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Optimal cryopreservation methodology for oocytes as measured by post-shipment cryosurvival: vitrification versus slow freezing
attached to the dish to form a confluent monolayer with scattered but adherent germ cells. In both media, aggregates gradually increased to a diameter of 100mm by D10, however, more SSC colonies were observed in the culture with serum. By D14, serumþ medium had twice as many SSCs. FE-J1 positive cells ranged from 0.2 – 2%. To have an enriched SSC population, dissociated testicular aggregates were magnetically sorted and cryopreserved in a system utilizing a biopolymer. CONCLUSIONS: Custom-tailored medium supplemented with growth factors was able to sustain and enrich germ cells for over two weeks. SSCs were able to reach post-meiotic stages following growth factor modulation. Propagation, meiotic differentiation, and cryostorage of spermatogonia stem cell may serve to recolonize seminiferous tubules of pre-pubertal individuals. Supported by: Institutional.
O-228 Wednesday, October 21, 2009 5:15 PM OPTIMAL CRYOPRESERVATION METHODOLOGY FOR OOCYTES AS MEASURED BY POST-SHIPMENT CRYOSURVIVAL: VITRIFICATION VERSUS SLOW FREEZING. L. Valluzzo, L. Chuang, F. Poleshchuk, C. Frank Sage, A. B. Copperman, J. Barritt. Reproductive Medicine Associates of New York, New York, NY; Extend Fertility, Woburn, MA; Department of OBGYN and Reproductive Science, Mount Sinai School of Medicine, New York, NY. OBJECTIVE: We set out to explore the impact of Nitrogen Vapor (NV) shipment on the recovery of viable human metaphase II oocytes cryopreserved by either vitrification or slow freezing methods. DESIGN: Prospective analysis. MATERIALS AND METHODS: 171 MII oocytes were included in the study: 80 utilized the Vitrification Cooling and Warming kit (Medicult, Denmark) with an open storage device (McGill Cryoleaf) and 91 were frozen by the Oocyte Freeze and Thaw kit (Medicult, Denmark) with a closed system (MVE straw). All were stored in liquid nitrogen (LN2) prior to and after shipment. The NV shipper was shown to maintain -196 C for >24 hours. 30 vitrified and 12 slow-frozen oocytes were transported in NV priority overnight by FedEx to New England Cryogenic Center, a long-term storage facility. Oocytes were stored in LN2 for 48 hours and then returned for thawing. Shipped oocytes were exposed to air 4 times. The transit time was 18 hours per shipment. Oocyte survival after 3 hours post-thaw was analyzed by chisquare. RESULTS: See table.
Vitrified, not Vitrified, Slow-frozen, Slow-frozen, shipped (%) shipped (%) not shipped (%) shipped (%) MII oocytes 50 Survival 3 hr 48 (96)* post-thaw
30 22 (73.3)*
79 68 (86)
12 10 (83.3)
* p¼0.009 CONCLUSIONS: Vitrified and slow-frozen oocytes that were not shipped had similar post-thaw survival rates (96% vs. 86%). Slow-frozen oocyte survival rate between the shipped and non-shipped was similar after 3 hours. In contrast, the survival rate of vitrified and shipped oocytes was significantly lower than the non-shipped after 3 hours(p¼0.009). Repeated exposure to air could have played a role in the decreased survival of vitrified oocytes. The small amount of vitrification media (1ml) could be ultra-sensitive to temperature changes as compared to the 200ml slow-freeze media. Our study showed that NV shipping between facilities can be deleterious to vitrified oocytes. Supported by: Medicult, Extend Fertility.
