- Email: [email protected]
Sodium hyaluronate (HA) supplementation of an ICE DMSO-free blastocyst (BL) vitrification solution supports high survival and implantation of PGS-BL
rates were 12.1% for fresh, 18.8% for shared, and 1.2% for frozen cycles (p¼0.02). The time from patient’s initial donor consult visit to cycle start was 217 days for fresh, 210 days for shared, and 172 days for frozen cycles (p¼0.006). Patient cost per transfer was $29,621 for fresh, $19,792 for shared, and $17,500 for frozen cycles. Patient cost per clinical pregnancy was $49,530 for fresh, $34,307 for shared, and $30,532 for frozen cycles. For the last 6 months of the study period, cycles utilizing frozen eggs comprised 61% of all donor egg cycles, indicating a high level of patient acceptance and ease of use.
(p<0.001). Blastocyst cell number analysis from zygote vitrification after 96h embryo development showed an improvement (p<0.05) for auto (n¼46), 993cells over manual (n¼35), 893cells. Fresh embryos (n¼46) were 1094cells. CONCLUSION: Osmotic shock is more accurately described as ‘‘% volume drop/sec’’. A minimum osmotic stress cryo-exchange profile made possible by microfluidics demonstrated that minimizing osmotic stress improved embryo development in comparison to manual pipetting vitrification protocols. Supported by: NIH GM096040.
% Frozen Donor Egg Cycles
Mar 2012 Apr May Jun Jul Aug Sep Oct Nov Dec Jan 2013 Feb
Frozen cycles (n)
Total cycles (n)
% Frozen cycles
0 3 2 1 3 1 14 10 17 5 14 13
25 19 14 23 12 18 23 23 24 7 23 20
0% 16% 14% 4% 25% 6% 61% 43% 71% 71% 61% 65%
CONCLUSION: Clinical pregnancy rates are similar between fresh, shared, and frozen cycles. Frozen donor egg cycles have significantly lower cancellation rates, shorter time to cycle start, and are more cost-effective than fresh and shared donor egg cycles. A frozen donor egg program can be successfully implemented within one year.
O-349 Wednesday, October 16, 2013 04:30 PM AUTOMATED MICROFLUIDIC GRADIENT CRYOPROTECTANT EXCHANGE PLATFORM FOR MURINE OOCYTE AND ZYGOTE VITRIFICATION REDUCES OSMOTIC STRESS AND IMPROVES EMBRYO DEVELOPMENTAL COMPETENCE. D. Lai,a,b J. Ding,b,c G. D. Smith,b,c,d,e S. Takayama.a,b,f,g aBiomedical Engineering, University of Michigan, Ann Arbor, MI; bReproductive Sciences Program, University of Michigan, Ann Arbor, MI; cObstetrics & Gynecology, University of Michigan, Ann Arbor, MI; dMolecular & Integrative Physiology, University of Michigan, Ann Arbor, MI; eUrology, University of Michigan, Ann Arbor, MI; fMacromolecular Science and Engineering, University of Michigan, Ann Arbor, MI; gNano-Bio and Chemical Enginering WCU Project, UNIST, Ulsan, Republic of Korea. OBJECTIVE: Redefine osmotic shock for vitrification and improve vitrification protocols by minimizing osmotic stress via cryo-exchange made possible by microfluidics. DESIGN: Negative control groups were non-cryopreserved (fresh) murine oocytes and zygotes. Experimental groups were oocytes and zygotes vitrified by pipetting (manual) with an equilibration phase, and a group of oocytes and zygotes vitrified by an automated microfluidic device (auto) delivering a mathematically derived minimal osmotic shock. Positive control group was designed for maximum osmotic shock (dunk) by directly exposing cells to vitrification solution (VS). All experimental groups used the same VS with permeable and impermeable solutes. Double-blind experiments were used. MATERIALS