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Trophectoderm biopsy – Age matters
MATERIALS AND METHODS: EB was performed on embryos with at least 4 cells and euploid embryos were either transferred or vitrified on day 5. TE biopsy was performed after a channel was made in the zona pellucida (ZP) on day 3 with a Research Instrument laser. On day 5 and 6 blastocysts that had expanded with differentiation were biopsied by removing herniated TE with the laser. Blastocysts were vitrified using Sage Vitrification Media and Biotech Cryolocks., A programmed thaw cycle was initiated using estrogen and progesterone the desired number (1-2) euploid of blasts were warmed and transferred. RESULTS: 94 patients had 870 embryos biopsied on day 3 biopsy. 6 patients had 105 blastocysts for TE biopsy. Biopsy results revealed 218/870 (25%) euploid embryos following day 3 biopsy versus 46/105 (44%) for TE biopsy (P¼0.0001 Fisher exact test). 37/94 (39%) patients had a positive hCG following day 3 biopsy compared to 8/12 (66%) frozen embryo transfers after TE biopsy (P¼0.0918). The implantation rate for day 3 biopsy was 47/ 115 (40%), compared to 8/19 (42%) for TE (NS). CONCLUSION: Day 3 EB takes place prior to or at the time of embryonic gene expression. Some aneuploidies do not allow embryos to progress beyond the cleavage stage. This may account for the higher euploidy rates of day 5/6 embryos. Studies have shown a greater incidence of mosaicism on day 3 that possibly corrects by the blastocyst stage. This can also account for higher euploidy rates of blastocysts and potentially a higher pregnancy and implantation rate. By screening out aneuploid embryos blastocyst culture may result in better embryo selection and better outcome compared with D3 embryos. Supported by: Grifo: Ferring 1 time consultant
DESIGN: Retrospective compilation of 24-aCGH blastocyst biopsy results from patients undergoing PGS between February 2009 and February 2011. Patients were stratified into 3 groups based on their age at the time of biopsy. The groups were as follows <35, 35-39 and >39. MATERIALS AND METHODS: Patients considered potential candidates for IVF and PGS due to prior pregnancy losses, previous failed IVF attempts and unexplained infertility were counseled extensively prior to proceeding. Patients who opted to undergo PGS testing had trophectoderm biopsy of their embryos via laser excision of 2 to 10 cells post retrieval on day 5, 6, or 7. Following biopsy, blastocysts were vitrified individually, and the biopsed cells were sent for 24-aCGH. RESULTS: To date, a total of 231 blastocysts were biopsied for PGS. Of these, 48.5% were euploid, 43.7% aneuploid and 7.8% failed to produce a signal. A significant difference in the euploidy rate was observed between <35 and >39 age groups and between 35-39 and >39 age groups. There was no difference in euploidy rate between <35 and 35-39. Many aneuploid embryos displayed more than one anomaly, the frequency of which increased with age.