OBJECTIVE: Our objective was to investigate the possible differences in laboratory and clinical outcomes between fresh and vitrified/warmed sibling oocytes. DESIGN: Prospective study. MATERIALS AND METHODS: Inclusion criteria were: 1, patients consented to the IRB approved study; 2, maternal age between 30-39 years (34.93.1); and 3, greater than 10 MII oocytes obtained. MII oocytes from every patient were randomly divided into two groups: vitrified/warmed (group A) and fresh sibling oocytes (group B). Cryopreservation of oocytes was performed by vitrification with 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. Oocytes were fertilized by ICSI 2-3 h after warming, and fresh sibling oocytes were also fertilized at the same time. On day 5, embryos were assessed and only embryos derived from vitrification were transferred. Results were analyzed by the Chi-square (P<0.05) statistical test. RESULTS: A total of 370 MII oocytes were retrieved from 21 study participants. One hundred eighty-four oocytes were allocated to group A and 186 oocytes was assigned to group B. One hundred forty-seven oocytes survived after vitrification and warming(79.9%). Fertilization rates were 66.3% and 73.1% (NS) in groups A and B. Blastocyst formation rates were 62% (76/ 122) and 56% (76/136; NS), in groups A and B. Fifty blastocysts were transferred to 21 patients (2.380.7); 13 out of 21 had positive hCG (61.9%). Eight patients had a clinical pregnancy (11 FCAs were detected; 22% implantation rate). To date, nine babies have been born from 6 deliveries, and the other 2 pregnancies are still ongoing. CONCLUSIONS: This study demonstrates a high survival, fertilization and embryo development after vitrification/warming of oocytes obtained from 30-39 year old IVF patients; these outcomes are very similar to those obtained using the sibling (fresh) oocytes. All together, these results suggest that the effect of vitrification is minimal on oocyte physiology, making its routine use applicable for patients of ages 30 to 39 years old. Supported by: Study Grant from EMD Serono.
CLINICAL FEMALE INFERTILITY AND GYNECOLOGY O-230 Wednesday, October 21, 2009 3:45 PM INTRAVENOUS IMMUNOGLOBULIN (IVIG) FOR TREATMENT OF IDIOPATHIC SECONDARY RECURRENT MISCARRIAGE (ISRM): A PROSPECTIVE, RANDOMIZED, DOUBLE-BLINDED, PLACEBO-CONTROLLED, MULTI-CENTERED TRIAL. M. D. Stephenson, W. Kutteh, S. Purkiss, C. Librach, E. Houlihan, P. Schultz. Obstetrics and Gynecology, University of Chicago, Chicago, IL; Obstetrics and Gynecology, University of Tennessee, Memphis, TN; Reproductive Medicine, British Columbia’s Women’s Hospital & Health Centre, Vancouver, BC, Canada; Department of Obstetrics and Gynaecology, Sunnybrook Health Sciences Centre & the Women’s College Hospital, University of Toronto, Toronto, ON, Canada. OBJECTIVE: Evaluate the efficacy of IVIG in couples with ISRM. Subanalysis of previous RCTs suggest this cohort may benefit from IVIG. DESIGN: RCT. MATERIALS AND METHODS: Entry criteria: (1) maternal age of 1845 yrs, (2) history of a pregnancy R20 wks followed by R3 unexplained miscarriages <20 wks, all with the present partner, (3) most recent conception took <1 yr, (4) negative for parental translocation, maternal thyroid disease, hyperprolactinemia, APS and uterine factors. No concomitant medication for RM was allowed. A sample size of 178 was calculated based on a treatment effect of 20%. An interim analysis was performed. IVIG (GamunexÒ or GamimuneÒ) 500 mg/kg, or equivalent volume of normal saline, was infused prior to ovulation, for a maximum of 6 cycles, and continued q4wks until 18-20 wks. Pregnancy was defined as a þve hCG at 4 wks. Primary outcome was an ongoing pregnancy R20 wks. Chi-square and t-test applied. RESULTS: 82 patients enrolled, of which 47 had an index pregnancy.
Subjects
IVIG (n¼23)
Placebo (n¼24)
O-229 Wednesday, October 21, 2009 5:30 PM COMPARISON OF LABORATORY AND CLINICAL OUTCOMES BETWEEN FRESH AND VITRIFIED/WARMED SIBLING OOCYTES OBTAINED FROM 30-39 YEAR OLD IVF PATIENTS. Z. P. Nagy, C.-C. Chang, D. P. Bernal, D. B. Shapiro, D. Mitchell-Leef, H. I. Kort. Reproductive Biology Associates, Atlanta, GA.
