Professor Darren Griffin

Professor of Genetics
Director, Centre for Interdisciplinary Studies of Reproduction

About

Professor Darren Griffin received his Bachelor of Science and Doctor of Science degrees from the University of Manchester and his PhD from University College London. After postdoctoral stints at Case Western Reserve University and the University of Cambridge he landed his first academic post at Brunel University, before settling at the University of Kent, where he’s been for the last 15+ years. He has worked under the mentorship of Professors Joy Delhanty, Christine Harrison, Terry Hassold, Alan Handyside and Malcolm Ferguson-Smith.
He is President of the International Chromosome and Genome Society, a Fellow of the Royal College of Pathologists, the Royal Society of Biology and the Royal Society of Arts, Manufacture and Commerce. He sits on the faculty of CoGen (controversies in genetics) and has previously sat on the board of the Preimplantation Genetic Diagnosis International Society (PGDIS), organising its annual meeting in 2014.
Darren is a world leader in cytogenetics. He performed the first successful cytogenetic preimplantation genetic diagnosis (sexing of IVF embryos) and, more recently, played a significant role in the development of Karyomapping a universal test for genetic disease in IVF, an approach he now applies to cattle. In a 30+ year scientific research career he has co-authored over 200 scientific publications, mainly on the cytogenetics of reproduction and evolution, most recently providing insight into the karyotypes of dinosaurs.
He is a prolific science communicator, making every effort to make scientific research publicly accessible (both his own and others) and is an enthusiastic proponent for the benefits of interdisciplinary research endeavour. He has supervised over 35 PhD students to completion and his work appears consistently in the national and international news. He currently runs a vibrant research lab of about 20 people (including a programme of externally supervised students) and maintains commercial interests in the outcomes of research findings, liaising with companies in the agricultural sector in the area of fertility screening. 

Darren is a member of the Centre for Interdisciplinary Studies of Reproduction (CISoR) 
He also regularly coordinates the International Chromosome Conferences and the Pig Breeders’ Round Table

Research Career 

  • 2013 Fellow of the Royal Society for the encouragement of Arts, Manufactures and Commerce (FRSA)
  • 2011-present President - International Chromosome and Genome Society (VP from 2008) 
  • 2008 Doctor of Science (DSc), University of Manchester 
  • 2007 Fellow of the Royal College of Pathologists (FRCPath) 
  • 2002 Postgraduate Certificate in Teaching and Learning in Higher Education, Brunel University (PGCertHE) 
  • 2000 Fellow of the Royal Society of Biology (CBiol, FRSB) 
  • 1992 Doctor of Philosophy (PhD), Human Genetics, University College London 
  • 1988 Bachelor of Science (BSc), Genetics and Cell Biology, University of Manchester 

ORCID: 0000-0001-7595-3226 

Research interests

The purpose of research in the Griffin lab is to make a significant contribution at the interface of molecular cytogenetics and genomics to the understanding of chromosome behaviour in an individual and evolutionary context. The research is highly impact driven with an aim to make key breakthroughs to advance diagnostics and improve agriculture. A key focus is widespread education and public engagement, both of the research findings and of the field in general.
Gross Genomic Changes in Human Gametes and Embryos Following early work performing the world’s first cytogenetic preimplantation genetic testing (PGT) case we operate a network of research activities with IVF units (e.g. London Women’s Clinic) and diagnostic companies such as Cooper and Igenomix. We played a significant role in developing “Karyomapping” a universal test for the diagnosis of genetic disease in human preimplantation embryos, which has been used to treat >10,000 patients. We are addressing fundamental questions about the incidence and origin and aetiology of chromosome abnormalities gametes and embryos and have a number of PhD candidates in Kent and around the world.
Non-Human IVF and Preimplantation Genetic Testing We established a pig and cattle IVF facility designed to generate genotyped IVF embryos, partly as a means of improving food production and also as a model to understand human IVF. Optimisation of protocols currently available for the production of cattle and pig embryos would be highly beneficial to the national and international breeding industry, as this technology has the potential to multiply the offspring of genetically superior animals thereby improving the production and selection of more efficient livestock.
Genome Reconstruction, Evolution & Comparative Cytogenomics of Terrestrial Vertebrates Drawing on initial work distinguishing avian chromosomes (particularly those too tiny to differentiate by classical means), we developed a means of taking sub-chromosomal sized scaffold-based assemblies and “upgrading” them to chromosome-level at a fraction of the cost. It has a number of applications in comparative genomics, developmental biology, molecular ecology, genome organization, and agriculture. Our research is currently focussed on generating chromosome-level assemblies in a range of avian, mammalian and reptilian species, studying the role of chromosome evolution. This has led to highly publicized studies, determining the overall genome structure (karyotype) of dinosaurs. 

Enterprise Activity in Genomic Screening See profile for Dr Becky O’Connor
Genomic and Cross-disciplinary Applications to the Poultry Industry See profile for Dr Mike Romanov 

Publicity, Broadcast and Outreach An increasing interest of the group has been education and publicity:

On “being a successful scientist”
https://www.youtube.com/watch?v=7F1ee3lFBWohttps://www.acs.org/content/acs/en/careers/college-to-career/video/successful-scientist-part1.html
 
http://blogs.nature.com/naturejobs/2011/01/31/how-to-be-a-successful-scientist/ 

https://www.sciencemag.org/custom-publishing/webinars/finding-your-way-science-and-psychology-success

Articles for “The Conversation”
https://theconversation.com/profiles/darren-griffin-436118/articles

Pallab Ghosh (BBC) covering our work on dinosaur evolution
https://www.dropbox.com/s/td7mzwvwb6j0buv/BBC_News_Channel-2018-08-26_16-24-11.mp4?dl=0 

“YouNome– your genome in 25 self-portraits” https://keithrobinsonpainting.com/Younome 

Publications

Showing 50 of 172 total publications in the Kent Academic Repository. View all publications.

