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MATERIALS AND METHODS: The experiments were carried out in primarily cultured rat skeletal muscle cells. Homogeneity of the skeletal muscle culture was confirmed using immunohistochemical staining for myosin heavy chain. Differentiated myotubes were stimulated by insulin for 20-30 min after 16-hour pre-exposure to low (20ng/ml) or high (200ng/ml) dose of testosterone. The phosphorylation of insulin receptor substrate-1 (IRS-1), Akt, mammalian target of rapamycin (mTOR), and p70 protein kinase (S6K) were compared to that from cells without pre-exposure to testosterone. Each experiment was replicated six times. Statistics were performed using the Student’s t-test. Results are presented as means ⫾ SD. P values ⬍ 0.05 are considered significant. RESULTS: Low-dose testosterone pre-exposure substantially increased IRS-1- Ser636/639 phosphorylation (fold change 3.09 ⫾ 1.53, p ⫽ 0.07) and significantly increased the mTOR-Ser2448 and S6K-Thr389 phosphorylation (fold change 1.40 ⫾ 0.14 and 1.52 ⫾ 0.19, respectively, p ⬍ 0.05) after insulin treatment. High-dose testosterone pre-exposure significantly increased the IRS-1- Ser636/639 (fold change 3.01 ⫾ 0.51, p ⬍ 0.05) phosphorylation by insulin. Akt phosphorylation showed no significant change in insulin stimulated phosphorylation in response to testosterone pre-exposure. CONCLUSION: Testosterone exposure significantly increased insulin stimulated phosphorylation of insulin signaling pathway molecules compared to non-T exposed cells. IRS-1 Ser636/639 phosphorylation is one of the known mechanisms of IR. Our finding represents the first data demonstrating the effect of T in this interaction. This data suggests a link between a hyperandrogenic, hyperinsulinemic environment and the development of insulin resistance through aberrant serine phosphorylation of IRS-1 Ser636/639, and may serve as a model for PCOS-related IR. Supported by: NIH/NIDK # R01 41973-15.

Wednesday, October 25, 2006 4:15 pm O-203 GENOTYPIC AND PHENOTYPIC CHARACTERISTICS OF MURINE BLASTOCYSTS PRODUCED BY IN VITRO FERTILIZATION. G. Giritharan, S. Talbi, M. Rosen, F. Di Sebastiano, A. T. Dobson, P. F. Rinaudo. Univ of California San Francisco, San Francisco, CA. OBJECTIVE: Mounting evidence suggests that in vitro fertilization and in vitro culture may not be completely benign. Prior studies have shown that gene expression and development is affected by the type of culture media and oxygen concentration present during in vitro culture from the zygote stage to the blastocyst stage, when compared to control embryos. This study was conducted to explore if in vitro fertilization and gamete manipulation have an additional effect on gene expression compared to simple culture from the zygote stage to the blastocyst stage in mouse preimplantation embryos using microarray technology. DESIGN: Comparative analysis of in vivo produced and in vitro produced embryos. MATERIALS AND METHODS: CF-1 female mice were injected with 5 IU PMSG and 42-46 h later with 5 IU hCG; some of the females were then bred to B6D2F1/J males overnight (group 1: IVC). The following morning, zygotes or/and oocytes were collected. Unfertilized oocytes were incubated in WM containing 15 mg/ml BSA and sperm obtained from cauda epididymis of male B6D2F1/J mice for 4 hrs (group 2: IVF). All embryos were cultured in Whitten Medium (WM) to the blastocyst stage under 20% CO2 in humidified air at 37° C. Control in vivo embryos were collected 4 days after fertilization (group 3: In vivo). Representative blastocysts from each treatment group were stained with propidium iodide and bisbenzamide, and the number of trophectoderm and inner cell mass cells was recorded. Total RNA was extracted and five embryo equivalents were used for reverse transcription followed by linear amplification of antisense cDNA strand, fragmentation and biotin labeling. The samples were hybridized to Affymetrix mouse 430 2.0 GeneChip. Each treatment was repeated 4 times. Pair-wise comparison was conducted between control and each treatment group using GeneSpring Software. RESULTS: IVF embryos had fewer total cell number (45) compared to IVC (52) or control (65) embryos but developed faster to the blastocyst stage. Gene expression analysis revealed that each treatment group clustered separately. IVF embryos had down-regulation of enzyme linked signaling pathways, steroid metabolism and embryo development pathways, and up

FERTILITY & STERILITY威

regulation of apoptosis and protein biosynthesis pathways compared to IVC embryos.

