomes also features the National Avian Research Facility and how it has developed into a resource for the study of avian biology, genetics, infection and disease. In this volume researchers interested in genetics, genomics and evolution will find detailed information that has not been available until now.
  • Damas, J. et al. (2015). Towards the construction of avian chromosome assemblies Griffin, D. K. et al. eds. Chromosome Research [Online] 23:378-379, Abstract O21. Available at: http://dx.doi.org/10.1007/s10577-014-9447-3.
    The advent of the next generation sequencing (NGS) made sequencing and scaffolding of an entire animal genome a routine procedure. As the result we face a fast increase in the number of animal genomes available due to the activities of large international genome sequencing initiatives e.g., Genome 10K (G10K) or smaller projects. However, the full informative power of a sequenced genome could only be achieved when it is assembled into chromosomes. Usually, a draft or nearly complete animal chromosome assembly is achieved through three steps: (i) constructing contigs based on read overlaps, (ii) merging contigs into scaffolds using pair-end reads, and (iii) mapping scaffolds on chromosomes with the use of physical or genetic maps. As the cost of mapping techniques is still much higher than sequencing, the genetic and physical maps are not available for the majority of the de novo sequenced genomes. To overcome this problem for assemblies that employ long-insert libraries (5 – 40 Kbp) we recently developed the reference-assisted chromosome assembly (RACA) algorithm (Kim et al., 2013). This method relies on both the raw sequencing data (reads) and comparative information; the latter is obtained from alignments between the target (de novo sequenced), a closely related (reference) and more distantly related (outgroup) genomes.

    Using RACA followed by the manual FISH or PCR verification steps we are reconstructing the chromosome organisation of 19 bird species sequenced by the G10K community. We use the publically available chicken (Gallus gallus) and zebra finch (Taeniopygia guttata) chromosome assemblies as either reference or outgroup for each reconstruction depending on their phylogenetic relationships with each target species. Initially, we established the optimal RACA parameters for a bird chromosome assembly reconstruction using the duck (Anas platyrhynchos) and budgerigar (Melopsittacus undulatus) super-scaffolds assembled with the support from physical maps. This step allowed us to test the reliability of RACA reconstructions for bird genomes. Due to a higher evolutionary conservation of the bird karyotype compared to the mammalian one, we have achieved ~97% accuracy of scaffold adjacencies in our predicted chromosome fragments compared to the ~93-96% accuracies reported for mammals (Kim et al., 2013). We detected ~4-28% of scaffolds in different target bird genomes that are either chimeric or containing genuine lineage-specific evolutionary breakpoint regions. Some of these scaffolds will be selected for follow up PCR or FISH verifications. All RACA reconstructions will become publicly available from our Evolution Highway comparative chromosome browser http://evolutionhighway.ncsa.uiuc.edu/birds/ and will be further utilised to study connections between the chromosome evolution, adaptation and phenotypic diversity in birds and other vertebrates.
  • Romanov, M. et al. (2015). Avian ancestral karyotype reconstruction and differential rates of inter- and intrachromosomal change in different lineages Griffin, D. K. et al. eds. Chromosome Research [Online] 23:414, Abstract P63. Available at: http://dx.doi.org/10.1007/s10577-014-9447-3.
    In birds, genome is organised into several large chromosomes (macrochromosomes) and many smaller chromosomes (microchromosomes) that usually constitute about 25 and 75 per cent of the karyotype, respectively. Cytogenetic and molecular cytogenetic evidence suggests that avian karyotype is remarkably stable in evolution, with exception of several clades. To date, at least 21 avian genomes have been sequenced and assembled at the chromosome or scaffold level with N50 greater than 2 Mb, thereby allowing cytogenomic studies of chromosome organisation and change. To understand the comparative organisation and evolution of several avian species, we aligned chromosomes and scaffolds using an interactive genome browser (Evolution Highway), identifying homologous synteny blocks (HSBs) and evolutionary breakpoint regions (EBRs). For ancestral karyotype reconstruction, we focused on six species (chicken, turkey, duck, zebra finch, ostrich, and budgerigar; N50 > 10Mb) and reconstructed avian ancestor chromosomes using an outgroup (Anole lizard). In particular, we addressed the following biological questions: (1) whether species-specific EBRs could represent recombination hotspots, and (2) whether entire microchromosomes could be considered as blocks of conserved synteny. Our study did not reveal a significant association between EBRs and recombination. With support from molecular cytogenetic mapping, we did find that microchromosomes are characterised by a high interchromosomal conservation in almost all birds studied, except ostrich and parrots (budgerigar). By analysing HSBs in six birds and using a lizard outgroup,, we reconstructed a tentative avian ancestral genome and chromosomal rearrangements that occurred in the major avian evolutionary lineages. We identified most intrachromosomal changes (mostly inversions) in the zebra finch clade (Passeriformes) since the time when it diverged from the sister group of parrots (Psittaciformes) 54MYA. Our data also suggest the fewest number of chromosomal changes in the chicken as compared to the dinosaur-like avian ancestor.
