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Preimplantation Genetic Diagnosis for Aneuploidy Screening in Patients With Poor Reproductive Outcome
generated by the standard FISH protocol. The total hybridization time is reduced significantly to approximately 1 hour instead of the requisite 5 hours thus allowing aneuploidy-screening cases to be completed in a much more time efficient manner. Supported by: University of Florida
P-521 Preimplantation Genetic Diagnosis for Aneuploidy Screening in Patients With Poor Reproductive Outcome. J. Kim, C. Lim, J. Jun, S. Cha, M. Koong, I. Kang. Dept. of Ob/Gyn, Sungkyunkwan Univ. School of Medicine, Samsung Cheil Hospital, Seoul, Republic of Korea; Laboratory of Reproductive Biology and Infertility, Samsung Cheil Hospital, Seoul, Republic of Korea. OBJECTIVE: Aneuploidies of embryos are related to implantation failure or miscarriage. The risk of aneuploid embryo increases in advanced maternal age or parental karyotype abnormality and it results in poor reproductive outcomes such as recurrent spontaneous abortion (RSA) or recurrent implantation failure (RIF). Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) can be applied to these patients with poor reproductive outcome for better ART outcome by selecting chromosomally normal embryos. The aim of this study is to evaluate the clinical outcome of PGD-AS and which group can get much benefit from PGD-AS among the patients expected to have poor reproductive outcome. DESIGN: Retrospective anaylsis of PGD data. MATERIALS AND METHODS: PGD-AS was performed in 48 cycles of 31 patients with poor reproductive outcome from May 1999 to December 2003. Patients were divided into 3 groups: group I - RIF more than 3 times (n⫽11, mean age ⫽42.2 yrs.), group II - RSA (ⱖ 2 times) associated with aneuploidy (n⫽19, mean age ⫽38.9 yrs.), group III - parental sex chromosome abnormality (n⫽18, mean age ⫽29.6 yrs.) including Turner syndrome (45,X/46,XX or 47,XXX/45,X/46,XX), Klinefelter syndrome (47,XXY or 47,XXY/46,XY or 48,XXXY/47,XXY/46,XY) and 47,XYY. Ovarian stimulation was done using GnRH agonist/FSH/HMG midluteal long protocol. Oocytes were inseminated by ICSI and the blastomeres were biopsied at 6-10 cell stage by drilling with Acid Tyrode’s. PGD was performed by using FISH for chromosome 13, 16, 18, 21, X and Y in group I and II, and chromosome X, Y and 18/17 in group III. The embryos diagnosed to be chromosomally normal were replaced 4 days after oocyte retrieval. RESULTS: Blastomere biopsy was successful in 481 embryos and FISH efficiency was 93.1%. The proportions of transferable embryos for the tested probes were 27.9%, 20.5% and 51.2%, respectively in each group showing higher normal rate in group III (group II vs. III, p⬍0.05). The number of transferred embryos was 3.9⫾1.5, 2.0⫾1.2 and 3.1⫾1.5 (mean⫾SD), respectively. The clinical pregnancy rate per transfer was 0%, 35.7% and 16.7%, respectively and significantly higher in group II (p⬍0.05). The overall pregnancy rate was 18.6 % (8/43). Only 2 biochemical pregnancies occurred in group I. Eight healthy babies (one twin pregnancy) were born with normal karyotype and one case was aborted with normal karyotype. CONCLUSION: Our data suggest that PGD-AS provides advantages in patients with poor reproductive outcome, especially with previous RSA associated with aneuploidy or sex chromosome abnormality. PGD-AS does not seem to be beneficial in RIF group in our study due to advanced maternal age and other detrimental factors involved in implantation. Further comprehensive analysis for other autosomes and in more cases of proper indication might be needed. Supported by: None
P-523 Cryopreservation of Human Embryos Prior to Biopsy and Pre-Implantation Genetic Diagnosis (PGD): Does Cell Stage Influence the Outcome? A. D. Dorfmann, M. E. Geltinger, M. A. Iwaszko, M. A. Simpkins, M. E. Sisson, S. R. Lincoln. Genetics and IVF Institute, Fairfax, VA. OBJECTIVE: In some situations where embryo biopsy and Pre-Implantation Genetic Diagnosis (PGD) are being performed, it is necessary to freeze some or all of the embryos created in the fresh IVF cycle. These clinical situations are: 1) Thin endometrial lining, 2) Ovarian Hyper Stimulation Syndrome (OHSS), or 3) Excessive embryos are created. Our purpose is to determine if cryopreserving embryos prior to biopsy and PGD is a feasible clinical alternative, and whether developmental stage has an impact on the outcome. DESIGN: A retrospective study comparing the survival rates of embryos and the pregnancy rates of patients in which embryos were cryopreserved at either the pronucleate stage or at early cleavage stages and then later biopsied for PGD. MATERIALS AND METHODS: All embryos were cryopreserved using standard controlled rate freezing and slow thawing methods. Embryos were individually cryopreserved and stored in straws. Mechanical biopsy was performed on day 3 on all embryos with ⱖ5 blastomeres. When possible, 2 blastomeres were removed for genetic diagnosis. A total of 171 pronuclear zygotes and 214 cleaved embryos were utilized from 23 and 29 patients respectively. RESULTS: Table 1 shows the number of thaw cycles, numbers of biopsies and transfers and the outcomes of the thaws at each developmental stage. There was no significant difference in any of these parameters among the 3 groups.