Article

  • Narushin, V., Romanov, M., Lu, G., Cugley, J. and Griffin, D. (2020). Digital imaging assisted geometry of chicken eggs using Hügelschäffer’s model. Biosystems Engineering [Online] 197:45-55. Available at: http://dx.doi.org/10.1016/j.biosystemseng.2020.06.008.
    Geometrical description of the egg shape is of a great importance in a variety of studies and can be instrumental in predicting quality traits of table and hatching poultry eggs. Importantly, developments of non-destructive oomorphological models can drive novel insights in engineering and physical science and lead to new egg-related technologies and egg sorting systems for poultry industry. We attempted to test the Hügelschäffer’s egg model according to which an egg profile curve can be transformed from an ellipse using a specific parameter w. For this purpose, two-dimensional digital imaging and follow-up image processing techniques of chicken eggs were employed. The formulae for recalculation of the egg volume and surface area were consequently deduced from the Hügelschäffer’s equation. Eventually, we refined the Hügelschäffer’s egg model and proved its applicability for defining the contours of hen’s eggs. For practical use in poultry industry and food engineering, the proposed non-destructive methodology can be contributory in defining accurately the contour of any avian egg and determining such characteristics of the egg shape as volume, surface area, etc., with an expected potential in designing automated systems in poultry industry and in egg-related applications in biology, physical science, engineering and other areas.
  • Archibald, A., Smith, T., Watson, M., Tuggle, C., Tseng, E., Talbot, R., Skinner, B., Schwartz, A., Schroeder, S., Schook, L., Sargent, C., Rund, L., Rosen, B., Rohrer, G., Phillippy, A., O’Connor, R., Nonneman, D., Martin, F., Manchanda, N., Liu, H., Koren, S., Kim, K., Izuogu, O., Hume, D., Howe, K., Hourlier, T., Hannum, G., Hall, R., Griffin, D., Girón, C., Flicek, P., Finlayson, H., Eory, L., Chow, W., Billis, K., Bickhart, D., Beiki, H., Aken, B., Affara, N. and Warr, A. (2020). An improved pig reference genome sequence to enable pig genetics and genomics research. GigaScience [Online] 9. Available at: https://doi.org/10.1093/gigascience%2Fgiaa051.
    Background: The domestic pig (Sus scrofa) is important both as a food source and as a biomedical model given its similarity in size, anatomy, physiology, metabolism, pathology, and pharmacology to humans. The draft reference genome (Sscrofa10.2) of a purebred Duroc female pig established using older clone-based sequencing methods was incomplete, and unresolved redundancies, short-range order and orientation errors, and associated misassembled genes limited its utility. Results: We present 2 annotated highly contiguous chromosome-level genome assemblies created with more recent long-read technologies and a whole-genome shotgun strategy, 1 for the same Duroc female (Sscrofa11.1) and 1 for an outbred, composite-breed male (USMARCv1.0). Both assemblies are of substantially higher (>90-fold) continuity and accuracy than Sscrofa10.2. Conclusions: These highly contiguous assemblies plus annotation of a further 11 short-read assemblies provide an unprecedented view of the genetic make-up of this important agricultural and biomedical model species. We propose that the improved Duroc assembly (Sscrofa11.1) become the reference genome for genomic research in pigs.
  • Kretschmer, R., Furo, I., Gomes, A., Kiazim, L., Gunski, R., Garnero, A., Pereira, J., Ferguson-Smith, M., de Oliveira, E., Griffin, D., de Freitas, T. and O’Connor, R. (2020). A Comprehensive Cytogenetic Analysis of Several Members of the Family Columbidae (Aves, Columbiformes). Genes [Online] 11:632. Available at: https://doi.org/10.3390/genes11060632.
    The Columbidae species (Aves, Columbiformes) show considerable variation in their diploid numbers (2n = 68–86), but there is limited understanding of the events that shaped the extant karyotypes. Hence, we performed whole chromosome painting (wcp) for paints GGA1-10 and bacterial artificial chromosome (BAC) probes for chromosomes GGA11-28 for Columbina passerina, Columbina talpacoti, Patagioenas cayennensis, Geotrygon violacea and Geotrygon montana. Streptopelia decaocto was only investigated with paints because BACs for GGA10-28 had been previously analyzed. We also performed phylogenetic analyses in order to trace the evolutionary history of this family in light of chromosomal changes using our wcp data with chicken probes and from Zenaida auriculata, Columbina picui, Columba livia and Leptotila verreauxi, previously published. G-banding was performed on all these species. Comparative chromosome paint and G-banding results suggested that at least one interchromosomal and many intrachromosomal rearrangements had occurred in the diversification of Columbidae species. On the other hand, a high degree of conservation of microchromosome organization was observed in these species. Our cladistic analysis, considering all the chromosome rearrangements detected, provided strong support for L. verreauxi and P. cayennensis, G. montana and G. violacea, C. passerina and C. talpacoti having sister taxa relationships, as well as for all Columbidae species analyzed herein. Additionally, the chromosome characters were mapped in a consensus phylogenetic topology previously proposed, revealing a pericentric inversion in the chromosome homologous to GGA4 in a chromosomal signature unique to small New World ground doves
  • Singchat, W., Sillapaprayoon, S., Muangmai, N., Baicharoen, S., Indananda, C., Duengkae, P., Peyachoknagul, S., O’Connor, R., Griffin, D. and Srikulnath, K. (2020). Do sex chromosomes of snakes, monitor lizards, and iguanian lizards result from multiple fission of an “ancestral amniote super-sex chromosome”?. Chromosome Research [Online]. Available at: https://doi.org/10.1007/s10577-020-09631-4.
    Sex chromosomes in some amniotes share linkage homologies with distantly related taxa in regions orthologous to squamate reptile chromosome 2 (SR2) and the snake W sex chromosome. Thus, the SR2 and W chromosomes may formerly have been part of a larger ancestral amniote super-sex chromosome. Comparison of various sex chromosomal linkage homologies in Toxicofera with those in other amniotes offers an excellent model to assess key cytological differences, to understand the mechanisms of amniote sex chromosome evolution in each lineage and the existence of an ancestral amniote super-sex chromosome. Chromosome maps of four species of Toxicofera were constructed using bacterial artificial chromosomes (BACs) derived from chicken and zebra finch libraries containing amniote sex chromosomal linkages. Different macrochromosome linkage homologies were highly conserved among Toxicofera, and at least two BACs (CH261-125F1 and CH261-40D6) showed partial homology with sex chromosomes of amniotes associated with SR2, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial linkage homologies. The present data also suggest a possible multiple fission mechanism of an ancestral super-sex chromosome, which resulted in further development of various sex chromosomal linkages of Toxicofera based on particular properties that favored the role of sex chromosomes.
  • Ellis, P. and Griffin, D. (2020). Form from Function, Order from Chaos in Male Germline Chromatin. Genes [Online] 11. Available at: https://doi.org/10.3390/genes11020210.
