thawing cycles

TABLE 1. Outcome of Frozen Embryo Transfer (FET) vs. Frozen Oocyte Transfer (FOT)

No. Cycles No. Thawed Survival Rate Pregnancy Rate Implantation Rate

Frozen Embryo (FET)

Frozen Oocyte (FOT)

214 1096 80.5% (882/1096) 47.2% (101/214) 16.4% (125/763)

70 471 92% (433/471) 54.3% (38/70) 20.4% (61/299)

CONCLUSIONS: According to our data from 2005 through 2007, this comparison study illustrates a favorable outcome using a new slow freeze protocol for human oocyte cryopreservation. We are pursing an expansion of the present study to further evaluate the efficacy of oocyte cryopreservation as an alternative to embryo freezing. Supported by: EMD Serono.

P-505 EMBRYO VITRIFICATION FOR ICSI CASES UNDERGOING COASTING DURING OVARIAN STIMULATION. N. F. Hanafi, A. H. Abo Ali, W. S. El Ghazaerly. Lecurer Med. Microbiol./Embryologist, Faculty of Medicine, Alexandria University, Alexandria, Egypt; Fertility Lab, MAdina Fertility Center, Alexandria, Egypt; Obstetric & Gynecology Department, Madian Fertility Center, Alexandria, Egypt. OBJECTIVE: To assess outcome of vitrified embryo transfer in later stimulated cycle, in cases undergoing coasting. DESIGN: A retrospective comparative study. MATERIALS AND METHODS: This study included 105 cases that followed a long agonist protocol for ovarian stimulation, and who revealed more than 3 cleaved (4A/8A) non fragmented embryos. these cases underwent vitrification of the supernumerary embryos using cryotip virtrification (Irvine Scientifica, USA). Cases were divided into 2 groups: group 1 included 36 case undergoing coasting during ovarian stimulation and group 2 included 69 cases not undergoing coasting. Cases underwent fresh embryo transfer followed by vitrified/thawed embryo transfer in case of negative pregnancy test. Comparison of pregnancy rates of coasting versus no coasting during ovarian stimulation of cases undergoing ICSI in their fresh and later frozen embryo transfers was performed. RESULTS: We found by comparing pregnancy in coasted and non coasted cases that coasting more than 2 days significantly decreased pregnancy in fresh embryo transfer.(c2¼ 4.105, P>0.05).It was found that 11 out of 36(30.5%) cases of fresh embryo transfer (ET) undergoing coasting more than 2 days got pregnant (positive foetal pulsation). While 12 out of 19 (63.2%) of later frozen embryo transfer got pregnant for cases undergoing coasting in the original stimulation cycle. This reveals that pregnancy rate significantly increased in later thawed embryo transfer. TABLE 1. Comparison of pregnancy rates of coasting versus no coasting during ovarian stimulation of cases undergoing ICSI in their fresh and later frozen embryo transfers

Pregnancy rate of cases

Fresh embryo transfer

Frozen embryo transfer in later cycle

With coasting Without coasting

30.5% (11/36) 69.5% (48/69)

63.2% (12/19) 17.5% (4/23)

CONCLUSIONS: Vitrification of all embryos for embryo transfer in later cycle may lead to marked improvement in the pregnancy rate in cases with severe coating as the later may have a negative impact on endometrium compromising implantation in presence of good quality embryos. Supported by: Al-Hasani,et al. Three years of routine vitrification of human zygotes: is it still fair to advocate slow-rate freezing? Reproductive BioMedicine Online Vol 14. No 3. 2007 288-293. Arslan M et al. Effect of coasting on the implantation potential of embryos transferred after cryopreservation and thawing. Fertil Steril. 2005 Oct;84(4):867-74.

