- Email: [email protected]
S9 PGD and HLA matching: a clinical update
S34
11th International Conference on Preimplantation Genetic Diagnosis
that TE biopsy is not detrimental to implantation regardless if replaced fresh or after vitrification, while day 3 biopsy produces worse results if replaced in a fresh cycle, but not if coupled with vitrification. When the right biopsy and transfer stage is used, implantation rates, per retrieval, are significantly higher than controls almost doubling, which is to be expected if 50% or more embryos are aneuploid. In addition, at least with TE biopsy, the maternal age effect disappears and if euploid TE-biopsied embryos are present and replaced, they implant at the same rate irrespective of maternal age. S8 PGD for de novo mutations (DNM) S. Rechitsky1 , E. Pomerantseva1 , T. Pakhalchuk1 , O. Verlinsky1 , A. Kuliev1 . 1 Reproductive Genetics Institute, Chicago, IL, USA Aim: PGD for DNM could not be performed previously, due to unavailability of family history and lack of any affected family member to identify the origin of mutation and trace the inheritance of the mutant and normal alleles in oocytes and embryos. We present the approaches for PGD of DNM, their accuracy and clinical outcome of the accumulated experience, as part of the world’s largest experience of PGD for Mendelian disorders Method: Strategies differed depending on the type of DNM inheritance, but general approach involved identification of DNM origin and search for relevant parental haplotypes and possible gonadal mosaicism. Polar body and/or embryo biopsy was performed depending of the mutation origin, with mutation testing and hyplotyping, combined with 24 chromosome testing in patients of advanced reproductive age. One of the important steps was single sperm typing, which was performed in approximately half of the patients. The other essential requirement was to identify the relevant linked markers in both parents even if only one is a DNM carrier. PB analysis was the method of choice in over one third of the cases to detect or confirm the maternal normal and mutant haplotypes. Results: 212 PGD cycles for DNM was performed, involving 120 patients with 49 different conditions, which is significant part of our overall experience of 2711 PGD cycles, resulting in transfer of 4,329 unaffected embryos, which yielded 8,756 clinical pregnancies and birth of 724 apparently healthy children (67 pregnancies still ongoing). Increasing number of cycles for patients of advanced reproductive age are currently performed together with 24 chromosome testing by array-CGH, with presently accumulated data for 59 PGD cycles, in which only unaffected embryos euploid for 24 chromosomes were transferred. The overall clinical outcome of PGD for DNM showed as high as 86.3% success rate of identifying unaffected embryos for transfer (183 of 212 initiated cycles), with the average 1.77 embryos per transfer (325 embryos transferred in 183 cycles), resulting in 48.6% pregnancy rate (89 clinical pregnancies) and birth of 83 unaffected children (14 pregnancies still ongoing), with no misdiagnosis observed in the follow up analysis. This is in accordance with the extremely high accuracy of over 99.975% per transfer in our overall PGD experience, resulting in birth of 724 unaffected apparently healthy children. As expected, the majority of cases involved DNM of dominant inheritance, in agreement with high mutation rate of dominant disorders. However, almost a similar proportion of DNM of dominant type was either of paternal (45%) or maternal (55%) origin, requiring the testing for the presence of DNM in both parents. On the other hand, all the cases of DNM of recessing inheritance were of paternal origin, but the number of cases is not sufficient for conclusions. Conclusion: The presented data show that PGD for DNM is an important addition to the practice of PGD for Mendelian diseases,
as it makes now possible to offer PGD to any couple at risk for producing the offspring with genetic disease, despite the traditional requirement of family data, which is not always available even in cases with known family history for the disease. So the data demonstrate feasibility of PGD for DNM, which may now be routinely performed with the accuracy of over 99%, using the established PGD strategy. S9 PGD and HLA matching: a clinical update S. Kahraman1 . 