- Email: [email protected]
The effect of embryo morphology on inner cell mass isolation and production of human embryonic stem cell lines
Objective: To study the effect on blastocyst formation and pregnancy rates of zona opening for PGD using three dimensional partial zona dissection or noncontact diode laser. Design: Prospective randomized study. Materials/Methods: A total of 75 IVF/ICSI-PGD cycles undertaken for repeated implantation failure were studied. Following fertilization of the oocytes by IVF or ICSI patients were randomized on the day of PGD to embryo biopsy performed by three dimensional partial zona dissection (n⫽37) or a 1.48 mu non-contact diode laser (Hamilton-Thorne Instruments)(n⫽38). After the biopsy the embryos were individually cultured in G2 medium until transfer. Chromosomes 13,16,18,21,22,X,and Y were analysed by multicolor FISH technique for aneuploidy screening. Blastocyst formation from euploid embryos and pregnancy rates after embryo transfer on day 5 were analysed and compared. Results: Mean age, duration of infertility, mean number of previous implantation failures, number of oocytes, 2PN fertilization rate, and cleavage rate were similar between groups. Number of G1⫹G2 embryos and 8 cell embryos were likewise similar. A total of 139 embryos were biopsied and 129 analysed in the mechanical and 142 embryos were biopsied and 130 analysed in the laser group. Aneuploidy rates were 60.4% and 62.3% respectively (NS). The rate of progression to the blastocyst stage in normal embryos was 54% and 53% in the two groups (NS). Mean number of blastocysts tranferred was 1.5 in the mechanical and 1.2 in the laser group (NS). Implantation and clinical pregnancy rates per embryo transfer were 15.2 and 23.3% in the mechanical and 16.3 and 21% in the laser group (NS). Conclusions: Zona opening for PGD by either three dimensional partial zona dissection or noncontact diode laser have similar impact on blastocyst formation and the occurrence of pregnancy in women undergoing IVF/ICSI for repeated implantation failures. Supported by: None.
variables, D-1 ROS levels were negatively correlated with fertilization rate at day 1, % embryo fragmentation at day 3, and with clinical pregnancy rate (Table). Conclusions: ROS are produced in day 1 culture media after ICSI. The antioxidant capacity of the culture system used could scavenge different amounts of ROS which appears to be variable among the cycles. Increasing levels of ROS in the culture system have a negative impact on the in vitro development of embryos, as well as pregnancy rates in ICSI cycles. Supported by: None. P-200 Detection of chromosomal aberrations by preimplantation genetic diagnosis (PGD). Sau W. Cheung, W. S. Wun, Steven C. Angus, M. L. Cooper, Andi A. Ybarra, George M. Grunert. Dynagene, Houston, TX; Obstetrical Gynecological Assoc, Houston, TX.
Univariate Spearman correlation of D1-ROS and parameters of ICSI cycle
Objective: To present the results obtained from our PGD program using Fluorescent in situ hybridization (FISH) for the detection of numerical and structural chromosome abnormalities prior to embryo transfer. Design: A prospective study of IVF patients undergoing blastocyst transfer after embryo biopsy for PGD. Materials/Methods: PGD was performed in 17 patients. Group 1 included 13 patients with an average age of 39.4 years; Group 2 included 3 patients who were translocation carriers and one patient whose spouse was mosaic for trisomy 8. The average age of this group was 34.1 years. Embryo biopsy was performed on all embryos with 5 cells or greater on day 3. All patient blastomeres were subjected to two rounds of multicolor FISH for chromosomes 13, 16, 18, 21, 22, X and Y (DNA Probes from Vysis). Patient blastomeres in Group 2 were subjected to an additional third round of multicolor FISH. A three-color scheme combination of telomeric probes for the long and short arm of one translocation chromosome (Vysis) and the telomeric probe for one arm of the other translocation chromosome (CytoCell) was used in two patients. A telomeric probe for chromosome 14q was used for the third patient with a 13;14 Robertsonian translocation. A centromeric probe for chromosome 8 was used for the patient at risk for chromosome 8 aneuploidy. Only one cell was biopsied and only normal or balanced embryos were transferred. All patients underwent genetic counseling prior to the PGD procedure. Results: A total of 169 embryos in 22 cycles were analyzed. In Group 1, 106 embryos were biopsied. Following FISH analysis, 18 embryos had either no nucleus or were undiagnosed. Out of the 88 embryos that were diagnosed, 49 (55.7%) embryos had a normal compliment of chromosomes 13, 16, 18, 21, 22, X and Y, and 39 (44.3%) were determined to be chromosomally abnormal, including both aneuploid (28) and chaotic (11). The efficiency of the technique is 83%. Fourteen transfers were performed, resulting in 3 pregnancies (one live birth, one ongoing pregnancy and one spontaneous miscarriage). Two patients had no transfer because of the lack of normal embryos. In Group 2, 63 embryos were biopsied. After the third round of FISH, 8 embryos had either no nucleus or were undiagnosed. Out of the 55 embryos that were diagnosed, 15 (27.3 %) embryos had a normal compliment of the chromosomes tested, and 40 (72.7%) were determined to be chromosomally abnormal, including one that was chaotic. The efficiency of the technique is 87.3%. Five transfers were performed, resulting in one ongoing pregnancy and one biochemical pregnancy. Conclusions: Multicolor FISH analysis of blastomeres for preimplantation testing is a feasible approach for aneuploidy screening. This approach is even more helpful in translocation carriers who have a high incidence of unbalanced translocations in human preimplantation embryos obtained following IVF. Supported by: NA.