AND METHODS: We derived a new definition for osmotic shock using mathematical modeling by Kedem-Katchalsky equations and used a microfluidic device for validation. Cells were vitrified according to their experimental groups and warmed manually. Oocytes were analyzed for t¼0h cryosurvival between dunk, manual and auto groups. Zygotes were cultured for 96h and cell number recorded for fresh, manual and auto groups. Statistical analyses were done using fisher’s exact test or student’s t-test. RESULTS: Osmotic shock is redefined as ‘‘% volume drop/sec’’ instead of ‘‘minimum cell volume’’. Each group had identical minimum oocyte volume but dramatically different shrinkage rate and cryosurvival: dunk (n¼38) 56.25.1%, manual (n¼80) 1000% (p<0.001), auto (n¼100) 1000%
FERTILITY & STERILITYÒ
O-350 Wednesday, October 16, 2013 04:45 PM OOCYTE SLOW FREEZING AND OOCYTE VITRIFICATION: DATA FROM THE ITALIAN ART REGISTER 2007-2011. P. E. Levi P. Patrizio,d E. Porcu,c V. Vigiliano,b R. De Luca,b Setti,a b a G. Scaravelli. Department of Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Clinical and Research Institute, Rozzano, Milan, Italy; bART Italian National Register, National Centre for Epidemiology, Surveillance and Health Promotion, National Health Institute, Rome, Italy; cInfertility and IVF Center S. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy; dDepartment of Obtetrics, Gynecology and Reproductive Science, Yale School of Medicine, New Heaven, CT. OBJECTIVE: To analyze slow freezing/rapid thawing or vitrification in one of the largest worldwide experience of oocyte cryopreservation. DESIGN: Retrospective analysis of data submitted to the registry by all centers performing ART in Italy during the period 2007-2011. MATERIALS AND METHODS: All cycles were analyzed from aggregated data and both pregnancy rate for started cycle and transfer were calculated for each year and in the total sample. Data considered any method of oocyte cryopreservation (slow freezing or vitrification). RESULTS: Across all ages a total of 14,328 cycles, 11,599 transfers and 1,850 pregnancies (12.9% per started cycle and 15.9% per transfer) were analyzed. Of these, 8,927 cycles, 7,276 transfers and 1,074 pregnancies were after slow freezing and 5,401 cycles, 4,323 transfers and 776 pregnancies after vitrification. A significantly higher pregnancy rate per started cycle was found likely due to the high statistical power of the sample (14.8 vs 18.0 p¼0.0000). The difference in pregnancy rate was 2.4% for started cycle and 3.2% per transfer higher. Pregnancy Rate / Started Cycle (%) Slow Years Freezing Vitrification 2007 2008 2009 2010 2011 Total
10.5 11.9 12.4 13.8 13.8 12.0
12.9 13.7 16.5 13.7 14.2 14.4
Pregnancy Rate / Transfer (%) p
0,1013 0,2148 0,0014 0,9577 0,7928 0,0001
Slow Freezing Vitrification 13.1 14.6 15.0 16.9 16.7 14.8
15.1 17.1 20.7 17.2 17.9 18.0
p 0,2364 0,1509 0,0002 0,8268 0,5082 0,0000
CONCLUSION: These data represent mean results of centers with leading experience in one or both cryopreservation protocols and data from centers with a limited number of cycles and poorer results either with slow freezing or vitrificaton. As for other ART procedures the results are not homogeneous among clinics and protocols, but confirm the clinical value of oocyte cryopreservation in infertile patients. Even with all these biases vitrification showed a small, but statistically significant higher performance.
