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A Non-Invasive Bioassay for Real-Time Measurement of Glucose Uptake by Single Embryos
collected from a PCOS patient were also exposed to insulin and the IGFs and VEGF secretion was evaluated. Results: IGF-I, IGF-II and insulin were shown to increase secreted VEGF by 3– 4 fold (ANOVA and Student’s t test; p,.05). Interestingly, both the baseline and the stimulated secretion of VEGF from the GCs of the PCOS patient were markedly increased. In fact there was a 3-fold increase in baseline VEGF secretion, a 9-fold increase by IGF-I, a 13-fold by IGF-II and a 4-fold stimulation by insulin as compared to secretion from cells of non-PCOS patients. Conclusions: We conclude that IGF-I, IGF-II and insulin increase the release of VEGF by luteinized human GCs. The exaggerated response of PCOS GCs to these factors may explain why patients with PCOS and insulin resistance develop OHSS. Additionally, these results give further support to the hypothesis that the intra-ovarian IGF system plays an important role in luteotropism. [Supported by NIH grant HD-31903].
REPRODUCTIVE LABORATORY TECHNOLOGY PROFESSIONAL GROUP Tuesday, October 24, 2000 2:00 P.M. O-101 A Non-Invasive Bioassay for Real-Time Measurement of Glucose Uptake by Single Embryos. 1,3J. R. Pepperell, 2L. Liu, 2,3D. L. Keefe, 1P. J. S. Smith. 1BioCurrents Research Center, and 2Laboratory for Reproductive Medicine, Marine Biological Laboratory, Woods Hole, MA; 3Department Ob/Gyn, Brown University Medical School, Providence, RI. Objectives: Improvements in human IVF culture conditions have enabled longer culture periods of embryos that has led to the transfer of 8-cell and later-stage embryos to patients. With long-term embryo culture, the potential exists to profile biochemical parameters in a single embryo in order to select for transfer, only embryos of good developmental potential. This approach has been limited, until now, by the inherently small sample size available for assay. Since uptake of energy substrates is indicative of a healthy respiring embryo, the objective of this study is to build a noninvasive, self-referencing glucose microelectrode to measure glucose uptake in embryos. Design: Techniques in electrode construction were used to make micro-, glucose oxidase-based electrodes. Measurement of glucose flux by the electrodes was assessed against a glucose-source micropipette, and mouse blastocysts. Materials and Methods: A carbon fiber electrode enclosed in a glass microcapillary was constructed with a diameter of approximately 10 mm. The fiber was electrochemically and chemically treated that enabled the attachment of biotin molecules to which avidin was subsequently bound. Biotinylated glucose oxidase was then bound to the avidin. Activity of glucose oxidase forms H2O2 that is measured amperometrically at 1.2V. The glucose-sensitive electrode was connected to a headstage attached to a computer-controlled micromanipulator that oscillated the electrode between two points 30 mm apart and established self-referencing. The difference in concentration between the two points was applied to Fick’s equation to calculate net glucose flux. Mouse 2-cell embryos were obtained from B6C3F1 crossed mice, and cultured in KSOM media to blastocyst stage. Blastocysts were placed in modified HTF with or without glucose (5mM) for glucose uptake measurement experiments. Results: The electrode responded linearly to increasing glucose concentrations (0 –35 mM). A source micropipette of diameter 5–10 mm containing 200 mM glucose in 0.3% agar was used in a bath of 2 mM glucose to established a gradient. The electrode measured the gradient and a net efflux from the source pipette. Measurement of glucose-dependent current adjacent to the zonae pellucidae of mouse blastocysts in the absence of glucose, showed no net change in the current, indicating no net flux of glucose. However, in the presence of 5 mM glucose, the glucose-dependent current indicated a net glucose influx in 4 out of 7 blastocysts. The morphologic appearance of the blastocysts where no flux was detected was poor. Conclusions: The self-referencing glucose electrode measures glucose flux. Current indicating glucose influx in some mouse blastocysts is consistent with glucose metabolism by these cells. This study suggests this tech-
FERTILITY & STERILITYt
nique may be applicable for assessing metabolism in single embryos that may be a predictor of embryo viability. Supported by P41 RR01395 to PJSS.