CONCLUSION: The processes of fertilization in vitro and gamete manipulation affect developmental characteristics and gene expression of the pre-implantation embryo. These effects are different and in addition to the changes present in the embryos cultured in vitro from the zygote stage to the blastocyst stage. Both IVF and IVC embryos are different from control in vivo embryos. Interestingly, the total number of genes changed after culture in vitro alone is higher than after in vitro fertilization and in vitro culture. These findings should be further investigated. Supported by: WRHR.

Wednesday, October 25, 2006 4:30 pm O-204 ANGIOTENSIN II (ANG II) REGULATES APOPTOSIS IN HUMAN GRANULOSA-LUTEIN CELLS (GL). E. Acosta, O. Pen˜a, F. Naftolin, ´ vila. Laboratorio de Biologı´a del Desarrollo, Departamento A. Palumbo, J. A de Bioquı´mica y Biologı´a Molecular, Univ of La Laguna, La Laguna, Tenerife, Spain; Dept of Obstetrics and Gynecology, New York Univ, NY, NY; Centro de Asistencia a la Reproduccio´n Humana de Canarias, La Laguna, Tenerife, Spain. OBJECTIVE: The human ovary has a complete and functional gonadotrophin-sensitive renin-angiotensin system and both types of AngII receptors, AT1 and AT2, are present in ovarian cells. We have shown that AngII is present in high concentrations in granulosa cells and follicular fluid of human preovulatory follicles and in luteal cells. AngII regulates ovulation and modulates steroidogenesis in rat luteal cells and human GL cells. Other investigators have shown that AngII regulates luteal cell apoptosis. Recently, we have shown that apoptosis of human GL cells correlates with several indicators of IVF outcome, such as embryo fragmentation and pregnancy rate, and is regulated by intraovarian growth factors. The aim of the present study was to test whether AngII could induce in vitro apoptosis of gonadotrophin-exposed human IVF GL cells. DESIGN: Following AngII exposure, apoptosis was assessed by caspace expression. Controls included the non-specific angiotensin receptor blocker Saralasin and the specific AT2 receptor (AT2-R) blocker CGP-42112A. MATERIALS AND METHODS: GL cells from 87 IVF-intra-cytoplasmic sperm injection (ICSI) cycles were analyzed. Ovarian stimulation was performed using recombinant FSH and LH or HMG. The pregnancy rate for these cycles was 56.32%. Following oocyte recovery GL cells were collected from pooled follicular fluid (FF), washed and purified using a 50% percoll gradient and anti-CD45-coated magnetic beads. Cells were cultured without serum for 48 hours in the presence or absence of 10-11, 10-9, 10-7M AngII, with and without Saralasin10-5 or CGP-42112A 10-5M. The percentage of apoptotic cells was determined under a fluorescence microscope by staining with 10␮m caspace FITV-VAD-FMK (a fluorescent marker for activated caspaces) and 0.5␮g/ml propidium iodide. Statistical analysis was carried out using the student-t test. RESULTS: AngII in concentrations present in human follicle fluid caused increased apoptosis of GL cells compared to untreated control cells (10-11M p⫽0.312; 10-9M p⬍0,05; 10-7M p⬍0,05, respectively). This effect was partially inhibited by both Saralasin and CGP-42112A (18.15% and 33.01%, respectively, p⬍0,05). CONCLUSION: In these preliminary studies on human GL cells Ang II at concentrations that are present in FF from human IVF cycles (J Soc Gynecol Investig. 1994 1:118-27) is proapoptotic. The partial blockade by angiotensin receptor antagonists is consistent with a receptor-mediated action. Ovarian AII has been linked to fertility, steroidogenesis and polycystic ovarian syndrome. Further studies are underway to determine the