O-351 Wednesday, October 16, 2013 05:00 PM SODIUM HYALURONATE (HA) SUPPLEMENTATION OF AN ICE DMSO-FREE BLASTOCYST (BL) VITRIFICATION SOLUTION SUPPORTS HIGH SURVIVAL AND IMPLANTATION OF PGSJ. Whitney,a S. Zozula,a N. Nugent,a BL. M. C. Schiewe,a R. E. Anderson,a J. J. Stachecki.b aART Lab, Southern California Institute
S107
for Reproductive Sciences, Newport Beach, CA; bInnovative Cryo Enterprises (ICE) LLC, Linden, NJ. OBJECTIVE: The aim of this study was to determine if the addition of HA to a BL vitrification (VTF) solution promotes membrane stabilization of cryopreserved cellular tissue. DESIGN: Prospective, randomized study. MATERIALS AND METHODS: In 2011, we began prospectively vitrified PGS cases with (+) or without (-) HA for Day 6 or D5 BLs, respectively. In 2012, we randomly assigned all PGS cases, by patient, to VTF +HA or -HA. Aseptic microSecure (mS)–VTF was applied to all PGS-BLs using ICE VTF-BL solutions/3-step dilution. mS-VTF is a FDA compliant method that offers superior QC characteristics, including high-security weld seals, insitu vapor storage, and dual-colored, tamperproof labeling. Thirty warming cycles were conducted and chi-squared analysis was performed to compare survival rates, pregnancy outcomes and implantation rates. RESULTS: The mean age (SE) of PGS patients was 35.1 1.6 (-HA, n¼13) and 35.41.4 (+HA, n¼17). mS-VTF resulted in over 99% survival of PGS-BLs independent of HA supplement. Furthermore, no difference (PR 0.2) in clinical pregnancy rates (+HA: 94.1%; -HA: 84.6%) or implantation rates (+HA: 87.5%; -HA: 72.0%) was determined to-date, eventhough the mean number of BLs transferred tended to be lower in the +HA group (1.33 vs 1.67). All single euploid BL-VETs (17 of 30) resulted in a pregnancy. CONCLUSION: In combination with ICE DMSO-free BL-VTF solutions, BL viability remains unchanged from its fresh-state. The addition of a large macromolecule like HA offers dual support by increasing both the viscosity of the VTF solution and supplemental cellular adhesion/membrane stabilization. If the trend toward higher implantation rates +HA becomes significant with a higher sample size, it would support the latter theory, as all BLs were cultured and transferred in the presence of HA. Overall, the success of mSVTF of BLs has greatly facilitated single ET and Day5/6 trophectoderm biopsy/PGS as practical ART options. O-352 Wednesday, October 16, 2013 05:15 PM A SIMPLIFIED COLLAPSING TECHNIQUE USING TREHALOSE DOES NOT COMPROMISE VITRIFICATION OUTCOMES. C. Valdespin,a M. Elhelaly,b S. Catt,c P. Chen,c A. Colin,a a a G. Barroso. Reproductive Health Research, Nascere Reproductive Centre, Mexico City, Mexico; bEmbryology Department, Al-ahram Fertility Centre, Mansoura, Egypt; cObstetrics and Gynaecology, Monash University, Melbourne, Victoria, Australia. OBJECTIVE: To evaluate if a hyperosmotic solution based on trehalose is as effective as the laser pulse technique for collapsing the blastocoele cavity of expanded blastocysts prior to vitrification. DESIGN: Experimental, prospective cohort-study. MATERIALS AND METHODS: A total of 329 expanded mouse blastocysts were assigned to 3 different groups: i) collapsing via laser pulse [LP], ii) collapsing via trehalose exposure pre-vitrification [TPV]), or iii) a control group. Vitrification was performed using a short protocol on Cryotops. After warming, survival, re-expansion, and hatching rates were evaluated. In addition, selected embryos from both collapsing groups were transferred to pseudo-pregnant mice, each recipient receiving embryos from both groups (TPV&LP) to assess implantation rate. The remaining cultured blastocysts were evaluated for re-expansion, and hatching rates at 24h post-warm. RESULTS: Blastocyst collapse prior to vitrification resulted in significantly higher survival rates in both collapsing groups (LP 97.3% [142/ 146] and TPV 98.9% [91/92]) when compared to the control group (86.8% [79/91], p¼0.01 in both). Similarly, re-expansion and hatching rates after 24 hours in both collapsing groups were higher than the control group (LP: 85.6% [125/146] and TPV 87.0% [80/92] vs 74.7% [69/91], p¼0.04 and p¼0.02, respectively); (LP 58.2% [46/79] and TPV 52.9% [74/140] vs 21.9% [14/64], p¼0.001 in both). There was non significant differences observed between both collapsing groups. Implantation sacs were observed in 4/6 uterine horns, 2 each from the LP and TPV transfers. CONCLUSION: In conclusion, collapsing expanded mouse blastocysts prior to vitrification using a short equilibration protocol is associated with higher survival, re-expansion, and hatching rates with proven implantation when compared to a control group. The use of TPV offers comparable results to LP; therefore, it provides an excellent option to use in a setting where there is no access to expensive devices and equipment. Supported by: Monash University.
