Better ongoing pregnancy rates from frozen embryo transfers when vitrified blastocysts are used

TABLE 1. Oocytes with single abnormalities Control Irregular (normal M-II) shape

Large PVS

n (%) 1399 (26.4) 480 (9.0) 515 (9.7) Cryo-survival (%) 1231 (87.9) 396 (82.5) 418 (81.1) Blast form (%) 652 (52.9) 201 (50.7) 204 (48.8) Good quality blasts (%) 402 (61.6) 119 (59.2) 122 (59.8) Hatching blasts (%) 185 (28.3) 57 (28.3) 53 (25.9)

Dark ZP 427 (8.0) 359 (84.0) 179 (49.8) 110 (61.4) 47 (26.2)

P-728

Dark cyto þ slight Vacuolar Central gran cyto granulation 421 (7.9) 108 (2.0) 82 (1.5) 338 (80.2) 49 (45.3) 36 (43.9) 165 (48.8) 12 (24.4) 8 (22.2) 94 (56.9) 1 (8.3) 0 40 (24.2) 0 0

TABLE 2. Oocytes with multiple abnormalities

n (%) Cryo-survival (%) Blast form (%) Good quality blasts (%) Hatching blasts (%)

Double extracytop abn.

Extra cyto þ cyto abn.

Only cyto abn.

Triple extra cyto abn.

Triple extracyto þ cyto abn.

630 (11.9) 496 (78.7) 241 (48.5) 135 (56.0) 56 (23.2)

418 (7.8) 194 (46.4) 52 (26.8) 10 (19.2) 0

177 (3.3) 55 (31.0) 8 (14.5) 0 0

394 (7.4) 282 (71.5) 127 (45.0) 69 (54.3) 28 (22.0)

241 (4.5) 90 (37.3) 16 (17.7) 0 0

CONCLUSIONS: Severe cytoplasmic abnormalities of the oocyte hinder the post-thaw development of the cryopreserved cleavage stage embryos. Extracytoplasmic defects, however, do not affect progression to the blastocyst stage. Supported by: None.

P-727 APPLICATION OF DIFFERENT OOCYTE VITRIFICATION PROTOCOLS, LASER ASSISTED ZONA SLITTING, AND TEMPERATURE ON POST-WARMING SURVIVAL OF BOVINE OOCYTE. L. Keskintepe, G. Sher, Y. Agca, M. Adamowicz, G. Maassarani. Sher Institute for Reproductive Medicine, Las Vegas, NV; Department of Pathobiology, University of Missouri, Columbia, MO. OBJECTIVE: Although embryo cryopreservation is a well established technology in human, murine and livestock, oocyte cryopreservation provides very limited application in ART in these species. Empirical improvements in conventional freezing and vitrification protocols and the use of ICSI for fertilization have led to an increasing number of live births. Oocyte cryopreservation can certainly be further improved by basic research. In an effort to improve oocyte vitrification conditions we evaluated three different vitrification protocols in combination with laser assisted hatching before vitrification. DESIGN: Prospective, randomized, in vitro experiments. MATERIALS AND METHODS: This study investigated effects of laser slitting on zona pellicuda (LSZP), vitrification temperatures (22 C vs. 37 C), and 3 different vitrification protocols (VTP 1, VTP 2, VTP 3). All vitrification solutions contained DMSO and ethylene glycol (EG). Non-selected oocytes were randomly allocated to eight different treatments: (1) Control, (2) VTP 1 þ LSZP þ 22 C, (3) VTP 1 þ LSZP þ 37 C, (4) VTP 2 þ LSZP þ 22 C, (5) VTP 2 þ LSZP þ 37 C, (6) VTP 3 þ LSZP þ 22 C, (7) VTP 3 þ LSZP þ 37 C. After warming bovine oocytes were cultured in Global-one medium with 10% synthetic serum supplement for 2 hrs before they were treated with 0.5 M sucrose solution for 5 min. Then, they were cultured another 24 hrs before they were treated with 0.5 sucrose solution again. RESULTS: Oocyte cryosurvival was evaluated after warming and sucrose treatment at 2 and 24 hr. A total of 1,210 oocytes were included. VTP 3 resulted in 32% more surviving oocytes than VTP 2 and VTP1 at 2 hr post warming sucrose treatment. Oocyte survivability at 24 hr sucrose treatment was 100% for Control and VTP 3, 68% for VTP 2 and 51% for VTP 1. No differences were found between control and VTP 3 for 2 hr and 24 hr sucrose treatment (P>0.05). There was a significant interaction (P<0.05) between LSZP and 37 C for oocyte survivability after warming; although trends were similar, the benefit of 37 C compared to 22 C was greater for treatments (P<0.05). CONCLUSIONS: Bovine oocytes represent an excellent model for vitrification studies for human oocyte. Laser zona slitting before vitrification enabled bovine oocytes more effectively equilibrated with cryoprotectants at 37 C. Vitrification protocol 3, which included stepwise addition of DMSO and EG, provided more efficient vitrification than other two protocols. Supported by: None.

