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Comparison of apoptosis in the mouse oocytes and the resultant embryos following vitrification and slow-freezing
are associated with higher pregnancy rates compared to those obtained post cancer treatment. The latter is the first comparative evidence that embryo cryopreservation preserves fertility in cancer patients. Supported by: None. P-697 VITRIFICATION OF ISOLATED PRE-ANTRAL FOLLICLES FROM THE MOUSE OVARY: COMPARISON OF VITRIFICATION PROTOCOLS. N. Desai, J. Szeptycki, F. Abdel-Hafez, J. Goldfarb. OB-GYN, Cleveland Clinic Fertility Center, Beachwood, OH. OBJECTIVE: Vitrification has not been extensively studied for ovarian follicle cryopreservation. The simplicity of the technique and the promising results seen with oocyte vitrification suggest that this technique may be very valuable for ovarian follicle banking. The mouse ovary provides an excellent system to assess the effect of vitrification on subsequent pre-antral follicle development and maturation. In the present study we applied two different vitrification protocols to mouse pre-antral follicles and studied subsequent maturation upon thawing. DESIGN: Pre-antral follicles from mouse ovaries were isolated and vitrified under different conditions. MATERIALS AND METHODS: Ovaries from 14 to 16 day old CB6F1 pups were removed and enzymatically digested with collagenase at 37 C. After digestion the ovarian tissue was rinsed several times before being placed in fresh medium for mechanical disaggregation of tissue using an Eppendorf pipettor. Pre-antral follicles with intact basement membranes were collected. Follicles were vitrified using either ethylene glycol (EG) alone (dela Pena et al, 2002) or else ethylene glycol in combination with dimethylsulphoxide (DMSO) (Lieberman et al, 2003). The cryoloop was used as the carrier (Hampton Research, LaGuna, CA). All vitrification and warming steps were performed at room temperature. Control and thawed follicles were cultivated in vitro on Transwell Inserts for 6–9 days at 37 C with 5.5% CO2, before final maturation with hCG. Outcome measures post-vitrification were as follows: 1) Post-thaw recovery 2) Antral cavity formation by the end of the cultre interval 3) Maturation rate as determined by germinal vesicle breakdown (GVBD) and progression to Metaphase II. Data was analyzed using the Chi square test. A P value <0.05 was considered statistically significant. RESULTS: Vitrification was effective in preserving both the oocyte and its surrounding granulosa cells. Basement membranes appeared to be intact and plated follicles maintained their structural integrity. TABLE
Total number Recovery Number plated Rate of antral cavity formation Ovulation rate GVBD MII maturation rate
ControlFresh
Vit Protocol A (EG)
Vit Protocol B (EG/DMSO)
233 NA 233 38% 85%** 48%** 42%*
83 100% 83 47% 52% 28% 28%
140 99% 138 50% 48% 30% 26%
*P%0.01, **P%0.001. CONCLUSIONS: Vitrification technology was easily applied to isolated follicles with excellent survival and growth in culture. Further optimization of culture conditions will be necessary to improve the rate of metaphase II maturation. Supported by: None. P-698 COMPARISON OF APOPTOSIS IN THE MOUSE OOCYTES AND THE RESULTANT EMBRYOS FOLLOWING VITRIFICATION AND SLOW-FREEZING. J. Y. J. Huang, H. Y. Chen, J. Y. S. Park, C. Woo, B. C. Jee, R.-C. Chian. Division of Reproductive Biology, Obstetrics and Gynecology, McGill University, Montreal, QC, Canada. OBJECTIVE: The objective is to evaluate the presence of DNA fragmentation and apoptosis in the oocytes and the resultant embryos following vitrification and slow-freezing methods. DESIGN: Animal model studies.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: Mature oocytes obtained from superovulated CD-1 mice were cryopreserved by vitrification and choline-base, sodium-depleted slow-freezing method. Vitrification was performed using the McGill Cryoleaf (Huang et al. Gynecol Onc 2007). Another group was frozen using choline-based, sodium-depleted slow-freezing procedure (Stachecki et al., Cryobiol 1998). The thawed oocytes were incubated for 1 hour before performing in-vitro fertilization (IVF) and in-vitro culture (IVC). DNA fragmentation was analyzed by TUNEL assay (Roche Diagnostics, Mannheim, Germany). Caspase activity was evaluated using carboxyfluorescein labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases (CaspaTag pan-caspase in situ assay kit, Chemicon International, Purchase, NY). The outcomes included: 1) post-thaw survival, 2) embryonic developmental potential following IVF-IVC, and 3) the presence of DNA fragmentation and caspase activity in the thawed oocytes and the 2-cells, 4-cells, morula, and blastocyst stage embryos. RESULTS: Based on 800 vitrified oocytes and 