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ASRM Abstracts
O-353 Wednesday, October 16, 2013 05:30 PM A NOVEL METHOD FOR HUMAN OOCYTE VITRIFICATION WITH A CLOSED DEVICE USING SUPER-COOLED AIR. S. Machac,a V. Hubinka,a M. Larman,b M. Koudelka.a aReprofit International s.r.o, Brno, Czech Republic; bVitrolife, Englewood, CO. OBJECTIVE: Human oocytes have been successfully vitrified with open systems that involve direct contact with liquid nitrogen. Concerns have been raised regarding the safety and sterility of this methodology. It is therefore highly desirable to use a device that does not utilize direct contact with liquid nitrogen during vitrification and storage. It is thought, however, that the high cooling rate afforded by direct liquid nitrogen contact is essential for human oocyte vitrification. DESIGN: Human oocytes were collected from donors and vitrified/ warmed for transfer into recipients. Following oocyte warming; fertilization, embryo development, implantation, biochemical pregnancy and clinical pregnancy rates were recorded. The results were compared retrospectively to data from non-vitrified oocytes within the same time period. MATERIALS AND METHODS: Almost 600 oocytes from 53 donors were vitrified using a newly developed vitrification medium specifically for oocytes (RapidVit and RapidWarm Oocyte: Vitrolife AB) and a closed vitrification device that uses a straw with super-cooled air (Rapid-iÔ: Vitrolife AB). RESULTS: Oocytes were collected from 53 oocyte donors (mean age of 24.6 4.0) and vitrified/warmed. The survival and fertilization rates were 94 % and 76 %, respectively. A mean number of 1.8 0.5 embryos were transferred on day 5/6 per recipient (mean age 40.4 4.6). The implantation rate was 34.4 % (33/96). The biochemical and clinical pregnancy rates were 65 % (33/51) and 51 % (26/51), respectively. The ongoing (>12 weeks) pregnancy rate was 49 % with 5 healthy live births reported so far. These results were not statistically different from outcomes with non-vitrified oocytes. CONCLUSION: This study demonstrates that, despite lower cooling rates, human oocytes can be vitrified without direct contact with liquid nitrogen in a closed device (Rapid-iÔ). A closed system that uses super-cooled air to vitrify removes the potential concerns regarding contamination from liquid nitrogen. Supported by: Vitrolife AB. O-354 Wednesday, October 16, 2013 05:45 PM SIGNIFICANT IMPROVEMENT IN PREGNANCY RATES FOLLOWING FROZEN EMBRYO TRANSFERS WHEN COMBINED WITH MICROARRAY-COMPARATIVE GENOMIC HYM. Surrey,a,b H. Danzer,a,b BRIDIZATION (aCGH). D. Hill,a S. Ghadir,a,b W. Chang,b J. Barritt.a aART Reproductive Center, Beverly Hills, CA; bSouthern California Reproductive Center, Beverly Hills, CA. OBJECTIVE: There is a growing body of evidence that embryo transfer (ET) of vitrified blastocysts in an unstimulated cycle improves implantation rates due to a more receptive uterine environment. Evidence is also growing that 24-chromosome screening to select euploid embryos improves implantation rates. This study looks at the effect of combining these two procedures on pregnancy rates by comparing outcomes of fresh versus warmed transfers of blastocysts that were screened by aCGH and those that were not. DESIGN: Retrospective data analysis at a private fertility clinic. MATERIALS AND METHODS: 827 sequential, non-randomized, ET’s performed over 11-months during 2012 – 2013 were evaluated. Fresh ET was performed at the blastocyst stage on patients with a mean age of 34.8 years. Warmed ET was performed in unstimulated cycles after blastocysts were vitrified then individually warmed using the CryotopÒ method (Kitazato). Trophectoderm biopsy followed by aCGH testing (Reprogenetics) was performed on Day 5 or 6 on patients with a mean age of 38.6 years. RESULTS: Significant improvement in clinical pregnancy (CP) was demonstrated in all warmed ETs following aCGH testing (*p<0.0216). A trend in CP increase was found for both: warmed ET vs. fresh ET without testing (^p<0.0858) and for warmed ET vs. fresh ET following aCGH testing (#p<0.0520).