  • O'Connor, R. et al. (2015). Reconstruction of the putative Saurian karyotype and the hypothetical chromosome rearrangements that occurred along the Dinosaur lineage Griffin, D. K. et al. eds. Chromosome Research [Online] 23:379-380, Abstract O22. Available at: http://dx.doi.org/10.1007/s10577-014-9447-3.
    Dinosaurs hold a unique place both in the history of the earth and the imagination of many. They dominated the terrestrial environment for around 170 million years during which time they diversified into at least 1000 different species. Reptilia, within which they are placed is one of the most remarkable vertebrate groups, consisting of two structurally and physiologically distinct lineages – the birds and the non-avian reptiles, of which there are 10,000 and 7,500 extant species respectively. The dinosaurs are without doubt the most successful group of vertebrate to have existed. They survived several mass extinction events before finally non-avian dinosaurs were defeated 66 million years ago in the Cretaceous-Paleogene extinction event, leaving the neornithes (modern birds) as their living descendants. Aside from the huge phenotypic diversity seen in this group, the birds and non-avian reptiles interestingly display similar karyotypic patterns (with the exception of crocodilians); with the characteristic pattern of macro and micro chromosomes, small genome size and few repetitive elements, suggesting that these were features exhibited in their common ancestor.

    In this study, the availability of multiple reptile genome sequences (including birds) on an interactive browser (Evolution Highway) allowed us to identify multi species homologous synteny blocks (msHSBs) between the putative avian ancestor (derived from six species of extant birds), the Lizard (Anolis carolensis) and the Snake (Boa constrictor). From these msHSBs we were able to produce a series of contiguous ancestral regions (CARs) representing the most likely ancestral karyotype of the Saurian (ancestor of archosaurs and lepidosaurs) that diverged from the mammalian lineage 280 mya. From this we have hypothesised the series of inter and intra-chromosomal rearrangements that have occurred along the dinosaur (archosaur) lineage to the ancestor of modern birds (100 mya) and along the lepidosaur lineage to the modern snake and lizard using the model of maximum parsimony.

    Our study shows that relatively few chromosomal rearrangements took place over this period with an average of one inter or intra-chromosomal (translocations and inversions respectively) rearrangement occurring approximately every 2 million years. The majority of these rearrangements appear to be intra-chromosomal suggesting an overall karyotypic stability, which is consistent with that of that of modern birds. Our results support the hypothesis that the characteristically avian genome was present in the saurian ancestor and that it has remained remarkably stable in the 280 million years since. It is credible therefore to suggest that this ‘avian-style’ genome may be one of the key factors in the success of this extraordinarily diverse animal group.
  • Coates, A. et al. (2015). Use of suboptimal sperm increases the risk of aneuploidy of the sex chromosomes in preimplantation blastocyst embryos. Fertility and Sterility [Online] 104:866-872. Available at: http://doi.org/10.1016/j.fertnstert.2015.06.033.
  • Griffin, D. et al. (2015). Avian cytogenetics goes functional, in: Third Report on Chicken Genes and Chromosomes 2015 Schmid, M., Smith, J. and Burt, D. W. eds. Cytogenetic and Genome Research [Online] 145:100-105. Available at: http://dx.doi.org/10.1159/000430927.
