- Email: [email protected]
4.004 New approaches in polar body analysis
Abstracts - PGDIS: 8th International Symposium on PGD
Materials/Methods: In this study, the fastFISH technique was tested using different probe mixtures in three sequential rounds on lymphocytes. Probes for chromosomes 13, 18 and 21 were used in the first round, those for chromosomes 15, 16 and 17 in the second round and for chromosomes 14 and 22 in the third round. Results: With lymphocytes, fastFISH gave 89% (177/200) efficiency when the probes for chromosomes 13, 18 and 21 were used. Probes for chromosomes 15, 16 and 17 had an efficiency of 88% (176/200) and using the probes for chromosomes 14 and 22 gave 87% (180/200) efficiency. The optimized protocol will be applied to human embryos. Conclusion: After optimization, this new technique could be applied as a PGS protocol, which would allow screening for eight autosomes and the release of results on the day after embryo biopsy. Embryo transfer may be possible on day 4 post insemination. 4.004 New approaches in polar body analysis Klehr-Martinelli M1, Seifert B2, Bals-Pratsch M2, Buchholz T1 1Centre for Polar Body Diagnosis, Gyn-Gen-Lehel, Munich, Germany; 2Center for Reproductive Medicine, Regensburg, Germany Chromosomal aneuploidies are the major cause of pregnancy loss or implantation failure. Therefore, many efforts have been made to establish assays for the detection of aneuploidies in preimplantation diagnosis. In past years, we have been able to gain much experience with polar body analysis by fluorescence in-situ hybridization (FISH). Both aneuploidy testing and translocation analysis are well established in our laboratory. The main disadvantage of this technique is that only a maximum of 10 chromosomes can be analysed during the restricted time frame due to the German embryo protection law. In order to be able to analyse all 23 chromosomes, an accurate and stable procedure to amplify a single cell to get a sufficient amount of DNA has to be carried out. Different approaches for single cell amplification were evaluated, for example DOP-PCR, linker adapter PCR or whole genome amplification with different kits. Benefits and downsides will be discussed. Amplified polar body DNA as well as amplified oocyte DNA was used for validation for new approaches in polar body analysis, i.e. conventional comparative genomic hybridization (CGH) as well as for different CGH-arrays. Conventional CGH was performed mainly in patients with translocations. The laborious CGH procedure can be performed within a minimum timeframe of 17 h, but it is dependent on the amount of oocytes per patient and it is still not applicable in a fresh IVF cycle. Computer-assisted analysis cuts the time from start to result to a maximum of 20 h by using a bac CGH-array. To detect possible aneuploidies of all chromosomes, we compared one array with 600 clones covering the whole genome to one array with a 1 MB resolution throughout the genome. Alterations in various features, i.e. amount of polar body and oocyte DNA, hybridization time, kind of reference DNA, with or without dye swab, were studied and will be discussed. Particular interest was given to the phenomenon of Y-chromosome hybridization, despite Y DNA in polar bodies and oocytes and strict contamination precautions. This peculiarity will be discussed in consideration of the literature.
