- Email: [email protected]
Comprehensive aneuploidy screening in single cells using microarray comparative genomic hybridization methods implications for preimplantation genetic diagnosis
Saint Barnabas, Livingston, NJ; Zouves Fertility Center, Daly City, CA; Coastal Fertility Medical Center, Irvine, CA; The Center for Reproductive Medicine, Englewood, CA; Highland Park IVF Laboratory-FCI, Highland Park, IL. OBJECTIVE: The purpose of this study was to determine in a large population of infertility patients whether Preimplantation Genetic Diagnosis (PGD) reduces spontaneous miscarriage and increases take-home baby rates. DESIGN: Multi-center, retrospective study involving five IVF centers with high volume of PGD procedures (>10% total cycles) sending slides to the same reference PGD laboratory. MATERIALS AND METHODS: The pregnancy, miscarriage, and live birth rates following PGD was retrospectively compared to non-PGD cycles obtained from the 2003–2005 SART database. PGD was performed using FISH with 9 chromosomes. RESULTS: Results are shown in the table. Overall the PGD results were significantly better for pregnancy rate (P<0.01), miscarriage rate (P<0.01) and pregnancy to term (P<0.001). The improvement in ART outcome was obvious in four of the five clinics.
IVF clinic
Age group
Non PGD cycles
Loss rate
Delivery rate
PGD cycles
Loss rate
Delivery rate
1 2 3 4 5 Total
38–42 38–42 38–42 38–42 38–42 38–42
505 210 1204 509 191 2619
27% 36% 34% 29% 25% 30%
35% 14% 12% 15% 17% 18%
70 72 120 236 208 706
22% 27% 15% 26% 16% 21%
40% 15% 23% 22% 25% 24%
CONCLUSIONS: The data suggests that PGD can significantly increase the chance of pregnancy to term in multiple IVF programs, while reducing the risk of miscarriage in women 38–42. Inter-clinic variation indicates that PGD is more effective in some IVF centers compared to others, suggesting that patient selection, follicular stimulation, culture systems, and biopsy may play important roles. Supported by: None.
Wednesday, October 17, 2007 3:00 pm O-230 NEW RECOMMENDATIONS FOR MALE FACTOR AND REPEATED PREGNANCY LOSS PATIENTS WHO ARE CONSIDERING IVF AND ARE AT RISK OF HAVING TRANSLOCATIONS. S. Munne, J. Fischer, T. Escudero, A. Warner, P. Colls, J. Cohen. Reprogenetics, Livingston, NJ; Reprogenetics, South San Francisco, CA. OBJECTIVE: To determine the pregnancy and miscarriage rate of translocation carriers after PGD and to draw recommendations for IVF programs. Patients requiring ICSI are at a six fold increased risk (3%) of carrying chromosome structural abnormalities (mostly translocations, but also inversions or others), and for couples suffering recurrent pregnancy loss (RPL) the risk is 5–9%. Because the majority of embryos from translocation carriers are abnormal (82%), IVF without PGD will likely result in failure or miscarriage. If patients that are carriers can be identified before IVF, both patients and IVF programs could benefit from this PGD application. DESIGN: Pregnancy, miscarriage and ongoing pregnancy after PGD in a group of translocation carriers were compared to their reproductive previous history as well as to expected loss rates. MATERIALS AND METHODS: Translocation and inversion carriers underwent IVF and PGD using FISH with probes binding to the chromosomes involved in the translocation. Two of these were either distal or proximal to the breakpoint of the translocation, and one or two other probes proximal or distal. RESULTS: A total of 458 cycles with known previous reproductive history and with at least 6 month follow-up after PGD were included. This group of 351 patients had 1145 pregnancies prior to their PGD cycle, of which 977 (85%) miscarried and 15% delivered. For this population (average age 35.5, mean 2.1 previous losses), the next pregnancy should result in 23% lost pregnancies irrespective if a translocation was present (Brigham et al.