Clinical Pregnancy Rates
Untested aCGH Tested
Fresh ET
Warmed ET
144/391 (37%)^ 35/82 (43%)#
107/245 (44%)* 62/109 (57%)*
Vol. 100, No. 3, Supplement, September 2013
(p<0.001). Blastocyst cell number analysis from zygote vitrification after 96h embryo development showed an improvement (p<0.05) for auto (n¼46), 993cells over manual (n¼35), 893cells. Fresh embryos (n¼46) were 1094cells. CONCLUSION: Osmotic shock is more accurately described as ‘‘% volume drop/sec’’. A minimum osmotic stress cryo-exchange profile made possible by microfluidics demonstrated that minimizing osmotic stress improved embryo development in comparison to manual pipetting vitrification protocols. Supported by: NIH GM096040.
% Frozen Donor Egg Cycles
Mar 2012 Apr May Jun Jul Aug Sep Oct Nov Dec Jan 2013 Feb
Frozen cycles (n)
Total cycles (n)
% Frozen cycles
0 3 2 1 3 1 14 10 17 5 14 13
25 19 14 23 12 18 23 23 24 7 23 20
0% 16% 14% 4% 25% 6% 61% 43% 71% 71% 61% 65%
CONCLUSION: Clinical pregnancy rates are similar between fresh, shared, and frozen cycles. Frozen donor egg cycles have significantly lower cancellation rates, shorter time to cycle start, and are more cost-effective than fresh and shared donor egg cycles. A frozen donor egg program can be successfully implemented within one year.
O-349 Wednesday, October 16, 2013 04:30 PM AUTOMATED MICROFLUIDIC GRADIENT CRYOPROTECTANT EXCHANGE PLATFORM FOR MURINE OOCYTE AND ZYGOTE VITRIFICATION REDUCES OSMOTIC STRESS AND IMPROVES EMBRYO DEVELOPMENTAL COMPETENCE. D. Lai,a,b J. Ding,b,c G. D. Smith,b,c,d,e S. Takayama.a,b,f,g aBiomedical Engineering, University of Michigan, Ann Arbor, MI; bReproductive Sciences Program, University of Michigan, Ann Arbor, MI; cObstetrics & Gynecology, University of Michigan, Ann Arbor, MI; dMolecular & Integrative Physiology, University of Michigan, Ann Arbor, MI; eUrology, University of Michigan, Ann Arbor, MI; fMacromolecular Science and Engineering, University of Michigan, Ann Arbor, MI; gNano-Bio and Chemical Enginering WCU Project, UNIST, Ulsan, Republic of Korea. OBJECTIVE: Redefine osmotic shock for vitrification and improve vitrification protocols by minimizing osmotic stress via cryo-exchange made possible by microfluidics. DESIGN: Negative control groups were non-cryopreserved (fresh) murine oocytes and zygotes. Experimental groups were oocytes and zygotes vitrified by pipetting (manual) with an equilibration phase, and a group of oocytes and zygotes vitrified by an automated microfluidic device (auto) delivering a mathematically derived minimal osmotic shock. Positive control group was designed for maximum osmotic shock (dunk) by directly exposing cells to vitrification solution (VS). All experimental groups used the same VS with permeable and impermeable solutes. Double-blind experiments were used. MATERIALS AND METHODS: We derived a new definition for osmotic shock using mathematical modeling by Kedem-Katchalsky equations and used a microfluidic device for validation. Cells were vitrified according to their experimental groups and warmed manually. Oocytes were analyzed for t¼0h cryosurvival between dunk, manual and auto groups. Zygotes were cultured for 96h and cell number recorded for fresh, manual and auto groups. Statistical analyses were done using fisher’s exact test or student’s t-test. RESULTS: Osmotic shock is redefined as ‘‘% volume drop/sec’’ instead of ‘‘minimum cell volume’’. Each group had identical minimum oocyte volume but dramatically different shrinkage rate and cryosurvival: dunk (n¼38) 56.25.1%, manual (n¼80) 1000% (p<0.001), auto (n¼100) 1000%
FERTILITY & STERILITYÒ