FERTILITY & STERILITYÒ

P-506 COMPACTION AND BLASTULATION AFTER THAWING AND OVERNIGHT CULTURE OF FROZEN DAY 3 EMBRYOS ARE RELIABLE PREDICTORS OF CLINICAL PREGNANCY. L. Van Landuyt, E. Van den Abbeel, H. Van de Velde, M. Camus, P. Devroey, J. Van der Elst. Centre for Reproductive Medicine, UZ Brussel, Brussels, Belgium. OBJECTIVE: Single embryo transfer (SET) has become daily practice in Belgium in view of a government funded program to reduce multiple pregnancies. This attitude tends to extend now to transfer of frozen embryos. Characterization of the single best frozen-thawed embryo for transfer has become a necessity. We analysed impact of embryo stage before freezing, cell loss and embryo development between thawing and transfer on clinical pregnancy rates in single frozen embryo transfers. DESIGN: Retrospective analysis of 526 single frozen day 3 embryo transfers carried out between January 2004 and October 2007. MATERIALS AND METHODS: Day 3 embryos with at least 6 blastomeres and %20% fragmentation were frozen using a slow-controlled DMSO protocol. Embryos surviving thawing were cultured overnight in Medicult BlastAssist medium. Embryo stage before freezing, immediately after thawing and at the moment of transfer was recorded. Clinical pregnancy was defined as a pregnancy with at least one gestational sac at seven weeks of gestation. Pregnancy rates were compared using Chi-squared test (p< 0.05). RESULTS: The overall clinical pregnancy rate was 20.0% (105/526). Embryo stage before freezing did not influence clinical pregnancy rates: 18.9% (14/74) for 6-cell embryos, 23.7% (27/114) for 7-cell embryos, 17.6% (43/245) for 8-cell embryos and 22.6% (21/93) for >8 cell embryos. The clinical pregnancy rate after transfer of frozen embryos with 100% intact cells was 19.9% (76/382). This was not significantly different compared to embryos with 1 blastomere (23.3%, 20/86) or 2 blastomeres (17.6%, 6/ 34) damaged. Three clinical pregnancies were obtained in the group of embryos with > 2 cells damaged (3/24, 12.5%) originating from transfer of embryos with 50% blastomere damage. Significantly higher clinical pregnancy rates were obtained after transfer of embryos with signs of compaction (71/317, 22.4%, p<0.001) or blastulation (21/62, 33.9%, p<0.0001) than embryos with >8 cells but without compaction (8/109, 7.3%). Embryos with %8 cells at transfer had a clinical pregnancy rate of 13.2% (5/38). CONCLUSIONS: Compaction and blastulation after thawing and overnight culture of frozen day 3 embryos are reliable predictors of clinical pregnancy. Loss of 1 or 2 blastomeres did not have a negative impact on clinical pregnancy rates. It is possible to obtain a clinical pregnancy after transfer of embryos with up to 50% of cell loss. Supported by: None.

P-507 EVALUATION OF BLASTOCYST RECUPERATION, IMPLANTATION AND PREGNANCY RATES AFTER VITRIFICATION/WARMING OR SLOW FREEZING/THAWING CYCLES. D. P. Bernal, C. C. Chang, L. F. Colturato, D. M. Leef, H. I. Kort, Z. P. Nagy. IVF Lab, Reproductive Biology Associates, Atlanta, GA; Reproductive Biology Associates, Atlanta, GA; Lab Director, Reproductive Biology Associates, Atlanta, GA. OBJECTIVE: To evaluate and to compare the efficiency of two blastocyst cryopreservation techniques, slow-freezing and vitrification, by measuring survival, recuperation, pregnancy and implantation rates in human IVF. DESIGN: Prospective, randomized study. MATERIALS AND METHODS: A total of 51 slow freezing/thawing cycles and 64 vitrification/warming cycles were performed by random distribution in the same period between January 2007 and April 2008 and results were analyzed. In the slow freezing group, blastocysts were cryopreserved using 9% glycerol and 0.2M sucrose (Quinn’s AdvantageÔ Blastocyst Freeze kit; SAGE, Biopharma, Trumbull, CT, USA). Blastocysts were vitrified using 15% ethylene glycol, 15% DMSO, and 0.5M sucrose using the CryolockÒ device (Biodiseno Ltda, Colombia). Blastocyst survival and expansion was assessed after thawing/warming, and embryos were transferred 2-3 h later. (Recuperation rate: number of blastocysts expanded/number of blastocyst thawed or warmed). Results were analyzed by Fisher’s exact tests using the P value at %0.05.

