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RESULTS: Until June 2005 we had performed 1.072 single blastocyst transfers. Of these, 522 (49%) women had a clinical pregnancy ( ⫽ implantation rate 49%) and 444 (41%) women were delivered. One or more blastocysts were frozen in 688 of these 1.072 cycles (64%). Until April 2006, we have thawed 303 of these cases; whereof 241 women had at least one viable blastocyst transferred. In these freeze/thaw cycles we transferred one or a maximum of two embryos, resulting in 68 (22% of all thawings and 28% of transfers) clinical pregnancies. A similar response in all freeze-thaw cycles would be expected. The 1.072 single blastocyst transfers ⫹ their resulting freeze/thaw cycles would then result in 673 clinical pregnancies (63% cumulative pregnancy rate of the cycles with an initial fresh single blastocyst transfer). CONCLUSION: Considering the large amount of cycles and the long time-span, we find the results reliable. We find them most encouraging and single blastocyst transfer is now performed in 80% of all blastocyst transfers at the clinic. It may be that in selected groups, single blastocyst tranfers and freeze/thaw of surplus good quality blastocysts is the optimal approach to fully use the pregnancy potential of one stimulated cycle, while at the same time avoiding duplex pregnancies. Supported by: None.
P-249 SUCCESSFUL CLINICAL PREGNANCY FOLLOWING THE TRANSFER OF EMBRYOS FROZEN AND THAWED TWICE: A CASE REPORT. D. Dasig, M. Dangcil Jr., W. Shen. Kaiser Permanente Center for Reproductive Health, Fremont, CA. OBJECTIVE: To report the ongoing clinical pregnancy of an IVF patient following the transfer of embryos frozen/thawed at the two pronucleus stage (2PN) then cultured to day 5 and frozen/thawed again at the blastocyst stage. DESIGN: Case Report. MATERIALS AND METHODS: An IVF cycle was performed using a normal ovarian stimulation protocol with GnRH agonist down regulation, recombinant FSH stimulation and recombinant hCG ovulation-induction. Half of oocytes retrieved underwent standard insemination and the remaining half was fertilized by intracytoplasmic sperm injection (ICSI). Embryo culture was performed using Quinn’s Advantage Cleavage Media (QAC) supplemented with 10% v/v Serum Substitute Supplement (SSS) in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 37o C. All resulting 2PN’s were cryopreserved using a standard propanediol and sucrose slow freezing protocol in a controlled-rate liquid nitrogen (LN2) freezer. 2PN’s were thawed with a multi-step thaw protocol in sucrose solutions at room temperature. The zygotes were then cultured again in QAC Medium for 48 hrs under similar culture conditions. Blastocyst culture was performed using Quinn’s Blastocyst Medium also in a humidified low oxygen environment at 37oC to Day 5 and Day 6. Blastocyst cryopreservation was performed on Day 6 on all remaining blastocysts of grade 3BB or better using a two-step slow-freezing protocol (Menezo) with glycerol and sucrose in a controlled rate LN2 freezer. Blastocysts were thawed with a two-step thaw protocol in sucrose solutions (Menezo). Recovery and re-expansion was conducted in Quinn’s Blastocyst Medium supplemented with 20% SSS in a humidified low oxygen environment at 37oC for two hours before embryo transfer. Laser assisted hatching was performed on all frozen embryos transferred. RESULTS: A 34-year-old female (gravida 0) with a diagnosis of polycystic ovarian disease and BMI of 37.6 underwent controlled ovarian hyperstimulation for IVF. The cycle resulted in 29 oocytes which were split between standard insemination and ICSI. 17- 2PN’s, 8 from standard insemination and 9 from ICSI, were observed on Day 1 all of which were cryopreserved due to risks associated with ovarian hyperstimulation syndrome. Since then, three FET cycles followed. First 5 zygotes from the IVF group were thawed yielding the transfer of 2 cleavage stage embryos and 3 embryos cultured to the blastocyst stage. One blastocyst (5AB) was refrozen. After a negative pregnancy test the patient underwent a second FET cycle. 5 zygotes from the ICSI group were thawed yielding the transfer of 3 cleavage stage embryos and 2 embryos cultured to the blastocyst stage. 2 blastocysts were frozen (4AA, 3BA). Again a negative pregnancy test ensued. The third FET cycle was composed of a blastocyst thaw using the one blastocyst (5AB) from the first FET cycle and both of the blastocysts (4AA,3BA) from the second FET cycle. All 3 blastocysts survived (90%, 70%, 70%) the thaw to be transferred following laser assisted hatching. A
FERTILITY & STERILITY威
positive pregnancy test resulted and subsequent 6 and 8 week ultrasound scans revealed a viable singleton intrauterine pregnancy. CONCLUSION: Human embryos are capable of withstanding more than one freeze/thaw cycle without impairing their ability to develop and implant. Furthermore, additional data relating to the favorable outcome of fetuses born from twice frozen/thawed embryos is needed before such practices are recommended routinely. Supported by: None.