O-350 Wednesday, October 16, 2013 04:45 PM OOCYTE SLOW FREEZING AND OOCYTE VITRIFICATION: DATA FROM THE ITALIAN ART REGISTER 2007-2011. P. E. Levi P. Patrizio,d E. Porcu,c V. Vigiliano,b R. De Luca,b Setti,a b a G. Scaravelli. Department of Gynecology, Division of Gynecology and Reproductive Medicine, Humanitas Clinical and Research Institute, Rozzano, Milan, Italy; bART Italian National Register, National Centre for Epidemiology, Surveillance and Health Promotion, National Health Institute, Rome, Italy; cInfertility and IVF Center S. Orsola-Malpighi University Hospital, University of Bologna, Bologna, Italy; dDepartment of Obtetrics, Gynecology and Reproductive Science, Yale School of Medicine, New Heaven, CT. OBJECTIVE: To analyze slow freezing/rapid thawing or vitrification in one of the largest worldwide experience of oocyte cryopreservation. DESIGN: Retrospective analysis of data submitted to the registry by all centers performing ART in Italy during the period 2007-2011. MATERIALS AND METHODS: All cycles were analyzed from aggregated data and both pregnancy rate for started cycle and transfer were calculated for each year and in the total sample. Data considered any method of oocyte cryopreservation (slow freezing or vitrification). RESULTS: Across all ages a total of 14,328 cycles, 11,599 transfers and 1,850 pregnancies (12.9% per started cycle and 15.9% per transfer) were analyzed. Of these, 8,927 cycles, 7,276 transfers and 1,074 pregnancies were after slow freezing and 5,401 cycles, 4,323 transfers and 776 pregnancies after vitrification. A significantly higher pregnancy rate per started cycle was found likely due to the high statistical power of the sample (14.8 vs 18.0 p¼0.0000). The difference in pregnancy rate was 2.4% for started cycle and 3.2% per transfer higher. Pregnancy Rate / Started Cycle (%) Slow Years Freezing Vitrification 2007 2008 2009 2010 2011 Total
10.5 11.9 12.4 13.8 13.8 12.0
12.9 13.7 16.5 13.7 14.2 14.4
Pregnancy Rate / Transfer (%) p
0,1013 0,2148 0,0014 0,9577 0,7928 0,0001
Slow Freezing Vitrification 13.1 14.6 15.0 16.9 16.7 14.8
15.1 17.1 20.7 17.2 17.9 18.0
p 0,2364 0,1509 0,0002 0,8268 0,5082 0,0000
CONCLUSION: These data represent mean results of centers with leading experience in one or both cryopreservation protocols and data from centers with a limited number of cycles and poorer results either with slow freezing or vitrificaton. As for other ART procedures the results are not homogeneous among clinics and protocols, but confirm the clinical value of oocyte cryopreservation in infertile patients. Even with all these biases vitrification showed a small, but statistically significant higher performance.
O-351 Wednesday, October 16, 2013 05:00 PM SODIUM HYALURONATE (HA) SUPPLEMENTATION OF AN ICE DMSO-FREE BLASTOCYST (BL) VITRIFICATION SOLUTION SUPPORTS HIGH SURVIVAL AND IMPLANTATION OF PGSJ. Whitney,a S. Zozula,a N. Nugent,a BL. M. C. Schiewe,a R. E. Anderson,a J. J. Stachecki.b aART Lab, Southern California Institute
S107
for Reproductive Sciences, Newport Beach, CA; bInnovative Cryo Enterprises (ICE) LLC, Linden, NJ. OBJECTIVE: The aim of this study was to determine if the addition of HA to a BL vitrification (VTF) solution promotes membrane stabilization of cryopreserved cellular tissue. DESIGN: Prospective, randomized study. MATERIALS AND METHODS: In 2011, we began prospectively vitrified PGS cases with (+) or without (-) HA for Day 6 or D5 BLs, respectively. In 2012, we randomly assigned all PGS cases, by patient, to VTF +HA or -HA. Aseptic microSecure (mS)–VTF was applied to all PGS-BLs using ICE VTF-BL solutions/3-step dilution. mS-VTF is a FDA compliant method that offers superior QC characteristics, including high-security weld seals, insitu vapor storage, and dual-colored, tamperproof labeling. Thirty warming cycles were conducted