FERTILITY & STERILITYÒ

IMPLANTATION RATE AND PREGNANCY RATES FOLLOWING ‘‘ALL FREEZE’’ CRYOPRESERVATION CYCLES COMPARED TO THOSE FOLLOWING CRYOPRESERVATION OF EXCESS EMBRYOS. D. Barad, N. Gleicher. The Center for Human Reproduction, New York, NY. OBJECTIVE: Most cryopreserved embryos are the product of excess embryo production. In such IVF cycles the best embryos are transferred fresh and the remaining embryos are cryopreserved. It is commonly believed that cryopreserved embryos have less potential than fresh embryos. However, it is not clear if this is because of their ‘‘second string’’ status or an effect of freezing. The object of this study is to compare implantation and pregnancy rates of embryos that are product of cycles in which all embryos were frozen to avoid hyperstimulation syndrome with cryopreserved ‘‘excess embryos.’’ DESIGN: Retrospective study. MATERIALS AND METHODS: Patients who had all embryos frozen to avoid severe hyperstimulation syndrome were identified from our database as the study group. Subsequent frozen embryo transfers were analyzed for implantation rate. The control group is comprised of patients with excess embryos cryopreserved. Implantation and pregnancy rates were calculated for both groups and compared by analysis of variance adjusted for age. Statistical analysis was performed using SPSS version 15.0. Continuous values are presented as mean  standard error. RESULTS: Implantation rate among 25 patients who had had all freeze cycles was 0.22  0.04 vs. 0.13  0.01 (P<0.04) for 800 patients who had transfer of thawed ‘‘excess embryos’’. Similar numbers of cryopreserved embryos were transferred in both groups (2.9  0.16 vs. 3.0  0.03; ns). Patients with previous all freeze were significantly younger that those with excess embryos 33.5  1.2 vs. 36  0.2 years (P<0.05), however implantation rates for cryopreserved embryos among all SART member clinics are similar to those of our control group. CONCLUSIONS: Embryos that are a product of an all freeze cycle have better implantation rates than those that are a product of excess embryo freezing. The fact that, in general, most cryopreserved embryos are not as high quality as those that are transferred in fresh cycles, may contribute in part to the lower implantation rate compared to fresh and all freeze cycles. Supported by: Foundatin for Reproductive Medicine. P-729 BETTER ONGOING PREGNANCY RATES FROM FROZEN EMBRYO TRANSFERS WHEN VITRIFIED BLASTOCYSTS ARE USED. A. L. Clifford, P. M. Jilbert, W. L. Gentry, M. A. Henry, C. C. Wegner. Center for Reproductive Biology of Indiana, LLC., Indianapolis, IN. OBJECTIVE: To determine if blastocyst cryopreservation by vitrification improves frozen embryo transfer (FET) clinical pregnancy rates compared to traditional slow freeze methods. Day 5 fresh blastocyst transfers result in good pregnancy rates (58% positive hCG and 51% fetal cardiac activity (FCA) at 6 weeks), but FET results are lower (32.8% positive hCG and 22% FCA at 6 weeks). DESIGN: A retrospective comparison of FET pregnancy rates from April 2006 (initiation of clinical vitrification) to May 2007 comparing vitrified to slowly frozen blastocysts. MATERIALS AND METHODS: Blastocysts were slowly frozen in 0.5 ml straws using Blast Freeze kits (Sage) as per kit instructions and frozen using a programmable freezer at a rate of 1 C/min to 7 C, seeded/held for 7 min, continued freezing at 0.3 C/min to 37 C, at 20 C/min to 100 C, before plunging in liquid nitrogen. Blastocysts were thawed using G-Thawkit Blast Medium (Vitrolife) as per kit instructions. Vitrification was performed by immersing blastocysts into medium containing 10% human serum albumin (HSA), 10% dimethylsulfoxide (DMSO), and 10% ethylene glycol (EG) for two min, then briefly rinsing in 20% DMSO, 20% EG, 10 mg/ml Ficoll, 0.65 M sucrose and 10% HSA, or alternatively, using a commercial kit (Irvine Scientific), before plunging into liquid nitrogen. Warming was performed stepwise in medium with 20% HSA and decreasing sucrose concentrations (1.0 M, 0.5 M, 0 M, Irvine Vit Kit Thaw). Blastocysts were incubated a minimum of 1 hour in equilibrated G2.3 Plus (Vitrolife) before placement in EmbryoGlue (Vitrolife) for transfer. RESULTS: Mean oocyte age at retrieval and embryos transferred were similar between slow freeze (32.9  4.02 yr, 2.5  1.1 embryos) and vitrification (30.7  5.23 yr, 3  0.89 embryos) groups. Patients in the slow freeze group were almost twice as likely (70%, n ¼ 30) to have had a prior