1 Istanbul Memorial Hospital, IVF and Reproductive Genetics Centre, Istanbul, Turkey PGD in combination with HLA typing may allow the birth of healthy children who may also be potential donors for their affected siblings. It has been more than ten years since the first successful PGD in combination with HLA typing for Fanconi anemia was reported, allowing successful hematopoietic reconstitution in affected sibling by transplantation of stem cells (Verlinsky et al., 2001). Since then, this technique has become a therapeutic tool and allowed dozens of children to be cured of life threatening disorders, both acquired diseases such as leukemia and single gene disorders such as thalassemia (Rechitsky et al., 2004, Kahraman et al., 2011; Kuliev et al., 2011). Between 2003 and 2011, 400 PGD cycles for HLA typing with or without mutation analysis were performed at Istanbul Memorial Hospital, IVF and Reproductive Genetics Center, Turkey. Of these, 320 were performed with mutation analysis and 80 cycles were performed for HLA typing only. The vast majority of cycles for HLA typing with mutation analysis were performed for beta-thalassemia. Most of the PGD cycles for HLA typing only were performed for leukemia. Earlier mutation analyses were done using minisequencing, more recent ones by restriction fragment length polymorphism (RFLP) combined with linked short tandem repeats (STR) marker analysis. HLA typing was performed using polymorphic STR markers scattered through the HLA gene cluster. A total of 3200 embryos were diagnosed of which 13.2% were found to be both unaffected and HLA matched. 36.2% ongoing pregnancy rate was achieved in the 246 cycles where suitable embryos were found for transfer. So far, 80 healthy HLA matched babies have been born. 35 affected siblings have already been cured by successful hematopoietic stem cell transplantation, cases include beta thalassemia, sickle cell anemia, Wiskott Aldrich Syndrome, Diamond Blackfan anemia, acute myeloid leukemia. Many others are awaiting an appropriate time for transplantation. Despite the low probability of finding suitable embryos for transfer in HLA typing groups, the data demonstrates that preimplantation HLA typing alone or in combination with mutation analysis is an effective therapeutic tool for the cure of an affected sibling. Factors affecting the outcomes of HLA typing cycles and stem cell transplantation details will be discussed. Reference(s) Verlinsky Y, Rechitsky S, Schoolcraft W et al, 2001. Preimplantation diagnosis for Fancini anemia combined with HLA matching. Journal of American Medical Association 285, 3130 3133 Rechitsky, S., Kuliev, A., Tur-Kaspa, I., Morris, R., Verlinsky, Y., 2004. Preimplantation genetic diagnosis with HLA matching. Reprod. Biomed. Online 2, 210 221. Kahraman S, Beyazyurek C, Ekmekci CG. Seven years of experience of preimplantation HLA typing: a clinical overview of 327 cycles. Reprod Biomed Online. 2011 Sep; 23(3): 363 71.
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.
11th International Conference on Preimplantation Genetic Diagnosis
that TE biopsy is not detrimental to implantation regardless if replaced fresh or after vitrification, while day 3 biopsy produces worse results if replaced in a fresh cycle, but not if coupled with vitrification. When the right biopsy and transfer stage is used, implantation rates, per retrieval, are significantly higher than controls almost doubling, which is to be expected if 50% or more embryos are aneuploid. In addition, at least with TE biopsy, the maternal age effect disappears and if euploid TE-biopsied embryos are present and replaced, they implant at the same rate irrespective of maternal age. S8 PGD for de novo mutations (DNM) S. Rechitsky1 , E. Pomerantseva1 , T. Pakhalchuk1 , O. Verlinsky1 , A. Kuliev1 . 1 Reproductive Genetics Institute, Chicago, IL, USA Aim: PGD for DNM could not be performed previously, due to unavailability of family history and lack of any affected family member to identify the origin of mutation and trace the inheritance of the mutant and normal alleles in oocytes and embryos. We present the approaches for PGD of DNM, their accuracy and clinical outcome of the accumulated experience, as part of the world’s largest experience of PGD for Mendelian disorders Method: Strategies differed depending on the type of DNM inheritance, but general approach involved identification of DNM origin and search for relevant parental haplotypes and possible gonadal mosaicism. Polar body and/or embryo biopsy was performed depending of the mutation origin, with mutation testing and hyplotyping, combined with 24 chromosome testing in patients of advanced reproductive age. One of the important steps was single sperm typing, which was performed in approximately half of the patients. The other essential requirement was to identify the relevant linked markers in both parents even if only one is a DNM carrier. PB analysis was the method of choice in over one third of the cases to detect or confirm the maternal normal and mutant haplotypes. Results: 212 PGD cycles for DNM was performed, involving 120 patients with 49 different conditions, which is significant part of our overall experience of 2711 PGD cycles, resulting in transfer of 4,329 unaffected embryos, which yielded 8,756 clinical pregnancies and birth of 724 apparently healthy children (67 pregnancies still ongoing). Increasing number of cycles for