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Dynamics of the human oocyte microrna transcriptome during maturation
P-463 Wednesday, October 21, 2009 MITOCHONDRIAL FUNCTION, QUANTITY AND DNA INTEGRITY IN HUMAN OOCYTES AT DIFFERENT STAGES OF MATURATION. H. E. Duran, F. Simsek-Duran, S. Oehninger, R. J. Swanson, H. Jones, F. Castora. Obstetrics and Gynecology, The Jones Institute for Reproductive Medicine, Eastern Virginia Medical School, Norfolk, VA; Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA; Biological Sciences, Old Dominion University, Norfolk, VA. OBJECTIVE: Human oocytes significantly vary in pregnancy potential. Differences in energy metabolism may contribute to this variation. We investigated ATP content and mitochondrial DNA (mtDNA) copy number and integrity in individual human oocytes at different stages of maturation. DESIGN: Experimental study. MATERIALS AND METHODS: A total of 41 oocytes obtained from 12 women 25-38 years of age during IVF cycles were examined. 22 were at prophase 1 (P1), 11 were at metaphase 1 (M1) and 6 were at the M2 stage, whereas 2 were degenerating. The main outcome parameters were ATP content and mtDNA copy number; a secondary outcome was mtDNA integrity, as assessed by the existence of the 5 Kb ‘‘common’’ mtDNA deletion. A chemiluminsecent luciferase/luciferin reaction was utilized to measure ATP content, whereas real-time PCR was used to quantify the total mtDNA copy number. Nested PCR was applied to detect the 5 Kb mtDNA deletion. RESULTS: The mean ATP content was 774 418 fmol 977 364 fmol , and 1957 516 fmol for P1, M1 and M2 oocytes respectively (p¼0.003 for P1-M2, p¼0.025 for M1-M2, p¼1.0 for P1-M1). Total mtDNA copy numbers were similar among the various stages of oocyte maturation (57,116 83,834, 323,372 521,503 and 77,834 115,755 for P1, M1 and M2 oocytes, respectively). mtDNA copy number correlated positively with the duration of incubation between oocyte retrieval and lysis for analysis (r¼0.43, p¼0.01); whereas ATP content did not. There was no effect of woman’s age on ATP content and mtDNA copy number. However, we observed the presence of the common deletion in the mtDNA in 6 (4 M1, 2 degenerating) of the 22 oocytes tested. CONCLUSIONS: ATP content increased with oocyte developmental progress from the P1 to M2 stage. In addition, mitochondrial number increased in parallel to the duration of in vitro culture. We speculate that these changes prepare the oocyte for an optimized mitochondrial redistribution during early embryogenesis. Supported by: Grant from the Jones Institute Foundation and the Dept. of Ob/Gyn, EVMS.
OOCYTE MATURATION P-464 Wednesday, October 21, 2009 DYNAMICS OF THE HUMAN OOCYTE MICRORNA TRANSCRIPTOME DURING MATURATION. A. Lonczak, X. Tao, K. Miller, R. Scott, N. Treff. Reproductive Medicine Associates of New Jersey, Morristown, NJ; UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: One of the major functions of micro ribonucleic acid (miRNA) is to negatively regulate gene expression through messenger RNA (mRNA) degradation. Since mRNA degradation is one of the hallmarks of oocyte maturation, it is logical to hypothesize that miRNAs play an important role in this process. Therefore, this study sought to characterize expression of miRNAs in human germinal vesicle (GV), meiosis I (MI), and meiosis II (MII) oocytes for the first time. DESIGN: Descriptive study. MATERIALS AND METHODS: Eight GV, MI, and MII oocytes were evaluated. TaqMan real time PCR arrays were used to study expression levels of 667 miRNAs, representing the entire Sanger miRNA database (miRBase version 10.0). GVoocytes were used as the reference sample to identify miRNAs with significant (p<0.05) differential expression in MI and MII oocytes using a Student’s t-test for each group. RESULTS: Seven miRNAs were significantly differentially expressed in the MI oocyte and 32 were significant in the MII oocyte relative to the GV stage. Two miRNAs were significantly downregulated in both MI and MII stage oocytes; hsa-miR-339-3p (-22 and -15 fold), and hsa-miR-744 (-140 and -40 fold), respectively. CONCLUSIONS: Analysis of the miRNA transcriptome has revealed novel miRNA expression patterns with significant regulation during oocyte
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maturation. Evaluation of their association with reproductive potential and expression at post-fertilization stages is ongoing. This report represents the first comprehensive analysis of miRNA expression in the human oocyte.
