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Maternal hyperglycemia adversely affects preovulatory oocyte metabolism
MATERIALS AND METHODS: The data consisted of 22 patients who had assisted hatching at the cleavage stage of development followed by blastocyst transfer and 43 patients who had blastocyst transfer without hatching. All patients were aged 37 or over and assisted hatching was carried out either mechanically or using acid tyrodes. Chi squared analysis was used to identify any significant differences in pregnancy rates, ongoing pregnancies and implantation rates. RESULTS: There was no statistically significant differences between the two groups in any of the categories tested. These included the number of positive pregnancy tests, ongoing pregnancies, number of implantation sacs per embryo transfered or the number of fetal hearts per embryo transfered. CONCLUSION: It would appear that there is no advantage to hatching embryos on day 3 and culturing them to blastocyst for patients over 37 years old. From the data it seems that this group of patients who have sufficient embryos of good enough quality to have a blastocyst transfer do not require the intervention of assisted hatching. Supported by: Milki AA, Hinckley MD, Behr B. Comparison of blastocyst transfer to day 3 transfer with assisted hatching in the older patient. Fertil Steril. 2002 Dec;78(6):1244 –7. Graham MC, Hoeger KM, Phipps WR. Initial IVF-ET experience with assisted hatching performed 3 days after retrieval followed by day 5 embryo transfer. Fertil Steril. 2000 Oct; 74(4):668 –71. Lanzendorf SE, Nehchiri F, Mayer JF, Oehninger S, Muasher SJ. A prospective, randomized, double-blind study for the evaluation of assisted hatching in patients with advanced maternal age. Hum Reprod. 1998 Feb;13(2):409 –13.
REPRODUCTIVE BIOLOGY: ANIMAL AND EXPERIMENTAL MODELS P-376 Effects of osteopontin on apoptosis and DNA repair during mouse embryo development in vitro. J. Lyon, H. Wang, D. Dasig, B. Behr. Stanford University Medical Center IVF Program, Palo Alto, CA. OBJECTIVE: Osteopontin (OPN) is a protein with a range of functions, found in many species and observed in reproductive tissues, various cell types, and secretions. The objective of this study is to evaluate the effect of OPN supplementation on apoptosis in the in vitro developed mouse blastocyst. DESIGN: Prospective, experimental study. MATERIALS AND METHODS: Mouse strain B6C3F1, 7– 8 week old females were superovulated and mated 48 hours post hCG injection. Onecell zygotes were harvested 16 –18 hours post-hCG, pooled, then randomly cultured in HTF, HTF ⫹ 10% SSS, or HTF ⫹ 0.125 ng/ml OPN. All groups were cultured under oil in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 37C. Total blastocyst rate for each group was calculated. Apoptosis evaluation was performed using the fluorescein in situ cell death detection TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) assay kit on the mouse blastocyst. Counterstaining with DAPI (4⬘,6-diamidino-2-phenylindole, dihydrochloride) allowed for the calculation of average cell count and average apoptotic rate per blastocyst. Data was analyzed by 2-way ANOVA based on logistic regression (blastocyst rates), Poisson regression (apoptotic rates), and Kruskal-Wallis test (total cell numbers). Statistically significant was considered to be p ⬍ 0.01. RESULTS: There was no significant difference in blastocyst development between the HTF (70.0%) and HTF ⫹ 0.125 ng/ml OPN (70.8%) culture groups. However, the blastocyst development in the culture group HTF ⫹ 10% SSS was significantly higher (87.7%) than the other two culture groups. In assessing apoptotic rate, culture group HTF ⫹ 0.125 ng/ml OPN had significantly lower apoptosis (2.6%) than the HTF culture group (4.6%). There was no significant difference in apoptotic rate between the culture groups HTF ⫹ 10% SSS (3.8%) and HTF ⫹ 0.125 ng/ml OPN. Also, the HTF ⫹ 0.125 ng/ml OPN culture group had significantly higher cell numbers (70.9) than the HTF ⫹ 10% SSS culture group (63.4). No significant difference in cell numbers between the HTF ⫹ 0.125 ng/ml OPN and the HTF culture groups (70.3). CONCLUSION: These results show for the first time that OPN supplemented HTF media can significantly increase mouse blastocyst cell numbers and reduce apoptosis in vitro. This was achieved despite the fact that these embryos were cultured in the absence of any other protein. The findings of
FERTILITY & STERILITY威
this preliminary experiment demonstrate the beneficial effects of OPN as a potential media supplement. Supported by: None.
