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P56 Array-based Preimplantation Genetic Diagnosis (PGD): first experiences
POSTERS Materials and Methods: There were 7 patients receiving PGD or PGS via array CGH since September of 2011. The indications were repeated abortion (2 couples), female X mosaicism (one couple), advanced maternal age (>37 yrs; two couples) and translocation carriers (two couples). The mean age is 35.3 years old (31 40). One cell from each day 3 embryo underwent whole genome amplification (WGA) using Surplex (BlueGnome Ltd, Cambridge, UK). WGA products and control genomic DNA were labeled in Cy3 and Cy5 fluorophores respectively. Both labeled DNAs were co-hybridizeed in a 24sure array or a 24sure plus array (BlueGnome Ltd, Cambridge, UK). 12 hour protocols with overnight hybridization were adopted. After washing, slides were scanned using Innoscan 710 (Innopsys, Carbonne, France). Data were analyzed by BlueFuse Multi software (BlueGnome Ltd, Cambridge, UK). Suitable embryos (normal or balanced) were transferred back on day 4. Abnormal embryos were fixed and analyzed with FISH via commercial probes (Vysis probes from Abbott, GH & Co. KG.). Results: A successful WGA was obtained in 44 out of 47 biopsied embryos (93.6%) and all amplified products were labeled successfully. A total of 33 embryos were diagnosed as abnormal or unbalanced in translocation cases (33/44; 75%). After transfer of 9 embryos in 5 cycles, there are 3 ongoing pregnancies including two pairs of twins (pregnancy rate 50% per OPU cycle or 60% per transfer cycle) with an implantation rate of 56%. 15 non-transferred embryos due to abnormal results were reanalyzed on day 4 by FISH with probes to chromosomes 13, 18, 21, X and Y. The mean number of blastomeres to detect was 5. Three of these showed euploidy for the probes used but the abnormalities revealed by array were not belonging to the probes we used. The others of these reanalyzed embryos were abnormal including aneuploidy, mosaicisms or other abnormality. The two aneuploid embryos were compatible with results of array CGH. Interestingly, there were 4 embryos with euploid mosaicism for probes to 13, 18, 21, X and Y. The three embryos without good WGA products revealed mosaicism by reanalysis with FISH. Conclusions: PGD/PGS on day 3 embryos via FISH has been challenged with high mosaicism rates at this stage and limited number of probes used. The preliminary experience of array CGH applied on PGS and PGD for day 3 embryos chromosome diseases showed promising clinical results. The encouraging data implied a place for day 3 embryo biopsy with array CGH for diagnostic assistance, especially for couples with limited number of embryos and wishing embryo transfer in fresh cycle. Further evaluation is needed for the impact of mosaicism on PGD/PGS with array CGH for day 3 embryos. Keywords: day 3 embryo biopsy, FISH, array CGH P55 Nuclear organisation in spermatozoa, a potential biomarker for male infertility? N. Millan1 , P. Lau1 , M. Hann1 , D. Hoffman2 , W. Maxson2 , S. Ory2 , H.G. Tempest1 . 1 Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, FL, USA, 2 IVF Florida Reproductive Associates, Margate, FL, USA Introduction: Infertility is common affecting 1 in 6 couples worldwide, with male factors accounting for ~50%. Diagnosis of male infertility is largely based on subjective measures of semen parameters (count, motility and morphology) as per the World Health Organization (WHO), which address a limited aspect of spermatogenesis and are insufficient for the assessment of male fertility and reproductive capabilities. There is a growing need to develop new assays to assess male reproductive potential. The aim of this study is to investigate whether there is organization of DNA within spermatozoa to determine if DNA organization may be a useful biomarker to improve our understanding of
S69 spermatogenic defects and potentially predict the success of assisted reproductive technologies (ART). Materials and Methods: Semen samples were obtained from nine subjects, undergoing a routine semen parameter assessment as per standard WHO guidelines. All subjects were normozoospermic. Fluorescent in situ hybridization was performed utilizing whole chromosome paints (WCP) for chromosomes 18, 19, 21, 22, X and Y (assessed in 100 cells/chromosome/ patient). Nuclear position was assessed by two methods: radial (internal peripheral location) and polar (position relative to head or tail). Chi-squared analysis tested existence of random or non-random organization. Radial organization was analysed by measuring and dividing the DNA content of the nucleus into five equal areas (based on DAPI fluorescence intensity; shell-1 interior shell-5 periphery) and measuring the amount of WCP fluorescence in each shell. Polar distribution was assessed by dividing the nucleus into three-regions and assigning the WCP to either the head, middle or tail. All