Table 1
Tx⫽Transfer; Bx⫽Biopsy; Clinical Pregnancy ⫽ Fetal Heart Beat on Ultrasound
P-522 Application of High Density Array-CGH in Detecting Embryo Chromosomal Abnormalities. A. Hellani, S. Coskun. King Faisal Hospital, Riyadh, Saudi Arabia; King Faisal Specialist Hospital, Riyadh, Saudi Arabia. OBJECTIVE: Detection of chromosomal abnormalities at the embryo level as a model for preimplantation genetic diagnosis applications using high density array-CGH on amplified embryos by multiple displacement amplification. DESIGN: Abnormalities in chromosomes of human embryos could reach up to 50%. Such high percentage urges the establishment of a technique
S340
capable of detecting chromosomal aneuploidy in addition to deletions and duplications. MATERIALS AND METHODS: 30 blocked embryos were amplified by multiple displacement amplification (MDA) and analysed by BACs DNA microarray with 1MB resolution. The amplified embryos were compared to XX normal karyotype single lymphocytes amplified with the same technique and using the same protocol. RESULTS: The analysis of the blocked embryos showed a high percentage of aneuploidies mainly in chromosomes 19, 18, 22, 8 and 21 in addition to sex chromosomes. Furthermore, deletions and duplications were detected in chromosomes 1 and 8. CONCLUSION: The high percentage of abnormalities in blocked embryos might explain the cause of embryo growth failure. Furthermore, the detection of deletions show the need for high density Array-CGH able to detect such deletions in addition to aneuploidy and other chromosomal gain or loss. Our results would open a new area in embryo diagnosis for better pregnancy outcome in preimplantation genetic diagnosis since any abnormality could be detected by our technique. Supported by: King Faisal Specialist Hospital and Research CenterDepartment of Genetics
Abstracts
CONCLUSION: Embryos from day 1, day 2 and day 3 were all used successfully. The most effective and efficient method from a logistical standpoint is to freeze embryos on day 1 or day 2, then thaw on the equivalent day in a subsequent frozen embryo transfer cycle prior to doing biopsy and genetic diagnosis. We conclude that embryos can be successfully frozen, thawed and biopsied when it is clinically necessary or advisable to do so; with very reasonable results. Supported by: None
Vol. 84, Suppl 1, September 2005
P-521 Preimplantation Genetic Diagnosis for Aneuploidy Screening in Patients With Poor Reproductive Outcome. J. Kim, C. Lim, J. Jun, S. Cha, M. Koong, I. Kang. Dept. of Ob/Gyn, Sungkyunkwan Univ. School of Medicine, Samsung Cheil Hospital, Seoul, Republic of Korea; Laboratory of Reproductive Biology and Infertility, Samsung Cheil Hospital, Seoul, Republic of Korea. OBJECTIVE: Aneuploidies of embryos are related to implantation failure or miscarriage. The risk of aneuploid embryo increases in advanced maternal age or parental karyotype abnormality and it results in poor reproductive outcomes such as recurrent spontaneous abortion (RSA) or recurrent implantation failure (RIF). Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) can be applied to these patients with poor reproductive outcome for better ART outcome by selecting chromosomally normal embryos. The aim of this study is to evaluate the clinical outcome of PGD-AS and which group can get much benefit from PGD-AS among the patients expected to have poor reproductive outcome. DESIGN: Retrospective anaylsis of PGD data. MATERIALS AND METHODS: PGD-AS was performed in 48 cycles of 31 patients with poor reproductive outcome from May 1999 to December 2003. Patients were divided into 3 groups: group I - RIF more than 3 times (n⫽11, mean age ⫽42.2 yrs.), group II - RSA (ⱖ 2 times) associated with aneuploidy (n⫽19, mean age ⫽38.9 yrs.), group III - parental sex chromosome abnormality (n⫽18, mean age ⫽29.6 yrs.) including Turner syndrome (45,X/46,XX or 47,XXX/45,X/46,XX), Klinefelter syndrome (47,XXY or 47,XXY/46,XY or 48,XXXY/47,XXY/46,XY) and 47,XYY. Ovarian stimulation was done using GnRH agonist/FSH/HMG midluteal long protocol. Oocytes were inseminated by ICSI and the blastomeres were biopsied at 6-10 cell stage by drilling with Acid Tyrode’s. PGD was performed by using FISH for chromosome 13, 16, 18, 21, X and Y in group I and II, and chromosome X, Y and 18/17 in group III. The embryos diagnosed to be chromosomally normal were replaced 4 days after oocyte retrieval. RESULTS: Blastomere biopsy was successful in 481 embryos and FISH efficiency was 93.1%. The proportions of transferable embryos for the tested probes were 27.9%, 20.5% and 51.2%, respectively in each group showing higher normal rate in group III (group II vs. III, p⬍0.05). The number of transferred embryos was 3.9⫾1.5, 2.0⫾1.2 and 3.1⫾1.5 (mean⫾SD), respectively. The clinical pregnancy rate per transfer