P-377 Maternal hyperglycemia adversely affects preovulatory oocyte metabolism. A. S. Chang, M. M. Chi, K. H. Moley. Washington University School of Medicine, St. Louis, St. Louis, MO. OBJECTIVE: Maternal diabetes (DM) is associated with an increased risk of miscarriages and congenital anomalies. It has been found to adversely affect the preovulatory oocyte by causing maturational delay and greater apoptosis in the supportive granulosa cells. The objective of this study was to examine if maternal hyperglycemia influenced preovulatory oocyte metabolism which could then, in turn, impact oocyte development. DESIGN: Metabolic analyses of oocytes. MATERIALS AND METHODS: Hyperglycemia was induced with streptozotocin injection in C57BL/6JXSLJ/JF1 mice 20 –23 days old. Mice were sequentially primed with equine chorionic gonadotropin (eCG) and hCG. Follicles were punctured and oocytes were collected at timed intervals after hCG injection (T⫽ 0, 2, 6h). Oocytes were freeze-dried and extract aliquots were obtained to measure ATP, 5⬘-AMP, glucose-6-phosphate dehydrogenase(G6PDH), adenylate kinase, -hydroxyacyl CoA dehydrogenase (BOAC), and alanine transaminase(SGOT). Statistical analysis was performed with Student’s t-test and ANOVA. RESULTS: ATP levels were significantly lower in diabetic denuded oocytes (24.3%) and cumulus enclosed oocyte complexes (58.5%) compared to controls (P⬍.01). 5⬘-AMP and G6PDH levels were not significantly different in the DM versus the control groups. Adenylate kinase activity was remarkably lower in the DM group (P⬍.05). Similarly, both BOAC and SGOT which are important enzymes in the lipid and amino acid metabolic pathways respectively were significantly less in the DM group. CONCLUSION: ATP levels were lower in diabetic denuded oocytes and complexes which may represent a decrease in overall oocyte health and energy. In hyperglycemia, there may be a loss of proper communication because several parameters were not upregulated as expected in this stress situation, including adenylate kinase and enzymes critical to lipid and amino acid metabolism. These alterations of metabolism may negatively affect the developing oocyte and result in problems in the preimplantation embryos and fetuses of diabetic mothers. Supported by: Supported in part by the ASRM Ortho/McNeil Research Grant and NICHD Cooperative Program on Female Health and Egg Quality, U01 HD044691.
P-378 High isoflavone soy protein does not alter menstrual cyclicity or ovarian function in fully mature, premenopausal monkeys. J. R. Kaplan, S. L. Berga, M. E. Wilson, T. B. Clarkson. Wake Forest University School of Medicine, Winston-Salem, NC; Emory University School of Medicine, Atlanta, GA; Yerkes National Primate Research Center, Emory University, Atlanta, GA. OBJECTIVE: Soy may increase menstrual cycle length or reduce ovarian hormones in women, thereby providing protection against breast cancer but potentially impairing fertility. The study reported here was designed to determine whether soy supplementation alters any aspect of ovarian function in premenopausal monkeys. DESIGN: A six month baseline period during which all subjects consumed an animal protein-based diet was followed by a 12 month treatment period involving random assignment of one-half of individuals to the diet deriving its protein from animal sources and the remainder to a diet deriving its protein from soy. Menstrual cyclicity and ovarian hormones were assessed during the baseline period and again following treatment. Predictor variables included protein source (animal vs. soy), and social status (because high social status in monkeys is generally associated with normal ovarian function while low status induces an ovarian deficient state). MATERIALS AND METHODS: Subjects were 96 fully adult, premenopausal cynomolgus monkeys (Macaca fascicularis) imported from Indonesia and housed in social groups of five or six individuals each. The experimental diets (animal source vs. soy) derived their protein entirely
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REPRODUCTIVE BIOLOGY: ANIMAL AND EXPERIMENTAL MODELS P-376 Effects of osteopontin on apoptosis and DNA repair during mouse embryo development in vitro. J. Lyon, H. Wang, D. Dasig, B. Behr. Stanford University Medical Center IVF Program, Palo Alto, CA. OBJECTIVE: Osteopontin (OPN) is a protein with a range of functions, found in many species and observed in reproductive tissues, various cell types, and secretions. The objective of this study is to evaluate the effect of OPN supplementation on apoptosis in the in vitro developed mouse blastocyst. DESIGN: Prospective, experimental study. MATERIALS AND METHODS: Mouse strain B6C3F1, 7– 8 week old females were superovulated and mated 48 hours post hCG injection. Onecell zygotes were harvested 16 –18 hours post-hCG, pooled, then randomly cultured in HTF, HTF ⫹ 10% SSS, or HTF ⫹ 0.125 ng/ml OPN. All groups were cultured under oil in a humidified atmosphere of 5% CO2, 5% O2 and 90% N2 at 37C. Total blastocyst rate for each group was calculated. Apoptosis evaluation was performed using the fluorescein in situ cell death detection TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) assay kit on the mouse blastocyst. Counterstaining with DAPI (4⬘,6-diamidino-2-phenylindole, dihydrochloride) allowed for the calculation of average cell count and average apoptotic rate per blastocyst. Data was analyzed by 2-way ANOVA based on logistic regression (blastocyst rates), Poisson regression (apoptotic rates), and Kruskal-Wallis test (total cell numbers). Statistically significant was considered to be p ⬍ 0.01. RESULTS: There was no significant difference in blastocyst development between the HTF (70.0%) and HTF ⫹ 0.125 ng/ml OPN (70.8%) culture groups. However, the blastocyst development in the culture group HTF ⫹ 10% SSS was significantly higher (87.7%) than the other two culture groups. In assessing apoptotic rate, culture group HTF ⫹ 0.125 ng/ml OPN had significantly lower apoptosis (2.6%) than the HTF culture group (4.6%). There was no significant difference in apoptotic rate between the culture groups HTF ⫹ 10% SSS (3.8%) and HTF ⫹ 0.125 ng/ml OPN. Also, the HTF ⫹ 0.125 ng/ml OPN culture group had significantly higher cell numbers (70.9) than the HTF ⫹ 10% SSS culture group (63.4). No significant difference in cell numbers between the HTF ⫹ 0.125 ng/ml OPN and the HTF culture groups (70.3). CONCLUSION: These results show for the first time that OPN supplemented HTF media can significantly increase mouse blastocyst cell numbers and reduce apoptosis in vitro. This was achieved despite the fact that these embryos were cultured in the absence of any other protein. The findings of
FERTILITY & STERILITY威
this preliminary experiment demonstrate the beneficial effects of OPN as a potential media supplement. Supported by: None.