patients of advanced reproductive age are currently performed together with 24 chromosome testing by array-CGH, with presently accumulated data for 59 PGD cycles, in which only unaffected embryos euploid for 24 chromosomes were transferred. The overall clinical outcome of PGD for DNM showed as high as 86.3% success rate of identifying unaffected embryos for transfer (183 of 212 initiated cycles), with the average 1.77 embryos per transfer (325 embryos transferred in 183 cycles), resulting in 48.6% pregnancy rate (89 clinical pregnancies) and birth of 83 unaffected children (14 pregnancies still ongoing), with no misdiagnosis observed in the follow up analysis. This is in accordance with the extremely high accuracy of over 99.975% per transfer in our overall PGD experience, resulting in birth of 724 unaffected apparently healthy children. As expected, the majority of cases involved DNM of dominant inheritance, in agreement with high mutation rate of dominant disorders. However, almost a similar proportion of DNM of dominant type was either of paternal (45%) or maternal (55%) origin, requiring the testing for the presence of DNM in both parents. On the other hand, all the cases of DNM of recessing inheritance were of paternal origin, but the number of cases is not sufficient for conclusions. Conclusion: The presented data show that PGD for DNM is an important addition to the practice of PGD for Mendelian diseases,
as it makes now possible to offer PGD to any couple at risk for producing the offspring with genetic disease, despite the traditional requirement of family data, which is not always available even in cases with known family history for the disease. So the data demonstrate feasibility of PGD for DNM, which may now be routinely performed with the accuracy of over 99%, using the established PGD strategy. S9 PGD and HLA matching: a clinical update S. Kahraman1 . 1 Istanbul Memorial Hospital, IVF and Reproductive Genetics Centre, Istanbul, Turkey PGD in combination with HLA typing may allow the birth of healthy children who may also be potential donors for their affected siblings. It has been more than ten years since the first successful PGD in combination with HLA typing for Fanconi anemia was reported, allowing successful hematopoietic reconstitution in affected sibling by transplantation of stem cells (Verlinsky et al., 2001). Since then, this technique has become a therapeutic tool and allowed dozens of children to be cured of life threatening disorders, both acquired diseases such as leukemia and single gene disorders such as thalassemia (Rechitsky et al., 2004, Kahraman et al., 2011; Kuliev et al., 2011). Between 2003 and 2011, 400 PGD cycles for HLA typing with or without mutation analysis were performed at Istanbul Memorial Hospital, IVF and Reproductive Genetics Center, Turkey. Of these, 320 were performed with mutation analysis and 80 cycles were performed for HLA typing only. The vast majority of cycles for HLA typing with mutation analysis were performed for beta-thalassemia. Most of the PGD cycles for HLA typing only were performed for leukemia. Earlier mutation analyses were done using minisequencing, more recent ones by restriction fragment length polymorphism (RFLP) combined with linked short tandem repeats (STR) marker analysis. HLA typing was performed using polymorphic STR markers scattered through the HLA gene cluster. A total of 3200 embryos were diagnosed of which 13.2% were found to be both unaffected and HLA matched. 36.2% ongoing pregnancy rate was achieved in the 246 cycles where suitable embryos were found for transfer. So far, 80 healthy HLA matched babies have been born. 35 affected siblings have already been cured by successful hematopoietic stem cell transplantation, cases include beta thalassemia, sickle cell anemia, Wiskott Aldrich Syndrome, Diamond Blackfan anemia, acute myeloid leukemia. Many others are awaiting an appropriate time for transplantation. Despite the low probability of finding suitable embryos for transfer in HLA typing groups, the data demonstrates that preimplantation HLA typing alone or in combination with mutation analysis is an effective therapeutic tool for the cure of an affected sibling. Factors affecting the outcomes of HLA typing cycles and stem cell transplantation details will be discussed. Reference(s) Verlinsky Y, Rechitsky S, Schoolcraft W et al, 2001. Preimplantation diagnosis for Fancini anemia combined with HLA matching. Journal of American Medical Association 285, 3130 3133 Rechitsky, S., Kuliev, A., Tur-Kaspa, I., Morris, R., Verlinsky, Y., 2004. Preimplantation genetic diagnosis with HLA matching. Reprod. Biomed. Online 2, 210 221. Kahraman S, Beyazyurek C, Ekmekci CG. Seven years of experience of preimplantation HLA typing: a clinical overview of 327 cycles. Reprod Biomed Online. 2011 Sep; 23(3): 363 71.
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.