P-250 SINGLE BLASTOCYST TRANSFER - ENCOURAGING RESULTS OF 1.072 CYCLES. U. Waldenstro¨m, S. Nilsson, A. Engstrom, D. Hellberg. IVF Falun, Falun, Sweden. OBJECTIVE: To prospectively compare pregnancy rates between Two Blastocyst and Single Blastocyst transfer in selected groups. DESIGN: Prospective non-randomised study MATERIALS AND METHODS: In Jan. 2002, we started blastocyst culture for all women ⬍39 years of age with ⫽⬎5 oocytes fertilised. If a high risk of twin pregnancy was calculated from the woman⬘s age and the embryo score; we performed single blastocyst transfer for that selected group of women. The other women had two blastocysts transferred, none had more. RESULTS: We present the results of transfers done until June 2005. Of 1.883 initiated blastocyst culture cycles, 1.725 (92%) had a blastocyst transfer performed; after exclusion of cycles leading to total freezing because of hyperstimulation and cases where no good blastocyst was formed. Single Blastocyst transfer was performed in 1.072 cycles, whereof 522 (49%) had a clinical pregnancy and 444 (41%) were delivered, whereof three were monozygote twin pregnancies.. In 688 (64%) of these cycles, one or more blastocysts were frozen. Two Blastocysts transfer was performed in 653 cycles, whereof 264 (40%) had a clinical pregnancy and 216 (33%) were delivered. In 157 (24%) of these cycles, one or more blastocysts were frozen. CONCLUSION: In a broadly selected group, the great majority of blastocyst cultures result in at least one good blastocyst. After a further simple selection of 62% of these cycles, a single blastocyst transfer result in most encouraging pregnancy rates. It also avoids the risks of twin pregnancies and results in many potential subsequent pregnancies from freeze cycles. At our clinic, it is now the preferred method in 80% of all blastocyst transfers. Considering the large material and the long time span, we find the results reliable. Supported by: None.
P-251 SELECTING EARLY STAGE EMBRYOS BY USING STRICT MORPHOLOGY GRADING PREDICTS HIGH QUALITY EMBRYOS AND IMPROVES FETAL HEARTBEAT RATES. S. T. McLellan, A. Finn, T. F. O’Leary, J. A. Hill III, L. A. Scott. Fertility Centers of New England, Reading, MA. OBJECTIVE: To determine if selecting early stage embryos using strict grading predicts high quality day three (D3) embryos and/or increases fetal heartbeat (FHB) rates. DESIGN: Included in this study were normally fertilized embryos that did not undergo pre-implantation genetic diagnosis and were part of a cohort of ⱖ4 embryos. Embryos in the developmental study group met the main inclusion criteria and were cultured to at least D3. For FHB analyses, embryos that participated in an embryo transfer (ET) cycle and met the main inclusion criteria were included. MATERIALS AND METHODS: Embryos were strictly graded on day one (D1), day two (D2), and D3. The embryologist selected embryos on D1 and/or D2, if any, considered to be of highest quality. Preferred characteristics on D1 were equal numbers of nucleolar precursor bodies (npbs; difference ⱕ2), symmetrical alignment of npbs, and overall good zygote appearance. D2 embryos having 2-4 cells of appropriate size, ⱕ10% fragmentation, absence of multinucleation in blastomeres, and good overall appearance were considered for selection. D3 embryos were given a grade using a scale of 1-5. Grade 1 had appropriate size blastomeres and no fragmentation. Grade 2 had appropriate cell sizes and ⱕ10% fragmentation.