subjects consented to participate in this research study approved by the local Institutional Review Board. Results: Our results demonstrate statistical significant evidence (p 0.05) of non-random positioning for all chromosomes tested. All chromosomes were located in an intermediate/ peripheral nuclear address (average shell location ranging from 3.93 to 4.13; stddev 0.12 0.24). Polar position analysis provided clear evidence of preferential localization with chromosomes ordered based on their average positions from the sperm head to tail (chromosomes X, 19, Y, 22, 21 and 18 respectively). All chromosomes exhibited evidence of non-random organization in all subjects except for gene-poor chromosomes 18 and 21. Chromosomes 18 and 21 demonstrated evidence of random organization in three subjects. Conclusion: This study provides evidence of non-random organization of chromosomes within the sperm nucleus (radial and polar). To date, assessment of polar organization appears more informative than radial organization. Polar analysis provided evidence of random distribution in three subjects for two gene-poor chromosomes. This finding may reflect important differences between the gene-content of chromosomes and the requirement to occupy specific regions during spermatogenesis, fertilization and early embryo development. Future studies will include: a larger cohort, more chromosomes and inclusion of infertile men. We plan to investigate the following: (i) are chromosomes strictly organized in sperm; (ii) can we observe altered organization in infertile males and is there any association between diminished semen parameters and/or reproductive outcomes. This could provide an understanding of the mechanistic basis of their infertility and allow individuals and clinicians to make more informed choices regarding ART. Keywords: spermatogenesis, infertility, chromosome organization P56 Array-based Preimplantation Genetic Diagnosis (PGD): first experiences K.-J. Heiliger1 , D. Gutknecht2 , C. Adelfalk1 , M. Bals-Pratsch2 , T. Buchholz1 . 1 Gyn-Gen-Lehel, Centre for Reproductive Genetics, Munich, Germany, 2 Profertilita, Centre for Fertiliy Medicine, Regensburg, Germany Introduction: Whole chromosome or partial chromosome aneuploidies are the major cause of pregnancy loss or implantation failure. Array-based Comparative Genomic Hybridization (arrayCGH) now offers the possibility to gain detailed information about the chromosomal constitution of embryos derived from paternal and maternal carriers of reciprocal or Robertsonian translocations. Material and Methods: The sample consists of up to 10 trophectoderm cells from a day 5 embryo. Embryos were vitrified
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11th International Conference on Preimplantation Genetic Diagnosis
straight after biopsy. Transfer of unaffected embryos is scheduled one or two cycles later. For translocation carriers we perform PGD on whole genome amplified (WGA) cells to receive sufficient DNA from the sample. DNA is hybridized overnight on a 1 Mb BAC array from PerkinElmer (ConstitutionalChip 4.0, HG18). This array contains 5,200 BAC clones covering the whole genome in a 650 kb spacing to detect gains or losses of chromosomes or parts of chromosomes. Results: Up to now we have performed 10 cases of PGD for Robertsonian and reciprocal translocations with a total of 22 embryos. WGA was successful in 21/22 samples (95%). In 60% of cases all embryos were abnormal and no transfer could be performed. High standards of contamination prophylaxis are met. The results achieved by using One Click analysis were clearly interpretable. Conclusion: In all cases we received definite results to determine whether the embryo is transferable or not. This method can be used to avoid pregnancies with non viable embryos for translocation carriers. However, the size of the chromosomal fragments which are translocated should not be smaller than 3 Mb. Keywords: PGD, arrayCGH, whole genome amplification, trophectoderm biopsy, translocation carrier
Results: A linked marker assay for ADRP was successfully designed and optimised. The assay consisted of two multiplexes each containing eight polymorphic markers that flank the NR2E3 gene. The paternal haplotypes were established by isolation of single sperm followed by WGA and linked marker analysis. Of the 21 sperm isolated, 19 amplified successfully. The seven samples with the most informative haplotypes were selected for direct sequencing of the causative mutation to determine phase and distinguish between the high and low risk haplotypes. The couple then underwent a successful PGD cycle. Conclusions: Haplotyping of single sperm cells can be used to identify the high risk haplotype in males where appropriate family members are not available or in cases of a de novo paternal mutation. We believe this is the first example of the application of this method for PGD in an ADRP case.