O-370 Wednesday, October 19, 2011 05:00 PM
1
EFFICIENCY OF DRY CULTURE SYSTEM IN HUMAN BLASTOCYST CULTURE. T. Okimura, M. Kuwayama, C. Mori, F. Aono, Y. Takehara, O. Kato. Kato Ladies Clinic, Shinjuku-ku, Tokyo, Japan; Repro-Support Medical Research Center, Shinjuku-ku, Tokyo, Japan. OBJECTIVE: Recently, a ‘‘dry culture system’’ which has no tray of water in the incubator has been designed to reduce the risk of microbial contamination for mammalian embryos culture. We conducted an experiment to evaluate the clinical efficiency of the dry culture system. DESIGN: Prospective data analysis of over 3,000 blastocyst culture cycles. MATERIALS AND METHODS: A total of 6,709 human 4- to 8-cell embryos of 3,618 IVF cycles from patients (from 24 to 42 years old) with informed consent were cultured in dry or humid incubators. The dry incubator was the EZ-Culture system (ASTEC, Japan). The conventional humid incubator containing a water tray was the Model AP30 (ASTEC, Japan). Normal 4- to 8-cell embryos were cultured for 4 days from Day 3 in Blastocyst culture medium (SAGE, USA). All the embryos were cultured in 20 ml droplets covered with mineral oil. The rates of development into blastocysts in both groups were determined on day 5 and 6, respectively, and compared. RESULTS: Of 2,597 4- to 8-cell embryos cultured in the dry incubator, 1,486 (57.2%) developed to blastocysts. And of 4,112 embryos cultured in the humid incubator, 2,398 (58.3%) developed to blastocysts. There was no significant difference in the rates between the two groups. However, excellent and good blastocysts (Grade 3AA–3BB; by the Gardner grading) rate of the dry incubator (21.3%, 262/1,228) was significantly higher (P<0.01) than those in the humid incubator group (15.5%, 296/1,915). CONCLUSION: The results suggest that development of human embryos in the dry culture system was identically effective to those of embryos cultured under conventional humidification conditions. We conclude that it may not be necessary to use a full humidified atmosphere for human embryos culture in droplets covered with mineral oil. The dry culture system used in the present study is not only efficient but also hygienic and simple to maintain. Thus, this dry incubation system seems to have advantages to reduce possible contamination as well as the maintenance costs.
O-371 Wednesday, October 19, 2011 05:15 PM TROPHECTODERM BIOPSY – AGE MATTERS. M. P. Portmann, L. S. Morrison, S. M. Carney, C. F. Boylan, R. F. Feinberg, G. Kovalevsky. Laboratory, Reproductive Associates of Delaware, Newark, DE. OBJECTIVE: To determine the difference in aneuploidy rate among patients in different age groups utilizing trophectoderm biopsy and 24 chromosome microarray comparative genomic hybridization (24-aCGH).
FERTILITY & STERILITYÒ
Biopsy Data # Blasts Biopsied Euploid Status Aneuploid State Failed DNA Amplification
2
<35
35-39
>39
Total
83 51 (61.4%)1 26 (31.3%) 6 (7.2%)
109 56 (51.4%)2 45 (41.3%) 8 (7.3%)
39 5 (12.8%)1,2 30 (76.9%) 4 (10.3%)
231 112 (48.5%) 101 (43.7%) 18 (7.8%)
P<0.01 P<0.01
CONCLUSION: Previous reports have suggested a relationship between age and aneuploidy rate. Utilizing trophectoderm biopsy with microarray CGH, the long held notion of increasing aneuploidy rate with age was confirmed in this small study.
O-372 Wednesday, October 19, 2011 05:30 PM RESCUE ICSI OF INSEMINATION 6 HOURS OOCYTES IN IVF CYCLES AFTER TOTAL FERTILIZATION FAILURE. N. Zhang, B. Wang, Z. Xu, H. Sun, Y. Hu. Reproductive Medicine, Drum Tower Hospital, Nanjing, Jiangsu, China. OBJECTIVE: Due to various factors, there are cases of total fertilization failure, and patients are forced to give up the cycle. After rescueICSI attempts, the developmental potential and implantation rate of embryos are low because of oocyte aging. Early signs of fertilization can be observed 6 h after the initial insemination, providing the possibility of performing rescue ICSI attempts as early as possible. DESIGN: A retrospective clinical study. MATERIALS AND METHODS: Of the 4014 conventional IVF cycles between January 2004 and December 2008,197 cycles involved total fertilization failure. 161 cycles of early rescue ICSI was performed on those oocytes in which a second polar body was not evident after insemination 6 h. 36 cycles provided the oocytes for 18 h rescue ICSI. Fertilization, implantation, pregnancy and take baby home rates were the main outcome measures compared between the 6 h and the 18 h group. A P value of <0.05 was considered statistically significant. RESULTS: Following early rescue ICSI, evidently fertilization was observed in 943 oocytes (75.7%) of 1245 metaphase IIoocytes. This was no significant different between 18 h group, in which 285 oocytes in metaphase II and 200(70.2%)showed evidence of fertilization. Grade I-II embryos rate in the 6 h vs 18 h was 67.4%(607/901)vs 45.1%(83/184)(P < 0.01). Clinical pregnancy rate in two groups was 55.9%(90/161)vs 16.7%(6/36)(P < 0.01). Implantation rate was 37.6%(121/322)vs 12.7%(9/71)(P < 0.01).Take baby home rate was 46.6%(75/161)vs 16.7%(6/36)(P < 0.05). After early rescue ICSI, 75 cases have succeeded delivery, and 97 babies were born. CONCLUSION: Identifying unfertilized oocytes within 6h relyed on the correct identification of polar bodies to indicate oocyte activation as the preliminary sign of fertilization before pronuclei developed. Rescue ICSI of unfertilized oocytes closer to the egg retrieval is more efficient. As results demonstrated, a higher ongoing and clinical PR was observed in 6 h rescue ICSI, to prove the method feasibility.