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P-249 SUCCESSFUL CLINICAL PREGNANCY FOLLOWING THE TRANSFER OF EMBRYOS FROZEN AND THAWED TWICE: A CASE REPORT. D. Dasig, M. Dangcil Jr., W. Shen. Kaiser Permanente Center for Reproductive Health, Fremont, CA. OBJECTIVE: To report the ongoing clinical pregnancy of an IVF patient following the transfer of embryos frozen/thawed at the two pronucleus stage (2PN) then cultured to day 5 and frozen/thawed again at the blastocyst stage. DESIGN: Case Report. MATERIALS AND METHODS: An IVF cycle was performed using a normal ovarian stimulation protocol with GnRH agonist down regulation, recombinant FSH stimulation and recombinant hCG ovulation-induction. Half of oocytes retrieved underwent standard insemination and the remaining half was fertilized by intracytoplasmic sperm injection (ICSI). Embryo culture was performed using Quinn’s Advantage Cleavage Media (QAC) supplemented with 10% v/v Serum Substitute Supplement (SSS) in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 37o C. All resulting 2PN’s were cryopreserved using a standard propanediol and sucrose slow freezing protocol in a controlled-rate liquid nitrogen (LN2) freezer. 2PN’s were thawed with a multi-step thaw protocol in sucrose solutions at room temperature. The zygotes were then cultured again in QAC Medium for 48 hrs under similar culture conditions. Blastocyst culture was performed using Quinn’s Blastocyst Medium also in a humidified low oxygen environment at 37oC to Day 5 and Day 6. Blastocyst cryopreservation was performed on Day 6 on all remaining blastocysts of grade 3BB or better using a two-step slow-freezing protocol (Menezo) with glycerol and sucrose in a controlled rate LN2 freezer. Blastocysts were thawed with a two-step thaw protocol in sucrose solutions (Menezo). Recovery and re-expansion was conducted in Quinn’s Blastocyst Medium supplemented with 20% SSS in a humidified low oxygen environment at 37oC for two hours before embryo transfer. Laser assisted hatching was performed on all frozen embryos transferred. RESULTS: A 34-year-old female (gravida 0) with a diagnosis of polycystic ovarian disease and BMI of 37.6 underwent controlled ovarian hyperstimulation for IVF. The cycle resulted in 29 oocytes which were split between standard insemination and ICSI. 17- 2PN’s, 8 from standard insemination and 9 from ICSI, were observed on Day 1 all of which were cryopreserved due to risks associated with ovarian hyperstimulation syndrome. Since then, three FET cycles followed. First 5 zygotes from the IVF group were thawed yielding the transfer of 2 cleavage stage embryos and 3 embryos cultured to the blastocyst stage. One blastocyst (5AB) was refrozen. After a negative pregnancy test the patient underwent a second FET cycle. 5 zygotes from the ICSI group were thawed yielding the transfer of 3 cleavage stage embryos and 2 embryos cultured to the blastocyst stage. 2 blastocysts were frozen (4AA, 3BA). Again a negative pregnancy test ensued. The third FET cycle was composed of a blastocyst thaw using the one blastocyst (5AB) from the first FET cycle and both of the blastocysts (4AA,3BA) from the second FET cycle. All 3 blastocysts survived (90%, 70%, 70%) the thaw to be transferred following laser assisted hatching. A
FERTILITY & STERILITY威
positive pregnancy test resulted and subsequent 6 and 8 week ultrasound scans revealed a viable singleton intrauterine pregnancy. CONCLUSION: Human embryos are capable of withstanding more than one freeze/thaw cycle without impairing their ability to develop and implant. Furthermore, additional data relating to the favorable outcome of fetuses born from twice frozen/thawed embryos is needed before such practices are recommended routinely. Supported by: None.