P57 Development of a preimplantation genetic haplotyping assay for autosomal dominant retinitis pigementosa and its use for single sperm analysis to establish phase
Background: Invasive procedures, associated with a 0.5% increased risk of miscarriage, are still the gold standard in prenatal diagnosis even though some non-invasive approaches already reached clinical applicability. Basically there are two main approaches in non-invasive prenatal diagnosis (NIPD), namely one based on the analysis of circulating fetal cells and one based on cell-free fetal DNA. The latter being closer to implementation into clinical routine, faces a drawback because cell-free DNA always contains a mixture of fetal and maternal DNA. This situation longs for sophisticated strategies especially with respect to analyses of fetal mutations being also present in the maternal background. Analysis based on circulating fetal cells suffers from the extreme rarity and the lack of suitable markers for detection and enrichment. Conversely, single cells represent whole genomic units and analysis on the single cell level allows excluding contamination. Thus, single cells may be forwarded to a panel of different molecular genetic and cytogenetic analyses. Methods: Here, we present a method allowing combined analysis of single cells by means of DNA typing, sequencing, and metaphase comparative genome hybridization (mCGH) post whole genome amplification of single cells (Kroneis et al., 2010, 2011). We established chimeric samples by spiking HT-29 cells (colon adenocarcinoma cell line) into peripheral blood mononuclear cells. Upon cytocentrifuged onto membrane coated slides and immunostaining for cytokeratin, single candidate chimeric cells (HT-29) were transferred to a PCR platform using laser microdissection and pressure catapulting. Thereafter, single cells were forwarded to low-volume on-chip isothermal whole genome amplification (WGA). Isothermal WGA aliquots of the target cells were subjected to 16plex DNA typing (PowerPlex 16 PCR System, Promega), sequencing, and mCGH. Results: DNA profiles of amplified single candidate target cells (n = 6) and cell pools (n = 7) allowed verification of the samples’ genetic origin. Selected single candidate cells (n = 3) forwarded to all downstream analysis showed high mean PCR efficiency of 97% (range 96 100%) in the DNA profiles. Sequencing of three haploid single nucleotide polymorphisms known to be present in the target cell population was successful in 20 of 32 allelic loci. Metaphase CGH with isothermal WGA products showed gains and losses comparable to the genomic data obtained from HT-29 cells.