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RESULTS: No differences were present in the clinical and laboratory parameters between the two groups prior to embryo freezing. Survival rates post thaw were 76% (110/145) for slow freezing group and 93% (159/171) for those blastocyst vitrified/warmed (P<0.0001). Recuperation rates post thaw were 50% (55/110) for slow freezing group and 81% (128/ 159) for vitrified blastocysts using CryolockÒ (P<0.0001). The average maternal age was 35.4 and 34.7 years for slow freezing and vitrification groups, respectively (NS). The mean number of embryos transferred was 2.2 and 2.5, respectively (NS). Clinical pregnancy rate after slow freeze/ thaw was 55% (37 patients with positive FCA’s out of 51 patients transferred) and 65% (56/62) after vitrification/warming (NS). Implantation rate per embryo thawed after slow freeze was 26% (37 FCA’s were detected out of 145 embryos thawed) and 33% (56/171) after vitrification (NS). CONCLUSIONS: The results of this study demonstrate that survival and recuperation rates after vitrification/warming of blastocysts were significantly higher than with the traditional slow freeze/thaw procedure. Improved survival of blastocyst stage embryos seemed to correlate with a trend to higher implantation and pregnancy rates following vitrification, however, the difference did not reach statistical significance. Further studies are necessary to confirm the consistent benefit of embryo freezing using the vitrification method. Supported by: None.

P-508 EFFICIENCY OF VITRIFICATION AT DIFFERENT EMBRYONIC STAGES COMPARED TO SLOW FREEZING. D. P. Bernal, G. Wright, T. A. Elliot, C. W. Elsner, A. A. Toledo, Z. P. Nagy. IVF Lab, Reproductive Biology Associates, Atlanta, GA; Reproductive Biology Associates, Atlanta, GA; Lab Director, Reproductive Biology Associates, Atlanta, GA. OBJECTIVE: To evaluate the clinical efficiency of vitrification, at various embryonic development stages, using the CryolockÒ device, and to compare it with the slow freezing technique. DESIGN: Retrospective analysis. MATERIALS AND METHODS: A total of 289 thawing cycles of slow freezing and 108 warming cycles of vitrification were performed parallel and data were analyzed. Zygotes and cleaved embryos were frozen and thawed by slow freezing protocol using Freeze-Kit1 and Thaw Kit1 (Vitrolife, Kungsbacka-Sweden) and day-5 embryos using the Blastocyst Freeze / Thaw Kit (SAGE, Biopharma, Trumbull, CT, USA). In the vitrification group, embryos at all stages were vitrified using the same vitrification protocol 15% ethylene glycol, 15% DMSO, and 0.5M sucrose with the CryolockÒ (Biodiseno Ltda, Colombia) and warming using series of solutions (1.0M/ 0.5M sucrose). Results were analyzed using One-way ANOVA and the Fisher’s exact tests using P at 0.05 level. TABLE 1. Survival, Pregnancy and Implantation rates after warming/thawing transferred cycles Rates Survival Rate

Pregnancy Rate (FCA)