Measure
P-199 Reactive oxygen species: a biological marker for early embryonic development in intracytoplasmic sperm injection (ICSI) cycles. Mohamed A. Bedaiwy, Tommaso Falcone, Kurt Miller, A. Abdel-Aleem, T. AlHussaini, Ashok Agarwal. Cleveland Clin Fdn, Cleveland, OH; Dept of Gynecology-Obstetrics, Assiut Sch of Medicine, Assiut, Egypt. Objective: Reactive oxygen species (ROS) are produced in the course of oxidative phosphorylation reactions in the mitochondria. There is growing evidence denoting various roles of ROS in different reproductive processes. The objective of this study is to examine the relationship of day 1 culture media ROS (D-1 ROS) levels and subsequent evolution of embryos in ICSI cycles. Design: A prospective study Materials/Methods: Oocytes (n ⫽1451) were denuded in preparation for ICSI by hyalourindase exposure and gentle aspiration in graduated pipettes. ICSI was performed in (n ⫽ 1120) M-2 oocytes approximately 42 hours post HCG administration. Fertilization and early culture were performed in HTF with 5% serum substitute supplement. D-1 ROS levels in the central well (sample) and the outer well (control) of each embryo culture dish were measured after overnight incubation by enhanced chemiluminescence assay using luminol as the probe. Average levels of ROS were calculated per cycle. Pregnancy and implantation were determined after transfer of embryos selected solely on the basis of embryo quality (cell number and fragmentation). Fertilization rate, day 3 and day 5 embryo quality parameters, and pregnancy were recorded for each cycle.
Fertilization rate Average cell no. at day 3 Embryo fragmentation at day 3 Blastocyst rate at days 5/6 Clinical pregnancy
r
P-value
P-201
⫺0.294 ⫺0.054 ⫺0.264 ⫺0.189 ⫺0.348
0.002 0.584 0.008 0.059 ⬍0.001
The effect of embryo morphology on inner cell mass isolation and production of human embryonic stem cell lines. Susan E. Lanzendorf, Catherine A. Boyd, D. L. Wright. The Jones Institute for Reproductive Medicine, Eastern Virginia Medical Sch, Norfolk, VA; Dept of Ob/Gyn, MA Gen Hosp, Boston, MA.
Results: Normal fertilization was achieved in 708/1120 oocytes (fertilization rate 63.2%). After controlling for all demographic and clinical
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Abstracts
Objective: To determine if embryo morphology at the blastocyst stage has an effect on isolation of the inner cell mass (ICM) and subsequent development into human embryonic stem (hES) cell colonies.