FERTILITY & STERILITYÒ
Mean # pregnancies R20 wks (SD) Mean # miscarriages (SD) Mean maternal age at miscarriage (SD) BMI (SD) Smoker
1.2 (0.4) 4.2 (1.3) 35 yrs (5.1) 26 (4.0) 1
1.4 (0.7) 5.1 (2.9) 33 yrs (4.7) 25 (4.7) 3
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O-228 Wednesday, October 21, 2009 5:15 PM OPTIMAL CRYOPRESERVATION METHODOLOGY FOR OOCYTES AS MEASURED BY POST-SHIPMENT CRYOSURVIVAL: VITRIFICATION VERSUS SLOW FREEZING. L. Valluzzo, L. Chuang, F. Poleshchuk, C. Frank Sage, A. B. Copperman, J. Barritt. Reproductive Medicine Associates of New York, New York, NY; Extend Fertility, Woburn, MA; Department of OBGYN and Reproductive Science, Mount Sinai School of Medicine, New York, NY. OBJECTIVE: We set out to explore the impact of Nitrogen Vapor (NV) shipment on the recovery of viable human metaphase II oocytes cryopreserved by either vitrification or slow freezing methods. DESIGN: Prospective analysis. MATERIALS AND METHODS: 171 MII oocytes were included in the study: 80 utilized the Vitrification Cooling and Warming kit (Medicult, Denmark) with an open storage device (McGill Cryoleaf) and 91 were frozen by the Oocyte Freeze and Thaw kit (Medicult, Denmark) with a closed system (MVE straw). All were stored in liquid nitrogen (LN2) prior to and after shipment. The NV shipper was shown to maintain -196 C for >24 hours. 30 vitrified and 12 slow-frozen oocytes were transported in NV priority overnight by FedEx to New England Cryogenic Center, a long-term storage facility. Oocytes were stored in LN2 for 48 hours and then returned for thawing. Shipped oocytes were exposed to air 4 times. The transit time was 18 hours per shipment. Oocyte survival after 3 hours post-thaw was analyzed by chisquare. RESULTS: See table.
Vitrified, not Vitrified, Slow-frozen, Slow-frozen, shipped (%) shipped (%) not shipped (%) shipped (%) MII oocytes 50 Survival 3 hr 48 (96)* post-thaw
30 22 (73.3)*
79 68 (86)
12 10 (83.3)
* p¼0.009 CONCLUSIONS: Vitrified and slow-frozen oocytes that were not shipped had similar post-thaw survival rates (96% vs. 86%). Slow-frozen oocyte survival rate between the shipped and non-shipped was similar after 3 hours. In contrast, the survival rate of vitrified and shipped oocytes was significantly lower than the non-shipped after 3 hours(p¼0.009). Repeated exposure to air could have played a role in the decreased survival of vitrified oocytes. The small amount of vitrification media (1ml) could be ultra-sensitive to temperature changes as compared to the 200ml slow-freeze media. Our study showed that NV shipping between facilities can be deleterious to vitrified oocytes. Supported by: Medicult, Extend Fertility.