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DESIGN: Retrospective compilation of 24-aCGH blastocyst biopsy results from patients undergoing PGS between February 2009 and February 2011. Patients were stratified into 3 groups based on their age at the time of biopsy. The groups were as follows <35, 35-39 and >39. MATERIALS AND METHODS: Patients considered potential candidates for IVF and PGS due to prior pregnancy losses, previous failed IVF attempts and unexplained infertility were counseled extensively prior to proceeding. Patients who opted to undergo PGS testing had trophectoderm biopsy of their embryos via laser excision of 2 to 10 cells post retrieval on day 5, 6, or 7. Following biopsy, blastocysts were vitrified individually, and the biopsed cells were sent for 24-aCGH. RESULTS: To date, a total of 231 blastocysts were biopsied for PGS. Of these, 48.5% were euploid, 43.7% aneuploid and 7.8% failed to produce a signal. A significant difference in the euploidy rate was observed between <35 and >39 age groups and between 35-39 and >39 age groups. There was no difference in euploidy rate between <35 and 35-39. Many aneuploid embryos displayed more than one anomaly, the frequency of which increased with age.
O-370 Wednesday, October 19, 2011 05:00 PM
1
EFFICIENCY OF DRY CULTURE SYSTEM IN HUMAN BLASTOCYST CULTURE. T. Okimura, M. Kuwayama, C. Mori, F. Aono, Y. Takehara, O. Kato. Kato Ladies Clinic, Shinjuku-ku, Tokyo, Japan; Repro-Support Medical Research Center, Shinjuku-ku, Tokyo, Japan. OBJECTIVE: Recently, a ‘‘dry culture system’’ which has no tray of water in the incubator has been designed to reduce the risk of microbial contamination for mammalian embryos culture. We conducted an experiment to evaluate the clinical efficiency of the dry culture system. DESIGN: Prospective data analysis of over 3,000 blastocyst culture cycles. MATERIALS AND METHODS: A total of 6,709 human 4- to 8-cell embryos of 3,618 IVF cycles from patients (from 24 to 42 years old) with informed consent were cultured in dry or humid incubators. The dry incubator was the EZ-Culture system (ASTEC, Japan). The conventional humid incubator containing a water tray was the Model AP30 (ASTEC, Japan). Normal 4- to 8-cell embryos were cultured for 4 days from Day 3 in Blastocyst culture medium (SAGE, USA). All the embryos were cultured in 20 ml droplets covered with mineral oil. The rates of development into blastocysts in both groups were determined on day 5 and 6, respectively, and compared. RESULTS: Of 2,597 4- to 8-cell embryos cultured in the dry incubator, 1,486 (57.2%) developed to blastocysts. And of 4,112 embryos cultured in the humid incubator, 2,398 (58.3%) developed to blastocysts. There was no significant difference in the rates between the two groups. However, excellent and good blastocysts (Grade 3AA–3BB; by the Gardner grading) rate of the dry incubator (21.3%, 262/1,228) was significantly higher (P<0.01) than those in the humid incubator group (15.5%, 296/1,915). CONCLUSION: The results suggest that development of human embryos in the dry culture system was identically effective to those of embryos cultured under conventional humidification conditions. We conclude that it may not be necessary to use a full humidified atmosphere for human embryos culture in droplets covered with mineral oil. The dry culture system used in the present study is not only efficient but also hygienic and simple to maintain. Thus, this dry incubation system seems to have advantages to reduce possible contamination as well as the maintenance costs.