Vol. 78, No. 3, Suppl. 1, September 2002
Design: Retrospective analysis of data following ICM isolation for production of hES cell lines. The grade of the embryo and ICM was compared to the rate of embryos producing a viable ICM following isolation and the rate at which colonies were initiated. Prior to initiation of the study, it was reviewed and approved by the Institutional Review Board at EVMS. Materials/Methods: Human blastocysts were evaluated using the following grading system: Grade 1-Expanded, ICM visible, no areas of degeneration or bodies within blastocoel; Grade 2-Expanded, ICM visible, areas of degeneration visible and/or bodies within blastocoel; Grade 3-Expanded, ICM not visible, no or various amounts of degeneration or inclusions. In addition, the ICM was evaluated separately from the rest of the embryo and graded as follows: Grade A-ICM compact, clear and shaped like a ball, no dark cells; Grade B-Same as A but with a small amount of dark cells; Grade C-ICM visible but not compact and spread across the inside of the trophoblast, no dark cells; Grade D-Same as C but with dark cells. Blastocysts were exposed to acidified medium or pronase for removal of the zona pellucida followed by immunosurgery for isolation of the ICM. Recovered ICMs were plated on radiated feeder cells and evaluated for 4 –11 days for the formation of colonies with the correct morphological characteristics for hES cell lines, including a high ratio of nucleus to cytoplasm and prominent nucleoli. Results: Rates of ICM isolation and colony formation between the embryo and ICM grades are presented in the tables below: Effect of embryo grade on ICM isolation and colony formation. Embryo Grade
ICMs per Blastocyst (%)
Colony per ICM (%)
23/23 (100) 31/37 (84) 3/30 (10)
8/23 (35) 5/31 (16) 1/3 (33)
ICM Grade
ICMs Isolated per Blastocyst (%)
Colony per ICM (%)
A B C D
17/17 (100) 14/15 (93) 12/17 (71) 9/11 (82)
6/17 (35) 4/14 (28) 1/12 (8) 0/9 (0)
1 2 3
Conclusions: Blastocysts with compact, round ICMs (Grades A and B) result in the most hES colonies following isolation. When visualized before treatment, an ICM is often recovered following immunosurgery (52/60, 87%). When they cannot be visualized (Grade 3), few (10%) contain a viable ICM but a colony may be initiated if one is recovered. As with infertility treatment, embryos of highest quality are the most successful and efforts should be made to optimize the culture system. Supported by: None.
P-202 Three round FISH aneuploidy screening test for 12 chromosomes using human blastomeres. Man Li, Kenneth C. Drury, Larisa Kovalinskaia, Robert Stan Williams. Univ of Florida, Gainesville, FL. Objective: To determine the efficiency of a three round FISH aneuploidy screening test for 12 chromosomes using human blastomeres. Design: Prospective randomized trials Materials/Methods: 49 blastomeres derived from 11 day 3 human embryos donated for PGD research from IVF patients were analyzed in this study. Dissociated blastomeres were fixed to glass slides using the following protocol: Rinse blastomere in 0.05%Tween 20 ⫹ 0.01N HCl and transfer in 1ul to a clean slide. At the moment the droplet is dry, apply a drop of 3:1 (methanol/acetic acid). Place slide in 100% methanol to dehydrate and perform FISH or store at –20C. FISH probe sets consisted of the following: (Round #1) Chromosome probes 2(CEP spectrum orange, Vysis), 15(CEP spectrumRed, Cytocell), X(CEP spectrum Green, Vysis), Y(CEP spectrumAqua, Vysis). (Round #2) Multivysion PB probe set (13 LSI spectrumRed, 16 CEP spectrumAqua, 18 CEP spectrumblue,21 LSI spectrumGreen, 22 LSI spectrum orange, Vysis). (Round #3) Chromosome probes 7(CEP spectrum Aqua, Vysis), 8(CEP spectrum green, Vysis) and 1(CEP spectrum orange, Vysis). Samples were denatured at 76C for 5 mins, hybridized, and
FERTILITY & STERILITY威
processed for analysis. First and 3rd rounds were hybidized for 1 to 2hrs and the 2nd round for 4 to 5 hrs. Between each round, the cover slips were gently washed off the slide by placing them in 0.4SSC at 72C for approximately 1 min. Results: Table 1. Efficiency (%) of 3 Round 12 Chromosomes FISH Analysis:(see attached table) Table 1. Efficiency (%) of 3-Round 12-Chromosome FISH Analysis: Chromosome # # Nuclei # Result Round No. Efficiency
1
2
7
8
13
15
16
18
21
22
X
Y
34 32 3rd 94.1
49 49 1st 100
34 34 3rd 100
34 34 3rd 100
49 46 2nd 93.9
49 48 1st 98.0
49 43 2nd 87.8
49 47 2nd 95.9
49 47 2nd 95.9
49 47 2nd 95.9
49 49 1st 100
49 49 1st 100
Conclusions: Discussion: The ability to assess the chromosomal normalcy of preimplantaion embryos is limited by the number of chromosomes that can be analyzed. Due to technical constraints only a limited number of chromosomes can be screened during a single round of hybridization. Therefore, multiple rounds are necessary to screen those chromosomes most likely to contribute to a poor outcome. We provide a multi-round FISH screening protocol that allows accurate analysis with high efficiency for 12 key chormosomes in a single blastomere. Conclusion(s): The efficiency of three round FISH and subsequent signal generation is influenced by multiple steps such as nuclei fixation, denaturation and hybridization temperature and time, stringency of slide wash, and removal of coverslips between each round. This multi-round FISH protocol can be used to screen 12 chromosomes during the analysis for chromosomal aneuploidy in single blastomeres from human embryos. Supported by: University of Florida.