Tuesday, October 24, 2000 2:15 P.M. O-102 Developmental Ability and Normality of Mouse Oocytes Reconstructed by Pronuclear Transfer. J. Mizuno, M. Kuwayama, F. Aono, O. Kato. R&D division, Kato Ladies Clinic, Tokyo, Japan. Objectives: It is suggested that there is a possibility to improve the quality and the developmental ability of oocytes from old patients by microinjection of intact ooplasm of young donors (Mol Hum Reprod, 1998;4:269 –280). Whole exchange of cytoplasm between patient and donor oocytes can be completed by using the pronuclear (PN) transfer technology, and this may maximize the improving of oocytes quality. This study was conducted to know the safety of the PN transfer technology using mice oocytes as an animal model. Design: Developmental ability of the reconstructed oocytes in vitro/in vivo, and morphological normality and reproductive ability of the youngs were examined for the embryos derived from female PN transferred, both 2PNs transferred and intact oocytes. Materials and Methods: F1(C57BL/6JxCBA) oocytes (n5361) were used as the source of the karyoplasts and the cytoplasts for the experiments. Female PN or both male and female PNs were removed out by microaspiration. The karyoplast was immediately inserted into the perivitelline space of a previously enucleated donor oocyte. Then oocytes were electrically fused by the double pulses of 1 kv/cm for 70 ms. After confirmation of the fusion of the oocytes, they were cultured for 4 days and observed the further in vitro development to the blastocyst stage. Some blastocysts were transferred to pseudo pregnant recipients. No. of pregnancies and youngs, and their morphological normality, birth weights were examined. Furthermore, they were mated each other at 6 weeks old and examined the pregnancies. Results: The fusion rates of the PN and 2PNs transferred oocytes were similar at high level (94 and 97%). The developmental rates to blastocysts of the PN and 2PNs groups were 63% and 60%, respectively, and were significantly lower than those of control (80%, P,0.05). The proportions of pregnancy after transfer of the blastocysts derived from PN, 2PNs transferred and control groups were 60, 50 and 60%, respectively, and there was no significantly difference among the groups. However, proportion of youngs (no. youngs/no. pregnancies) of the PN and 2PNs transferred groups were lower (31 and 36%) than those in control (54%, P,0.05). Birth weights of the young and placenta weights were normal in all the groups (average weight of youngs 1.1760.12g and placenta 0.8960.01g). All of the youngs derived from PN/2PNs transferred and control embryos were morphologically normal at birth and matured to adult mice. All the female adults became pregnant after mating with the male adults. Conclusion: The results suggested that the continuous treatments of micro manipulation and electric stimulation slightly decrease of developmental ability in of the oocytes vitro/in vivo. However, the developmental rates of PN and 2PNs transferred oocytes were still acceptable for the practical use for the experiments, and morphological and reproductive normality of the youngs indicate that the PN(s) transfer technology of the present study may have value for the further studies for human ART.
Tuesday, October 24, 2000 2:30 P.M. O-103 Nuclear and Cytoplasmic Maturation of Human Oocytes Cultured in a Serum and Albumin Free Chemically Defined Medium. 1,2Z. Yang, 2 A. K. Goff, 3Y. Zhang, 3H. Zhao, 4J. Smitz, 1J. Kustin. 1Washington Center for Reproductive Medicine, Bellevue, WA, USA; 2University of Montreal, Quebec, Canada; 3Harbin Medical University, Harbin, China; 4 Dutch-Speaking Free University, Brussels, Belgium. Objectives: It has been suggested that use of a medium containing serum or albumin for culture of human ova may confound outcome of results and
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REPRODUCTIVE LABORATORY TECHNOLOGY PROFESSIONAL GROUP Tuesday, October 24, 2000 2:00 P.M. O-101 A Non-Invasive Bioassay for Real-Time Measurement of Glucose Uptake by Single Embryos. 1,3J. R. Pepperell, 2L. Liu, 2,3D. L. Keefe, 1P. J. S. Smith. 1BioCurrents Research Center, and 2Laboratory for Reproductive Medicine, Marine Biological Laboratory, Woods Hole, MA; 3Department Ob/Gyn, Brown University Medical School, Providence, RI. Objectives: Improvements in human IVF culture conditions have enabled longer culture periods of embryos that has led to the transfer of 8-cell and later-stage embryos to patients. With long-term embryo culture, the potential exists to profile biochemical parameters in a single embryo in order to select for transfer, only embryos of good developmental potential. This approach has been limited, until now, by the inherently small sample size available for assay. Since uptake of energy substrates is indicative of a healthy respiring embryo, the objective of this study is to build a noninvasive, self-referencing glucose microelectrode to measure glucose uptake in embryos. Design: Techniques in electrode construction were used to make micro-, glucose oxidase-based electrodes. Measurement of glucose flux by the electrodes was assessed against a glucose-source micropipette, and mouse blastocysts. Materials and Methods: A carbon fiber electrode enclosed in a glass microcapillary was constructed with a diameter of approximately 10 mm. The fiber was electrochemically and chemically treated that enabled the attachment of biotin molecules to which avidin was subsequently bound. Biotinylated glucose oxidase was then bound to the avidin. Activity of glucose oxidase forms H2O2 that is measured amperometrically at 1.2V. The glucose-sensitive electrode was connected to a headstage attached to a computer-controlled micromanipulator that oscillated the electrode between two points 30 mm apart and established self-referencing. The difference in concentration between the two points was applied to Fick’s equation to calculate net glucose flux. Mouse 2-cell embryos were obtained from B6C3F1 crossed mice, and cultured in KSOM media to blastocyst stage. Blastocysts were placed in modified HTF with or without glucose (5mM) for glucose uptake measurement experiments. Results: The electrode responded linearly to increasing glucose concentrations (0 –35 mM). A source micropipette of diameter 5–10 mm containing 200 mM glucose in 0.3% agar was used in a bath of 2 mM glucose to established a gradient. The electrode measured the gradient and a net efflux from the source pipette. Measurement of glucose-dependent current adjacent to the zonae pellucidae of mouse blastocysts in the absence of glucose, showed no net change in the current, indicating no net flux of glucose. However, in the presence of 5 mM glucose, the glucose-dependent current indicated a net glucose influx in 4 out of 7 blastocysts. The morphologic appearance of the blastocysts where no flux was detected was poor. Conclusions: The self-referencing glucose electrode measures glucose flux. Current indicating glucose influx in some mouse blastocysts is consistent with glucose metabolism by these cells. This study suggests this tech-
FERTILITY & STERILITYt
nique may be applicable for assessing metabolism in single embryos that may be a predictor of embryo viability. Supported by P41 RR01395 to PJSS.
