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S11 Chromosomal mosaicism in the cleavage stage embryo revisited
SESSIONS, Session 2
The cleavage and blastocyst stage embryo: the evolving genome
Kuliev A, Pakhalchuk T, Verlinsky O, Rechitsky S. Preimplantation genetic diagnosis for hemoglobinopathies. Hemoglobin. 2011; 35(5 6): 547 55.
Session 2 The cleavage and blastocyst stage embryo: the evolving genome S10 Cleavage stage and blastocyst biopsy A.H. Handyside1 . 1 London Bridge Fertility, Gynaecology and Genetics Centre, London and Institute of Integrative and Comparative Biology, University of Leeds, Leeds, UK In the late 1960’s, Gardner and Edwards pioneered the use of micromanipulation to remove trophectoderm cells from rabbit blastocysts to identify the sex, by staining for the inactive X chromosome in females. At the time, this work was directed towards predetermining the sex of offspring in domestic species because of the commercial advantages. However, they also recognised that a similar approach with human embryos might be used for the avoidance of X linked diseases, which typically only affect males. Twenty years later, with the development of in vitro fertilisation (IVF) methods for infertility treatment, which provided access to human oocytes and embryos, and the polymerase chain reaction (PCR), which allowed short fragments of DNA to be amplified over a millionfold, preimplantation genetic diagnosis (PGD) of genetic defects became a practical possibility. Biopsy of trophectoderm was possible using mechanical dissection of partially hatched human blastocysts but pregnancy rates following blastocyst transfers were low in the media in use then. Alternatively, the use of acid Tyrode’s solution to make an opening in the zona pellucida, which had been used in attempts to assist fertilisation, allowed the development of a method for aspirating single cleavage stage blastomeres, prior to compaction. By biopsying embryos at the 6- to 10-cell stage early on day 3, following insemination, it was then possible to perform the genetic analysis of the single cell within 12 24h before selection and transfer. The development of these methods, their subsequent refinement and their advantages and disadvantages will be reviewed. S11 Chromosomal mosaicism in the cleavage stage embryo revisited L. Wilton1 . 1 Preimplantation Genetics, Melbourne IVF, Australia Chromosomal mosaicism is well documented in early human embryos and a recent review reported that more than 70% of embryos were mosaic with almost 60% having the diploid/aneuploid mosaicism that is said to confound PGD for aneuploidy (Van Echten-Arends et al 2011). However, these observations seem incongruous with the very low misdiagnosis rate of 0.07% after PGD for aneuploidy (Wilton et al., 2009). The vast majority of studies that have reported chromosomal mosaicism in embryos have assessed poor quality or known aneuploid embryos and used FISH to analyse only a few chromosomes. Many blastomeres reported to be euploid would be affected with aneuploidy of chromosomes that were not included in the analysis meaning that diploid/aneuploid mosaicism would have been over-estimated in most of these studies. The advent of 24 chromosome analysis using microarray technology provides new opportunities to accurately determine the true extent of mosaicism by enumerating all 24 chromosomes. We have used this approach to obtain baseline information about mosaicism by analyzing multiple cells from good quality embryos from young patients who have been successful in ART treatment. This has demonstrated that only 24% of embryos had diploid/aneuploid
S35
mosaicism and within these embryos only a minority of cells was aneuploid. Hence the risk of biopsying a cell that was not representative of the rest of the embryo (as might happen in PGD for aneuploidy) could be estimated at only 7%. We are now using a similar approach to assess chromosomal mosaicism in embryos from older women. It would appear that previous studies have over-estimated chromosomal mosaicism in human embryos. Most likely reasons for this include the inherent limitations of FISH technology and the fact that the overwhelming majority of embryos examined were known to be aneuploid. Reference(s) Van Echten-Arends et al., 2011. Hum Reprod Update 17: 620. Wilton et al., 2009. Hum Reprod 24: 1221. S12 Chromosomal instability in the human preimplantation embryo T. Voet1,4 , N. Van der Aa1 , M. Zamani Esteki1 , P. Kumar1 , E. Vanneste1,2 , C. Melotte1 , P. Konings3 , S. Debrock2 , J.-P. Fryns1 , Y. Moreau3 , T. D’Hooghe2 , M.R. Stratton4 , P.J. Campbell4 , J.R. Vermeesch1 . 