    It is now over 10 years since the first avian genome [International Chicken Genome Sequencing Consortium, 2004] and the first complete avian karyotype [Masabanda et al., 2004] were both published; however, until 2014, avian cytogenetics has focused heavily on descriptive studies [e.g. Griffin et al., 2007, 2008; Skinner et al., 2009; Völker et al., 2010] with less attention to its functional relevance. Last year, however, saw 2 landmark efforts in the chromosomal studies of birds: a special issue of Chromosome Research in April and the announcement of recently completed sequences of multiple new avian genomes in Science and the BMC journals (taking the total number sequenced to over 50) in December. Studying the chromosomes of birds is, perhaps for the first time, telling us more about avian biology, function and evolution than it ever has...

    Conclusions.
    The most recent advances in avian cytogenetics have culminated in great promise not only for the study of bird karyotypes, but also for providing insight into the mechanisms of chromosome evolution in general. New avenues for investigation include gene regulation; for instance, it will become necessary to map accurately the physical location of polyadenylation and transcription start sites, important reference points that define promoters and post-transcriptional regulation. It will also become possible to sequence full-length transcripts, to allow accurate identification of alternate splicing events and their controlling elements. The ENCODE (Encyclopedia of DNA Elements) project has helped to define functional elements of the human genome, including those aforementioned as well as other chromatin signals, e.g. active chromatin, enhancers, insulators, methylation domains, etc. An effort of agENCODE is underway to include agriculturally important birds such as chicken, turkey, duck, quail, and perhaps ostrich. The study of cytogenetics will be essential here in helping to define higher-order structures in nuclear organization that show regulatory interactions within and between chromosomes. Finally, reconstruction of evolutionary events allows us to study genome organization and function not only in extant but, by extrapolation, in extinct species also. Reconstruction of avian-reptilian ancestral karyotypes will allow us to define chromosomal rearrangements in long-dead species that have captured the public imagination. Here be dragons!
  • Griffin, D. et al. (2015). Avian chromonomics goes functional Griffin, D. K. et al. eds. Chromosome Research [Online] 23:367, Abstract S32. Available at: http://dx.doi.org/10.1007/s10577-014-9447-3.
    Whole chromosomes (and sub-chromosomal homologous synteny blocks (HSBs)) have great significance in molecular studies of genome evolution. In birds, our ability to define chromosomes and HSBs precisely has however been impeded by a near intractable karyotype and so has focused primarily on comparative molecular cytogenetics (zoo- FISH) of the largest chromosomes (1-10+Z). Availability of multiple avian genome sequence assemblies has however allowed us, for the first time, to identify chromosomal syntenies across species. In recent work we have made use of comparative maps for 20+ avian genome assemblies (plus out-groups) and presented them on “Evolution Highway” an open-access, interactive freely available comparative chromosome browser designed to store and visualise comparative chromosome maps. This browser (http://evolutionhighway.ncsa.uiuc.edu) is used to visualize comparative genome organization and to identify and visualize the different types of evolutionary breakpoint regions (EBRs) in chromosomes, e.g., lineage specific, ordinal, superordinal, and reuse. Comparative analysis of all available genomes is providing insight into the mechanisms of chromosome change through correlation of EBRs with transposable elements and non-allelic homologous recombination. Gene ontology analysis is revealing interesting correlations with avian specific phenotype and function. Focus on six genomes (chicken, turkey, duck, zebra finch, ostrich and budgerigar) with both the largest N50s and supporting molecular cytogenetic information, has allowed us to assemble a putative ancestral avian karyotype and identify the key changes that led to the gross genome organization of representatives in the major avian clades (Palaeognathae, Galliformes, Anseriformes and Neoaves). We describe, for the first time, numerous inter- chromosomal rearrangements in a Paleoganthaeous bird (the ostrich), plus rearrangements in the budgerigar (Psattaciformes) and 15 other species. Intra- chromosomal evolutionary change in all species studied, can be derived, most parsimoniously, by a series of inversions, inter-chromosomal rearrangements by fissions and fusions. Increased chromosome rearrangement is associated with differentiation in certain clades, with the most intrachromosomal changes (primarily inversions) occurring in the zebra finch (Passeriformes) since its divergence from its sister group, the Psittaciformes 54MYA, This is coincident with the evolution of passerine-specific phenotypes e.g. vocal learning. Results also suggest that the Galloanserae (especially chicken) underwent the fewest changes compared to the ancestral karyotype; notably these birds appear, from fossil evidence, to be the most similar to ancient avian ancestors. We thus present the most comprehensive analysis of chromosomal rearrangements in birds to date and draw novel conclusions about their mechanisms of origin and association with avian-specific phenotypic features.