Abstracts
Wednesday, October 17, 2007 3:15 pm O-231
TABLE. Results
S86
1999, Human Reprod 14:2868) to 68% lost pregnancies (Sugiuga-Ogasawara et al. 2004, Fertil Steril. 81: 367) for patients with translocations. After PGD, 30.3% (139/458) of retrievals resulted in a pregnancy, of which only 12% were lost and the rest delivered or are ongoing in their third trimester (27%). The difference between observed and expected miscarriage rate are highly significant (P<0.005 to P<0.001, respectively). CONCLUSIONS: The chance of translocation carriers to become pregnant and maintain the pregnancy to term is significantly higher after PGD and requires less IVF attempts. We recommend that all patients requiring ICSI for male factor infertility and couples with a history of RPL are karyotyped before IVF to identify if they are carriers of translocations. Unidentified carriers undergoing IVF may contribute to lower the overall success of an IVF program and be cycling unsuccessfully multiple times. Supported by: None.
OLIGO FLUORESCENCE IN SITU HYBRIDIZATION (OLIGOFISH), A NEW STRATEGY FOR ENUMERATING CHROMOSOMES IN INTERPHASE NUCLEI. J. Aurich-Costa, L. Zamechek, P. Keenan, S. Bradley. One Cell Systems, Inc., Cambridge, MA. OBJECTIVE: Development of FISH probes using labeled oligonucleotides (ODNs) for 5 color chromosome (Chr.) enumeration in interphase nuclei. DESIGN: 10–20 ODN for Chr. X, 15, 17 and 20 a-satellite repeats and for Chr. Y alpha 3 repeat were designed, 50 end labeled, and tested individually on human metaphases. Only ODNs exhibiting signal specific for the target region were selected for each Chr. cocktail. For each Chr., ODNs were mixed together and hybridized on human cells and signal to noise ratio (S/N), sensitivity and specificity were assessed. Only probes exhibiting S/N >2 and sensitivity and specificity R98% were included in each Chr. specific cocktail. Next, we selected 5 fluors that could be simultaneously assessed using epifluorescence. To rank fluor intensity, Chr. Y probe was labeled with all 5 fluors, and S/N was assessed. S/N obtained for each probe labeled with A568, was inversely matched with the ranking of the fluors. Finally, all the probes were mixed together, and varying hybridization times were tested until the shortest time giving the same S/N was found. MATERIALS AND METHODS: Cytogenetic Slides from 5 chromosomally normal males were prepared by harvesting peripheral blood as previously described. ODNs’ sequences were selected in regions where the satellite 3 pentamer or the a-satellite consensus sequence was underrepresented. Sequences were compared to the whole human genome database (NCBI) using BLAST. In situ hybridization was performed as previously described. Fluorescence microscopy and digital image analysis were performed using a Nikon Eclipse E600 microscope equipped with a Photometrics cooled CCD camera. Images were captured using IP Lab Spectrum software. Analytical Specificity and Sensitivity were calculated according to the Standards and Guidelines for Clinical Genetic Laboratories of the American College of Medical Genetics. S/N was calculated on 30 interphase nuclei. Images were segmented into signal and noise regions. S/N was obtained by dividing signal level by noise level. RESULTS: We designed specific ODNs for 5 Chr. After probes were labeled with the 5 fluors and combined, all probes exhibited S/N >2. The shortest hybridization time was determined at 5min. CONCLUSIONS: FISH ODN probes’ short length permits rapid hybridization, a significant advantage for time critical procedures such as enumeration of chromosome in interphase nuclei for preimplantation genetic diagnostics. Supported by: NIH grant: 1R43 HD052597–01.
Wednesday, October 17, 2007 3:30 pm O-232 COMPREHENSIVE ANEUPLOIDY SCREENING IN SINGLE CELLS USING MICROARRAY COMPARATIVE GENOMIC HYBRIDIZATION METHODS IMPLICATIONS FOR PREIMPLANTATION GENETIC DIAGNOSIS. S. Munne, N. M. Steuerwald, D. Wells, S. Bethea, N. J. Nowak, J. Cohen. Reprogenetics, Livingston, NY; A.R.T.
