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P-212
OBJECTIVE: Preimplantation genetic diagnosis (PGD) has recently been introduced to screen embryos for chromosomal abnormalities and involves the use of acidified Tyrode solution and blastomere biopsy. The aim of this study was to assess the efficacy of Tyrode-assisted blastomere biopsy on 4and 6-cell mouse embryos with regards to in-vitro development and hatching rate of blastocysts. DESIGN: Prospective, comparative, in-vitro animal study. MATERIALS AND METHODS: Mouse embryos at the 4- and 6-cell stage were divided into three groups and were (1) treated with acidified Tyrode (AT) to digest a hole in the zona pellucida (ZP), (2) treated with AT and biopsied by removing one or two blastomeres, respectively, and re-inserting them into their original ZP, and (3) treated with AT and biopsied by removing one or two blastomeres, respectively. Operated embryos from the three experimental groups were cultured in-vitro to the blastocyst stage. In-vitro development and hatching rate of mouse blastocysts derived from AT treatment and blastomere biopsy was documented and compared for the three experimental groups. Non-operated embryos were cultured under the same conditions and served as controls. RESULTS: The results are summarized in Table 1. Operated 4-cell embryos in groups 1, 2 and 3 showed a blastocyst developmental rate of 88.8%, 84.4% and 87.5% and a hatching rate of 85.0%, 80.0% and 82.5%, respectively. Operated 6-cell embryos in groups 1, 2 and 3 showed a blastocyst developmental rate of 89.5%, 83.3% and 85.7% and a hatching rate of 84,8%, 77.1% and 80.0%, respectively. For the non-operated control embryos, the blastocyst development rate was 90.2% with a hatching rate of 78.2%. There was no significant difference in the blastocyst developmental or hatching potential when either one blastomere was biopsied from 4-cell embryos or two blastomeres were biopsied from 6-cell embryos. However, blastocysts derived from both the operated 4- and 6-cell embryos in the three groups showed an elevated hatching rate when compared to blastocysts derived from the control group. Moreover, these blastocysts hatched 10-12hr earlier than the control blastocysts.
sol, Cyprus; Kentucky Center for Reproductive Medicine, Lexington, KY; Andrology Institute of America, Lexington, KY.
OBJECTIVE: We have previously shown that splitting of mouse embryos has provided a high success rate of twin blastocysts. Therefore, the objectives for this study were to determine if serial splitting of early mouse embryos is feasible and, if so, to assess the developmental potential of the multiple split embryos under in-vitro culture conditions. DESIGN: Prospective in-vitro animal study. MATERIALS AND METHODS: One, two and three blastomeres were biopsied from 2-, 4- and 6-cell embryos, respectively, and inserted into empty zona pellucida recipients (first split). These embryos were cultured to reach their original cell number status and then were split again (second split). Once these embryos regained their original cell status, they were split again (third split). Blastocyst development of embryos split serially at the 2-, 4- and 6-cell stage were recorded and compared. RESULTS: The success rate of blastocyst development after splitting 2-,4- and 6-cell embryos was 74.3%, 75.0% and 66.6%, respectively, for the first split group, and 71.8%, 62.6, and 48.4%, respectively, for the second split group. Concerning the third split, only 48.4%, 38.1% and 10.6% developed into blastocysts, respectively, showing poor morphological quality and reduced cell numbers. To evaluate the efficacy of the first, second and third embryo splitting with regard to blastocyst formation, we calculated the embryo formation or multiplication rate which is defined by the number of blastocysts derived from the original number of embryos split expressed in percentage form. The multiplication rate for 2-, 4- and 6-cell stage embryo splitting was 138%, 141%, and 121% following the first split, 230%, 188% and 140% following the second split and 228%, 160% and only 38% following the third split, respectively (Figure 1).
a
One and two blastomeres were biopsied from 4-cell and 6-cell embryos, respectively. b Hatching index is defined as the number of blastocysts hatched from the number of blastocysts developed expressed as a percentage. CONCLUSION: Our data clearly show that the AT treatment without (group 1) and with blastomere biopsy (groups 2 and 3) imposed on 4and 6-cell mouse embryos does not affect the overal blastocyst development. Moreover, in all three experimental groups, the blastocyst hatching rate was higher than in the non-operated control group, indicating an additional positive effect that may yield to higher implantation rates. When considering our results with regards to human preimplantation genetic diagnosis (PGD), the blastomere biopsy procedure may offer an additive beneficial hatching effect in patients undergoing IVF/ PGD. Supported by: None.