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relevance of these results to the outcome of IVF cycles and other clinical scenarios. Supported by: This study was supported by grant PI030667 from Fondo Investigaciones Sanitarias, Spain. EA is supported by an educational award from Fondo Investigaciones Sanitarias, Spain and OP is supported by an educational award from CajaCanarias, Spain.

(1cell) the average respiration rate was 0.15 nl/hr over a 4 hour measuring period. Eight of these embryos were subsequently transferred to the EmbryoScope where the respiration rate was recorded as 0.1 nl Oxygen/h. The similarity of average respiration rates for mouse embryos in the EmbryoScope and those measured by closed respirometry in glass vials indicate that the respiration rate calculations EmbryoScope are correct. The average respiration rates for human GV oocytes was 0.4 nl/hour, for M1 it was 0.55 and for M11 it was 0.7. The rate of respiration increased over time as the oocytes matured. Respiration rates for cleaving embryos averaged 0.5 and had a steady state through cleavage and a marginal increase as they formed blastocysts. As embryos arrested or became attretic, the rates decreased, which acted as a control for the measurements. CONCLUSION: This is the first report of a real time non-invasive measurements of respiration in human oocytes and embryos. With more samples and the ability to look at different cohorts of oocytes and embryos, the signifigance of differences for selection purposes could be realized. Supported by: None.

MENOPAUSE SPECIAL INTEREST GROUP Wednesday, October 25, 2006 3:00 pm O-206

Wednesday, October 25, 2006 4:45 pm O-205 NON-INVASIVE, REAL-TIME RESPIRATIONS MEASUREMENTS OF HUMAN OOCYTES AND EMBRYOS: A FUTURE MEANS OF EMBRYO SELECTION? L. Scott, A. Finn, K. De Legge, J. Hill, N. Ramsing. Fertility Centers of New England, Reading, MA; Unisense, Aahus, Denmark. OBJECTIVE: Maturing oocytes go through many changes from the GV to mature metaphase 11 phase. Early embryos are also very active as cleavge and gene activation begin. Respiration rate measurements, or oxygen consumption, which measures metabolism, could be used as an noninvasive means to asses these activities and for selecting oocytes and embryos with increased implantation potential. An EmbryoScope has been developed which allows automated, accurate, oxygen consumption measurements on single oocytes and embryos within a controlled incubator environment that is non-invasive and clinically compatible. The objective of this pre-clinical study was to use the EmbryoScope to document respiration rates in donated/discarded human oocytes and embryos. DESIGN: The EmbryoScope uses amperometric microelectrodes and steady state diffusive oxygen flux to embryos in glass trays. The methodology was validated by closed respirometry data using optical oxygen microsensors made by Presens, Germany placed in glass vials with a volume of about 20 ul and a long (8 mm) narrow (0.5 - 0.8 mm diam) opening. The glass trays used in the system and the system as a whole was validated and tested using mouse embryos grown from the 1-cell to hatching blastocyst stage. Finally immature oocytes on the day of retrieval (D0) from ICSI patients; immature and non-fertilized M11 oocytes, 3PN and 1PN embryos from standard insemination patients on the day after ER (D1) and donated cryopreserved and thawed cleaving human embryos were used for respiration measurements. MATERIALS AND METHODS: Informed consent for the use of all human material was obtained. No IRB was sought since the samples were not clinical. To validate the rates with human oocytes 1, 2 and 3 M1 oocytes were used in some wells. RESULTS: 40/47 (85%) of mouse embryos reached the blastocyst stage and 31 (60%) hatched in the glass trays. Control TC dishes gave 19/28 (68%) blastocyst formation and 15 hatching (51%). The rates measured in the EmbryoScope on single mouse embryos and on groups of 1, 2, 3 and 5 embryos gave average respiration rates of 0.26 nl O2/h (with low std/dev). From the closed system experiments with 20 freshly thawed mouse embryos