P-377 Maternal hyperglycemia adversely affects preovulatory oocyte metabolism. A. S. Chang, M. M. Chi, K. H. Moley. Washington University School of Medicine, St. Louis, St. Louis, MO. OBJECTIVE: Maternal diabetes (DM) is associated with an increased risk of miscarriages and congenital anomalies. It has been found to adversely affect the preovulatory oocyte by causing maturational delay and greater apoptosis in the supportive granulosa cells. The objective of this study was to examine if maternal hyperglycemia influenced preovulatory oocyte metabolism which could then, in turn, impact oocyte development. DESIGN: Metabolic analyses of oocytes. MATERIALS AND METHODS: Hyperglycemia was induced with streptozotocin injection in C57BL/6JXSLJ/JF1 mice 20 –23 days old. Mice were sequentially primed with equine chorionic gonadotropin (eCG) and hCG. Follicles were punctured and oocytes were collected at timed intervals after hCG injection (T⫽ 0, 2, 6h). Oocytes were freeze-dried and extract aliquots were obtained to measure ATP, 5⬘-AMP, glucose-6-phosphate dehydrogenase(G6PDH), adenylate kinase, -hydroxyacyl CoA dehydrogenase (BOAC), and alanine transaminase(SGOT). Statistical analysis was performed with Student’s t-test and ANOVA. RESULTS: ATP levels were significantly lower in diabetic denuded oocytes (24.3%) and cumulus enclosed oocyte complexes (58.5%) compared to controls (P⬍.01). 5⬘-AMP and G6PDH levels were not significantly different in the DM versus the control groups. Adenylate kinase activity was remarkably lower in the DM group (P⬍.05). Similarly, both BOAC and SGOT which are important enzymes in the lipid and amino acid metabolic pathways respectively were significantly less in the DM group. CONCLUSION: ATP levels were lower in diabetic denuded oocytes and complexes which may represent a decrease in overall oocyte health and energy. In hyperglycemia, there may be a loss of proper communication because several parameters were not upregulated as expected in this stress situation, including adenylate kinase and enzymes critical to lipid and amino acid metabolism. These alterations of metabolism may negatively affect the developing oocyte and result in problems in the preimplantation embryos and fetuses of diabetic mothers. Supported by: Supported in part by the ASRM Ortho/McNeil Research Grant and NICHD Cooperative Program on Female Health and Egg Quality, U01 HD044691.
P-378 High isoflavone soy protein does not alter menstrual cyclicity or ovarian function in fully mature, premenopausal monkeys. J. R. Kaplan, S. L. Berga, M. E. Wilson, T. B. Clarkson. Wake Forest University School of Medicine, Winston-Salem, NC; Emory University School of Medicine, Atlanta, GA; Yerkes National Primate Research Center, Emory University, Atlanta, GA. OBJECTIVE: Soy may increase menstrual cycle length or reduce ovarian hormones in women, thereby providing protection against breast cancer but potentially impairing fertility. The study reported here was designed to determine whether soy supplementation alters any aspect of ovarian function in premenopausal monkeys. DESIGN: A six month baseline period during which all subjects consumed an animal protein-based diet was followed by a 12 month treatment period involving random assignment of one-half of individuals to the diet deriving its protein from animal sources and the remainder to a diet deriving its protein from soy. Menstrual cyclicity and ovarian hormones were assessed during the baseline period and again following treatment. Predictor variables included protein source (animal vs. soy), and social status (because high social status in monkeys is generally associated with normal ovarian function while low status induces an ovarian deficient state). MATERIALS AND METHODS: Subjects were 96 fully adult, premenopausal cynomolgus monkeys (Macaca fascicularis) imported from Indonesia and housed in social groups of five or six individuals each. The experimental diets (animal source vs. soy) derived their protein entirely
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