P-465 Wednesday, October 21, 2009 THE EFFECT OF ANTIOXIDANT AGENT DURING IN VITRO MATURATION (IVM) OF MURINE OOCYTES ON SURVIVAL, FERTILIZATION AND EARLY EMBRYONIC DEVELOPMENT FOLLOWING VITRIFICATION. X. Yu, J. Yan, K. Rao, X. He, S. Tan, R.-C. Chian. Department of Obstetrics and Gynecology, Mcgill University, Montreal, QC, Canada; Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China; First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China. OBJECTIVE: It has been reported that there are differences between in vitro and in vivo matured oocytes for their survival rates following vitrification The purpose of this study is to determine the competency of cryodamage resistance of in vitro matured oocytes in the presence of cysteamine during in vitro maturation (IVM). DESIGN: Animal-model study. MATERIALS AND METHODS: Female mice (CD-1) were stimulated with 10IU PMSG. Oocytes were collected after 48h and the collected oocytes were divided into following groups: (1) Cumulus-oocyte-complex (COC); (2) denuded groups. Both COC and denuded oocytes were cultured in IVM medium containing 75mIU/mL FSH and LH, 10% FBS for 16 h in the presence or absence of cysteamine (200mM). Following culture, the maturated oocytes were either vitrified or inseminated immediately. Vitrification and thawing procedures were performed with McGill Cryoleaf as reported previously. Vitrified oocytes were thawed and inseminated on the other day. Fertilization rate were recorded 24 h after insemination, and early embryonic development were assessed. Statistical differences were considered significant at p<0.05. RESULTS: The presence of cysteamine during IVM significantly increased maturation rate in COC group but had no significant difference in denuded group. The presence of cyseamine during IVM does not improve the survival rate of in vitro matured oocytes with or without cumulus cells during IVM. However, the presence of cumulus cells during IVM had better fertilization rate (p<0.05) compared to the denuded oocytes after vitrificationthawing. The presence of cysteamine during IVM significantly reduced the fragmentation rate in cumulus-intact group compared to cumulus-free group following vitrification-thawing. CONCLUSIONS: The presence of cysteamine during IVM has beneficial effect on both COC and denuded oocytes in terms of their competency of subsequent fertilization and early embryonic development following vitrification. Supported by: Vitrification-kits were provided by MediCult company (Demark).
P-466 Wednesday, October 21, 2009 OOCYTE MATURATION RATE: AN INDICATOR OF CLINICAL OUTCOME. C. A. Guerrero, D. M. Bookout, S. J. Chantilis, A. J. Rodriguez, R. A. Kaufmann, D. G. Hammitt. ARTS, Texas Health Presbyterian Dallas, Dallas, TX; ARTS, Texas Health Presbyterian Plano, Plano, TX; ARTS, Texas Health Harris Methodist Fort Worth, Fort Worth, TX. OBJECTIVE: Indirect markers to assess the developmental potential of an oocyte cohort after controlled ovarian hyperstimulation are limited. Our objective was to evaluate the effect of in vivo oocyte maturation rate on implantation and birth rates. DESIGN: Data were prospectively collected over a 6-year period. Patients were divided into four groups based on the percentage of mature oocytes. Clinical and laboratory end points were analyzed using t-test and Fisher’s exact test. MATERIALS AND METHODS: Patients undergoing ICSI (n¼2191) %37 years old were included. Oocyte maturation was assessed by extrusion of the first polar body at ICSI, 6-10 hours post retrieval. Injected oocytes were cultured in sequential G-1/G-2Ô media under low oxygen tension (5%). Epididymal or testicular sperm, donor oocytes and patients with no mature oocytes were excluded. RESULTS: There was a higher implantation, clinical pregnancy and birth rate as the number of retrieved mature oocytes increased. No difference was
Vol. 92., No. 3, Supplement, September 2009
OOCYTE MATURATION P-464 Wednesday, October 21, 2009 DYNAMICS OF THE HUMAN OOCYTE MICRORNA TRANSCRIPTOME DURING MATURATION. A. Lonczak, X. Tao, K. Miller, R. Scott, N. Treff. Reproductive Medicine Associates of New Jersey, Morristown, NJ; UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ. OBJECTIVE: One of the major functions of micro ribonucleic acid (miRNA) is to negatively regulate gene expression through messenger RNA (mRNA) degradation. Since mRNA degradation is one of the hallmarks of oocyte maturation, it is logical to hypothesize that miRNAs play an important role in this process. Therefore, this study sought to characterize expression of miRNAs in human germinal vesicle (GV), meiosis I (MI), and meiosis II (MII) oocytes for the first time. DESIGN: Descriptive study. MATERIALS AND METHODS: Eight GV, MI, and MII oocytes were evaluated. TaqMan real time PCR arrays were used to study expression levels of 667 miRNAs, representing the entire Sanger miRNA database (miRBase version 10.0). GVoocytes were used as the reference sample to identify miRNAs with significant (p<0.05) differential expression in MI and MII oocytes using a Student’s t-test for each group. RESULTS: Seven miRNAs were significantly differentially expressed in the MI oocyte and 32 were significant in the MII oocyte relative to the GV stage. Two miRNAs were significantly downregulated in both MI and MII stage oocytes; hsa-miR-339-3p (-22 and -15 fold), and hsa-miR-744 (-140 and -40 fold), respectively. CONCLUSIONS: Analysis of the miRNA transcriptome has revealed novel miRNA expression patterns with significant regulation during oocyte
S218
Abstracts
maturation. Evaluation of their association with reproductive potential and expression at post-fertilization stages is ongoing. This report represents the first comprehensive analysis of miRNA expression in the human oocyte.