Vol. 88, Suppl 1, September 2007
Institute of New York and New Jersey, Livingston, NY; Department of Biology, University of North Carolina at Charlotte, Charlotte, NC; Department of OB/GYN and Reproductive Sciences, Yale University School of Medicine, New Haven, CT; Department of Biochemistry, New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY; Department of Cancer Prevention and Population Science, Roswell Park Cancer Institute, Buffalo, NY; Empire Genomics, Buffalo, NY. OBJECTIVE: To develop clinically applicable DNA microarray methods for comprehensive identification of genomic imbalances (ie aneuploidy) in isolated cells. DESIGN: Array comparative genomic hybridization (array CGH) together with whole genome amplification (WGA) were used to screen for copy number aberrations in single fibroblast cells derived from various aneuploid cell lines. MATERIALS AND METHODS: DNA was extracted from single fibroblasts and amplified using degenerate oligonucleotide primed PCR (DOPPCR). Test and reference DNA were labeled with Cy5 and Cy3 fluorescent dyes, respectively. The labeled DNAs were co-precipitated and hybridized to bacterial artificial chromosome (BAC)-arrays spotted with clones specific for every chromosome at an average resolution of 0.5 megabases. The array was then scanned and Cy5/Cy3 intensity ratios calculated. Ratio values close to 1 indicate a balanced chromosomal status whereas a ratio >1 or <1 denotes a corresponding gain or a loss of genetic material in the test sample. RESULTS: DOP-PCR mediated DNA amplification and array CGH permitted reproducible genomic profiling of single cells. Trisomies of chromosomes 13, 15, 16, 18, and 21 along with balanced ploidy levels of the remaining chromosomes were identified in single aneuploid fibroblast cells. Sex mismatched reference DNA was also accurately determined.
tive Sciences, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: The combination of whole-genome amplification (WGA) and microarray technology is emerging as a solution to the limitations of current methods for PGD. However, a direct comparison of available microarray platforms has yet to be conducted. The objective of this study is to directly compare the performance of a bacterial artificial chromosome (BAC) array to a single nucleotide polymorphism (SNP) based array. DESIGN: Prospective, randomized, and blinded. MATERIALS AND METHODS: Single cells from cultures with known abnormalities including trisomies for 8, 9, 13, 15, 16 and 21, 18, and X, a monosomy 21, and a normal female were obtained (Coriell). WGA was performed with a modification to the GenomePlex system (Sigma-Aldrich). Nine amplified samples were divided such that genome-wide 250K SNP genotyping microarray (Affymetrix), and a BAC 32K Array (Microarray Core Facility, UCSF) could be completed on the same WGA product. Thus, any differences in the results should be attributable to the differences in the microarrays themselves. Log2 ratios were developed using copy number analysis tool 4.0.1 (Affymetrix) for SNP arrays, and using SPROC (Jain et al. Genome Research 12 2002) for BAC arrays. Aneuploidy was assigned to chromosomes with Log2 ratios outside a defined range. RESULTS: The percentage of individual chromosomes diagnosed correctly was 98.6% for BAC and 100% for SNP array data. The percentage of aneuploidy chromosomes diagnosed correctly was 100% for both BAC and SNP arrays. The clinical accuracy of assigning the correct karyotypes to each cell was decreased in BAC (66%) compared to SNP (100%) arrays. In addition, overall standard deviation from the mean of log2 ratios (noise) was significantly higher (P<0.01) with BAC (0.253) compared to SNP (0.172) arrays. Examples of BAC and SNP array analysis are presented in the following figure.
Figure 1.
CONCLUSIONS: Conventional PGD techniques, which rely on fluorescence in situ hybridization (FISH) analysis, usually screen <1/2 of the human chromosomes and consequently many oocytes or embryos carrying lethal abnormalities are not identified. Array CGH together with whole genome amplification allows comprehensive aneuploidy testing and detection of unbalanced translocations in single cells in a manner compatible with transfer during an IVF cycle. Sufficient DNA is also available following amplification to permit testing for single gene defects. This diagnostic may lead to improved IVF outcomes as all aneuploidies can be avoided and could potentially result in a reduction of the multiple birth rate as pregnancy rates could be maintained with fewer embryos transferred. Supported by: Supported in part by NIH grant 5R44HD044292 to ART Institute of NY/NJ and N.M.S.