1 Center for Human Genetics, KULeuven, Leuven, Belgium, 2 Leuven University Fertility Center, UZ Gasthuisberg, Leuven, Belgium, 3 ESAT, KULeuven, Heverlee, Belgium, 4 Wellcome Trust Sanger Institute, Hinxton-Cambridge, UK Recently, we demonstrated chromosome instability (CIN) in human cleavage stage embryogenesis following in vitro fertilization (IVF). CIN not necessarily undermines normal human development (i.e. when remaining normal diploid blastomeres develop the embryo proper), however it can spark a spectrum of conditions, including loss of conception, genetic disease and genetic variation development. To study embryonic CIN further we have developed new methods based on highresolution microarray as well as next-generation sequencing technology that characterize the genome of a single human cell. We delivered proof-of-principle for detecting various types of structural variants, including Mb- to Kb-sized duplications and deletions, in single human (tumor) cells by low coverage pairedend sequencing and mapping. Based on the copy number changes that were detected by singlecell microarray analysis of multiple blastomeres of the same embryo, it was hypothesized that chromosome breakages and fusions occur frequently in human cleavage stage embryos and instigate subsequent breakage-fusion-bridge cycles. In addition, we hypothesized that the DNA breaks present in spermatozoa could trigger this CIN. To test these hypotheses, we genotyped both parents as well as 93 blastomeres from 24 IVF embryos and developed a novel SNP-array based algorithm to determine the parental origin of (aberrant) loci in single cells. Paternal as well as maternal alleles were commonly rearranged in the blastomeres indicating that sperm-specific DNA-breaks do not explain the majority of these structural variants. In addition, single-cell genome sequencing together with parent-of-origin SNP-array and microarray-guided FISH analyses demonstrate that breakage-fusion-bridge cycles as well as more complex rearrangements are sparked in the human cleavage stage embryo. Our data provide evidence that the human cleavage stage embryo is likely an important source of constitutional chromosomal disorders. The developed single-cell genome analysis methods are generic and will deliver novel insights in embryo and tumor genome research.
The cleavage and blastocyst stage embryo: the evolving genome
Kuliev A, Pakhalchuk T, Verlinsky O, Rechitsky S. Preimplantation genetic diagnosis for hemoglobinopathies. Hemoglobin. 2011; 35(5 6): 547 55.
Session 2 The cleavage and blastocyst stage embryo: the evolving genome S10 Cleavage stage and blastocyst biopsy A.H. Handyside1 . 1 London Bridge Fertility, Gynaecology and Genetics Centre, London and Institute of Integrative and Comparative Biology, University of Leeds, Leeds, UK In the late 1960’s, Gardner and Edwards pioneered the use of micromanipulation to remove trophectoderm cells from rabbit blastocysts to identify the sex, by staining for the inactive X chromosome in females. At the time, this work was directed towards predetermining the sex of offspring in domestic species because of the commercial advantages. However, they also recognised that a similar approach with human embryos might be used for the avoidance of X linked diseases, which typically only affect males. Twenty years later, with the development of in vitro fertilisation (IVF) methods for infertility treatment, which provided access to human oocytes and embryos, and the polymerase chain reaction (PCR), which allowed short fragments of DNA to be amplified over a millionfold, preimplantation genetic diagnosis (PGD) of genetic defects became a practical possibility. Biopsy of trophectoderm was possible using mechanical dissection of partially hatched human blastocysts but pregnancy rates following blastocyst transfers were low in the media in use then. Alternatively, the use of acid Tyrode’s solution to make an opening in the zona pellucida, which had been used in attempts to assist fertilisation, allowed the development of a method for aspirating single cleavage stage blastomeres, prior to compaction. By biopsying embryos at the 6- to 10-cell stage early on day 3, following insemination, it was then possible to perform the genetic analysis of the single cell within 12 24h before selection and transfer. The development of these methods, their subsequent refinement and their advantages and disadvantages will be reviewed. S11 Chromosomal mosaicism in the cleavage stage embryo revisited L. Wilton1 . 