was 0%, 35.7% and 16.7%, respectively and significantly higher in group II (p⬍0.05). The overall pregnancy rate was 18.6 % (8/43). Only 2 biochemical pregnancies occurred in group I. Eight healthy babies (one twin pregnancy) were born with normal karyotype and one case was aborted with normal karyotype. CONCLUSION: Our data suggest that PGD-AS provides advantages in patients with poor reproductive outcome, especially with previous RSA associated with aneuploidy or sex chromosome abnormality. PGD-AS does not seem to be beneficial in RIF group in our study due to advanced maternal age and other detrimental factors involved in implantation. Further comprehensive analysis for other autosomes and in more cases of proper indication might be needed. Supported by: None
P-523 Cryopreservation of Human Embryos Prior to Biopsy and Pre-Implantation Genetic Diagnosis (PGD): Does Cell Stage Influence the Outcome? A. D. Dorfmann, M. E. Geltinger, M. A. Iwaszko, M. A. Simpkins, M. E. Sisson, S. R. Lincoln. Genetics and IVF Institute, Fairfax, VA. OBJECTIVE: In some situations where embryo biopsy and Pre-Implantation Genetic Diagnosis (PGD) are being performed, it is necessary to freeze some or all of the embryos created in the fresh IVF cycle. These clinical situations are: 1) Thin endometrial lining, 2) Ovarian Hyper Stimulation Syndrome (OHSS), or 3) Excessive embryos are created. Our purpose is to determine if cryopreserving embryos prior to biopsy and PGD is a feasible clinical alternative, and whether developmental stage has an impact on the outcome. DESIGN: A retrospective study comparing the survival rates of embryos and the pregnancy rates of patients in which embryos were cryopreserved at either the pronucleate stage or at early cleavage stages and then later biopsied for PGD. MATERIALS AND METHODS: All embryos were cryopreserved using standard controlled rate freezing and slow thawing methods. Embryos were individually cryopreserved and stored in straws. Mechanical biopsy was performed on day 3 on all embryos with ⱖ5 blastomeres. When possible, 2 blastomeres were removed for genetic diagnosis. A total of 171 pronuclear zygotes and 214 cleaved embryos were utilized from 23 and 29 patients respectively. RESULTS: Table 1 shows the number of thaw cycles, numbers of biopsies and transfers and the outcomes of the thaws at each developmental stage. There was no significant difference in any of these parameters among the 3 groups.
Table 1
Tx⫽Transfer; Bx⫽Biopsy; Clinical Pregnancy ⫽ Fetal Heart Beat on Ultrasound
P-522 Application of High Density Array-CGH in Detecting Embryo Chromosomal Abnormalities. A. Hellani, S. Coskun. King Faisal Hospital, Riyadh, Saudi Arabia; King Faisal Specialist Hospital, Riyadh, Saudi Arabia. OBJECTIVE: Detection of chromosomal abnormalities at the embryo level as a model for preimplantation genetic diagnosis applications using high density array-CGH on amplified embryos by multiple displacement amplification. DESIGN: Abnormalities in chromosomes of human embryos could reach up to 50%. Such high percentage urges the establishment of a technique
S340
capable of detecting chromosomal aneuploidy in addition to deletions and duplications. MATERIALS AND METHODS: 30 blocked embryos were amplified by multiple displacement amplification (MDA) and analysed by BACs DNA microarray with 1MB resolution. The amplified embryos were compared to XX normal karyotype single lymphocytes amplified with the same technique and using the same protocol. RESULTS: The analysis of the blocked embryos showed a high percentage of aneuploidies mainly in chromosomes 19, 18, 22, 8 and 21 in addition to sex chromosomes. Furthermore, deletions and duplications were detected in chromosomes 1 and 8. CONCLUSION: The high percentage of abnormalities in blocked embryos might explain the cause of embryo growth failure. Furthermore, the detection of deletions show the need for high density Array-CGH able to detect such deletions in addition to aneuploidy and other chromosomal gain or loss. Our results would open a new area in embryo diagnosis for better pregnancy outcome in preimplantation genetic diagnosis since any abnormality could be detected by our technique. Supported by: King Faisal Specialist Hospital and Research CenterDepartment of Genetics
Abstracts
CONCLUSION: Embryos from day 1, day 2 and day 3 were all used successfully. The most effective and efficient method from a logistical standpoint is to freeze embryos on day 1 or day 2, then thaw on the equivalent day in a subsequent frozen embryo transfer cycle prior to doing biopsy and genetic diagnosis. We conclude that embryos can be successfully frozen, thawed and biopsied when it is clinically necessary or advisable to do so; with very reasonable results. Supported by: None
Vol. 84, Suppl 1, September 2005