P-203 Blastocyst transfer after preimplantation genetic diagnosis. A. E. Jones, G. W. Wright, C. A. Davidson-Garcia, A. A. Toledo, D. Mitchell-Leef, J. D. Wininger. Reproductive Biology Assoc, Atlanta, GA. Objective: Historically, embryo transfers following embryo biopsy and preimplantation diagnosis (PGD) were performed on day 3 or 4. With the development of sequential media and blastocyst transfer, patients undergoing PGD now have the option of a day 4 or day 5 transfer. The objective of this study was to compare the pregnancy and implantation rates between patients who received a day 4 or day 5 embryo transfer following PGD for aneuploidy Design: Retrospective Analysis Materials/Methods: Embryo biopsy was performed in Ca2⫹/Mg2⫹ -free medium on day 3 and a diagnosis was determined using fluorescent in situ hybridization (FISH) for chromosomes X,Y,13,18 and 21. On the morning of day 4 all biopsied embryos were transferred into blastocyst culture medium. Patients who did not want their embryos cultured until day 5 had an embryo transfer on day 4. Embryos which were not transferred remained in culture until day 6. Results: Blastocyst transfer was offered to 50 patients post preimplantation genetic diagnosis. Twenty-two patients chose to have a day 5 transfer, while the remaining 40 requested to have a day 4 transfer. In the blastocyst transfer group 56% (13/23) of the patients had a positive pregnancy test of which 52% (12/23) are ongoing. The average age in this group was 38 and the implantation rate was 27% (16/59). Patients who received a day 4 embryo transfer had a 33.3%% (9/27) pregnancy rate of which 26% (7/27) are ongoing. The average age in this group was 39 and the implantation rate was 12% (8/66). Conclusions: The removal of a cell or cells from a day 3 embryo for the purpose of PGD does not preclude the subsequent in vitro development of that embryo to the blastocyst stage. The transfer of blastocysts instead of day 4 embryos post PGD may allow the embryo to better withstand the trauma of embryo transfer procedure. In this study blastocysts replaced to the uterus had a higher implantation rate than embryos transferred on day 4. Culture to the blastocyst stage may allow for selection of embryos for ET when multiple chromosomally normal embryos are available, or allow extra time for performing the genetic diagnosis or rechecking results. Many smaller IVF centers cannot justify the cost of equipment needed for PGD, though a micromanipulator is available. Cells removed from embryos can
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variables, D-1 ROS levels were negatively correlated with fertilization rate at day 1, % embryo fragmentation at day 3, and with clinical pregnancy rate (Table). Conclusions: ROS are produced in day 1 culture media after ICSI. The antioxidant capacity of the culture system used could scavenge different amounts of ROS which appears to be variable among the cycles. Increasing levels of ROS in the culture system have a negative impact on the in vitro development of embryos, as well as pregnancy rates in ICSI cycles. Supported by: None. P-200 Detection of chromosomal aberrations by preimplantation genetic diagnosis (PGD). Sau W. Cheung, W. S. Wun, Steven C. Angus, M. L. Cooper, Andi A. Ybarra, George M. Grunert. Dynagene, Houston, TX; Obstetrical Gynecological Assoc, Houston, TX.