    Spermatogenesis requires radical restructuring of germline chromatin at multiple stages, involving co-ordinated waves of DNA methylation and demethylation, histone modification, replacement and removal occurring before, during and after meiosis. This Special Issue has drawn together papers addressing many aspects of chromatin organization and dynamics in the male germ line, in humans and in model organisms. Two major themes emerge from these studies: the first is the functional significance of nuclear organisation in the developing germline; the second is the interplay between sperm chromatin structure and susceptibility to DNA damage and mutation. The consequences of these aspects for fertility, both in humans and other animals, is a major health and social welfare issue and this is reflected in these nine exciting manuscripts.
  • Narushin, V., Lu, G., Cugley, J., Romanov, M. and Griffin, D. (2020). A 2-D imaging-assisted geometrical transformation method for non-destructive evaluation of the volume and surface area of avian eggs. Food Control [Online]. Available at: https://dx.doi.org/10.1016/j.foodcont.2020.107112.
    Egg volume and surface area are reliable predictors of quality traits for both table and hatching chicken eggs. A new non-destructive technique for the fast and accurate evaluation of these two egg variables is addressed in the present study. The proposed method is based on the geometrical transformation of actual egg contour into a well-known geometrical figure which shape most of all resembles the examined egg. The volume and surface area of an examined egg were recomputed using the formulae appropriate for three figures including sphere, ellipsoid, and egg-shape ovoid. The method of the geometrical transformation includes the measurements of the egg length and the area of the examined eggs. These variables were determined using two-dimensional (2-D) digital imaging and image processing techniques. The geometrical transformation approach is proven to be reliable to turn the studied chicken eggs into the three chosen ovoid models, with the best prediction being shown for the ellipsoid and egg-shape ovoid, whilst the former was slightly more preferable. Depending on the avian species studied, we hypothesise that it would be more suitable to use the sphere model for more round shaped eggs and the egg-shaped ovoid model if the examined eggs are more conical. The choice of the proposed transformation technique would be applicable not only for the needs of poultry industry but also in ornithological, basically zoological studies when handling the varieties of eggs of different shapes. The experimental results show that the method proposed is accurate, reliable, robust and fast when coupled and assisted with the digital imaging and image processing techniques, and can serve as a basis for developing an appropriate instrumental technology and bringing it into the practice of poultry enterprises and hatcheries.
  • Jennings, R., Griffin, D. and O’Connor, R. (2020). A new Approach for Accurate Detection of Chromosome Rearrangements That Affect Fertility in Cattle. Animals [Online] 10:114. Available at: https://doi.org/10.3390/ani10010114.
    Globally, cattle production has more than doubled since the 1960s, with widespread use of artificial insemination (AI) and an emphasis on a small pool of high genetic merit animals. Selecting AI bulls with optimal fertility is, therefore, vital, as impaired fertility reduces genetic gains and production, resulting in heavy financial and environmental losses. Chromosome translocations, particularly the 1;29 Robertsonian translocation, are a common cause of reduced fertility; however, reciprocal translocations are significantly underreported due to the difculties inherent in analysing cattle chromosomes. Based on our porcine work, we have developed an approach for the unambiguous detection of Robertsonian and reciprocal translocations, using a multiple-hybridization probe detection strategy. We applied this method on the chromosomes of 39 bulls, detecting heterozygous and homozygous 1;29 translocations and a 12;23 reciprocal translocation in a total of seven animals. Previously, karyotype analysis was the only method of diagnosing chromosomal rearrangements in cattle, and was time-consuming and error-prone. With calving rates of only 50–60%, it is vital to reduce further fertility loss in order to maximise productivity. The approach developed here identifies abnormalities that DNA sequencing will not, and has the potential to lead to long-term gains, delivering meat and milk products in a more cost-effective and environmentally-responsible manner to a growing population.
  • Taylor, T., Stankewicz, T., Katz, S., Patrick, J., Johnson, L. and Griffin, D. (2019). Preliminary assessment of aneuploidy rates between the polar, mid and mural trophectoderm. Zygote [Online]:1-4. Available at: https://doi.org/10.1017/S0967199419000637.
    The objective of this study is to compare aneuploidy rates between three distinct areas of the human trophectoderm: mural, polar and a region in between these two locations termed the ‘mid’ trophectoderm. This is a cohort study on in vitro fertilization (IVF) patients undergoing comprehensive chromosome screening at the blastocyst stage at a private IVF clinic. All embryos underwent assisted hatching on day 3 with blastocyst biopsy and comprehensive chromosome screening. Biopsied blastocysts were divided into three groups depending on which area (polar, mid, or mural) of the trophectoderm was protruding from the zona pellucida and biopsied. Aneuploidy rates were significantly higher with cells from the polar region of the trophectoderm (56.2%) compared with cells removed from the mural region of the trophectoderm (30.0%; P = 0.0243). A comparison of all three areas combined also showed a decreasing trend, but this did not reach clinical significance, polar (56.2%), mid (47.4%) and mural trophectoderm (30.0%; P = 0.1859). The non-concordance demonstrated between polar and mural trophectoderm can be attributed to biological occurrences including chromosomal mosaicism or procedural differences between embryologists.
  • Viotti, M., Victor, A., Griffin, D., Groob, J., Brake, A., Zouves, C. and Barnes, F. (2019). Estimating Demand for Germline Genome Editing: An In Vitro Fertilization Clinic Perspective. The CRISPR Journal [Online] 2:304-315. Available at: https://doi.org/10.1089/crispr.2019.0044.
    Germline genome editing (GGE) holds the potential to mitigate or even eliminate human heritable genetic disease, but also carries genuine risks if not appropriately regulated and performed. It also raises fears in some quarters of apocalyptic scenarios of designer babies that could radically change human reproduction. Clinical need and the availability of alternatives are key considerations in the ensuing ethical debate. Writing from the perspective of a fertility clinic, we offer a realistic projection of the demand for GGE. We lay out a framework proposing that GGE, hereditary genetic disorders, and in vitro fertilization are fundamentally entwined concepts. We note that the need for GGE to cure heritable genetic disease is typically grossly overestimated, mainly due to the underappreciated role of preimplantation genetic testing. However, we might still find applications for GGE in the correction of chromosomal abnormalities in early embryos, but techniques for that purpose do not yet exist.
