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Enhanced survival of human failed to fertilize oocytes cryopreserved using intra- and extracellular sugars and dimethylsulfoxide
groups: 1) ovarian tissue without cryopreservation (control, group I), 2) ovarian tissue vitrified with VFS-40 (vitrification, group II), and 3) ovarian tissue slowly frozen with DMSO (slow-freezing, group III). Thawing was carried out at room temperature. VEGF levels were checked in ovarian tissues of 3 groups recovered on day 7 after cryopreservation, also checked on day 14 after autotransplantation. RESULTS: In cryopreserved ovarian tissues, VEGF protein levels were significantly decreased in group II and III than group I (control) (p<0.05), whereas there were no significant difference between group II and III. In autotransplanted ovarian tissue, VEGF protein levels showed similar pattern in cryopreserved ovarian tissue. Primary and antral follicular density in autotransplanted ovarian tissue were significantly decreased in group II and III than group I (p<0.05). No significant differences were found in the density of primary and antral follicles in both groups. CONCLUSIONS: These result showed that VEGF may be damaged by cryopreservation. However method of cryopreservation showed no impact on the levels of VEGF damage in cryopreserved and autotransplanted ovarian tissues. Disruption of VEGF expression induced by cryopreservation seems to affect the angiogenesis and folliculogensis of the autotransplanted ovarian tissue. Future studies are needed to improve the preservation of VEGF after cryopreservation of ovarian tissues. Supported by: None. P-489 TIME COURSE OF HUMAN OOCYTE ATP RECOVERY AFTER VITRIFICATION AND THAWING. S. Manipalviratn, Z.-B. Tong, B. Stegmann, E. Widra, J. Carter, A. DeCherney. Reproductive Biology and Medicine Branch, NICHD/NIH, Bethesda, MD; Department of Obstetrics and Gynecology, Georgetown University Hospital, Washington, DC; Shady Grove Reproductive Science Center, Rockville, MD. OBJECTIVE: To evaluate the effect of vitrification and thawing and postthaw incubation time on oocyte ATP concentration. DESIGN: Prospective match-controlled laboratory study. MATERIALS AND METHODS: Oocytes discarded due to nuclear immaturity (GV or MI) from IVF / ICSI cycles were studied. Only oocytes from women with more than 3 discarded oocytes that matured to the MII stage in-vitro were used. One oocyte from each patient was randomly selected as a self-control (group A: non-vitrified/thawed). Oocytes in group A were lysed in ultrapure water and then stored at 80 C for further ATP analysis. The others underwent vitrification using 15% ethylene glycol, 15% dimethyl sulphoxide and 0.5 M sucrose as cryoprotectants. Post-thawed surviving oocytes were randomly separated into 2 groups which differed in post-thaw incubation time, 90-minutes (group B) or 180-minutes (group C) before lysing in ultrapure water and then stored at 80 C for further ATP analysis. ATP concentrations were compared between oocytes in group A, B and C. Intracellular ATP concentrations were measured using a luciferin-luciferase bioluminescent assay. Oocyte ATP analysis was performed in batches of 30-40 oocytes. Oocytes from the same women were analyzed within the same batch. Student t-test was used as appropriate, and analyses were controlled for multiple measures. RESULTS: 208 discarded oocytes from 40 women were included in this study. Of 168 oocytes that underwent vitrification/thawing, 118 oocytes survived, a 70.2% survival rate. There were 40, 57 and 61 oocytes in group A, B and C, respectively. The mean ATP concentrations were 0.420.29, 0.110.14 and 0.350.25 pmole/oocyte respectively for groups A, B and C (P < .001). Coefficient of variation of luciferin-luciferase bioluminescent assay is less than 5%. CONCLUSIONS: Vitrification and thawing were shown to have a significant negative impact on oocyte ATP concentration. Incubation up to 180 minutes after thawing allowed oocytes to regain ATP concentration to some extent though this level was still lower than their non-vitrified/thawed counterparts. Supported by: Intramural program, Reproductive Biology and Medicine Branch, NICHD, NIH.
P-490 ENHANCED SURVIVAL OF HUMAN FAILED TO FERTILIZE OOCYTES CRYOPRESERVED USING INTRA- AND EXTRACELLULAR SUGARS AND DIMETHYLSULFOXIDE. A. Younis, D. Carnovale, A. Eroglu, W. Butler. Department of Obstetrics and Gynecology, Mercer University School of Medicine, Macon, GA; Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, Augusta, GA.