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Abstracts

BAZEDOXIFENE COMBINED WITH CONJUGATED ESTROGENS: A NOVEL ALTERNATIVE TO TRADITIONAL HORMONE THERAPIES. D. Van Duren, S. Ronkin, J. Pickar, G. Constantine. Menox BV, Nijmegen, The Netherlands; Wyeth Pharmaceuticals, Collegeville, PA. OBJECTIVE: To examine the effects of bazedoxifene (BZA; 5, 10, or 20 mg) combined with conjugated estrogens (CE; 0.3 or 0.625 mg) on the endometrium in healthy postmenopausal women. DESIGN: This multicenter, randomized, double-blind, controlled pilot study explored 6 doses of BZA/CE administered daily for 84 days. Placebo, CE 0.3 mg, CE 0.625 mg, CE/MPA (CE 0.625 mg with medroxyprogesterone acetate 2.5 mg), and BZA 5 mg served as controls. Subjects were at least 1 year postmenopausal, with uterus intact, and had an average of at least 4 hot flushes/day. The primary outcome was endometrial thickness as measured by transvaginal ultrasound at Day 84. Other assessments included the mean weekly number of hot flushes and the incidence of amenorrhea (defined as no bleeding or spotting over the 84-day treatment period). MATERIALS AND METHODS: Endometrial thickness was assessed by transvaginal ultrasound at screening, Day 28, and Day 84. Number of hot flushes and bleeding/spotting episodes were recorded in a daily diary. Safety assessments were based on reports of AEs, physical examinations, and laboratory determinations. RESULTS: The safety population was composed of 408 randomized patients who received at least 1 dose of study drug. The intent-to-treat population included 397 women who were randomized, received at least 1 dose of study drug, and had at least 1 post-baseline evaluation of endometrial thickness. No differences were found between the groups in mean age, race, BMI, or years since last menstrual period at baseline. As expected, CE alone increased endometrial thickness. Adding BZA to CE resulted in a dose-related decrease in thickness; for each CE dose, change in endometrial thickness decreased as bazedoxifene dose increased. The change in endometrial thickness was significantly lower at Day 84 in the BZA 20 mg/CE 0.3 mg (0.11 mm; p ⫽ 0.049), BZA 10 mg/CE 0.625 mg (1.47 mm; p ⫽ 0.019), and in the BZA 20 mg/CE 0.625 mg groups (0.66 mm; p ⬍ 0.001) as compared to the respective CE-alone groups (CE 0.3 mg ⫽ 1.09 mm and CE 0.625 mg ⫽ 2.68 mm). In addition, the changes in endometrial thickness for both 20 mg BZA combinations (20 mg BZA/CE 0.3 mg, p ⫽ 0.986; 20 mg BZA/CE 0.625 mg, p ⫽ 0.273) were not statistically different from placebo (0.06 mm at Day 84). As expected, there was a significant reduction of hot flushes in the CE 0.3 mg, CE 0.625 mg, and CE 0.625/MPA 2.5 mg groups as compared to placebo. All BZA/CE 0.625 mg combination groups and the BZA 5 mg/CE 0.3 mg group demonstrated a statistically significant reduction of hot flushes vs placebo (p ⬍ 0.001). The decrease was not statistically significant for the 10 and 20 mg BZA/CE 0.3 mg groups vs placebo (p ⫽ 0.061, and 0.297, respectively). Amenorrhea was maintained by 91.9% of placebo-treated subjects. Treatment with BZA 5, 10, or 20 mg combined with 0.625 CE was associated with amenorrhea over 84 days in 86.1%, 84.2%, and 88.6% of subjects, respectively. Treatment with BZA 5,

Vol. 86, Suppl 2, September 2006