1 Preimplantation Genetics, Melbourne IVF, Australia Chromosomal mosaicism is well documented in early human embryos and a recent review reported that more than 70% of embryos were mosaic with almost 60% having the diploid/aneuploid mosaicism that is said to confound PGD for aneuploidy (Van Echten-Arends et al 2011). However, these observations seem incongruous with the very low misdiagnosis rate of 0.07% after PGD for aneuploidy (Wilton et al., 2009). The vast majority of studies that have reported chromosomal mosaicism in embryos have assessed poor quality or known aneuploid embryos and used FISH to analyse only a few chromosomes. Many blastomeres reported to be euploid would be affected with aneuploidy of chromosomes that were not included in the analysis meaning that diploid/aneuploid mosaicism would have been over-estimated in most of these studies. The advent of 24 chromosome analysis using microarray technology provides new opportunities to accurately determine the true extent of mosaicism by enumerating all 24 chromosomes. We have used this approach to obtain baseline information about mosaicism by analyzing multiple cells from good quality embryos from young patients who have been successful in ART treatment. This has demonstrated that only 24% of embryos had diploid/aneuploid
S35
mosaicism and within these embryos only a minority of cells was aneuploid. Hence the risk of biopsying a cell that was not representative of the rest of the embryo (as might happen in PGD for aneuploidy) could be estimated at only 7%. We are now using a similar approach to assess chromosomal mosaicism in embryos from older women. It would appear that previous studies have over-estimated chromosomal mosaicism in human embryos. Most likely reasons for this include the inherent limitations of FISH technology and the fact that the overwhelming majority of embryos examined were known to be aneuploid. Reference(s) Van Echten-Arends et al., 2011. Hum Reprod Update 17: 620. Wilton et al., 2009. Hum Reprod 24: 1221. S12 Chromosomal instability in the human preimplantation embryo T. Voet1,4 , N. Van der Aa1 , M. Zamani Esteki1 , P. Kumar1 , E. Vanneste1,2 , C. Melotte1 , P. Konings3 , S. Debrock2 , J.-P. Fryns1 , Y. Moreau3 , T. D’Hooghe2 , M.R. Stratton4 , P.J. Campbell4 , J.R. Vermeesch1 . 1 Center for Human Genetics, KULeuven, Leuven, Belgium, 2 Leuven University Fertility Center, UZ Gasthuisberg, Leuven, Belgium, 3 ESAT, KULeuven, Heverlee, Belgium, 4 Wellcome Trust Sanger Institute, Hinxton-Cambridge, UK Recently, we demonstrated chromosome instability (CIN) in human cleavage stage embryogenesis following in vitro fertilization (IVF). CIN not necessarily undermines normal human development (i.e. when remaining normal diploid blastomeres develop the embryo proper), however it can spark a spectrum of conditions, including loss of conception, genetic disease and genetic variation development. To study embryonic CIN further we have developed new methods based on highresolution microarray as well as next-generation sequencing technology that characterize the genome of a single human cell. We delivered proof-of-principle for detecting various types of structural variants, including Mb- to Kb-sized duplications and deletions, in single human (tumor) cells by low coverage pairedend sequencing and mapping. Based on the copy number changes that were detected by singlecell microarray analysis of multiple blastomeres of the same embryo, it was hypothesized that chromosome breakages and fusions occur frequently in human cleavage stage embryos and instigate subsequent breakage-fusion-bridge cycles. In addition, we hypothesized that the DNA breaks present in spermatozoa could trigger this CIN. To test these hypotheses, we genotyped both parents as well as 93 blastomeres from 24 IVF embryos and developed a novel SNP-array based algorithm to determine the parental origin of (aberrant) loci in single cells. Paternal as well as maternal alleles were commonly rearranged in the blastomeres indicating that sperm-specific DNA-breaks do not explain the majority of these structural variants. In addition, single-cell genome sequencing together with parent-of-origin SNP-array and microarray-guided FISH analyses demonstrate that breakage-fusion-bridge cycles as well as more complex rearrangements are sparked in the human cleavage stage embryo. Our data provide evidence that the human cleavage stage embryo is likely an important source of constitutional chromosomal disorders. The developed single-cell genome analysis methods are generic and will deliver novel insights in embryo and tumor genome research.