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Abstracts
OBJECTIVE: ASRM guidelines recommended application of current oocyte cryopreservation techniques as experimental. Alternative novel approaches for oocyte cryopreservation technique such as the trehalose microinjection may offer both infertile and fertile women a better alternative protocol to preserve fertility. We report here our preliminary results of preclinical cryopreservation protocol using failed to fertilized (FTF) MII stage oocytes. DESIGN: Discarded human oocytes, obtained from IVF patients under informed consent and IRB approval, were randomly distributed into three groups: (i) control (in 0.5 M dimethylsulfoxide (DMSO) without trehalose injection or extracellular raffinose), (ii) extracellular sugar (raffinose) þ DMSO, and (iii) intra- and extracellular sugar (trehalose and raffinose, respectively) þ DMSO. MATERIALS AND METHODS: We prospectively investigated the survival of human FTF oocytes after cooling to LN2 temperature. Oocytes were exposed to extracellular 0.3M raffinose, then microinjected with trehalose at an intracellular concentration of approximately 0.1 M. The control had no sugar treatment. Oocytes were cryopreserved in straws in Hepes medium containing 10% FBS þ 0.3M Raffinose þ 0.5M dimethylsulphoxide (DMSO). Straws were seeded at 6 C, cooled at 1 C/min to 60 C, then 5 C/min to 135 C, followed by plunging to liquid nitrogen. Oocytes were thawed in a water bath at 31-35 C for 10 sec and stepwise diluted in 0.1 M Galactose solutions. Oocytes were defined as having survived based on the morphological criteria of having an intact zona pellucida, intact oolemma, and a refractive cytoplasm after 24 hour of culture. RESULTS: Eighty-eight percent (14/16) of trehalose micro-injected, cryopreserved human oocytes survived after thawing. Sixty-seven percent (8/12) of non-injected oocytes cryopreserved in the presence of raffinose survived. Forty-six percent (6/13) of non-sugar treated cryopreserved oocytes survived thawing process. CONCLUSIONS: Trehalose injected oocytes cryopreserved in the presence of raffinose had higher survival rate compared to non-injected oocytes frozen in the presence of extracellular sugar. The non-sugar treated oocytes has lowest survival rate. This preliminary data with small number oocytes support the use of intra- and extracellular sugars as an alternative approach for cryopreservation of human oocytes. Clinical studies are needed to demonstrate fertilization and live births from oocytes cryopreserved using the method of microinjection of trehalose. Supported by: None.
P-491 CRYOPRESERVATION OF SEMEN COLLECTED REMOTELYAND PROCESSED AFTER OVERNIGHT SHIPMENT. E. J. Woods, B. C. Perry, L. A. Larson, J. W. Niehaus, M. A. Byers. General BioTechnology, LLC., Indianapolis, IN. OBJECTIVE: This study was designed to begin optimizing a process of cryopreserving extended human sperm shipped via overnight courier service. DESIGN: A remote semen collection kit was developed for the purposes of client depositor banking. The kit was developed to allow men to avail themselves of sperm banking services who might be physically and geographically unable to present themselves at the lab. Donors collected samples off site and shipped them to the lab for evaluation, cryopreservation and post thaw analysis. MATERIALS AND METHODS: The collection kit included a modified Ham’s F-10 buffered extender along with a refrigeration system. Semen samples were produced by men at their homes and transported to the lab overnight in the kit by overnight courier. Upon arrival at the lab, count and motility were scored and the semen was processed and frozen as per our standard cryopreservation protocol (TEST-yolk/glycerol cryoprotectant followed by rapid cooling in a vapor nitrogen shipper and plunging into LN2). Frozen samples were thawed after a minimum of 1 week, washed, and motility was again evaluated. The mean values from 10 donors were compared utilizing ANOVA. RESULTS: Upon arrival at the lab, the mean motility was 5712%. Post thaw, motility dropped to 198% (33% of the prefreeze value), which was significantly different from prefreeze motility (p<0.05). CONCLUSIONS: Collection of semen at a location remote from the processing lab can result in viable cryopreserved samples. Supported by: General BioTechnology, LLC.