900 slow-freezing oocytes, vitrification resulted in a significantly higher survival rates than the slowfreezing method (88.9 5.8% vs. 69.4 8.7%, P<0.05 in 10 replicates). Following IVF-IVC, the cleavage and blastocyst rates of vitrified oocytes were significantly higher than those of slow-freezing oocytes (63.6% vs. 39.9% and 30.5% vs. 19.1% respectively, P<0.05). Compared to vitrification, the slow-freezing method resulted in greater DNA fragmentation in the thawed oocytes (84.0% vs. 65.8%, P<0.05) and embryos at 4-cells stage (12.5% vs. 0.0%, P<0.05), morula stage (77.8% vs. 0.0%, P<0.05), and blastocyst stage (36.3% vs. 10.0%, P<0.05). Caspase activities were similar between vitrified- and slow-freezing oocytes but were significantly greater than fresh oocytes (12% and 14% vs. 2%, P<0.05). In 2-cells, 4-cells stage embryos, the caspase activities were similar to fresh embryos. CONCLUSIONS: Vitrification of mouse oocytes results in less DNA fragmentation in the thawed oocytes and the resultant embryos compared to the choline-based slow-freezing method. The reduced embryonic development of frozen-thawed oocytes may be related to DNA fragmentation and activation of caspase. Supported by: Research grant from Ton Yen General Hospital, Taiwan R.O.C. P-699 OOCYTE CRYOPRESERVATION FOR FERTILITY PRESERVATION: THE FIRST 149 PATIENT CONSULTS AND THEIR SUBSEQUENT CYCLE OUTCOMES. M. Luna, J. Barritt, M. Howard, C. Jones, B. Sandler, A. B. Copperman. Reproductive Endocrinology, Reproductive Medicine Associates of New York; Mount Sinai School of Medicine, New York, NY; Extend Fertility, Woburn, MA. OBJECTIVE: Elective fertility preservation through oocyte cryopreservation (OC) has recently become a viable option for women. Adoption of this technology has been slow and cautious, with careful attention paid to the medical, ethical, and psychological parameters surrounding its introduction. We set out to evaluate a cohort of patients presenting a request for OC at a single facility. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Patients presenting for elective fertility preservation between January 2005–March 2007. Variables analyzed included age, day 3 FSH, cancellation rate, number of retrieved oocytes, and number of cryopreserved oocytes. Patients were categorized by age groups: A <35; B 35–37; C 38–40; D >40. Statistical analysis was performed using X2 and Student’s t-test. RESULTS: Initial consultations for 149 patients, with 62 (41.6%) proceeding to initiate a total of 84 OC cycles. Forty three patients underwent 1 cycle, 16 attempted 2 cycles, and 3 initiated 3 cycles. Sixteen (19.1%) cycles were cancelled after starting stimulation due to poor ovarian response (<4 dominant follicles), 75% of which occurred in the first cycle and 25% in a second cycle. Two patients were cancelled twice. The mean age of patients who completed an OC cycle was 37.8 3.3. The mean age of those cancelled was 38.7 1.8 (NS). The mean day 3 FSH of patients who completed a cycle (8.8 4.1 IU/L) was significantly lower than those cancelled (15.6 7.5 IU/ L, P<0.0001). In the 68 completed cycles, the mean oocytes retrieved was 13.0 7.2 and the mean cryopreserved oocytes was 12.8 6.9. A negative correlation was identified between oocyte age and mean number of frozen oocytes: A:14.1 7.3; B:13.6 6.4; C:12.9 8.9; D:10.7 4.1. CONCLUSIONS: Nearly 60% of patients who completed an initial consultation for OC do not proceed to a stimulation cycle. This utilization rate is notably low, considering that it is being identified in a self-motivated, selected, and educated patient population. In those patients who did proceed to a stimulation protocol, almost 20% were cancelled due to low oocyte
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Total number Recovery Number plated Rate of antral cavity formation Ovulation rate GVBD MII maturation rate
ControlFresh
Vit Protocol A (EG)
Vit Protocol B (EG/DMSO)
233 NA 233 38% 85%** 48%** 42%*
83 100% 83 47% 52% 28% 28%
140 99% 138 50% 48% 30% 26%
*P%0.01, **P%0.001. CONCLUSIONS: Vitrification technology was easily applied to isolated follicles with excellent survival and growth in culture. Further optimization of culture conditions will be necessary to improve the rate of metaphase II maturation. Supported by: None. P-698 COMPARISON OF APOPTOSIS IN THE MOUSE OOCYTES AND THE RESULTANT EMBRYOS FOLLOWING VITRIFICATION AND SLOW-FREEZING. J. Y. J. Huang, H. Y. Chen, J. Y. S. Park, C. Woo, B. C. Jee, R.-C. Chian. Division of Reproductive Biology, Obstetrics and Gynecology, McGill University, Montreal, QC, Canada. OBJECTIVE: The objective is to evaluate the presence of DNA fragmentation and apoptosis in the oocytes and the resultant embryos following vitrification and slow-freezing methods. DESIGN: Animal model studies.