  • Surai, P., Kochish, I., Romanov, M. and Griffin, D. (2019). Nutritional modulation of the antioxidant capacities in poultry: the case of vitamin E. Poultry Science [Online] 98:4030-4041. Available at: https://dx.doi.org/10.3382/ps/pez072.
    Commercial poultry production is associated with a range of stresses, including environmental, technological, nutritional, and internal/biological ones, responsible for decreased productive and reproductive performance of poultry. At the molecular level, most of them are associated with oxidative stress and damages to important biological molecules. Poultry feed contains a range of feed-derived and supplemented antioxidants and, among them, vitamin E is considered as the “headquarters” of the antioxidant defense network. It is well-established that dietary supplementation of selenium, vitamin E, and carotenoids can modulate antioxidant defenses in poultry. The aim of the present paper is to present evidence related to modulation of the antioxidant capacities in poultry by vitamin E. Using 3 model systems including poultry breeders/males, semen, and chicken embryo/postnatal chickens, the possibility of modulation of the antioxidant defense mechanisms has been clearly demonstrated. It was shown that increased vitamin E supplementation in the breeder's or cockerel's diet increased their resistance to various stresses, including high polyunsaturated fatty acids (PUFA), mycotoxin, or heat stress. Increased vitamin E supplementation of poultry males was shown to be associated with significant increases in α-tocopherol level in semen associated with an increased resistance to oxidative stress imposed by various external stressors. Similarly, increased vitamin E concentration in the egg yolk due to dietary supplementation was shown to be associated with increased α-tocopherol concentration in the tissues of the developing embryos and newly hatched chicks resulting in increased antioxidant defenses and decreased lipid peroxidation. Furthermore, increased vitamin E transfer from the feed to egg yolk and further to the developing embryo was shown to be associated with upregulation of antioxidant enzymes reflecting antioxidant system regulation and adaptation. The role of vitamin E in cell signaling and gene expression as well as in interaction with microbiota and maintaining gut health in poultry awaits further investigation.
  • Victor, A., Griffin, D., Gardner, D., Brake, A., Zouves, C., Barnes, F. and Viotti, M. (2019). Births from embryos with highly elevated levels of mitochondrial DNA. Reproductive BioMedicine Online [Online] 39:403-412. Available at: https://dx.doi.org/10.1016/j.rbmo.2019.03.214.
    Measurement of mtDNA copy number might not provide any advantage in embryo prioritization and could lead to a deselection of blastocysts that would result in healthy pregnancies and births. Furthermore, the quantification of mitochondrial functional output in a model of cellular stress might suggest that mitochondria are not clear targets for biomarker identification as it relates to blastocyst viability. Any suggested link between mtDNA levels, mitochondria or their output with blastocyst transfer outcome requires further validation.
  • Laptev, G., Filippova, V., Kochish, I., Yildirim, E., Ilina, L., Dubrovin, A., Brazhnik, E., Novikova, N., Novikova, O., Dmitrieva, M., Smolensky, V., Surai, P., Griffin, D. and Romanov, M. (2019). Examination of the expression of immunity genes and bacterial profiles in the caecum of growing chickens infected with Salmonella Enteritidis and fed a phytobiotic. Animals [Online] 9:615. Available at: https://dx.doi.org/10.3390/ani9090615.
    This study was performed to investigate the differential expression of eight immunity genes and the bacterial profiles in the caecum of growing chickens challenged with Salmonella enterica serovar Enteritidis (SE) at 1 and 23 days post inoculation (dpi) in response to SE infection at 19 days of age and administration of the phytobiotic Intebio. Following infection, the genes CASP6 and IRF7 were upregulated by greater than twofold. Chicks fed Intebio showed at 1 dpi upregulation of AvBD10, IL6, IL8L2, CASP6 and IRF7. At 23 dpi, expression of AvBD11, IL6, IL8L2, CASP6 and IRF7 lowered in the experiment subgroups as compared with the control. Examination of the caecal contents at 1 dpi demonstrated a significant decrease in the microbial biodiversity in the infected subgroup fed normal diet. Bacterial content of Lactobacillus and Bacillus declined, while that of Enterobacteriaceae rose. In the infected subgroup fed Intebio, a pronounced change in composition of the microflora was not observed. In the early infection stages, the phytobiotic seemed to promote response to infection. Subsequently, an earlier suppression of the inflammatory reaction took place in chickens fed Intebio. Thus, use of Intebio as a drug with phytobiotic activity in chickens, including those infected with Salmonella, proved to be promising.
  • Deakin, J., Potter, S., O’Neill, R., Ruiz-Herrera, A., Cioffi, M., Eldridge, M., Fukui, K., Marshall Graves, J., Griffin, D., Grutzner, F., Kratochvíl, L., Miura, I., Rovatsos, M., Srikulnath, K., Wapstra, E. and Ezaz, T. (2019). Chromosomics: Bridging the Gap between Genomes and Chromosomes. Genes [Online] 10:627. Available at: https://doi.org/10.3390/genes10080627.
    The recent advances in DNA sequencing technology are enabling a rapid increase in the number of genomes being sequenced. However, many fundamental questions in genome biology remain unanswered, because sequence data alone is unable to provide insight into how the genome is organised into chromosomes, the position and interaction of those chromosomes in the cell, and how chromosomes and their interactions with each other change in response to environmental stimuli or over time. The intimate relationship between DNA sequence and chromosome structure and function highlights the need to integrate genomic and cytogenetic data to more comprehensively understand the role genome architecture plays in genome plasticity. We propose adoption of the term ‘chromosomics’ as an approach encompassing genome sequencing, cytogenetics and cell biology, and present examples of where chromosomics has already led to novel discoveries, such as the sex-determining gene in eutherian mammals. More importantly, we look to the future and the questions that could be answered as we enter into the chromosomics revolution, such as the role of chromosome rearrangements in speciation and the role more rapidly evolving regions of the genome, like centromeres, play in genome plasticity. However, for chromosomics to reach its full potential, we need to address several challenges, particularly the training of a new generation of cytogeneticists, and the commitment to a closer union among the research areas of genomics, cytogenetics, cell biology and bioinformatics. Overcoming these challenges will lead to ground-breaking discoveries in understanding genome evolution and function
  • McCallie, B., Parks, J., Trahan, G., Jones, K., Coate, B., Griffin, D., Schoolcraft, W. and Katz-Jaffe, M. (2019). Compromised global embryonic transcriptome associated with advanced maternal age. Journal of Assisted Reproduction and Genetics [Online]. Available at: https://doi.org/10.1007/s10815-019-01438-5.
    Purpose To investigate the global transcriptome and associated embryonic molecular networks impacted with advanced maternal age (AMA).