S349

pregnancy, compared to the vitrification group (36%, n ¼ 11). 54.5% (6/11) of patients had a positive hCG result from transfers using vitrified embryos compared to 26.6% (8/30) of patients from transfers using slowly frozen blastocysts. Likewise, more patients had positive FCA at 6 weeks in the vitrified group (40%, 4/10) compared to the slow freeze group (13.8%, 4/29). FCA is pending for one patient in each group. CONCLUSIONS: These early outcomes suggest that blastocyst vitrification may improve FET pregnancy rates. The Center’s first live birth from vitrified blastocyst transfer occurred in April 2007 and several other pregnancies are currently ongoing. Supported by: None. P-730 COMPARISON OF SPERMATOGENESIS AND STEROIDOGENESIS OF CRYOPRESERVED NEONATAL MOUSE TESTICULAR TISSUE – VITRIFICATION VS. CONTROLLED SLOW-RATE FREEZING. C. Yildiz, B. Mullen, K. A. Jarvi, K. C. Lo. Urology, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada; Pathology, Mount Sinai Hospital, University of Toronto, Toronto, ON, Canada. OBJECTIVE: Ectopic allo- or xeno-grafting of frozen testicular tissue is a promising new approach that can be used to preserve testicular function in mutant animals and cancer patients for future fertility. Rescue of genetic resources and valuable mutant strain associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The aim of present study was to improve new mouse testicular tissue cryopreservation protocols by comparing spermatogenesis and testosterone levels of grafted neonatal testicular tissue after exposed to different vitrification protocols and slow-rate freezing. DESIGN: Experimental basic animal study. MATERIALS AND METHODS: Neonatal mouse testes were frozen using 0.7 M DMSO þ 5% FBS with a computer controlled slow rate freezer. Three modified vitrification protocols were used: (1) DAP 213 (2 M DMSO þ 1 M Acetamid þ 3 M Propylene glycol (PG)), (2) 40% Ethylene glycol (EG) þ 18% Ficoll þ 0.35 M Sucrose, and (3) 15% EG þ 15% PG þ 0.5 M Sucrose solutions. Frozen-thawed testicular tissues were grafted onto castrated immunodeficient Ncr-nude mice. Fresh neonatal testes were grafted to serve as the positive control. Animals were sacrificed, and histological analyses of grafted tissue and serum hormonal assays were performed 12 weeks post-transplantation. RESULTS: Survival of testicular grafts is superior in the control and computer controlled slow rate freezing groups comparing to the other 3 vitrification groups (P<0.05). Spermatogenesis from the slow rate freezing groups is also comparable to the controls. Testosterone production from the slow rate freezing group is significantly higher than the vitrification protocols tested (P<0.01). There were no significant differences between controlled slowrate freezing technique and freshly grafted control. CONCLUSIONS: Our data shows a clear advantage of the computer controlled slow-rate freezing protocol over the standard vitrification techniques for neonatal mouse testis cryopreservation. Further modifications and improvement in the vitrification process is required before it can be applied to testicular tissue. Supported by: Canadian Urological Association Scholarship Foundation. P-731 PARALLEL COMPARISON OF PARTHENOGENETIC DEVELOPMENT FOLLOWING OOCYTE CRYOPRESERVATION VITRIFICATION VS. SLOW FREEZING. C.-C. Chang, L.-Y. Sung, C. X. Tian, X. Yang, H. I. Kort, Z. P. Nagy. Reproductive Biology Associates, Atlanta, GA; Center for Regenerative Biology, University of Connecticut, Storrs, CT. OBJECTIVE: Cryopreservation is known to have significant effects on zona pellucida, which can compromise the fertilization of oocyte. After cryopreservation, it is not clear if the compromised embryo development is due to direct cryo-injuries impacting oocyte quality, or simply it is just due to failed or incomplete fertilization. Therefore, parthenogenetic embryo development may be utilized to reflect the real oocyte quality following oocyte cryopreservation. The objective of our study was to evaluate the effect of two cryopreservation methods on oocyte quality. DESIGN: Comparative study to evaluate the effect of vitrification and slow freezing on oocyte quality. MATERIALS AND METHODS: Oocytes collected from 8-wk-old BDF1 mice with standard superovulation. Oocytes were then cryopreserved by vitrification (15% Ethylene Glycol þ 15% DMSO þ 0.5 M Sucrose) and slow