Wednesday, October 17, 2007 3:45 pm O-233 GENOME-WIDE CHROMOSOME ANEUPLOIDY ASSESSMENT ON SINGLE CELLS USING TWO TYPES OF ARRAYS – SNP BASED ARRAYS ARE MORE ACCURATE AND LESS VARIABLE THAN BAC ARRAYS. N. R. Treff, R. T. Scott, Jr. Reproductive Medicine Associates of New Jersey, Morristown, NJ; Obstetrics, Gynecology, and Reproduc-
FERTILITY & STERILITYÒ
Figure 1.
S87
IVF clinic
Age group
Non PGD cycles
Loss rate
Delivery rate
PGD cycles
Loss rate
Delivery rate
1 2 3 4 5 Total
38–42 38–42 38–42 38–42 38–42 38–42
505 210 1204 509 191 2619
27% 36% 34% 29% 25% 30%
35% 14% 12% 15% 17% 18%
70 72 120 236 208 706
22% 27% 15% 26% 16% 21%
40% 15% 23% 22% 25% 24%
CONCLUSIONS: The data suggests that PGD can significantly increase the chance of pregnancy to term in multiple IVF programs, while reducing the risk of miscarriage in women 38–42. Inter-clinic variation indicates that PGD is more effective in some IVF centers compared to others, suggesting that patient selection, follicular stimulation, culture systems, and biopsy may play important roles. Supported by: None.
Wednesday, October 17, 2007 3:00 pm O-230 NEW RECOMMENDATIONS FOR MALE FACTOR AND REPEATED PREGNANCY LOSS PATIENTS WHO ARE CONSIDERING IVF AND ARE AT RISK OF HAVING TRANSLOCATIONS. S. Munne, J. Fischer, T. Escudero, A. Warner, P. Colls, J. Cohen. Reprogenetics, Livingston, NJ; Reprogenetics, South San Francisco, CA. OBJECTIVE: To determine the pregnancy and miscarriage rate of translocation carriers after PGD and to draw recommendations for IVF programs. Patients requiring ICSI are at a six fold increased risk (3%) of carrying chromosome structural abnormalities (mostly translocations, but also inversions or others), and for couples suffering recurrent pregnancy loss (RPL) the risk is 5–9%. Because the majority of embryos from translocation carriers are abnormal (82%), IVF without PGD will likely result in failure or miscarriage. If patients that are carriers can be identified before IVF, both patients and IVF programs could benefit from this PGD application. DESIGN: Pregnancy, miscarriage and ongoing pregnancy after PGD in a group of translocation carriers were compared to their reproductive previous history as well as to expected loss rates. MATERIALS AND METHODS: Translocation and inversion carriers underwent IVF and PGD using FISH with probes binding to the chromosomes involved in the translocation. Two of these were either distal or proximal to the breakpoint of the translocation, and one or two other probes proximal or distal. RESULTS: A total of 458 cycles with known previous reproductive history and with at least 6 month follow-up after PGD were included. This group of 351 patients had 1145 pregnancies prior to their PGD cycle, of which 977 (85%) miscarried and 15% delivered. For this population (average age 35.5, mean 2.1 previous losses), the next pregnancy should result in 23% lost pregnancies irrespective if a translocation was present (Brigham et al.
Abstracts
Wednesday, October 17, 2007 3:15 pm O-231
TABLE. Results
S86
1999, Human Reprod 14:2868) to 68% lost pregnancies (Sugiuga-Ogasawara et al. 2004, Fertil Steril. 81: 367) for patients with translocations. After PGD, 30.3% (139/458) of retrievals resulted in a pregnancy, of which only 12% were lost and the rest delivered or are ongoing in their third trimester (27%). The difference between observed and expected miscarriage rate are highly significant (P<0.005 to P<0.001, respectively). CONCLUSIONS: The chance of translocation carriers to become pregnant and maintain the pregnancy to term is significantly higher after PGD and requires less IVF attempts. We recommend that all patients requiring ICSI for male factor infertility and couples with a history of RPL are karyotyped before IVF to identify if they are carriers of translocations. Unidentified carriers undergoing IVF may contribute to lower the overall success of an IVF program and be cycling unsuccessfully multiple times. Supported by: None.