Univariate Spearman correlation of D1-ROS and parameters of ICSI cycle
Objective: To present the results obtained from our PGD program using Fluorescent in situ hybridization (FISH) for the detection of numerical and structural chromosome abnormalities prior to embryo transfer. Design: A prospective study of IVF patients undergoing blastocyst transfer after embryo biopsy for PGD. Materials/Methods: PGD was performed in 17 patients. Group 1 included 13 patients with an average age of 39.4 years; Group 2 included 3 patients who were translocation carriers and one patient whose spouse was mosaic for trisomy 8. The average age of this group was 34.1 years. Embryo biopsy was performed on all embryos with 5 cells or greater on day 3. All patient blastomeres were subjected to two rounds of multicolor FISH for chromosomes 13, 16, 18, 21, 22, X and Y (DNA Probes from Vysis). Patient blastomeres in Group 2 were subjected to an additional third round of multicolor FISH. A three-color scheme combination of telomeric probes for the long and short arm of one translocation chromosome (Vysis) and the telomeric probe for one arm of the other translocation chromosome (CytoCell) was used in two patients. A telomeric probe for chromosome 14q was used for the third patient with a 13;14 Robertsonian translocation. A centromeric probe for chromosome 8 was used for the patient at risk for chromosome 8 aneuploidy. Only one cell was biopsied and only normal or balanced embryos were transferred. All patients underwent genetic counseling prior to the PGD procedure. Results: A total of 169 embryos in 22 cycles were analyzed. In Group 1, 106 embryos were biopsied. Following FISH analysis, 18 embryos had either no nucleus or were undiagnosed. Out of the 88 embryos that were diagnosed, 49 (55.7%) embryos had a normal compliment of chromosomes 13, 16, 18, 21, 22, X and Y, and 39 (44.3%) were determined to be chromosomally abnormal, including both aneuploid (28) and chaotic (11). The efficiency of the technique is 83%. Fourteen transfers were performed, resulting in 3 pregnancies (one live birth, one ongoing pregnancy and one spontaneous miscarriage). Two patients had no transfer because of the lack of normal embryos. In Group 2, 63 embryos were biopsied. After the third round of FISH, 8 embryos had either no nucleus or were undiagnosed. Out of the 55 embryos that were diagnosed, 15 (27.3 %) embryos had a normal compliment of the chromosomes tested, and 40 (72.7%) were determined to be chromosomally abnormal, including one that was chaotic. The efficiency of the technique is 87.3%. Five transfers were performed, resulting in one ongoing pregnancy and one biochemical pregnancy. Conclusions: Multicolor FISH analysis of blastomeres for preimplantation testing is a feasible approach for aneuploidy screening. This approach is even more helpful in translocation carriers who have a high incidence of unbalanced translocations in human preimplantation embryos obtained following IVF. Supported by: NA.
Measure
P-199 Reactive oxygen species: a biological marker for early embryonic development in intracytoplasmic sperm injection (ICSI) cycles. Mohamed A. Bedaiwy, Tommaso Falcone, Kurt Miller, A. Abdel-Aleem, T. AlHussaini, Ashok Agarwal. Cleveland Clin Fdn, Cleveland, OH; Dept of Gynecology-Obstetrics, Assiut Sch of Medicine, Assiut, Egypt. Objective: Reactive oxygen species (ROS) are produced in the course of oxidative phosphorylation reactions in the mitochondria. There is growing evidence denoting various roles of ROS in different reproductive processes. The objective of this study is to examine the relationship of day 1 culture media ROS (D-1 ROS) levels and subsequent evolution of embryos in ICSI cycles. Design: A prospective study Materials/Methods: Oocytes (n ⫽1451) were denuded in preparation for ICSI by hyalourindase exposure and gentle aspiration in graduated pipettes. ICSI was performed in (n ⫽ 1120) M-2 oocytes approximately 42 hours post HCG administration. Fertilization and early culture were performed in HTF with 5% serum substitute supplement. D-1 ROS levels in the central well (sample) and the outer well (control) of each embryo culture dish were measured after overnight incubation by enhanced chemiluminescence assay using luminol as the probe. Average levels of ROS were calculated per cycle. Pregnancy and implantation were determined after transfer of embryos selected solely on the basis of embryo quality (cell number and fragmentation). Fertilization rate, day 3 and day 5 embryo quality parameters, and pregnancy were recorded for each cycle.
Fertilization rate Average cell no. at day 3 Embryo fragmentation at day 3 Blastocyst rate at days 5/6 Clinical pregnancy
r
P-value
P-201
⫺0.294 ⫺0.054 ⫺0.264 ⫺0.189 ⫺0.348
0.002 0.584 0.008 0.059 ⬍0.001
The effect of embryo morphology on inner cell mass isolation and production of human embryonic stem cell lines. Susan E. Lanzendorf, Catherine A. Boyd, D. L. Wright. The Jones Institute for Reproductive Medicine, Eastern Virginia Medical Sch, Norfolk, VA; Dept of Ob/Gyn, MA Gen Hosp, Boston, MA.