P-465 Wednesday, October 21, 2009 THE EFFECT OF ANTIOXIDANT AGENT DURING IN VITRO MATURATION (IVM) OF MURINE OOCYTES ON SURVIVAL, FERTILIZATION AND EARLY EMBRYONIC DEVELOPMENT FOLLOWING VITRIFICATION. X. Yu, J. Yan, K. Rao, X. He, S. Tan, R.-C. Chian. Department of Obstetrics and Gynecology, Mcgill University, Montreal, QC, Canada; Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China; First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China. OBJECTIVE: It has been reported that there are differences between in vitro and in vivo matured oocytes for their survival rates following vitrification The purpose of this study is to determine the competency of cryodamage resistance of in vitro matured oocytes in the presence of cysteamine during in vitro maturation (IVM). DESIGN: Animal-model study. MATERIALS AND METHODS: Female mice (CD-1) were stimulated with 10IU PMSG. Oocytes were collected after 48h and the collected oocytes were divided into following groups: (1) Cumulus-oocyte-complex (COC); (2) denuded groups. Both COC and denuded oocytes were cultured in IVM medium containing 75mIU/mL FSH and LH, 10% FBS for 16 h in the presence or absence of cysteamine (200mM). Following culture, the maturated oocytes were either vitrified or inseminated immediately. Vitrification and thawing procedures were performed with McGill Cryoleaf as reported previously. Vitrified oocytes were thawed and inseminated on the other day. Fertilization rate were recorded 24 h after insemination, and early embryonic development were assessed. Statistical differences were considered significant at p<0.05. RESULTS: The presence of cysteamine during IVM significantly increased maturation rate in COC group but had no significant difference in denuded group. The presence of cyseamine during IVM does not improve the survival rate of in vitro matured oocytes with or without cumulus cells during IVM. However, the presence of cumulus cells during IVM had better fertilization rate (p<0.05) compared to the denuded oocytes after vitrificationthawing. The presence of cysteamine during IVM significantly reduced the fragmentation rate in cumulus-intact group compared to cumulus-free group following vitrification-thawing. CONCLUSIONS: The presence of cysteamine during IVM has beneficial effect on both COC and denuded oocytes in terms of their competency of subsequent fertilization and early embryonic development following vitrification. Supported by: Vitrification-kits were provided by MediCult company (Demark).
P-466 Wednesday, October 21, 2009 OOCYTE MATURATION RATE: AN INDICATOR OF CLINICAL OUTCOME. C. A. Guerrero, D. M. Bookout, S. J. Chantilis, A. J. Rodriguez, R. A. Kaufmann, D. G. Hammitt. ARTS, Texas Health Presbyterian Dallas, Dallas, TX; ARTS, Texas Health Presbyterian Plano, Plano, TX; ARTS, Texas Health Harris Methodist Fort Worth, Fort Worth, TX. OBJECTIVE: Indirect markers to assess the developmental potential of an oocyte cohort after controlled ovarian hyperstimulation are limited. Our objective was to evaluate the effect of in vivo oocyte maturation rate on implantation and birth rates. DESIGN: Data were prospectively collected over a 6-year period. Patients were divided into four groups based on the percentage of mature oocytes. Clinical and laboratory end points were analyzed using t-test and Fisher’s exact test. MATERIALS AND METHODS: Patients undergoing ICSI (n¼2191) %37 years old were included. Oocyte maturation was assessed by extrusion of the first polar body at ICSI, 6-10 hours post retrieval. Injected oocytes were cultured in sequential G-1/G-2Ô media under low oxygen tension (5%). Epididymal or testicular sperm, donor oocytes and patients with no mature oocytes were excluded. RESULTS: There was a higher implantation, clinical pregnancy and birth rate as the number of retrieved mature oocytes increased. No difference was
Vol. 92., No. 3, Supplement, September 2009