FERTILITY & STERILITYÒ
MATERIALS AND METHODS: Mature oocytes obtained from superovulated CD-1 mice were cryopreserved by vitrification and choline-base, sodium-depleted slow-freezing method. Vitrification was performed using the McGill Cryoleaf (Huang et al. Gynecol Onc 2007). Another group was frozen using choline-based, sodium-depleted slow-freezing procedure (Stachecki et al., Cryobiol 1998). The thawed oocytes were incubated for 1 hour before performing in-vitro fertilization (IVF) and in-vitro culture (IVC). DNA fragmentation was analyzed by TUNEL assay (Roche Diagnostics, Mannheim, Germany). Caspase activity was evaluated using carboxyfluorescein labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases (CaspaTag pan-caspase in situ assay kit, Chemicon International, Purchase, NY). The outcomes included: 1) post-thaw survival, 2) embryonic developmental potential following IVF-IVC, and 3) the presence of DNA fragmentation and caspase activity in the thawed oocytes and the 2-cells, 4-cells, morula, and blastocyst stage embryos. RESULTS: Based on 800 vitrified oocytes and 900 slow-freezing oocytes, vitrification resulted in a significantly higher survival rates than the slowfreezing method (88.9 5.8% vs. 69.4 8.7%, P<0.05 in 10 replicates). Following IVF-IVC, the cleavage and blastocyst rates of vitrified oocytes were significantly higher than those of slow-freezing oocytes (63.6% vs. 39.9% and 30.5% vs. 19.1% respectively, P<0.05). Compared to vitrification, the slow-freezing method resulted in greater DNA fragmentation in the thawed oocytes (84.0% vs. 65.8%, P<0.05) and embryos at 4-cells stage (12.5% vs. 0.0%, P<0.05), morula stage (77.8% vs. 0.0%, P<0.05), and blastocyst stage (36.3% vs. 10.0%, P<0.05). Caspase activities were similar between vitrified- and slow-freezing oocytes but were significantly greater than fresh oocytes (12% and 14% vs. 2%, P<0.05). In 2-cells, 4-cells stage embryos, the caspase activities were similar to fresh embryos. CONCLUSIONS: Vitrification of mouse oocytes results in less DNA fragmentation in the thawed oocytes and the resultant embryos compared to the choline-based slow-freezing method. The reduced embryonic development of frozen-thawed oocytes may be related to DNA fragmentation and activation of caspase. Supported by: Research grant from Ton Yen General Hospital, Taiwan R.O.C. P-699 OOCYTE CRYOPRESERVATION FOR FERTILITY PRESERVATION: THE FIRST 149 PATIENT CONSULTS AND THEIR SUBSEQUENT CYCLE OUTCOMES. M. Luna, J. Barritt, M. Howard, C. Jones, B. Sandler, A. B. Copperman. Reproductive Endocrinology, Reproductive Medicine Associates of New York; Mount Sinai School of Medicine, New York, NY; Extend Fertility, Woburn, MA. OBJECTIVE: Elective fertility preservation through oocyte cryopreservation (OC) has recently become a viable option for women. Adoption of this technology has been slow and cautious, with careful attention paid to the medical, ethical, and psychological parameters surrounding its introduction. We set out to evaluate a cohort of patients presenting a request for OC at a single facility. DESIGN: Retrospective analysis. MATERIALS AND METHODS: Patients presenting for elective fertility preservation between January 2005–March 2007. Variables analyzed included age, day 3 FSH, cancellation rate, number of retrieved oocytes, and number of cryopreserved oocytes. Patients were categorized by age groups: A <35; B 35–37; C 38–40; D >40. Statistical analysis was performed using X2 and Student’s t-test. RESULTS: Initial consultations for 149 patients, with 62 (41.6%) proceeding to initiate a total of 84 OC cycles. Forty three patients underwent 1 cycle, 16 attempted 2 cycles, and 3 initiated 3 cycles. Sixteen (19.1%) cycles were cancelled after starting stimulation due to poor ovarian response (<4 dominant follicles), 75% of which occurred in the first cycle and 25% in a second cycle. Two patients were cancelled twice. The mean age of patients who completed an OC cycle was 37.8 3.3. The mean age of those cancelled was 38.7 1.8 (NS). The mean day 3 FSH of patients who completed a cycle (8.8 4.1 IU/L) was significantly lower than those cancelled (15.6 7.5 IU/ L, P<0.0001). In the 68 completed cycles, the mean oocytes retrieved was 13.0 7.2 and the mean cryopreserved oocytes was 12.8 6.9. A negative correlation was identified between oocyte age and mean number of frozen oocytes: A:14.1 7.3; B:13.6 6.4; C:12.9 8.9; D:10.7 4.1. CONCLUSIONS: Nearly 60% of patients who completed an initial consultation for OC do not proceed to a stimulation cycle. This utilization rate is notably low, considering that it is being identified in a self-motivated, selected, and educated patient population. In those patients who did proceed to a stimulation protocol, almost 20% were cancelled due to low oocyte
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