Results: Normal fertilization was achieved in 708/1120 oocytes (fertilization rate 63.2%). After controlling for all demographic and clinical
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Abstracts
Objective: To determine if embryo morphology at the blastocyst stage has an effect on isolation of the inner cell mass (ICM) and subsequent development into human embryonic stem (hES) cell colonies.
Vol. 78, No. 3, Suppl. 1, September 2002
Design: Retrospective analysis of data following ICM isolation for production of hES cell lines. The grade of the embryo and ICM was compared to the rate of embryos producing a viable ICM following isolation and the rate at which colonies were initiated. Prior to initiation of the study, it was reviewed and approved by the Institutional Review Board at EVMS. Materials/Methods: Human blastocysts were evaluated using the following grading system: Grade 1-Expanded, ICM visible, no areas of degeneration or bodies within blastocoel; Grade 2-Expanded, ICM visible, areas of degeneration visible and/or bodies within blastocoel; Grade 3-Expanded, ICM not visible, no or various amounts of degeneration or inclusions. In addition, the ICM was evaluated separately from the rest of the embryo and graded as follows: Grade A-ICM compact, clear and shaped like a ball, no dark cells; Grade B-Same as A but with a small amount of dark cells; Grade C-ICM visible but not compact and spread across the inside of the trophoblast, no dark cells; Grade D-Same as C but with dark cells. Blastocysts were exposed to acidified medium or pronase for removal of the zona pellucida followed by immunosurgery for isolation of the ICM. Recovered ICMs were plated on radiated feeder cells and evaluated for 4 –11 days for the formation of colonies with the correct morphological characteristics for hES cell lines, including a high ratio of nucleus to cytoplasm and prominent nucleoli. Results: Rates of ICM isolation and colony formation between the embryo and ICM grades are presented in the tables below: Effect of embryo grade on ICM isolation and colony formation. Embryo Grade
ICMs per Blastocyst (%)
Colony per ICM (%)
23/23 (100) 31/37 (84) 3/30 (10)
8/23 (35) 5/31 (16) 1/3 (33)
ICM Grade
ICMs Isolated per Blastocyst (%)
Colony per ICM (%)
A B C D
17/17 (100) 14/15 (93) 12/17 (71) 9/11 (82)
6/17 (35) 4/14 (28) 1/12 (8) 0/9 (0)
1 2 3
Conclusions: Blastocysts with compact, round ICMs (Grades A and B) result in the most hES colonies following isolation. When visualized before treatment, an ICM is often recovered following immunosurgery (52/60, 87%). When they cannot be visualized (Grade 3), few (10%) contain a viable ICM but a colony may be initiated if one is recovered. As with infertility treatment, embryos of highest quality are the most successful and efforts should be made to optimize the culture system. Supported by: None.
P-202 Three round FISH aneuploidy screening test for 12 chromosomes using human blastomeres. Man Li, Kenneth C. Drury, Larisa Kovalinskaia, Robert Stan Williams. Univ of Florida, Gainesville, FL. Objective: To determine the efficiency of a three round FISH aneuploidy screening test for 12 chromosomes using human blastomeres. Design: Prospective randomized trials Materials/Methods: 49 blastomeres derived from 11 day 3 human embryos donated for PGD research from IVF patients were analyzed in this study. Dissociated blastomeres were fixed to glass slides using the following protocol: Rinse blastomere in 0.05%Tween 20 ⫹ 0.01N HCl and transfer in 1ul to a clean slide. At the moment the droplet is dry, apply a drop of 3:1 (methanol/acetic acid). Place slide in 100% methanol to dehydrate and perform FISH or store at –20C. FISH probe sets consisted of the following: (Round #1) Chromosome probes 2(CEP spectrum orange, Vysis), 15(CEP spectrumRed, Cytocell), X(CEP spectrum Green, Vysis), Y(CEP spectrumAqua, Vysis). (Round #2) Multivysion PB probe set (13 LSI spectrumRed, 16 CEP spectrumAqua, 18 CEP spectrumblue,21 LSI spectrumGreen, 22 LSI spectrum orange, Vysis). (Round #3) Chromosome probes 7(CEP spectrum Aqua, Vysis), 8(CEP spectrum green, Vysis) and 1(CEP spectrum orange, Vysis). Samples were denatured at 76C for 5 mins, hybridized, and
FERTILITY & STERILITY威