5.001 Origin of autosomal monosomy in early stages of preimplantation embryo development Artukhova VG1, Lebedev IN2, Markova EV1, Serebrennikova OA1, Svetlakov AV1 1Center for Reproductive Medicine, P.O. Box 2714, Russia, Krasnoyarsk, 660037; Tel/Fax: (3912) 640895, e-mail: [email protected]; 2Research Institute of Medical Genetics, Tomsk Scientific Center of the Siberian Branch of the Russian Academy of Medical Sciences, nab. R. Ushaiki 10, Tomsk, 634050, Russia; e-mail: [email protected] Objective: Recent data from molecular cytogenetics studies of first–trimester spontaneous abortions with cell culture failures have revealed an unusually high incidence of autosomal monosomies. This type of aneuploidy is a very rare finding among human miscarriages owing to elimination of such embryos prior to implantation. Previously published data indicates that all spontaneous abortions with autosomal monosomies had mosaic karyotypes with normal cell line. It was suggested that mosaic monosomies can arise as a result of post-zygotic anaphase lagging in somatic embryo cells rather than meiotic non-disjunction. The aim of the present investigation was to study mechanisms of autosomal monosomies origin during early stage of preimplantation development. Materials/Methods: A retrospective analysis of fluorescence in-situ hybridization (FISH) results of the first and second polar bodies (PB1 and PB2) and blastomeres biopsies for 784 embryos received from 88 IVF–PGD cycles at the Centre for Reproductive Medicine, Krasnoyarsk, Russia was performed. The PB1 and PB2 biopsy was done in 12–14 h after insemination. The biopsy of blastomeres was performed on day 3. DNA probes for chromosomes 13, 16, 18, 21, 22 (Vysis) were used for PB and blastomeres testing. FISH signals were interpreted by FISH View software (ASI). Results: The incidence of autosomal monosomies in blastomeres was 23% among all detected chromosomal abnormalities. Analysis of corresponding PB for monosomic embryos has shown that 12 from 58 ones had euploid PB1 and PB2 karyotypes (21%). At the same time the incidence on euploid PB for embryos with autosomal trisomy was 7% only (P < 0.01). Normal karyotype in both PB of monosomic embryos was observed for autosomes 13, 16, 18 and 21 with different frequencies. However, all embryos with monosomy 22 had aneuploid PB. Conclusion: Our data provide evidence that autosomal monosomies are more frequent results of post-zygotic mitotic errors in chromosome segregation than trisomies. This finding confirms the hypothesis about somatic origin of monosomy, which allows embryo survival during early preand post-implantation stages of development in the mosaic state. 5.002 Aneuploidy rates in blood cells and oocytes Zotova N, Artukhova V, Markova E, Serebrennikova O, Svetlakov A Centre for Reproductive Medicine, PO Box 2714, Krasnoyarsk, Russia [email protected] Objective: Differentiation between spontaneous aneuploidy rates, low-level mosaicism, and mosaicism are topical problems of cytogenetic diagnosis of infertility. There are opinions that low-level mosaicism can be associated with
S-39 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008
Materials/Methods: In this study, the fastFISH technique was tested using different probe mixtures in three sequential rounds on lymphocytes. Probes for chromosomes 13, 18 and 21 were used in the first round, those for chromosomes 15, 16 and 17 in the second round and for chromosomes 14 and 22 in the third round. Results: With lymphocytes, fastFISH gave 89% (177/200) efficiency when the probes for chromosomes 13, 18 and 21 were used. Probes for chromosomes 15, 16 and 17 had an efficiency of 88% (176/200) and using the probes for chromosomes 14 and 22 gave 87% (180/200) efficiency. The optimized protocol will be applied to human embryos. Conclusion: After optimization, this new technique could be applied as a PGS protocol, which would allow screening for eight autosomes and the release of results on the day after embryo biopsy. Embryo transfer may be possible on day 4 post insemination. 4.004 New approaches in polar body analysis Klehr-Martinelli M1, Seifert B2, Bals-Pratsch M2, Buchholz T1 1Centre for Polar Body Diagnosis, Gyn-Gen-Lehel, Munich, Germany; 2Center for Reproductive Medicine, Regensburg, Germany Chromosomal aneuploidies are the major cause of pregnancy loss or implantation failure. Therefore, many efforts have been made to establish assays for the detection of aneuploidies in preimplantation diagnosis. In past years, we have been able to gain much experience with polar body analysis by fluorescence in-situ hybridization (FISH). Both aneuploidy testing and translocation analysis are well established in our laboratory. The main disadvantage of this technique is that only a maximum of 10 chromosomes can be analysed during the restricted time frame due to the German embryo protection law. In order to be able to analyse all 23 chromosomes, an accurate and stable procedure to amplify a single cell to get a sufficient amount of DNA has to be carried out. Different approaches for single cell amplification were evaluated, for example DOP-PCR, linker adapter PCR or whole genome amplification with different kits. Benefits and downsides will be discussed. Amplified polar body DNA as well as amplified oocyte DNA was used for validation for new approaches in polar body analysis, i.e. conventional comparative genomic hybridization (CGH) as well as for different CGH-arrays. Conventional CGH was performed mainly in patients with translocations. The laborious CGH procedure can be performed within a minimum timeframe of 17 h, but it is dependent on the amount of oocytes per patient and it is still not applicable in a fresh IVF cycle. Computer-assisted analysis cuts the time from start to result to a maximum of 20 h by using a bac CGH-array. To detect possible aneuploidies of all chromosomes, we compared one array with 600 clones covering the whole genome to one array with a 1 MB resolution throughout the genome. Alterations in various features, i.e. amount of polar body and oocyte DNA, hybridization time, kind of reference DNA, with or without dye swab, were studied and will be discussed. Particular interest was given to the phenomenon of Y-chromosome hybridization, despite Y DNA in polar bodies and oocytes and strict contamination precautions. This peculiarity will be discussed in consideration of the literature.