and chi-squared analysis was performed to compare survival rates, pregnancy outcomes and implantation rates. RESULTS: The mean age (SE) of PGS patients was 35.1 1.6 (-HA, n¼13) and 35.41.4 (+HA, n¼17). mS-VTF resulted in over 99% survival of PGS-BLs independent of HA supplement. Furthermore, no difference (PR 0.2) in clinical pregnancy rates (+HA: 94.1%; -HA: 84.6%) or implantation rates (+HA: 87.5%; -HA: 72.0%) was determined to-date, eventhough the mean number of BLs transferred tended to be lower in the +HA group (1.33 vs 1.67). All single euploid BL-VETs (17 of 30) resulted in a pregnancy. CONCLUSION: In combination with ICE DMSO-free BL-VTF solutions, BL viability remains unchanged from its fresh-state. The addition of a large macromolecule like HA offers dual support by increasing both the viscosity of the VTF solution and supplemental cellular adhesion/membrane stabilization. If the trend toward higher implantation rates +HA becomes significant with a higher sample size, it would support the latter theory, as all BLs were cultured and transferred in the presence of HA. Overall, the success of mSVTF of BLs has greatly facilitated single ET and Day5/6 trophectoderm biopsy/PGS as practical ART options. O-352 Wednesday, October 16, 2013 05:15 PM A SIMPLIFIED COLLAPSING TECHNIQUE USING TREHALOSE DOES NOT COMPROMISE VITRIFICATION OUTCOMES. C. Valdespin,a M. Elhelaly,b S. Catt,c P. Chen,c A. Colin,a a a G. Barroso. Reproductive Health Research, Nascere Reproductive Centre, Mexico City, Mexico; bEmbryology Department, Al-ahram Fertility Centre, Mansoura, Egypt; cObstetrics and Gynaecology, Monash University, Melbourne, Victoria, Australia. OBJECTIVE: To evaluate if a hyperosmotic solution based on trehalose is as effective as the laser pulse technique for collapsing the blastocoele cavity of expanded blastocysts prior to vitrification. DESIGN: Experimental, prospective cohort-study. MATERIALS AND METHODS: A total of 329 expanded mouse blastocysts were assigned to 3 different groups: i) collapsing via laser pulse [LP], ii) collapsing via trehalose exposure pre-vitrification [TPV]), or iii) a control group. Vitrification was performed using a short protocol on Cryotops. After warming, survival, re-expansion, and hatching rates were evaluated. In addition, selected embryos from both collapsing groups were transferred to pseudo-pregnant mice, each recipient receiving embryos from both groups (TPV&LP) to assess implantation rate. The remaining cultured blastocysts were evaluated for re-expansion, and hatching rates at 24h post-warm. RESULTS: Blastocyst collapse prior to vitrification resulted in significantly higher survival rates in both collapsing groups (LP 97.3% [142/ 146] and TPV 98.9% [91/92]) when compared to the control group (86.8% [79/91], p¼0.01 in both). Similarly, re-expansion and hatching rates after 24 hours in both collapsing groups were higher than the control group (LP: 85.6% [125/146] and TPV 87.0% [80/92] vs 74.7% [69/91], p¼0.04 and p¼0.02, respectively); (LP 58.2% [46/79] and TPV 52.9% [74/140] vs 21.9% [14/64], p¼0.001 in both). There was non significant differences observed between both collapsing groups. Implantation sacs were observed in 4/6 uterine horns, 2 each from the LP and TPV transfers. CONCLUSION: In conclusion, collapsing expanded mouse blastocysts prior to vitrification using a short equilibration protocol is associated with higher survival, re-expansion, and hatching rates with proven implantation when compared to a control group. The use of TPV offers comparable results to LP; therefore, it provides an excellent option to use in a setting where there is no access to expensive devices and equipment. Supported by: Monash University.