  • Romanov, M. and Griffin, D. (2015). The use of avian BAC libraries and clones, in: Third Report on Chicken Genes and Chromosomes 2015 Schmid, M., Smith, J. and Burt, D. W. eds. Cytogenetic and Genome Research [Online] 145:94-96. Available at: http://dx.doi.org/10.1159/000430927.
    High-density gridded libraries of large-insert clones using bacterial artificial chromosome (BAC) and other vectors are essential tools for genetic and genomic research in chicken and other avian species... Taken together, these studies demonstrate that applications of large-insert clones and BAC libraries derived from birds are, and will continue to be, effective tools to aid high-throughput and state-of-the-art genomic efforts and the important biological insight that arises from them.
  • Natesan, S. et al. (2014). Live birth after PGD with confirmation by a comprehensive approach (karyomapping) for simultaneous detection of monogenic and chromosomal disorders. Reproductive BioMedicine Online [Online] 29:600-605. Available at: http://dx.doi.org/10.1016/j.rbmo.2014.07.007.
  • Taylor, T. et al. (2014). Outcomes of blastocysts biopsied and vitrified once versus those cryopreserved twice for euploid blastocyst transfer. Reproductive BioMedicine Online [Online] 29:59-64. Available at: http://dx.doi.org/10.1016/j.rbmo.2014.03.001.
    Trophectoderm biopsy with comprehensive chromosome screening (CCS) has been shown to increase implantation and pregnancy rates. Some patients desire CCS on previously cryopreserved blastocysts, resulting in blastocysts that are thawed/warmed, biopsied, vitrified and then warmed again. The effect of two cryopreservation procedures and two thawing/warming procedures on outcomes has not been effectively studied. Cycles were divided into two groups: group 1 patients underwent a cryopreserved embryo transfer with euploid blastocysts that were vitrified and warmed once; group 2 patients had a cryopreserved embryo transfer of a euploid blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified and warmed. Groups 1 and 2 included 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years, respectively (not significantly different). Blastocyst survival in group 1 (114/116, 98.3%) and survival of second warming in group 2 (21/24, 87.5%) was significantly different (P = 0.0354). There was no difference between biochemical (68.2% and 62.5%) and clinical (61.2% and 56.3%) pregnancy rates, implantation rate (58.4% and 52.4%) and live birth/ongoing pregnancy rate (54.0% and 47.6%) between groups 1 and 2, respectively. Although it is unconventional to thaw/warm, biopsy, revitrify and rewarm blastocysts for cryopreserved embryo transfer, the results indicate that outcomes are not compromised.

    Trophectoderm biopsy and screening the embryos for chromosomal abnormalities has been reported to increase implantation and pregnancy rates. There is a category of patients requesting chromosomal screening on previously cryopreserved blastocysts. This scenario requires blastocysts to be thawed/warmed, biopsied, cryopreserved, and thawed/warmed again. The effect of double cryopreservation procedures and double thawing/warming procedures on pregnancy is unknown. Patients were divided into two groups, group 1 underwent a cryopreserved embryo transfer with a chromosomally normal blastocyst that was vitrified and warmed once and group 2 included patients that had a cryopreserved embryo transfer of a chromosomally normal blastocyst that was cryopreserved, thawed/warmed, biopsied, vitrified, and rewarmed. A total of 85 and 17 women aged 35.6 ± 3.9 and 35.3 ± 4.9 years were included in groups 1 and 2, respectively. The survival rate for group 1 (114 of 116, 98.3%) compared with the second warming for group 2 (21 of 24, 87.5%) was significantly higher. There was no difference between biochemical (68.2% and 62.5%), and clinical pregnancies (61.2% and 56.3%), implantation (58.4% and 52.4%), and live birth/ongoing rates (54.0% and 47.6%) between groups 1 and 2. Although it is unconventional to twice cryopreserve and twice thaw/warm a blastocyst, our results indicate that outcomes are not compromised.
  • Ishishita, S. et al. (2014). Chromosome size-correlated and chromosome size-uncorrelated homogenization of centromeric repetitive sequences in New World quails. Chromosome Research [Online] 22:15-34. Available at: http://dx.doi.org/10.1007/s10577-014-9402-3.