    Methods Blastocysts derived from donor oocyte IVF cycles with no male factor infertility (< 30 years of age) and AMA blastocysts (≥ 42 years) with no other significant female factor infertility or male factor infertility were collected with informed patient consent. RNA sequencing libraries were prepared using the SMARTer® Ultra® Low Kit (Clontech Laboratories) and
    sequenced on the Illumina HiSEQ 4000. Bioinformatics included Ingenuity® Pathway Analysis (Qiagen) with ViiA™7 qPCR utilized for gene expression validation (Applied Biosystems).

    Results A total of 2688 significant differentially expressed transcripts were identified to distinguish the AMA blastocysts from young, donor controls. 2551 (95%) of these displayed decreased transcription in the blastocysts from older women. Pathway analysis revealed three altered molecular signaling networks known to be critical for embryo and fetal development: CREBBP, ESR1, and SP1. Validation of genes within these networks confirmed the global decreased transcription observed in AMA blastocysts (P < 0.05).

    Conclusions A significant, overall decreased global transcriptome was observed in blastocysts from AMA women. The ESR1/SP1/CREBBP pathway, in particular, was found to be a highly significant upstream regulator impacting biological processes that are vital during embryonic patterning and pre-implantation development. These results provide evidence that AMA embryos are compromised on a cell signaling level which can repress the embryo’s ability to proliferate and implant, contributing to a deterioration of reproductive outcomes.
  • Kudinov, A., Dementieva, N., Mitrofanova, O., Stanishevskaya, O., Fedorova, E., Larkina, T., Mishina, A., Plemyashov, K., Griffin, D. and Romanov, M. (2019). Genome-wide association studies targeting the yield of extraembryonic fluid and production traits in Russian White chickens. BMC Genomics [Online] 20:270. Available at: https://doi.org/10.1186/s12864-019-5605-5.
    Background: The Russian White is a gene pool breed, registered in 1953 after crossing White Leghorns with local populations and, for 50 years, selected for cold tolerance and high egg production (EL). The breed has great potential in meeting demands of local food producers, commercial farmers and biotechnology sector of specific pathogen-free (SPF) eggs, the former valuing the breed for its egg weight (EW), EL, age at first egg (AFE), body weight (BW), and the latter for its yield of extraembryonic fluid (YEF) in 12.5-day embryos, ratio of extraembryonic fluid to egg weight, and embryo mass. Moreover, its cold tolerance has been presumably associated with day-old chick down colour (DOCDC) – white rather than yellow, the genetic basis of these traits being however poorly understood.

    Results: We undertook genome-wide association studies (GWASs) for eight performance traits using single nucleotide polymorphism (SNP) genotyping of 146 birds and an Illumina 60KBeadChip. Several suggestive associations (p < 5.16*10− 5) were found for YEF, AFE, BW and EW. Moreover, on chromosome 2, an association with the white DOCDC was found where there is an linkage disequilibrium block of SNPs including genes that are responsible not for colour, but for immune resistance.

    Conclusions: The obtained GWAS data can be used to explore the genetics of immunity and carry out selection for increasing YEF for SPF eggs production.
  • Griffin, D., Larkin, D. and O’Connor, R. (2019). Time lapse: A glimpse into prehistoric genomics. European Journal of Medical Genetics [Online]. Available at: https://doi.org/10.1016/j.ejmg.2019.03.004.
    For the purpose of this review, ‘time-lapse’ refers to the reconstruction of ancestral (in this case dinosaur) karyotypes using genome assemblies of extant species. Such reconstructions are only usually possible when genomes are assembled to ‘chromosome level’ i.e. a a complete representation of all the sequences, correctly ordered contiguously on each of the chromosomes. Recent paleontological evidence is very clear that birds are living dinosaurs, the latest example of dinosaurs emerging from a catastrophic extinction event. Non-avian dinosaurs (ever present in the public imagination through art, and broadcast media) emerged some 240 million years ago and have displayed incredible phenotypic diversity. Here we report on our recent studies to infer the overall karyotype of the Theropod dinosaur lineage from extant avian chromosome level genome assemblies. Our work first focused on determining the likely karyotype of the avian ancestor (most likely a chicken-sized, two-legged, feathered, land dinosaur from the Jurassic period) finding karyotypic similarity to the chicken. We then took the work further to determine the likely karyotype of the bird-lizard ancestor and the chromosomal changes (chiefly translocations and inversions) that occurred between then and modern birds. A combination of bioinformatics and cross-species fluorescence in situ hybridization (zoo-FISH) uncovered a considerable number of translocations and fissions from a ‘lizard-like’ genome structure of 2n = 36–46 to one similar to that of soft-shelled turtles (2n = 66) from 275 to 255 million years ago (mya). Remarkable karyotypic similarities between some soft-shelled turtles and chicken suggests that there were few translocations from the bird-turtle ancestor (plus ∼7 fissions) through the dawn of the dinosaurs and pterosaurs, through the theropod linage and on to most to modern birds. In other words, an avian-like karyotype was in place about 240mya when the dinosaurs and pterosaurs first emerged. We mapped 49 chromosome inversions from then to the present day, uncovering some gene ontology enrichment in evolutionary breakpoint regions. This avian-like karyotype with its many (micro)chromosomes provides the basis for variation (the driver of natural selection) through increased random segregation and recombination. It may therefore contribute to the ability of dinosaurs to survive multiple extinction events, emerging each time as speciose and diverse.
  • Turner, K., Lynch, C., Rouse, H., Vasu, V. and Griffin, D. (2019). Direct Single-Cell Analysis of Human Polar Bodies and Cleavage-Stage Embryos Reveals No Evidence of the Telomere Theory of Reproductive Ageing in Relation to Aneuploidy Generation. Cells [Online] 8:163. Available at: https://doi.org/10.3390/cells8020163.
    Reproductive ageing in women, particularly after the age of 35, is associated with an exponential increase in the proportion of chromosomally abnormal oocytes produced. Several hypotheses have attempted to explain this observation, including the ‘limited oocyte pool’ hypothesis and the ‘two-hit’ hypothesis, the latter explaining that a depletion in oocyte quality with age results from the multiple opportune stages for errors to occur in meiosis. Recently however, the telomere theory of reproductive ageing in women has been proposed. This suggests that shortened telomeres in oocytes of women of advanced maternal age render oocytes unable to support fertilization and embryogenesis. Despite a credible rationale for the telomere theory of reproductive ageing in women, very few studies have assessed telomere length directly in human oocytes or preimplantation embryos. Therefore, we directly assessed relative telomere length in first polar bodies and blastomeres from cleavage stage (day 3) embryos. In both cell types we tested the hypothesis that (1) older women have shorter telomeres and (2) chromosomally abnormal (aneuploid) gametes/embryos have shorter telomeres. In all cases, we found no evidence of altered telomere length associated with age-related aneuploidy.
  • Fowler, K., Mandawala, A. and Griffin, D. (2019). The role of chromosome segregation and nuclear organisation in human subfertility. Biochemical Society Transactions [Online] 47:425-432. Available at: https://doi.org/10.1042/BST20180231.
    Spermatogenesis is central to successful sexual reproduction, producing large numbers of haploid motile male gametes. Throughout this process, a series of equational and reductional chromosome segregation precedes radical repackaging of the haploid genome. Faithful chromosome segregation is thus crucial, as is an ordered spatio-temporal ‘dance’ of packing a large amount of chromatin into a very small space. Ergo, when the process goes wrong, this is associated with an improper chromosome number, nuclear position and/or chromatin damage in the sperm head. Generally, screening for overall DNA damage is relatively commonplace in clinics, but aneuploidy assessment is less so and nuclear organisation studies form the basis of academic research. Several studies have focussed on the role of chromosome segregation, nuclear organisation and analysis of sperm morphometry in human subfertility observing significant alterations in some cases, especially of the sex chromosomes. Importantly, sperm DNA damage has been associated with infertility and both extrinsic (e.g. lifestyle) and intrinsic (e.g. reactive oxygen species levels) factors, and while some DNA-strand breaks are repaired, unexpected breaks can cause differential chromatin packaging and further breakage. A ‘healthy’ sperm nucleus (with the right number of chromosomes, nuclear organisation and minimal DNA damage) is thus an essential part of reproduction. The purpose of this review is to summarise state of the art in the fields of sperm aneuploidy assessment, nuclear organisation and DNA damage studies.
  • Miller, D., Pavitt, S., Sharma, V., Forbes, G., Hooper, R., Bhattacharya, S., Kirkman-Brown, J., Coomarasamy, A., Lewis, S., Cutting, R., Brison, D., Pacey, A., West, R., Brian, K., Griffin, D. and Khalaf, Y. (2019). Physiological, hyaluronan-selected intracytoplasmic sperm injection for infertility treatment (HABSelect): a parallel, two-group, randomised trial. The Lancet [Online] 393:416-422. Available at: https://doi.org/10.1016/S0140-6736(18)32989-1.
    Background
    Sperm selection strategies aimed at improving success rates of intracytoplasmic sperm injection (ICSI) include binding to hyaluronic acid (herein termed hyaluronan). Hyaluronan-selected sperm have reduced levels of DNA damage and aneuploidy. Use of hyaluronan-based sperm selection for ICSI (so-called physiological ICSI [PICSI]) is reported to reduce the proportion of pregnancies that end in miscarriage. However, the effect of PICSI on livebirth rates is uncertain. We aimed to investigate the efficacy of PICSI versus standard ICSI for improving livebirth rates among couples undergoing fertility treatment.