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ASRM Abstracts
O-353 Wednesday, October 16, 2013 05:30 PM A NOVEL METHOD FOR HUMAN OOCYTE VITRIFICATION WITH A CLOSED DEVICE USING SUPER-COOLED AIR. S. Machac,a V. Hubinka,a M. Larman,b M. Koudelka.a aReprofit International s.r.o, Brno, Czech Republic; bVitrolife, Englewood, CO. OBJECTIVE: Human oocytes have been successfully vitrified with open systems that involve direct contact with liquid nitrogen. Concerns have been raised regarding the safety and sterility of this methodology. It is therefore highly desirable to use a device that does not utilize direct contact with liquid nitrogen during vitrification and storage. It is thought, however, that the high cooling rate afforded by direct liquid nitrogen contact is essential for human oocyte vitrification. DESIGN: Human oocytes were collected from donors and vitrified/ warmed for transfer into recipients. Following oocyte warming; fertilization, embryo development, implantation, biochemical pregnancy and clinical pregnancy rates were recorded. The results were compared retrospectively to data from non-vitrified oocytes within the same time period. MATERIALS AND METHODS: Almost 600 oocytes from 53 donors were vitrified using a newly developed vitrification medium specifically for oocytes (RapidVit and RapidWarm Oocyte: Vitrolife AB) and a closed vitrification device that uses a straw with super-cooled air (Rapid-iÔ: Vitrolife AB). RESULTS: Oocytes were collected from 53 oocyte donors (mean age of 24.6 4.0) and vitrified/warmed. The survival and fertilization rates were 94 % and 76 %, respectively. A mean number of 1.8 0.5 embryos were transferred on day 5/6 per recipient (mean age 40.4 4.6). The implantation rate was 34.4 % (33/96). The biochemical and clinical pregnancy rates were 65 % (33/51) and 51 % (26/51), respectively. The ongoing (>12 weeks) pregnancy rate was 49 % with 5 healthy live births reported so far. These results were not statistically different from outcomes with non-vitrified oocytes. CONCLUSION: This study demonstrates that, despite lower cooling rates, human oocytes can be vitrified without direct contact with liquid nitrogen in a closed device (Rapid-iÔ). A closed system that uses super-cooled air to vitrify removes the potential concerns regarding contamination from liquid nitrogen. Supported by: Vitrolife AB. O-354 Wednesday, October 16, 2013 05:45 PM SIGNIFICANT IMPROVEMENT IN PREGNANCY RATES FOLLOWING FROZEN EMBRYO TRANSFERS WHEN COMBINED WITH MICROARRAY-COMPARATIVE GENOMIC HYM. Surrey,a,b H. Danzer,a,b BRIDIZATION (aCGH). D. Hill,a S. Ghadir,a,b W. Chang,b J. Barritt.a aART Reproductive Center, Beverly Hills, CA; bSouthern California Reproductive Center, Beverly Hills, CA. OBJECTIVE: There is a growing body of evidence that embryo transfer (ET) of vitrified blastocysts in an unstimulated cycle improves implantation rates due to a more receptive uterine environment. Evidence is also growing that 24-chromosome screening to select euploid embryos improves implantation rates. This study looks at the effect of combining these two procedures on pregnancy rates by comparing outcomes of fresh versus warmed transfers of blastocysts that were screened by aCGH and those that were not. DESIGN: Retrospective data analysis at a private fertility clinic. MATERIALS AND METHODS: 827 sequential, non-randomized, ET’s performed over 11-months during 2012 – 2013 were evaluated. Fresh ET was performed at the blastocyst stage on patients with a mean age of 34.8 years. Warmed ET was performed in unstimulated cycles after blastocysts were vitrified then individually warmed using the CryotopÒ method (Kitazato). Trophectoderm biopsy followed by aCGH testing (Reprogenetics) was performed on Day 5 or 6 on patients with a mean age of 38.6 years. RESULTS: Significant improvement in clinical pregnancy (CP) was demonstrated in all warmed ETs following aCGH testing (*p<0.0216). A trend in CP increase was found for both: warmed ET vs. fresh ET without testing (^p<0.0858) and for warmed ET vs. fresh ET following aCGH testing (#p<0.0520).
Clinical Pregnancy Rates
Untested aCGH Tested
Fresh ET
Warmed ET
144/391 (37%)^ 35/82 (43%)#
107/245 (44%)* 62/109 (57%)*
Vol. 100, No. 3, Supplement, September 2013