J. McLuskey1 , J. Pagan1 , S. Pickering2 , P. Renwick3 , R. Scorio2 , S. Morton1 , C. McAtamney2 , N. Carroll1 , J. Thong2 , N. Williams1 , M. Porteous1 , J. Warner1 . 1 South East of Scotland Clinical Genetics Service, Western General Hospital, Edinburgh, UK, 2 Edinburgh Fertility and Reproductive Endocrine Centre, Royal Infirmary of Edinburgh, UK, 3 GSTS Pathology, DNA laboratory, Guy’s Hospital, London, UK Introduction: Autosomal dominant retinitis pigmentosa (ADRP) is one of a group of inherited eye disorders characterised by progressive loss of peripheral and night vision, often followed by loss of central vision leading to incurable blindness. A patient affected with ADRP, with a mutation identified in the NR2E3 gene, was referred to the clinic, with his partner, for preimplantation genetic diagnosis (PGD). Preimplantation genetic haplotyping (PGH) assays are used in Edinburgh to determine high or low risk embryos, but in this case no additional family members were available to ascertain the high risk haplotype. As PGD for ADRP had not previously been performed in the UK a new licence from the Human Fertilisation and Embryology Authority (HFEA) was obtained. We describe the development of a new haplotyping assay and its use on single sperm to establish phase in this family. Materials and Methods: The UCSC Genome Browser was used to identify potentially polymorphic sequences in the region surrounding the NR2E3 gene on chromosome 15. PCR primers were selected to amplify these sequences and multiplex assays using Qiagen Multiplex buffer were optimised using control DNA. Standard semen preparation was performed and individual sperm were isolated using micromanipulation. Sperm cells were lysed and neutralised and whole genome amplification (WGA) by multiplex displacement amplification (MDA) was performed (Qiagen RepliG kit). Multiplex PCR amplifications and Sanger sequencing of exon 2 of the NR2E3 gene were performed on the WGA products. High and low risk haplotypes were determined. PGD cycles were performed using intracytoplasmic sperm injection (ICSI) and successfully fertilised zygotes were cultured. On day three post-fertilisation, a single blastomere was biopsied from each embryo, lysed, neutralised and DNA was amplified by MDA. Embryo haplotypes were scored.
P58 Combined molecular genetic and cytogenetic analyses at the single cell level Th. Kroneis1 , J.B. Geigl2 , A. El-Heliebi1 , E. Petek2 , P. Sedlmayr1 . 1 Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Graz, Austria, 2 Institute of Human Genetics, Medical University of Graz, Graz, Austria
S69 spermatogenic defects and potentially predict the success of assisted reproductive technologies (ART). Materials and Methods: Semen samples were obtained from nine subjects, undergoing a routine semen parameter assessment as per standard WHO guidelines. All subjects were normozoospermic. Fluorescent in situ hybridization was performed utilizing whole chromosome paints (WCP) for chromosomes 18, 19, 21, 22, X and Y (assessed in 100 cells/chromosome/ patient). Nuclear position was assessed by two methods: radial (internal peripheral location) and polar (position relative to head or tail). Chi-squared analysis tested existence of random or non-random organization. Radial organization was analysed by measuring and dividing the DNA content of the nucleus into five equal areas (based on DAPI fluorescence intensity; shell-1 interior shell-5 periphery) and measuring the amount of WCP fluorescence in each shell. Polar distribution was assessed by dividing the nucleus into three-regions and assigning the WCP to either the head, middle or tail. All subjects consented to participate in this research study approved by the local Institutional Review Board. Results: Our results demonstrate statistical significant evidence (p 0.05) of non-random positioning for all chromosomes tested. All chromosomes were located in an intermediate/ peripheral nuclear address (average shell location ranging from 3.93 to 4.13; stddev 0.12 0.24). Polar position analysis provided clear evidence of preferential localization with chromosomes ordered based on their average positions from the sperm head to tail (chromosomes X, 19, Y, 22, 21 and 18 respectively). All chromosomes exhibited evidence of non-random