    Methods
    This parallel, two-group, randomised trial included couples undergoing an ICSI procedure with fresh embryo transfer at 16 assisted conception units in the UK. Eligible women (aged 18–43 years) had a body-mass index of 19–35 kg/m2 and a follicle-stimulating hormone (FSH) concentration of 3·0–20·0 mIU/mL or, if no FSH measurement was available, an anti-müllerian hormone concentration of at least 1·5 pmol/L. Eligible men (aged 18–55 years) had not had a vasovasostomy or been treated for cancer in the 24 months before recruitment and were able, after at least 3 days of sexual abstinence, to produce freshly ejaculated sperm for the treatment cycle. Couples were randomly assigned (1:1) with an online system to receive either PICSI or a standard ICSI procedure. The primary outcome was full-term (?37 weeks' gestational age) livebirth, which was assessed in all eligible couples who completed follow-up. This trial is registered, number ISRCTN99214271.

    Findings
    Between Feb 1, 2014, and Aug 31, 2016, 2772 couples were randomly assigned to receive PICSI (n=1387) or ICSI (n=1385), of whom 2752 (1381 in the PICSI group and 1371 in the ICSI group) were included in the primary analysis. The term livebirth rate did not differ significantly between PICSI (27·4% [379/1381]) and ICSI (25·2% [346/1371]) groups (odds ratio 1·12, 95% CI 0·95–1·34; p=0·18). There were 56 serious adverse events in total, including 31 in the PICSI group and 25 in the ICSI group; most were congenital abnormalities and none were attributed to treatment.

    Interpretation
    Compared with ICSI, PICSI does not significantly improve term livebirth rates. The wider use of PICSI, therefore, is not recommended at present.
  • Turner, K., Silvestri, G., Black, D., Dobson, G., Smith, C., Handyside, A., Sinclair, K. and Griffin, D. (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://dx.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.
  • Turner, K., Vasu, V. and Griffin, D. (2019). Telomere Biology and Human Phenotype. Cells [Online] 8:73. Available at: https://doi.org/10.3390/cells8010073.
    Telomeres are nucleoprotein structures that cap the end of each chromosome arm and function to maintain genome stability. The length of telomeres is known to shorten with each cell division and it is well-established that telomere attrition is related to replicative capacity in vitro. Moreover, telomere loss is also correlated with the process of aging in vivo. In this review, we discuss the mechanisms that lead to telomere shortening and summarise telomere homeostasis in humans throughout a lifetime. In addition, we discuss the available evidence that shows that telomere shortening is related to human aging and the onset of age-related disease.
  • Victor, A., Griffin, D., Brake, A., Tyndall, J., Murphy, A., Lepkowsky, L., Lal, A., Zouves, C., Barnes, F., McCoy, R. and Viotti, M. (2019). Assessment of aneuploidy concordance between clinical trophectoderm biopsy and blastocyst. Human Reproduction [Online] 34:181-192. Available at: https://doi.org/10.1093/humrep/dey327.
    STUDY QUESTION
    Is a clinical trophectoderm (TE) biopsy a suitable predictor of chromosomal aneuploidy in blastocysts?

    SUMMARY ANSWER
    In the analyzed group of blastocysts, a clinical TE biopsy was an excellent representative of blastocyst karyotype in cases of whole chromosome aneuploidy, but in cases of only segmental (sub-chromosomal) aneuploidy, a TE biopsy was a poor representative of blastocyst karyotype.