Vol. 90, Suppl 1, September 2008
P-490 ENHANCED SURVIVAL OF HUMAN FAILED TO FERTILIZE OOCYTES CRYOPRESERVED USING INTRA- AND EXTRACELLULAR SUGARS AND DIMETHYLSULFOXIDE. A. Younis, D. Carnovale, A. Eroglu, W. Butler. Department of Obstetrics and Gynecology, Mercer University School of Medicine, Macon, GA; Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, Augusta, GA.
S272
Abstracts
OBJECTIVE: ASRM guidelines recommended application of current oocyte cryopreservation techniques as experimental. Alternative novel approaches for oocyte cryopreservation technique such as the trehalose microinjection may offer both infertile and fertile women a better alternative protocol to preserve fertility. We report here our preliminary results of preclinical cryopreservation protocol using failed to fertilized (FTF) MII stage oocytes. DESIGN: Discarded human oocytes, obtained from IVF patients under informed consent and IRB approval, were randomly distributed into three groups: (i) control (in 0.5 M dimethylsulfoxide (DMSO) without trehalose injection or extracellular raffinose), (ii) extracellular sugar (raffinose) þ DMSO, and (iii) intra- and extracellular sugar (trehalose and raffinose, respectively) þ DMSO. MATERIALS AND METHODS: We prospectively investigated the survival of human FTF oocytes after cooling to LN2 temperature. Oocytes were exposed to extracellular 0.3M raffinose, then microinjected with trehalose at an intracellular concentration of approximately 0.1 M. The control had no sugar treatment. Oocytes were cryopreserved in straws in Hepes medium containing 10% FBS þ 0.3M Raffinose þ 0.5M dimethylsulphoxide (DMSO). Straws were seeded at 6 C, cooled at 1 C/min to 60 C, then 5 C/min to 135 C, followed by plunging to liquid nitrogen. Oocytes were thawed in a water bath at 31-35 C for 10 sec and stepwise diluted in 0.1 M Galactose solutions. Oocytes were defined as having survived based on the morphological criteria of having an intact zona pellucida, intact oolemma, and a refractive cytoplasm after 24 hour of culture. RESULTS: Eighty-eight percent (14/16) of trehalose micro-injected, cryopreserved human oocytes survived after thawing. Sixty-seven percent (8/12) of non-injected oocytes cryopreserved in the presence of raffinose survived. Forty-six percent (6/13) of non-sugar treated cryopreserved oocytes survived thawing process. CONCLUSIONS: Trehalose injected oocytes cryopreserved in the presence of raffinose had higher survival rate compared to non-injected oocytes frozen in the presence of extracellular sugar. The non-sugar treated oocytes has lowest survival rate. This preliminary data with small number oocytes support the use of intra- and extracellular sugars as an alternative approach for cryopreservation of human oocytes. Clinical studies are needed to demonstrate fertilization and live births from oocytes cryopreserved using the method of microinjection of trehalose. Supported by: None.
P-491 CRYOPRESERVATION OF SEMEN COLLECTED REMOTELYAND PROCESSED AFTER OVERNIGHT SHIPMENT. E. J. Woods, B. C. Perry, L. A. Larson, J. W. Niehaus, M. A. Byers. General BioTechnology, LLC., Indianapolis, IN. OBJECTIVE: This study was designed to begin optimizing a process of cryopreserving extended human sperm shipped via overnight courier service. DESIGN: A remote semen collection kit was developed for the purposes of client depositor banking. The kit was developed to allow men to avail themselves of sperm banking services who might be physically and geographically unable to present themselves at the lab. Donors collected samples off site and shipped them to the lab for evaluation, cryopreservation and post thaw analysis. MATERIALS AND METHODS: The collection kit included a modified Ham’s F-10 buffered extender along with a refrigeration system. Semen samples were produced by men at their homes and transported to the lab overnight in the kit by overnight courier. Upon arrival at the lab, count and motility were scored and the semen was processed and frozen as per our standard cryopreservation protocol (TEST-yolk/glycerol cryoprotectant followed by rapid cooling in a vapor nitrogen shipper and plunging into LN2). Frozen samples were thawed after a minimum of 1 week, washed, and motility was again evaluated. The mean values from 10 donors were compared utilizing ANOVA. RESULTS: Upon arrival at the lab, the mean motility was 5712%. Post thaw, motility dropped to 198% (33% of the prefreeze value), which was significantly different from prefreeze motility (p<0.05). CONCLUSIONS: Collection of semen at a location remote from the processing lab can result in viable cryopreserved samples. Supported by: General BioTechnology, LLC.
Vol. 90, Suppl 1, September 2008