processed for analysis. First and 3rd rounds were hybidized for 1 to 2hrs and the 2nd round for 4 to 5 hrs. Between each round, the cover slips were gently washed off the slide by placing them in 0.4SSC at 72C for approximately 1 min. Results: Table 1. Efficiency (%) of 3 Round 12 Chromosomes FISH Analysis:(see attached table) Table 1. Efficiency (%) of 3-Round 12-Chromosome FISH Analysis: Chromosome # # Nuclei # Result Round No. Efficiency
1
2
7
8
13
15
16
18
21
22
X
Y
34 32 3rd 94.1
49 49 1st 100
34 34 3rd 100
34 34 3rd 100
49 46 2nd 93.9
49 48 1st 98.0
49 43 2nd 87.8
49 47 2nd 95.9
49 47 2nd 95.9
49 47 2nd 95.9
49 49 1st 100
49 49 1st 100
Conclusions: Discussion: The ability to assess the chromosomal normalcy of preimplantaion embryos is limited by the number of chromosomes that can be analyzed. Due to technical constraints only a limited number of chromosomes can be screened during a single round of hybridization. Therefore, multiple rounds are necessary to screen those chromosomes most likely to contribute to a poor outcome. We provide a multi-round FISH screening protocol that allows accurate analysis with high efficiency for 12 key chormosomes in a single blastomere. Conclusion(s): The efficiency of three round FISH and subsequent signal generation is influenced by multiple steps such as nuclei fixation, denaturation and hybridization temperature and time, stringency of slide wash, and removal of coverslips between each round. This multi-round FISH protocol can be used to screen 12 chromosomes during the analysis for chromosomal aneuploidy in single blastomeres from human embryos. Supported by: University of Florida.
P-203 Blastocyst transfer after preimplantation genetic diagnosis. A. E. Jones, G. W. Wright, C. A. Davidson-Garcia, A. A. Toledo, D. Mitchell-Leef, J. D. Wininger. Reproductive Biology Assoc, Atlanta, GA. Objective: Historically, embryo transfers following embryo biopsy and preimplantation diagnosis (PGD) were performed on day 3 or 4. With the development of sequential media and blastocyst transfer, patients undergoing PGD now have the option of a day 4 or day 5 transfer. The objective of this study was to compare the pregnancy and implantation rates between patients who received a day 4 or day 5 embryo transfer following PGD for aneuploidy Design: Retrospective Analysis Materials/Methods: Embryo biopsy was performed in Ca2⫹/Mg2⫹ -free medium on day 3 and a diagnosis was determined using fluorescent in situ hybridization (FISH) for chromosomes X,Y,13,18 and 21. On the morning of day 4 all biopsied embryos were transferred into blastocyst culture medium. Patients who did not want their embryos cultured until day 5 had an embryo transfer on day 4. Embryos which were not transferred remained in culture until day 6. Results: Blastocyst transfer was offered to 50 patients post preimplantation genetic diagnosis. Twenty-two patients chose to have a day 5 transfer, while the remaining 40 requested to have a day 4 transfer. In the blastocyst transfer group 56% (13/23) of the patients had a positive pregnancy test of which 52% (12/23) are ongoing. The average age in this group was 38 and the implantation rate was 27% (16/59). Patients who received a day 4 embryo transfer had a 33.3%% (9/27) pregnancy rate of which 26% (7/27) are ongoing. The average age in this group was 39 and the implantation rate was 12% (8/66). Conclusions: The removal of a cell or cells from a day 3 embryo for the purpose of PGD does not preclude the subsequent in vitro development of that embryo to the blastocyst stage. The transfer of blastocysts instead of day 4 embryos post PGD may allow the embryo to better withstand the trauma of embryo transfer procedure. In this study blastocysts replaced to the uterus had a higher implantation rate than embryos transferred on day 4. Culture to the blastocyst stage may allow for selection of embryos for ET when multiple chromosomally normal embryos are available, or allow extra time for performing the genetic diagnosis or rechecking results. Many smaller IVF centers cannot justify the cost of equipment needed for PGD, though a micromanipulator is available. Cells removed from embryos can
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