Tuesday, October 24, 2000 2:15 P.M. O-102 Developmental Ability and Normality of Mouse Oocytes Reconstructed by Pronuclear Transfer. J. Mizuno, M. Kuwayama, F. Aono, O. Kato. R&D division, Kato Ladies Clinic, Tokyo, Japan. Objectives: It is suggested that there is a possibility to improve the quality and the developmental ability of oocytes from old patients by microinjection of intact ooplasm of young donors (Mol Hum Reprod, 1998;4:269 –280). Whole exchange of cytoplasm between patient and donor oocytes can be completed by using the pronuclear (PN) transfer technology, and this may maximize the improving of oocytes quality. This study was conducted to know the safety of the PN transfer technology using mice oocytes as an animal model. Design: Developmental ability of the reconstructed oocytes in vitro/in vivo, and morphological normality and reproductive ability of the youngs were examined for the embryos derived from female PN transferred, both 2PNs transferred and intact oocytes. Materials and Methods: F1(C57BL/6JxCBA) oocytes (n5361) were used as the source of the karyoplasts and the cytoplasts for the experiments. Female PN or both male and female PNs were removed out by microaspiration. The karyoplast was immediately inserted into the perivitelline space of a previously enucleated donor oocyte. Then oocytes were electrically fused by the double pulses of 1 kv/cm for 70 ms. After confirmation of the fusion of the oocytes, they were cultured for 4 days and observed the further in vitro development to the blastocyst stage. Some blastocysts were transferred to pseudo pregnant recipients. No. of pregnancies and youngs, and their morphological normality, birth weights were examined. Furthermore, they were mated each other at 6 weeks old and examined the pregnancies. Results: The fusion rates of the PN and 2PNs transferred oocytes were similar at high level (94 and 97%). The developmental rates to blastocysts of the PN and 2PNs groups were 63% and 60%, respectively, and were significantly lower than those of control (80%, P,0.05). The proportions of pregnancy after transfer of the blastocysts derived from PN, 2PNs transferred and control groups were 60, 50 and 60%, respectively, and there was no significantly difference among the groups. However, proportion of youngs (no. youngs/no. pregnancies) of the PN and 2PNs transferred groups were lower (31 and 36%) than those in control (54%, P,0.05). Birth weights of the young and placenta weights were normal in all the groups (average weight of youngs 1.1760.12g and placenta 0.8960.01g). All of the youngs derived from PN/2PNs transferred and control embryos were morphologically normal at birth and matured to adult mice. All the female adults became pregnant after mating with the male adults. Conclusion: The results suggested that the continuous treatments of micro manipulation and electric stimulation slightly decrease of developmental ability in of the oocytes vitro/in vivo. However, the developmental rates of PN and 2PNs transferred oocytes were still acceptable for the practical use for the experiments, and morphological and reproductive normality of the youngs indicate that the PN(s) transfer technology of the present study may have value for the further studies for human ART.
Tuesday, October 24, 2000 2:30 P.M. O-103 Nuclear and Cytoplasmic Maturation of Human Oocytes Cultured in a Serum and Albumin Free Chemically Defined Medium. 1,2Z. Yang, 2 A. K. Goff, 3Y. Zhang, 3H. Zhao, 4J. Smitz, 1J. Kustin. 1Washington Center for Reproductive Medicine, Bellevue, WA, USA; 2University of Montreal, Quebec, Canada; 3Harbin Medical University, Harbin, China; 4 Dutch-Speaking Free University, Brussels, Belgium. Objectives: It has been suggested that use of a medium containing serum or albumin for culture of human ova may confound outcome of results and
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