    Many families of centromeric repetitive DNA sequences isolated from Struthioniformes, Galliformes, Falconiformes, and Passeriformes are localized primarily to microchromosomes. However, it is unclear whether chromosome size-correlated homogenization is a common characteristic of centromeric repetitive sequences in Aves. New World and Old World quails have the typical avian karyotype comprising chromosomes of two distinct sizes, and C-positive heterochromatin is distributed in centromeric regions of most autosomes and the whole W chromosome. We isolated six types of centromeric repetitive sequences from three New World quail species (Colinus virginianus, CVI; Callipepla californica, CCA; and Callipepla squamata, CSQ; Odontophoridae) and one Old World quail species (Alectoris chukar, ACH; Phasianidae), and characterized the sequences by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. The 385-bp CVI-MspI, 591-bp CCA-BamHI, 582-bp CSQ-BamHI, and 366-bp ACH-Sau3AI fragments exhibited tandem arrays of the monomer unit, and the 224-bp CVI-HaeIII and 135-bp CCA-HaeIII fragments were composed of minisatellite-like and microsatellite-like repeats, respectively. ACH-Sau3AI was a homolog of the chicken nuclear membrane repeat sequence, whose homologs are common in Phasianidae. CVI-MspI, CCA-BamHI, and CSQ-BamHI showed high homology and were specific to the Odontophoridae. CVI-MspI was localized to microchromosomes, whereas CVI-HaeIII, CCA-BamHI, and CSQ-BamHI were mapped to almost all chromosomes. CCA-HaeIII was localized to five pairs of macrochromosomes and most microchromosomes. ACH-Sau3AI was distributed in three pairs of macrochromosomes and all microchromosomes. Centromeric repetitive sequences may be homogenized in chromosome size-correlated and -uncorrelated manners in New World quails, although there may be a mechanism that causes homogenization of centromeric repetitive sequences primarily between microchromosomes, which is commonly observed in phasianid birds.

Book section

  • Griffin, D. and Ellis, P. (2018). The Human Y-chromosome: Evolutionary Directions and Implications for the Future of “Maleness”. in: Palermo, G. D. and Sills, E. S. eds. Intracytoplasmic Sperm Injection. Springer, pp. 183-192. Available at: https://doi.org/10.1007/978-3-319-70497-5_13.
    The human Y chromosome represents an iconic image of “maleness,” and mutation, deletion, or rearrangement of the Y often lead to attendance in infertility clinics. Its evolutionary history is however also one of gene loss, inversion, and heterochromatin accumulation. There is little argument that the Y chromosome once had the size and gene density of its partner, the X chromosome, and is thus now only a shadow of its former self. The question however revolves around whether we are observing the Y at a point on its way to oblivion, or whether it has evolved effective mechanisms to cling on to life indefinitely. There are two schools of thought: The first is that the Y has persisted for hundreds of millions of years and is going nowhere. It can, it is asserted, outsmart genetic decay without regular meiotic crossing over, and the majority of its genes show signs of evolutionary selection. Palindromic sequences along its length with near 100% identity ensure self-recombination. During its history, it has added at least eight different genes, some of which have expanded in copy number, and the Y has lost no genes since humans and chimpanzees diverged ~6 million years ago. The counterargument is that the Y chromosome is subject to higher rates of variation and inefficient selection and is degrading irreversibly. The Y chromosome in other mammals has undergone lineage-specific degradation and has already disappeared entirely in some rodent lineages, such as spiny rats and mole voles. The argument goes that there is virtually nothing left of the original human Y and that the added part of the chromosome is in fact degrading rapidly. An interesting aside to what should be really only a phenomenon of interest to evolutionary cytogeneticists is that the story often gets conflated in the popular press to assume that the alleged Y chromosome demise automatically means the demise of males. Fear not, it doesn’t. Males are here to stay, and the argument is about this strange looking chromosome alone. Everyone agrees that the Y has degraded significantly, it is now well established that it has evolved some clever mechanisms to put the brakes on. The prevailing question is how effective those brakes actually are. Even experts can’t agree and a straw poll at the 2011 International Chromosome Conference suggested an even split overall, but with more men favoring the “Y remaining” model and more women the “Y leaving” scenario.