O-371 Wednesday, October 19, 2011 05:15 PM TROPHECTODERM BIOPSY – AGE MATTERS. M. P. Portmann, L. S. Morrison, S. M. Carney, C. F. Boylan, R. F. Feinberg, G. Kovalevsky. Laboratory, Reproductive Associates of Delaware, Newark, DE. OBJECTIVE: To determine the difference in aneuploidy rate among patients in different age groups utilizing trophectoderm biopsy and 24 chromosome microarray comparative genomic hybridization (24-aCGH).
FERTILITY & STERILITYÒ
Biopsy Data # Blasts Biopsied Euploid Status Aneuploid State Failed DNA Amplification
2
<35
35-39
>39
Total
83 51 (61.4%)1 26 (31.3%) 6 (7.2%)
109 56 (51.4%)2 45 (41.3%) 8 (7.3%)
39 5 (12.8%)1,2 30 (76.9%) 4 (10.3%)
231 112 (48.5%) 101 (43.7%) 18 (7.8%)
P<0.01 P<0.01
CONCLUSION: Previous reports have suggested a relationship between age and aneuploidy rate. Utilizing trophectoderm biopsy with microarray CGH, the long held notion of increasing aneuploidy rate with age was confirmed in this small study.
O-372 Wednesday, October 19, 2011 05:30 PM RESCUE ICSI OF INSEMINATION 6 HOURS OOCYTES IN IVF CYCLES AFTER TOTAL FERTILIZATION FAILURE. N. Zhang, B. Wang, Z. Xu, H. Sun, Y. Hu. Reproductive Medicine, Drum Tower Hospital, Nanjing, Jiangsu, China. OBJECTIVE: Due to various factors, there are cases of total fertilization failure, and patients are forced to give up the cycle. After rescueICSI attempts, the developmental potential and implantation rate of embryos are low because of oocyte aging. Early signs of fertilization can be observed 6 h after the initial insemination, providing the possibility of performing rescue ICSI attempts as early as possible. DESIGN: A retrospective clinical study. MATERIALS AND METHODS: Of the 4014 conventional IVF cycles between January 2004 and December 2008,197 cycles involved total fertilization failure. 161 cycles of early rescue ICSI was performed on those oocytes in which a second polar body was not evident after insemination 6 h. 36 cycles provided the oocytes for 18 h rescue ICSI. Fertilization, implantation, pregnancy and take baby home rates were the main outcome measures compared between the 6 h and the 18 h group. A P value of <0.05 was considered statistically significant. RESULTS: Following early rescue ICSI, evidently fertilization was observed in 943 oocytes (75.7%) of 1245 metaphase IIoocytes. This was no significant different between 18 h group, in which 285 oocytes in metaphase II and 200(70.2%)showed evidence of fertilization. Grade I-II embryos rate in the 6 h vs 18 h was 67.4%(607/901)vs 45.1%(83/184)(P < 0.01). Clinical pregnancy rate in two groups was 55.9%(90/161)vs 16.7%(6/36)(P < 0.01). Implantation rate was 37.6%(121/322)vs 12.7%(9/71)(P < 0.01).Take baby home rate was 46.6%(75/161)vs 16.7%(6/36)(P < 0.05). After early rescue ICSI, 75 cases have succeeded delivery, and 97 babies were born. CONCLUSION: Identifying unfertilized oocytes within 6h relyed on the correct identification of polar bodies to indicate oocyte activation as the preliminary sign of fertilization before pronuclei developed. Rescue ICSI of unfertilized oocytes closer to the egg retrieval is more efficient. As results demonstrated, a higher ongoing and clinical PR was observed in 6 h rescue ICSI, to prove the method feasibility.
S109