Implantation Rate/ Embryo transf

Implantation Rate/ Embryo Thawed

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Abstracts

Vitrification % Slow Freezing %

P Value

2PN Cleaved Blastocyst All 2PN Cleaved Blastocyst All 2PN

97 90 93 94 59 50 65 61 28

85 75 76 80 43 43 55 45 23

<0.0001 0.0044 <0.0001 <0.0001 NS NS NS 0.0025 NS

Cleaved Blastocyst All 2PN

32 36 33 14

22 33 24 10

NS NS 0.0032 NS

Cleaved Blastocyst All

26 33 23

12 26 12

0.0021 NS <0.0001

As an additional parameter for embryos at the cleavage cell stage, the number of intact cells (compared to the lysed cells) was also noted. After vitrification, 93% of the original (before freezing) cells remained intact, and after slow freezing 68% (P<0.0001). CONCLUSIONS: The results of this study demonstrate that vitrification at all different stages of human embryo development provides high survival, high pregnancy, implantation rates and many of these outcomes are superior compared to slow-freezing protocol. This study also shows that CryolockÒ can function successfully as a carrier device for vitrification in the daily routine laboratory use. These outcomes provide strong evidence on adopting vitrification as a standard protocol for embryo cryopreservation for all developmental stages. Supported by: None.

P-509 SURVIVAL AND CLINICAL OUTCOME OF DAY-3 CRYOPRESERVED EMBRYOS IS CONSIDERABLY IMPROVED BY VITRIFICATION COMPARED WITH SLOW COOLING METHOD. A. Cobo, M. Marcos, M. J. de los Santos, A. Tejera, A. Pellicer, J. Remohi. IVI-Valencia, Valencia, Spain. OBJECTIVE: The replacement of cryopreserved embryos can increase the options of success in ‘‘in vitro’’ fertilization cycles. However, is widely accepted that thawed embryos have indeed a lower potential to implant with current cryopreservation methods, due to low survival rates, which involve the lysis of different extensions of the embryo. More efficient cryopreservation methods would increase survival rates and overall outcome. The aim of this study was to evaluate the outcome of embryos vitrified by Cryotop method and slow cooling method. DESIGN: Retrospective cohort study. MATERIALS AND METHODS: This study included women undergoing cryotransfer on day-3 embryos that were cryopreserved by classical slow cooling protocol (Group 1) or vitrified by Cryotop method (Group 2). For endometrium preparation all women underwent hormonal replacement therapy after down regulation with depot GnRHa in the luteal phase of the previous cycle. Increasing doses of estradiol valerate up to 6 mg/day were employed until an endometrial thickness of R7 mm was observed. Then, micronized vaginal progesterone was added (400 mgs/day, Day 1). Embryos were replaced 2 days later on day 3. Two-tailed student t-test and c2 test were used for mean values and proportions respectively. RESULTS: Results are summarized in the following table:

No. thawed/warmed embryos Mean age Survival rate No. of intact survived embryos x (%) Mean cell number of frozen/vitrified embryos Mean fragmentation of frozen/ vitrified embryos No. of transfers (%) Mean number of embryos transferred Clinical pregnancy rate / cycle Implantation rate Miscarriage rate Ectopic pregnancy rate Ongoing pregnancy rate/cycle

Group 1 (n¼573)

Group 2 (n¼238)

1968 38.2 (SD¼ 5.3) 1046 (53.2) 375 (35.9) 7.5(SD¼1.2)

563 38.1 (SD¼5.6) 513 (91.1)* 496 (96.7)* 7.6 (SD¼1.1)

6.8 (SD¼4.9)

7.6 (SD¼5.9)

449 (78.4) 1.4 (SD¼0.9)

231 (97)* 1.8 (SD¼0.6)*

166 (28.9) 25.2 36 (21.6) 5 (3.0) 125 (21.8)

111 (46.6)* 31.0 9 (8.1)* 4 (3.6) 98 (41.2)*

* p<0.05. x embryos showing 100% cell survival. CONCLUSIONS: This study shows clearly that the Cryotop Vitrification method provides improved embryo survival rates with a higher proportion of intact survived embryos that should be responsible of the better clinical outcome observed. We believe that this methodology is extremely valuable for establishing more efficient cryopreservation programs and will increase the success per started cycle. Supported by: None.

Vol. 90, Suppl 1, September 2008