    WHAT IS KNOWN ALREADY
    Due to the phenomenon of chromosomal mosaicism, concern has been expressed about the possibility of discarding blastocysts classified as aneuploid by preimplantation genetic testing for aneuploidy (PGT-A) that in fact contain a euploid inner cell mass (ICM). Previously published studies investigating karyotype concordance between TE and ICM have examined small sample sizes and/or have utilized chromosomal analysis technologies superseded by Next Generation Sequencing (NGS). It is also known that blastocysts classified as mosaic by PGT-A can result in healthy births. TE re-biopsy of embryos classified as aneuploid can potentially uncover new instances of mosaicism, but the frequency of such blastocysts is currently unknown.

    STUDY DESIGN, SIZE, DURATION
    For this study, 45 patients donated 100 blastocysts classified as uniform aneuploids (non-mosaic) using PGT-A by NGS (n = 93 whole chromosome aneuploids, n = 7 segmental aneuploids). In addition to the original clinical TE biopsy used for PGT-A, each blastocyst was subjected to an ICM biopsy as well as a second TE biopsy. All biopsies were processed for chromosomal analysis by NGS, and karyotypes were compared to the original TE biopsy.

    PARTICIPANTS/MATERIALS, SETTING, METHODS
    The setting for this study was a single IVF center with an in-house PGT-A program and associated research laboratory.

    MAIN RESULTS AND THE ROLE OF CHANCE
    When one or more whole chromosomes were aneuploid in the clinical TE biopsy, the corresponding ICM was aneuploid in 90 out of 93 blastocysts (96.8%). When the clinical TE biopsy contained only segmental (sub-chromosomal) aneuploidies, the ICM was aneuploid in three out of seven cases (42.9%). Blastocysts showing aneuploidy concordance between clinical TE biopsy and ICM were also aneuploid in a second TE biopsy in 86 out of 88 cases (97.7%). In blastocysts displaying clinical TE–ICM discordance, a second TE biopsy was aneuploid in only two out of six cases (33.3%).

    LIMITATIONS, REASONS FOR CAUTION
    All embryos in this study had an initial classification of ‘aneuploid’ and not ‘euploid’ or ‘mosaic’. Therefore, the findings of this study refer specifically to a TE biopsy predicting aneuploidy in the remaining blastocyst, and cannot be extrapolated to deduce the ability of a TE biopsy to predict euploidy in the blastocyst. No conclusions should be drawn from this study about the ability of a mosaic TE biopsy to predict the karyotype of the corresponding blastocyst. Caution should be exercised in generalizing the findings of the sample group of this study to the general IVF blastocyst population. The segmental aneuploidy group only contained seven samples.

    WIDER IMPLICATIONS OF THE FINDINGS
    The high rate of intra-blastocyst concordance observed in this study concerning whole chromosome aneuploidy contributes experimental evidence to the validation of PGT-A at the blastocyst stage. Concomitantly, the results suggest potential clinical value in reassessing blastocysts deemed aneuploid by TE re-biopsy in select cases, particularly in instances of segmental aneuploidies. This could impact infertility treatment for patients who only have blastocysts classified as aneuploid by PGT-A available.

    STUDY FUNDING/COMPETING INTEREST(S)
    This study was supported by the Zouves Foundation for Reproductive Medicine and Zouves Fertility Center. The authors have no competing interest to disclose.
  • Victor, A., Tyndall, J., Brake, A., Lepkowsky, L., Murphy, A., Griffin, D., McCoy, R., Barnes, F., Zouves, C. and Viotti, M. (2019). One hundred mosaic embryos transferred prospectively in a single clinic: exploring when and why they result in healthy pregnancies. Fertility and Sterility [Online] 111:280-293. Available at: https://doi.org/10.1016/j.fertnstert.2018.10.019.
    Objective
    To investigate the parameters of mosaicism and the biological mechanisms leading to healthy pregnancies from mosaic embryo transfers.

    Design
    Prospective study.

    Setting
    IVF center and associated research laboratory.

    Patient(s)
    Fifty-nine patients.

    Intervention(s)
    Embryos underwent blastocyst-stage preimplantation genetic testing for aneuploidy by next-generation sequencing. Trophectoderm biopsies containing 20%–80% abnormal cells were deemed mosaic, and corresponding blastocysts were transferred. Mosaic embryos donated to research were examined for karyotype concordance in multiple biopsies and assessed for cell proliferation and death by immunofluorescence and computational quantitation.

    Main Outcome Measure(s)
    Chemical start of pregnancy, implantation, fetal heartbeat, and birth.

    Result(s)
    Globally, mosaic embryos showed inferior clinical outcomes compared with euploid embryos. Aneuploid cell percentage in trophectoderm biopsies did not correlate with outcomes, but type of mosaicism did, as embryos with single mosaic segmental aneuploidies fared better than all other types. Mosaic blastocysts generated from oocytes retrieved at young maternal ages (?34 years) showed better outcomes than those retrieved at older maternal ages. Mosaic embryos displayed low rates of karyotype concordance between multiple biopsies and showed significant elevation of cell proliferation and death compared with euploid embryos.

    Conclusion(s)
    After euploid embryos, mosaic embryos can be considered for transfer, prioritizing those of the single segmental mosaic type. If a patient has mosaic embryos available that were generated at different ages, preference should be given to those made at younger ages. Intrablastocyst karyotype discordance and differential cell proliferation and death might be reasons that embryos classified as mosaic can result in healthy pregnancies and babies.
  • Singchat, W., O’Connor, R., Tawichasri, P., Suntronpong, A., Sillapaprayoon, S., Suntrarachun, S., Muangmai, N., Baicharoen, S., Peyachoknagul, S., Chanhome, L., Griffin, D. and Srikulnath, K. (2018). Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent “a hypothetical ancestral super-sex chromosome” or random distribution?. BMC Genomics [Online] 19. Available at: https://doi.org/10.1186/s12864-018-5293-6.
    Background
    Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes.

    Results
    We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes.