P-250 SINGLE BLASTOCYST TRANSFER - ENCOURAGING RESULTS OF 1.072 CYCLES. U. Waldenstro¨m, S. Nilsson, A. Engstrom, D. Hellberg. IVF Falun, Falun, Sweden. OBJECTIVE: To prospectively compare pregnancy rates between Two Blastocyst and Single Blastocyst transfer in selected groups. DESIGN: Prospective non-randomised study MATERIALS AND METHODS: In Jan. 2002, we started blastocyst culture for all women ⬍39 years of age with ⫽⬎5 oocytes fertilised. If a high risk of twin pregnancy was calculated from the woman⬘s age and the embryo score; we performed single blastocyst transfer for that selected group of women. The other women had two blastocysts transferred, none had more. RESULTS: We present the results of transfers done until June 2005. Of 1.883 initiated blastocyst culture cycles, 1.725 (92%) had a blastocyst transfer performed; after exclusion of cycles leading to total freezing because of hyperstimulation and cases where no good blastocyst was formed. Single Blastocyst transfer was performed in 1.072 cycles, whereof 522 (49%) had a clinical pregnancy and 444 (41%) were delivered, whereof three were monozygote twin pregnancies.. In 688 (64%) of these cycles, one or more blastocysts were frozen. Two Blastocysts transfer was performed in 653 cycles, whereof 264 (40%) had a clinical pregnancy and 216 (33%) were delivered. In 157 (24%) of these cycles, one or more blastocysts were frozen. CONCLUSION: In a broadly selected group, the great majority of blastocyst cultures result in at least one good blastocyst. After a further simple selection of 62% of these cycles, a single blastocyst transfer result in most encouraging pregnancy rates. It also avoids the risks of twin pregnancies and results in many potential subsequent pregnancies from freeze cycles. At our clinic, it is now the preferred method in 80% of all blastocyst transfers. Considering the large material and the long time span, we find the results reliable. Supported by: None.
P-251 SELECTING EARLY STAGE EMBRYOS BY USING STRICT MORPHOLOGY GRADING PREDICTS HIGH QUALITY EMBRYOS AND IMPROVES FETAL HEARTBEAT RATES. S. T. McLellan, A. Finn, T. F. O’Leary, J. A. Hill III, L. A. Scott. Fertility Centers of New England, Reading, MA. OBJECTIVE: To determine if selecting early stage embryos using strict grading predicts high quality day three (D3) embryos and/or increases fetal heartbeat (FHB) rates. DESIGN: Included in this study were normally fertilized embryos that did not undergo pre-implantation genetic diagnosis and were part of a cohort of ⱖ4 embryos. Embryos in the developmental study group met the main inclusion criteria and were cultured to at least D3. For FHB analyses, embryos that participated in an embryo transfer (ET) cycle and met the main inclusion criteria were included. MATERIALS AND METHODS: Embryos were strictly graded on day one (D1), day two (D2), and D3. The embryologist selected embryos on D1 and/or D2, if any, considered to be of highest quality. Preferred characteristics on D1 were equal numbers of nucleolar precursor bodies (npbs; difference ⱕ2), symmetrical alignment of npbs, and overall good zygote appearance. D2 embryos having 2-4 cells of appropriate size, ⱕ10% fragmentation, absence of multinucleation in blastomeres, and good overall appearance were considered for selection. D3 embryos were given a grade using a scale of 1-5. Grade 1 had appropriate size blastomeres and no fragmentation. Grade 2 had appropriate cell sizes and ⱕ10% fragmentation.
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