5.001 Origin of autosomal monosomy in early stages of preimplantation embryo development Artukhova VG1, Lebedev IN2, Markova EV1, Serebrennikova OA1, Svetlakov AV1 1Center for Reproductive Medicine, P.O. Box 2714, Russia, Krasnoyarsk, 660037; Tel/Fax: (3912) 640895, e-mail: [email protected]; 2Research Institute of Medical Genetics, Tomsk Scientific Center of the Siberian Branch of the Russian Academy of Medical Sciences, nab. R. Ushaiki 10, Tomsk, 634050, Russia; e-mail: [email protected] Objective: Recent data from molecular cytogenetics studies of first–trimester spontaneous abortions with cell culture failures have revealed an unusually high incidence of autosomal monosomies. This type of aneuploidy is a very rare finding among human miscarriages owing to elimination of such embryos prior to implantation. Previously published data indicates that all spontaneous abortions with autosomal monosomies had mosaic karyotypes with normal cell line. It was suggested that mosaic monosomies can arise as a result of post-zygotic anaphase lagging in somatic embryo cells rather than meiotic non-disjunction. The aim of the present investigation was to study mechanisms of autosomal monosomies origin during early stage of preimplantation development. Materials/Methods: A retrospective analysis of fluorescence in-situ hybridization (FISH) results of the first and second polar bodies (PB1 and PB2) and blastomeres biopsies for 784 embryos received from 88 IVF–PGD cycles at the Centre for Reproductive Medicine, Krasnoyarsk, Russia was performed. The PB1 and PB2 biopsy was done in 12–14 h after insemination. The biopsy of blastomeres was performed on day 3. DNA probes for chromosomes 13, 16, 18, 21, 22 (Vysis) were used for PB and blastomeres testing. FISH signals were interpreted by FISH View software (ASI). Results: The incidence of autosomal monosomies in blastomeres was 23% among all detected chromosomal abnormalities. Analysis of corresponding PB for monosomic embryos has shown that 12 from 58 ones had euploid PB1 and PB2 karyotypes (21%). At the same time the incidence on euploid PB for embryos with autosomal trisomy was 7% only (P < 0.01). Normal karyotype in both PB of monosomic embryos was observed for autosomes 13, 16, 18 and 21 with different frequencies. However, all embryos with monosomy 22 had aneuploid PB. Conclusion: Our data provide evidence that autosomal monosomies are more frequent results of post-zygotic mitotic errors in chromosome segregation than trisomies. This finding confirms the hypothesis about somatic origin of monosomy, which allows embryo survival during early preand post-implantation stages of development in the mosaic state. 5.002 Aneuploidy rates in blood cells and oocytes Zotova N, Artukhova V, Markova E, Serebrennikova O, Svetlakov A Centre for Reproductive Medicine, PO Box 2714, Krasnoyarsk, Russia [email protected] Objective: Differentiation between spontaneous aneuploidy rates, low-level mosaicism, and mosaicism are topical problems of cytogenetic diagnosis of infertility. There are opinions that low-level mosaicism can be associated with
S-39 Reproductive BioMedicine Online, Vol. 16, Suppl. 3, April 2008