OLIGO FLUORESCENCE IN SITU HYBRIDIZATION (OLIGOFISH), A NEW STRATEGY FOR ENUMERATING CHROMOSOMES IN INTERPHASE NUCLEI. J. Aurich-Costa, L. Zamechek, P. Keenan, S. Bradley. One Cell Systems, Inc., Cambridge, MA. OBJECTIVE: Development of FISH probes using labeled oligonucleotides (ODNs) for 5 color chromosome (Chr.) enumeration in interphase nuclei. DESIGN: 10–20 ODN for Chr. X, 15, 17 and 20 a-satellite repeats and for Chr. Y alpha 3 repeat were designed, 50 end labeled, and tested individually on human metaphases. Only ODNs exhibiting signal specific for the target region were selected for each Chr. cocktail. For each Chr., ODNs were mixed together and hybridized on human cells and signal to noise ratio (S/N), sensitivity and specificity were assessed. Only probes exhibiting S/N >2 and sensitivity and specificity R98% were included in each Chr. specific cocktail. Next, we selected 5 fluors that could be simultaneously assessed using epifluorescence. To rank fluor intensity, Chr. Y probe was labeled with all 5 fluors, and S/N was assessed. S/N obtained for each probe labeled with A568, was inversely matched with the ranking of the fluors. Finally, all the probes were mixed together, and varying hybridization times were tested until the shortest time giving the same S/N was found. MATERIALS AND METHODS: Cytogenetic Slides from 5 chromosomally normal males were prepared by harvesting peripheral blood as previously described. ODNs’ sequences were selected in regions where the satellite 3 pentamer or the a-satellite consensus sequence was underrepresented. Sequences were compared to the whole human genome database (NCBI) using BLAST. In situ hybridization was performed as previously described. Fluorescence microscopy and digital image analysis were performed using a Nikon Eclipse E600 microscope equipped with a Photometrics cooled CCD camera. Images were captured using IP Lab Spectrum software. Analytical Specificity and Sensitivity were calculated according to the Standards and Guidelines for Clinical Genetic Laboratories of the American College of Medical Genetics. S/N was calculated on 30 interphase nuclei. Images were segmented into signal and noise regions. S/N was obtained by dividing signal level by noise level. RESULTS: We designed specific ODNs for 5 Chr. After probes were labeled with the 5 fluors and combined, all probes exhibited S/N >2. The shortest hybridization time was determined at 5min. CONCLUSIONS: FISH ODN probes’ short length permits rapid hybridization, a significant advantage for time critical procedures such as enumeration of chromosome in interphase nuclei for preimplantation genetic diagnostics. Supported by: NIH grant: 1R43 HD052597–01.
Wednesday, October 17, 2007 3:30 pm O-232 COMPREHENSIVE ANEUPLOIDY SCREENING IN SINGLE CELLS USING MICROARRAY COMPARATIVE GENOMIC HYBRIDIZATION METHODS IMPLICATIONS FOR PREIMPLANTATION GENETIC DIAGNOSIS. S. Munne, N. M. Steuerwald, D. Wells, S. Bethea, N. J. Nowak, J. Cohen. Reprogenetics, Livingston, NY; A.R.T.