S350

Abstracts

freezing method (choline chloride substitution media with 1.5 M 1, 2 Propanediol þ 0.3 M Sucrose), respectively. 2 h after thawing, oocytes were subjected to parthenogenetic activation by exposure to activation medium (Ca2þ-free CZB), 10 mM strontium chloride and 5 mg/ml Cytochalasin B for 6 h. After activation, oocytes were incubated in KSOM medium. To examine the total cell number, the parthenogenetic blastocysts were fixed at 100 h post activation and stained with DAPI. Fisher’s exact test, P<.05. RESULTS: A total of 209 oocytes were studied. There were 3 groups included: group 1) fresh control oocytes (n ¼ 55), group 2) vitrification (n ¼ 70), group 3) slow freezing (n ¼ 84). The survival rates of thawed oocytes were 95.7% (67/70) in group 2 vs. 84.5% (71/84) in group 3 (P<.05). After 6 h of activation, two pronuclei were found in all of oocytes among three groups (group 1 ¼ 55; group 2 ¼ 67; group 3 ¼ 71). The cleavage rate was not significantly different (98.2% (54/55) vs. 98.5% (66/67) vs. 95.8% (68/71)). However, the blastocyst rate in group 3 (29.6%; 21/71) was significantly lower than group 1 (90.9%; 50/55) and group 2 (68.7%; 46/67) (P<.05). The average cell number of embryos that developed to blastocyst were group 1 (54.2), group 2 (36.9), and group 3 (35.7). CONCLUSIONS: This observation indicates that the cryopreserved oocytes can be activated efficiently as fresh control oocytes. However, the oocyte quality can be impaired by the cryopreservation process in accordance with parthenogenetic embryo development. The significant difference of blastocyst yield reflects the fact that the vitrification method appears to have less detrimental effect on oocyte quality. Supported by: None.

P-732 VITRIFICATION OF ISOLATED OVARIAN FOLLICLES FROM THE MOUSE OVARY: DOES TEMPERATURE OF CRYOPROTECTANT EXPOSURE REALLY MATTER? F. Abdel-Hafez, N. Desai, J. Goldfarb. OB/GYN, Cleveland Clinic Fertility Center, Beachwood, OH. OBJECTIVE: Vitrification of ovarian follicles is a promising fertility preservation method. The cryo-sensitivity of immature follicles has not been extensively studied. This study explores the effect of temperature during vitrification on ovarian follicle survival and subsequent in-vitro maturation. DESIGN: Mouse pre-antral ovarian follicles were vitrified and matured in vitro. MATERIALS AND METHODS: Ovaries from 14 to 16 day old CB6F1 pups were removed and enzymatically digested with collagenase. Isolated follicles were divided into 3 groups : A) Control-not frozen B) Vitrification at room temperature C) Vitrification at 37 C. An ethylene-glycol based protocol (dela Pena 2002) was used for vitrification. Follicles were loaded on to a nylon mesh and immediately plunged into a vial containing liquid nitrogen. All follicles were thawed at room temperature. Vitrified-thawed follicles and controls were matured in vitro for 6–9 days at 37 with 5.5% CO2. Follicles were examined daily for antral cavity formation and growth. Final maturation was triggered with hCG and ovulated oocytes were examined for GVBD and progression to metaphase II. Mature metaphase II oocytes were fixed and stained to study meiotic spindle morphology. Outcome measures examined were, post-thaw survival, growth in culture, antral cavity formation, ovulation, GVBD and maturation to metaphase II. Differences between treatments were analyzed using the Chi square test. P value of <0.05 was considered significant. RESULTS: A total of 1473 pre-antral follicles were isolaed from mouse ovaries for this study. The non-frozen control group containing 621 follicles had significantly more follicles with continued growth during the culture interval. No significant differences were observed with vitrifiaction at room temperature vs. 37 C. Meiotic spindle morphology was not different between treatments. TABLE 1.

Total follicles Recovery rate Number plated Growth in culture Ovulation rate GVBD MII maturation Normal meiotic spindle

Control-Fresh

Vit-Room Temp

Vit-37 C

621 NA 621 82%* 58% 52% 29% 74%

430 97% 396 62% 65% 48% 25% 64%

422 95% 375 62% 56% 46% 23% 55%

*P value%0.001.

Vol. 88, Suppl 1, September 2007