organization in all subjects except for gene-poor chromosomes 18 and 21. Chromosomes 18 and 21 demonstrated evidence of random organization in three subjects. Conclusion: This study provides evidence of non-random organization of chromosomes within the sperm nucleus (radial and polar). To date, assessment of polar organization appears more informative than radial organization. Polar analysis provided evidence of random distribution in three subjects for two gene-poor chromosomes. This finding may reflect important differences between the gene-content of chromosomes and the requirement to occupy specific regions during spermatogenesis, fertilization and early embryo development. Future studies will include: a larger cohort, more chromosomes and inclusion of infertile men. We plan to investigate the following: (i) are chromosomes strictly organized in sperm; (ii) can we observe altered organization in infertile males and is there any association between diminished semen parameters and/or reproductive outcomes. This could provide an understanding of the mechanistic basis of their infertility and allow individuals and clinicians to make more informed choices regarding ART. Keywords: spermatogenesis, infertility, chromosome organization P56 Array-based Preimplantation Genetic Diagnosis (PGD): first experiences K.-J. Heiliger1 , D. Gutknecht2 , C. Adelfalk1 , M. Bals-Pratsch2 , T. Buchholz1 . 1 Gyn-Gen-Lehel, Centre for Reproductive Genetics, Munich, Germany, 2 Profertilita, Centre for Fertiliy Medicine, Regensburg, Germany Introduction: Whole chromosome or partial chromosome aneuploidies are the major cause of pregnancy loss or implantation failure. Array-based Comparative Genomic Hybridization (arrayCGH) now offers the possibility to gain detailed information about the chromosomal constitution of embryos derived from paternal and maternal carriers of reciprocal or Robertsonian translocations. Material and Methods: The sample consists of up to 10 trophectoderm cells from a day 5 embryo. Embryos were vitrified
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11th International Conference on Preimplantation Genetic Diagnosis
straight after biopsy. Transfer of unaffected embryos is scheduled one or two cycles later. For translocation carriers we perform PGD on whole genome amplified (WGA) cells to receive sufficient DNA from the sample. DNA is hybridized overnight on a 1 Mb BAC array from PerkinElmer (ConstitutionalChip 4.0, HG18). This array contains 5,200 BAC clones covering the whole genome in a 650 kb spacing to detect gains or losses of chromosomes or parts of chromosomes. Results: Up to now we have performed 10 cases of PGD for Robertsonian and reciprocal translocations with a total of 22 embryos. WGA was successful in 21/22 samples (95%). In 60% of cases all embryos were abnormal and no transfer could be performed. High standards of contamination prophylaxis are met. The results achieved by using One Click analysis were clearly interpretable. Conclusion: In all cases we received definite results to determine whether the embryo is transferable or not. This method can be used to avoid pregnancies with non viable embryos for translocation carriers. However, the size of the chromosomal fragments which are translocated should not be smaller than 3 Mb. Keywords: PGD, arrayCGH, whole genome amplification, trophectoderm biopsy, translocation carrier
Results: A linked marker assay for ADRP was successfully designed and optimised. The assay consisted of two multiplexes each containing eight polymorphic markers that flank the NR2E3 gene. The paternal haplotypes were established by isolation of single sperm followed by WGA and linked marker analysis. Of the 21 sperm isolated, 19 amplified successfully. The seven samples with the most informative haplotypes were selected for direct sequencing of the causative mutation to determine phase and distinguish between the high and low risk haplotypes. The couple then underwent a successful PGD cycle. Conclusions: Haplotyping of single sperm cells can be used to identify the high risk haplotype in males where appropriate family members are not available or in cases of a de novo paternal mutation. We believe this is the first example of the application of this method for PGD in an ADRP case.