P-212 IN-VITRO BLASTOCYST DEVELOPMENT FROM SERIALLY SPLIT MOUSE EMBRYOS: FUTURE IMPLICATIONS FOR HUMAN ART. K. Illmensee, K. Kaskar, P. M. Zavos. Reprogen Ltd., Limas-
FERTILITY & STERILITY威
Figure 1. Graphic representation of blastocyst development resulting from first, second and third split of 2-, 4- and 6-cell mouse embryos.
CONCLUSION: These data depict very clearly that the first and second splitting of 2-, 4- and 6-cell embryos increases the multiplication rates for successful blastocyst development and thereby increases the number of embryos available for potential transfer. However, an additional third split created a negative and adverse effect on multiplying the number of blastocysts obtained. Furthermore, blastocysts originating from a third embryo split showed inferior morphology. Blastocyst multiplication derived from two serial embryo splittings may therefore be considered for further investigations concerning the developmental invivo potential of these blastocysts. Embryo splitting may also have multiple beneficial applications in human ART. Supported by: None.
S211
sol, Cyprus; Kentucky Center for Reproductive Medicine, Lexington, KY; Andrology Institute of America, Lexington, KY.
OBJECTIVE: We have previously shown that splitting of mouse embryos has provided a high success rate of twin blastocysts. Therefore, the objectives for this study were to determine if serial splitting of early mouse embryos is feasible and, if so, to assess the developmental potential of the multiple split embryos under in-vitro culture conditions. DESIGN: Prospective in-vitro animal study. MATERIALS AND METHODS: One, two and three blastomeres were biopsied from 2-, 4- and 6-cell embryos, respectively, and inserted into empty zona pellucida recipients (first split). These embryos were cultured to reach their original cell number status and then were split again (second split). Once these embryos regained their original cell status, they were split again (third split). Blastocyst development of embryos split serially at the 2-, 4- and 6-cell stage were recorded and compared. RESULTS: The success rate of blastocyst development after splitting 2-,4- and 6-cell embryos was 74.3%, 75.0% and 66.6%, respectively, for the first split group, and 71.8%, 62.6, and 48.4%, respectively, for the second split group. Concerning the third split, only 48.4%, 38.1% and 10.6% developed into blastocysts, respectively, showing poor morphological quality and reduced cell numbers. To evaluate the efficacy of the first, second and third embryo splitting with regard to blastocyst formation, we calculated the embryo formation or multiplication rate which is defined by the number of blastocysts derived from the original number of embryos split expressed in percentage form. The multiplication rate for 2-, 4- and 6-cell stage embryo splitting was 138%, 141%, and 121% following the first split, 230%, 188% and 140% following the second split and 228%, 160% and only 38% following the third split, respectively (Figure 1).
a
One and two blastomeres were biopsied from 4-cell and 6-cell embryos, respectively. b Hatching index is defined as the number of blastocysts hatched from the number of blastocysts developed expressed as a percentage. CONCLUSION: Our data clearly show that the AT treatment without (group 1) and with blastomere biopsy (groups 2 and 3) imposed on 4and 6-cell mouse embryos does not affect the overal blastocyst development. Moreover, in all three experimental groups, the blastocyst hatching rate was higher than in the non-operated control group, indicating an additional positive effect that may yield to higher implantation rates. When considering our results with regards to human preimplantation genetic diagnosis (PGD), the blastomere biopsy procedure may offer an additive beneficial hatching effect in patients undergoing IVF/ PGD. Supported by: None.
P-212 IN-VITRO BLASTOCYST DEVELOPMENT FROM SERIALLY SPLIT MOUSE EMBRYOS: FUTURE IMPLICATIONS FOR HUMAN ART. K. Illmensee, K. Kaskar, P. M. Zavos. Reprogen Ltd., Limas-
FERTILITY & STERILITY威
Figure 1. Graphic representation of blastocyst development resulting from first, second and third split of 2-, 4- and 6-cell mouse embryos.
CONCLUSION: These data depict very clearly that the first and second splitting of 2-, 4- and 6-cell embryos increases the multiplication rates for successful blastocyst development and thereby increases the number of embryos available for potential transfer. However, an additional third split created a negative and adverse effect on multiplying the number of blastocysts obtained. Furthermore, blastocysts originating from a third embryo split showed inferior morphology. Blastocyst multiplication derived from two serial embryo splittings may therefore be considered for further investigations concerning the developmental invivo potential of these blastocysts. Embryo splitting may also have multiple beneficial applications in human ART. Supported by: None.
S211