Conference or workshop item

  • Caujolle, S. et al. (2018). Assessing embryo development using swept source optical coherence tomography. in: Podoleanu, A. G. H. and Bang, O. eds. Second Canterbury Conference on Optical Coherence Tomography, 2017, Canterbury, United Kingdom. SPIE, p. . Available at: https://doi.org/10.1117/12.2282912.
    A detailed assessment of embryo development would assist biologists with selecting the most suitable embryos for transfer leading to higher pregnancy rates. Currently, only low resolution microscopy is employed to perform this assessment. Although this method delivers some information on the embryo surface morphology, no specific details are shown related to its inner structure. Using a Master-Slave Swept-Source Optical Coherence Tomography (SS-OCT), images of bovine embryos from day 7 after fertilization were collected from different depths. The dynamic changes inside the embryos were examined, in detail and in real-time from several depths. To prove our ability to characterize the morphology, a single embryo was imaged over 26 hours. The embryo was deprived of its life support environment, leading to its death. Over this period, clear morphological changes were observed.
  • Martell, H. et al. (2015). Assembling and comparing avian genomes by molecular cytogenetics. in: 2nd Bioinformatics Student Symposium. The Genome Analysis Centre, Norwich, UK: International Society of Computational Biology - Student Council - Regional Student Group UK; The Genome Analysis Centre, Norwich, UK. Available at: https://www.iscb.org/cms_addon/conferences/ismb2016/posterlist.php?cat=B.
    There has been a recent explosion in avian genomics. In December 2014 the Beijing Genomics Institute in collaboration with a number of labs worldwide (including Kent) released 48 new de-novo avian genome sequences in a special edition of Science. This has led to a complete re-evaluation of the phylogenetic tree of birds and presents the opportunity to study avian comparative genomics in far more detail than before. Most of these genome sequences however exist only as “scaffolds” i.e. the depth of sequence and length of read produces contiguous fragments of sub-chromosomal size. This impedes insight into overall genome structure, which is particularly challenging, as one of the most interesting biological features of birds is the peculiarity of their karyotype. This project is an on-going effort to map scaffold assemblies to avian chromosomes using a combination of bioinformatics and Fluorescent in situ Hybridization (FISH). This has traditionally been a very time-consuming and costly procedure, however a combination of bioinformatic approaches coupled with novel hardware innovation has deconstructed the FISH protocol and re-invented it as a high throughput, cheaper procedure. Initial work has helped to reconstruct Pigeon and Peregrine Falcon genomes and will ultimately provide insight into various unanswered questions pertaining to avian gross genome rearrangement. These include why the unique overall genomic structure of birds is so evolutionarily conserved, why intra and inter-chromosomal rearrangements happen (e.g. in response to the development of traits such as vocal learning) and what the karyotypes of extinct species such as dinosaurs may have looked like.
  • Romanov, M. et al. (2015). Comparative cytogenomics enhanced with bioinformatic tools provides further insights into genome evolution of birds and other amniotes. in: 2nd Annual Food, Nutrition and Agriculture Genomics Congress. London, UK: Oxford Global Conferences Ltd, p. Abstract 5. Available at: http://www.foodgenomics-congress.com/.
    Recent generation of multiple avian genome sequences enables further comparative analysis of their karyotypes, genome structure, and evolutionary changes. Using FISH and bioinformatic tools (e.g., Evolution Highway browser), we explore genome alignment for the sequenced birds, reptiles and mammals to infer ancestral genome organisation based on the information about their homologous synteny blocks and evolutionary breakpoints (EBRs). In EBRs shared between the birds, boa and opossum, we found over 6,000 human-chicken orthologous genes and examined their gene ontology (GO) categories. Four significant gene clusters were identified that embraced GO terms for phosphorylation (important for turning enzymes on and off and other metabolic pathways), and protein transport and maintenance at specific cell location. They were also enriched with genes of key cellular components (e.g. membranes, endoplasmic reticulum, Golgi membrane). Since chromosomal rearrangements occurred in EBRs may lead to new gene variants and, therefore, function changes, we suggest that the observed EBR enrichment could play a crucial part in evolution of metabolic networks and rates in higher vertebrates, including birds, adapted to various environments. We also developed a pipeline for analyzing cross-species FISH data including tools for semi-automated hybridization signal and FLpter identification, and comparative cytogenetic map construction.