    Conclusions
    The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes.
  • O’Connor, R., Kiazim, L., Skinner, B., Fonseka, G., Joseph, S., Jennings, R., Larkin, D. and Griffin, D. (2018). Patterns of microchromosome organization remain highly conserved throughout avian evolution. Chromosoma [Online]. Available at: https://doi.org/10.1007/s00412-018-0685-6.
    The structure and organization of a species genome at a karyotypic level, and in interphase nuclei, have broad functional
    significance. Although regular sized chromosomes are studied extensively in this regard, microchromosomes, which are present
    in many terrestrial vertebrates, remain poorly explored. Birds have more cytologically indistinguishable microchromosomes (~
    30 pairs) than other vertebrates; however, the degree to which genome organization patterns at a karyotypic and interphase level
    differ between species is unknown. In species where microchromosomes have fused to other chromosomes, they retain genomic
    features such as gene density and GC content; however, the extent to which they retain a central nuclear position has not been
    investigated. In studying 22 avian species from 10 orders, we established that, other than in species where microchromosomal
    fusion is obvious (Falconiformes and Psittaciformes), there was no evidence of microchromosomal rearrangement, suggesting an
    evolutionarily stable avian genome (karyotypic) organization. Moreover, in species where microchromosomal fusion has occurred,
    they retain a central nuclear location, suggesting that the nuclear position of microchromosomes is a function of their
    genomic features rather than their physical size
  • O’Connor, R., Kiazim, L., Skinner, B., Fonseka, G., Joseph, S., Jennings, R., Larkin, D. and Griffin, D. (2018). Patterns of microchromosome organization remain highly conserved throughout avian evolution. Chromosoma [Online] 128:21-29. Available at: https://doi.org/10.1007/s00412-018-0685-6.
    The structure and organization of a species genome at a karyotypic level, and in interphase nuclei, have broad functional significance. Although regular sized chromosomes are studied extensively in this regard, microchromosomes, which are present in many terrestrial vertebrates, remain poorly explored. Birds have more cytologically indistinguishable microchromosomes (~ 30 pairs) than other vertebrates; however, the degree to which genome organization patterns at a karyotypic and interphase level differ between species is unknown. In species where microchromosomes have fused to other chromosomes, they retain genomic features such as gene density and GC content; however, the extent to which they retain a central nuclear position has not been investigated. In studying 22 avian species from 10 orders, we established that, other than in species where microchromosomal fusion is obvious (Falconiformes and Psittaciformes), there was no evidence of microchromosomal rearrangement, suggesting an evolutionarily stable avian genome (karyotypic) organization. Moreover, in species where microchromosomal fusion has occurred, they retain a central nuclear location, suggesting that the nuclear position of microchromosomes is a function of their genomic features rather than their physical size.
  • O’Connor, R., Farré, M., Joseph, S., Damas, J., Kiazim, L., Jennings, R., Bennett, S., Slack, E., Allanson, E., Larkin, D. and Griffin, D. (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.
  • Joseph, S., O’Connor, R., Al Mutery, A., Watson, M., Larkin, D. and Griffin, D. (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.
  • Damas, J., Kim, J., Farré, M., Griffin, D. and Larkin, D. (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.
  • Fowler, K., Mandawala, A., Griffin, D., Walling, G. and Harvey, S. (2018). The production of pig preimplantation embryos in vitro: Current progress and future prospects. Reproductive Biology [Online] 18:203-211. Available at: https://dx.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.
  • Griffin, D., O’Connor, R., Romanov, M., Damas, J., Farré, M., Martell, H., Kiazim, L., Jennings, R., Mandawala, A., Joseph, S., Fowler, K., Slack, E., Allanson, E., Ferguson-Smith, M., Barrett, P., Valenzuela, N. and Larkin, D. (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.
  • 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.
  • O’Connor, R., Romanov, M., Kiazim, L., Barrett, P., Farré, M., Damas, J., Ferguson-Smith, M., Valenzuela, N., Larkin, D. and Griffin, D. (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., McCallie, B., Patton, A., Al-Safi, Z., Polotsky, A., Griffin, D., Schoolcraft, W. and Katz-Jaffe, M. (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.
  • Caujolle, S., Cernat, R., Silvestri, G., Marques, M., Bradu, A., Feuchter, T., Robinson, G., Griffin, D. and Podoleanu, A. (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.
  • Saretzki, G., Vasu, V., Turner, K., George, S., Greenall, J., Slijepcevic, P. and Griffin, D. (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.
  • McCallie, B., Parks, J., Griffin, D., Schoolcraft, W. and Katz-Jaffe, M. (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).
  • 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., Fonseka, G., Frodsham, R., Archibald, A., Lawrie, M., Walling, G. and Griffin, D. (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.
  • 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., Fishel, S., Gordon, T., Yaron, Y., Grifo, J., Hourvitz, A., Rechitsky, S., Elson, J., Blazek, J., Fiorentino, F., Treff, N., Munne, S., Leong, M., Schmutzler, A., Vereczkey, A., Ghobara, T., Nánássy, L., Large, M., Hamamah, S., Anderson, R., Gianaroli, L. and Wells, D. (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.
  • Saintas, E., Abrahams, L., Ahmed, G., Ajakaiye, A., AlHumaidi, A., Ashmore-Harris, C., Clark, I., Dura, U., Fixmer, C., Ike-Morris, C., Mato Prado, M., Mccullough, D., Mishra, S., Schoeler, K., Timur, H., Williamson, M., Alatsatianos, M., Bahsoun, B., Blackburn, E., Hogwood, C., Lithgow, P., Rowe, M., Yiangou, L., Rothweiler, F., Cinatl jr, J., Zehner, R., Baines, A., Garrett, M., Gourlay, C., Griffin, D., Gullick, W., Hargreaves, E., Howard, M., Lloyd, D., Rossman, J., Smales, C., Tsaousis, A., von der Haar, T., Wass, M. and Michaelis, M. (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.
  • Hornak, M., Kubicek, D., Broz, P., Hulinska, P., Hanzalova, K., Griffin, D., Machatkova, M. and Rubes, J. (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.
  • Damas, J., O’Connor, R., Farré, M., Lenis, V., Martell, H., Mandawala, A., Fowler, K., Joseph, S., Swain, M., Griffin, D. and Larkin, D. (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.
  • Coates, A., Bankowski, B., Kung, A., Griffin, D. and Munne, S. (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.
  • Victor, A., Brake, A., Tyndall, J., Griffin, D., Zouves, C., Barnes, F. and Viotti, M. (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.
  • Taylor, T., Griffin, D., Katz, S., Crain, J., Johnson, L. and Gitlin, S. (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.

Book section

  • Caujolle, S., Cernat, R., Silvestri, G., Marques, M., Bradu, A., Feuchter, T., Robinson, G., Griffin, D. and Podoleanu, A. (2018). Assessing embryo development using swept source optical coherence tomography. In: Podoleanu, A. G. and Bang, O. eds. 2nd Canterbury Conference on OCT With Emphasis on Broadband Optical Sources. SPIE. 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.
  • 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.
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