OBJECTIVE: Our objective was to investigate the possible differences in laboratory and clinical outcomes between fresh and vitrified/warmed sibling oocytes. DESIGN: Prospective study. MATERIALS AND METHODS: Inclusion criteria were: 1, patients consented to the IRB approved study; 2, maternal age between 30-39 years (34.93.1); and 3, greater than 10 MII oocytes obtained. MII oocytes from every patient were randomly divided into two groups: vitrified/warmed (group A) and fresh sibling oocytes (group B). Cryopreservation of oocytes was performed by vitrification with 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. Oocytes were fertilized by ICSI 2-3 h after warming, and fresh sibling oocytes were also fertilized at the same time. On day 5, embryos were assessed and only embryos derived from vitrification were transferred. Results were analyzed by the Chi-square (P<0.05) statistical test. RESULTS: A total of 370 MII oocytes were retrieved from 21 study participants. One hundred eighty-four oocytes were allocated to group A and 186 oocytes was assigned to group B. One hundred forty-seven oocytes survived after vitrification and warming(79.9%). Fertilization rates were 66.3% and 73.1% (NS) in groups A and B. Blastocyst formation rates were 62% (76/ 122) and 56% (76/136; NS), in groups A and B. Fifty blastocysts were transferred to 21 patients (2.380.7); 13 out of 21 had positive hCG (61.9%). Eight patients had a clinical pregnancy (11 FCAs were detected; 22% implantation rate). To date, nine babies have been born from 6 deliveries, and the other 2 pregnancies are still ongoing. CONCLUSIONS: This study demonstrates a high survival, fertilization and embryo development after vitrification/warming of oocytes obtained from 30-39 year old IVF patients; these outcomes are very similar to those obtained using the sibling (fresh) oocytes. All together, these results suggest that the effect of vitrification is minimal on oocyte physiology, making its routine use applicable for patients of ages 30 to 39 years old. Supported by: Study Grant from EMD Serono.
CLINICAL FEMALE INFERTILITY AND GYNECOLOGY O-230 Wednesday, October 21, 2009 3:45 PM INTRAVENOUS IMMUNOGLOBULIN (IVIG) FOR TREATMENT OF IDIOPATHIC SECONDARY RECURRENT MISCARRIAGE (ISRM): A PROSPECTIVE, RANDOMIZED, DOUBLE-BLINDED, PLACEBO-CONTROLLED, MULTI-CENTERED TRIAL. M. D. Stephenson, W. Kutteh, S. Purkiss, C. Librach, E. Houlihan, P. Schultz. Obstetrics and Gynecology, University of Chicago, Chicago, IL; Obstetrics and Gynecology, University of Tennessee, Memphis, TN; Reproductive Medicine, British Columbia’s Women’s Hospital & Health Centre, Vancouver, BC, Canada; Department of Obstetrics and Gynaecology, Sunnybrook Health Sciences Centre & the Women’s College Hospital, University of Toronto, Toronto, ON, Canada. OBJECTIVE: Evaluate the efficacy of IVIG in couples with ISRM. Subanalysis of previous RCTs suggest this cohort may benefit from IVIG. DESIGN: RCT. MATERIALS AND METHODS: Entry criteria: (1) maternal age of 1845 yrs, (2) history of a pregnancy R20 wks followed by R3 unexplained miscarriages <20 wks, all with the present partner, (3) most recent conception took <1 yr, (4) negative for parental translocation, maternal thyroid disease, hyperprolactinemia, APS and uterine factors. No concomitant medication for RM was allowed. A sample size of 178 was calculated based on a treatment effect of 20%. An interim analysis was performed. IVIG (GamunexÒ or GamimuneÒ) 500 mg/kg, or equivalent volume of normal saline, was infused prior to ovulation, for a maximum of 6 cycles, and continued q4wks until 18-20 wks. Pregnancy was defined as a þve hCG at 4 wks. Primary outcome was an ongoing pregnancy R20 wks. Chi-square and t-test applied. RESULTS: 82 patients enrolled, of which 47 had an index pregnancy.
Subjects
IVIG (n¼23)
Placebo (n¼24)
O-229 Wednesday, October 21, 2009 5:30 PM COMPARISON OF LABORATORY AND CLINICAL OUTCOMES BETWEEN FRESH AND VITRIFIED/WARMED SIBLING OOCYTES OBTAINED FROM 30-39 YEAR OLD IVF PATIENTS. Z. P. Nagy, C.-C. Chang, D. P. Bernal, D. B. Shapiro, D. Mitchell-Leef, H. I. Kort. Reproductive Biology Associates, Atlanta, GA.
FERTILITY & STERILITYÒ
Mean # pregnancies R20 wks (SD) Mean # miscarriages (SD) Mean maternal age at miscarriage (SD) BMI (SD) Smoker
1.2 (0.4) 4.2 (1.3) 35 yrs (5.1) 26 (4.0) 1
1.4 (0.7) 5.1 (2.9) 33 yrs (4.7) 25 (4.7) 3
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