Vol. 88, Suppl 1, September 2007
Institute of New York and New Jersey, Livingston, NY; Department of Biology, University of North Carolina at Charlotte, Charlotte, NC; Department of OB/GYN and Reproductive Sciences, Yale University School of Medicine, New Haven, CT; Department of Biochemistry, New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, NY; Department of Cancer Prevention and Population Science, Roswell Park Cancer Institute, Buffalo, NY; Empire Genomics, Buffalo, NY. OBJECTIVE: To develop clinically applicable DNA microarray methods for comprehensive identification of genomic imbalances (ie aneuploidy) in isolated cells. DESIGN: Array comparative genomic hybridization (array CGH) together with whole genome amplification (WGA) were used to screen for copy number aberrations in single fibroblast cells derived from various aneuploid cell lines. MATERIALS AND METHODS: DNA was extracted from single fibroblasts and amplified using degenerate oligonucleotide primed PCR (DOPPCR). Test and reference DNA were labeled with Cy5 and Cy3 fluorescent dyes, respectively. The labeled DNAs were co-precipitated and hybridized to bacterial artificial chromosome (BAC)-arrays spotted with clones specific for every chromosome at an average resolution of 0.5 megabases. The array was then scanned and Cy5/Cy3 intensity ratios calculated. Ratio values close to 1 indicate a balanced chromosomal status whereas a ratio >1 or <1 denotes a corresponding gain or a loss of genetic material in the test sample. RESULTS: DOP-PCR mediated DNA amplification and array CGH permitted reproducible genomic profiling of single cells. Trisomies of chromosomes 13, 15, 16, 18, and 21 along with balanced ploidy levels of the remaining chromosomes were identified in single aneuploid fibroblast cells. Sex mismatched reference DNA was also accurately determined.
tive Sciences, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: The combination of whole-genome amplification (WGA) and microarray technology is emerging as a solution to the limitations of current methods for PGD. However, a direct comparison of available microarray platforms has yet to be conducted. The objective of this study is to directly compare the performance of a bacterial artificial chromosome (BAC) array to a single nucleotide polymorphism (SNP) based array. DESIGN: Prospective, randomized, and blinded. MATERIALS AND METHODS: Single cells from cultures with known abnormalities including trisomies for 8, 9, 13, 15, 16 and 21, 18, and X, a monosomy 21, and a normal female were obtained (Coriell). WGA was performed with a modification to the GenomePlex system (Sigma-Aldrich). Nine amplified samples were divided such that genome-wide 250K SNP genotyping microarray (Affymetrix), and a BAC 32K Array (Microarray Core Facility, UCSF) could be completed on the same WGA product. Thus, any differences in the results should be attributable to the differences in the microarrays themselves. Log2 ratios were developed using copy number analysis tool 4.0.1 (Affymetrix) for SNP arrays, and using SPROC (Jain et al. Genome Research 12 2002) for BAC arrays. Aneuploidy was assigned to chromosomes with Log2 ratios outside a defined range. RESULTS: The percentage of individual chromosomes diagnosed correctly was 98.6% for BAC and 100% for SNP array data. The percentage of aneuploidy chromosomes diagnosed correctly was 100% for both BAC and SNP arrays. The clinical accuracy of assigning the correct karyotypes to each cell was decreased in BAC (66%) compared to SNP (100%) arrays. In addition, overall standard deviation from the mean of log2 ratios (noise) was significantly higher (P<0.01) with BAC (0.253) compared to SNP (0.172) arrays. Examples of BAC and SNP array analysis are presented in the following figure.
Figure 1.
CONCLUSIONS: Conventional PGD techniques, which rely on fluorescence in situ hybridization (FISH) analysis, usually screen <1/2 of the human chromosomes and consequently many oocytes or embryos carrying lethal abnormalities are not identified. Array CGH together with whole genome amplification allows comprehensive aneuploidy testing and detection of unbalanced translocations in single cells in a manner compatible with transfer during an IVF cycle. Sufficient DNA is also available following amplification to permit testing for single gene defects. This diagnostic may lead to improved IVF outcomes as all aneuploidies can be avoided and could potentially result in a reduction of the multiple birth rate as pregnancy rates could be maintained with fewer embryos transferred. Supported by: Supported in part by NIH grant 5R44HD044292 to ART Institute of NY/NJ and N.M.S.
Wednesday, October 17, 2007 3:45 pm O-233 GENOME-WIDE CHROMOSOME ANEUPLOIDY ASSESSMENT ON SINGLE CELLS USING TWO TYPES OF ARRAYS – SNP BASED ARRAYS ARE MORE ACCURATE AND LESS VARIABLE THAN BAC ARRAYS. N. R. Treff, R. T. Scott, Jr. Reproductive Medicine Associates of New Jersey, Morristown, NJ; Obstetrics, Gynecology, and Reproduc-
FERTILITY & STERILITYÒ
Figure 1.
S87