P57 Development of a preimplantation genetic haplotyping assay for autosomal dominant retinitis pigementosa and its use for single sperm analysis to establish phase
Background: Invasive procedures, associated with a 0.5% increased risk of miscarriage, are still the gold standard in prenatal diagnosis even though some non-invasive approaches already reached clinical applicability. Basically there are two main approaches in non-invasive prenatal diagnosis (NIPD), namely one based on the analysis of circulating fetal cells and one based on cell-free fetal DNA. The latter being closer to implementation into clinical routine, faces a drawback because cell-free DNA always contains a mixture of fetal and maternal DNA. This situation longs for sophisticated strategies especially with respect to analyses of fetal mutations being also present in the maternal background. Analysis based on circulating fetal cells suffers from the extreme rarity and the lack of suitable markers for detection and enrichment. Conversely, single cells represent whole genomic units and analysis on the single cell level allows excluding contamination. Thus, single cells may be forwarded to a panel of different molecular genetic and cytogenetic analyses. Methods: Here, we present a method allowing combined analysis of single cells by means of DNA typing, sequencing, and metaphase comparative genome hybridization (mCGH) post whole genome amplification of single cells (Kroneis et al., 2010, 2011). We established chimeric samples by spiking HT-29 cells (colon adenocarcinoma cell line) into peripheral blood mononuclear cells. Upon cytocentrifuged onto membrane coated slides and immunostaining for cytokeratin, single candidate chimeric cells (HT-29) were transferred to a PCR platform using laser microdissection and pressure catapulting. Thereafter, single cells were forwarded to low-volume on-chip isothermal whole genome amplification (WGA). Isothermal WGA aliquots of the target cells were subjected to 16plex DNA typing (PowerPlex 16 PCR System, Promega), sequencing, and mCGH. Results: DNA profiles of amplified single candidate target cells (n = 6) and cell pools (n = 7) allowed verification of the samples’ genetic origin. Selected single candidate cells (n = 3) forwarded to all downstream analysis showed high mean PCR efficiency of 97% (range 96 100%) in the DNA profiles. Sequencing of three haploid single nucleotide polymorphisms known to be present in the target cell population was successful in 20 of 32 allelic loci. Metaphase CGH with isothermal WGA products showed gains and losses comparable to the genomic data obtained from HT-29 cells.
J. McLuskey1 , J. Pagan1 , S. Pickering2 , P. Renwick3 , R. Scorio2 , S. Morton1 , C. McAtamney2 , N. Carroll1 , J. Thong2 , N. Williams1 , M. Porteous1 , J. Warner1 . 1 South East of Scotland Clinical Genetics Service, Western General Hospital, Edinburgh, UK, 2 Edinburgh Fertility and Reproductive Endocrine Centre, Royal Infirmary of Edinburgh, UK, 3 GSTS Pathology, DNA laboratory, Guy’s Hospital, London, UK Introduction: Autosomal dominant retinitis pigmentosa (ADRP) is one of a group of inherited eye disorders characterised by progressive loss of peripheral and night vision, often followed by loss of central vision leading to incurable blindness. A patient affected with ADRP, with a mutation identified in the NR2E3 gene, was referred to the clinic, with his partner, for preimplantation genetic diagnosis (PGD). Preimplantation genetic haplotyping (PGH) assays are used in Edinburgh to determine high or low risk embryos, but in this case no additional family members were available to ascertain the high risk haplotype. As PGD for ADRP had not previously been performed in the UK a new licence from the Human Fertilisation and Embryology Authority (HFEA) was obtained. We describe the development of a new haplotyping assay and its use on single sperm to establish phase in this family. Materials and Methods: The UCSC Genome Browser was used to identify potentially polymorphic sequences in the region surrounding the NR2E3 gene on chromosome 15. PCR primers were selected to amplify these sequences and multiplex assays using Qiagen Multiplex buffer were optimised using control DNA. Standard semen preparation was performed and individual sperm were isolated using micromanipulation. Sperm cells were lysed and neutralised and whole genome amplification (WGA) by multiplex displacement amplification (MDA) was performed (Qiagen RepliG kit). Multiplex PCR amplifications and Sanger sequencing of exon 2 of the NR2E3 gene were performed on the WGA products. High and low risk haplotypes were determined. PGD cycles were performed using intracytoplasmic sperm injection (ICSI) and successfully fertilised zygotes were cultured. On day three post-fertilisation, a single blastomere was biopsied from each embryo, lysed, neutralised and DNA was amplified by MDA. Embryo haplotypes were scored.
P58 Combined molecular genetic and cytogenetic analyses at the single cell level Th. Kroneis1 , J.B. Geigl2 , A. El-Heliebi1 , E. Petek2 , P. Sedlmayr1 . 1 Institute of Cell Biology, Histology & Embryology, Medical University of Graz, Graz, Austria, 2 Institute of Human Genetics, Medical University of Graz, Graz, Austria