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Wednesday, October 25, 2006 3:15 pm O-269 DEVELOPMENT OF A SENSITIVE ASSAY TO MEASURE LACTATE PRODUCTION OF PREIMPLANTATION EMBRYOS. C. L. Bormann, L. M. Cabrera, C. N. Chisolm, Y. S. Heo, S. Takayama, G. D. Smith. Univ of Michigan, Ann Arbor, MI. OBJECTIVE: Reliable indicators of embryo viability are rate of glucose/ pyruvate consumption and lactate production. The ability to non-invasively quantify embryo metabolism within culture media is a valuable tool. Current non-invasive methods of measuring embryo metabolism are based on generation or utilization of reduced pyridine nucleotides, NADH and NADPH. This method requires excitation of NADH/NADPH with ultraviolet (UV) light which can damage embryo DNA thus requiring manual removal of media, nanoliter pipetting, and dilutions that can introduce quantitative errors. Long-term goals of these studies are to develop metabolic assays that can be performed in microfluidic embryo culture and analysis platforms that remove manual media manipulation and dilution, do not use UV light, and are “embryo-safe”. Study objectives were to optimize a system of measuring single embryo-produced lactate that would be compatible with microfluidic culture without use of UV light detection. DESIGN: Experimental study. MATERIALS AND METHODS: Lactate assays were developed using Amplex UltraRed (AR), which is a colorless, stable and versatile fluorogenic substrate for horseradish peroxidase (HRP). In this assay, lactate oxidase (LOX) reacts with L-lactate to form pyruvate and H2O2. In the presence of HRP, H2O2 reacts with AR reagent in a 1:1 stoichiometry to generate a red-fluorescent oxidation product, resorfin. Amplex UltraRed lactate assays were optimized with 100 ␮M AR reagent, 0.2 U/ml HRP and 2U/ml LOX in three different reaction buffers (1X reaction buffer, glycinehydrazine buffer, and 1X reaction buffer containing glycine and hydrazine) at pH 6.5, 7.5, and 8.5 over a span of 0-3 h. Standard curves of L-lactate in KSOM were established using 10-fold serial dilutions from 10nM to10mM. Lactate was measured in presence or absence of pyruvate and glucose to rule out interference from other substrate sources. A 3X3 cross-over design was used to compare assay sensitivity under varying concentrations of AR reagent (100, 10, and 1 ␮M) and HRP (0.2, 0.02, and 0.002 U/ml). Lactate was quantified with an excitation filter at 535 nm and an emission filter at 580 nm. Two-cell embryos from B6SJL-F2 mice were individually cultured in 10 ␮l KSOM w/o L-lactate for 3 h at 37°C in 5% CO2 in air. Embryo culture media was assayed at a 1:1 ratio with AR. Triplicate standard curves measuring 10-fold dilutions of lactate were performed each time embryos were assessed for lactate. RESULTS: The optimum buffer conditions for AR lactate assays was 1X reaction buffer at pH 7.5. There was no effect of glucose or pyruvate on AR lactate assay sensitivity. Reducing AR reagent to 10 ␮M and HRP to 0.002 U/ml greatly reduced background fluorescence and decreased the limit of detection to 600 nM lactate. Individual 2-cell embryo lactate production was measured at 44.9 ⫾ 4.4 pmol/ 3h. CONCLUSION: Amplex UltraRed is a sensitive reagent that can be used to measure the production of lactate in the picomole range. These results provide a new method to non-invasively quantify preimplantation embryo metabolism in the absence of a UV light source. Future studies will focus on applying this technology to a microfluidic embryo culture and analysis system for real-time monitoring of embryo metabolism. Supported by: NIH-HD049607; USDA-2005-35203-16148; NIH T32 Training Grant-HD07048.

OBJECTIVE: More than 100,000 assisted reproductive technology (ART) cycles are started yearly in the U.S. Partly due to our inability to select the best embryo(s) to be transferred, a mean number of 3.1 embryos are transferred in ART cycles using fresh nondonor oocytes. This leads to a 34.3% overall pregnancy rate, 29.0% of which result in multiple-infant live births. Similarly, a mean number of 2.5 embryos are transferred in ART cycles using fresh donor oocytes, achieving a 50.4% pregnancy rate, 44.7% of which result in multiple-infant live births (SART 2002). These statistics have remained essentially unchanged for several years and reflect a need for improvement over our current embryo selection methodology that is based on cleavage rates and morphology. In this study, we hypothesized that, embryos that result in pregnancy may be different in their metabolomic profile compared to embryos that do not, and that the difference may be detected by the rapid, non-invasive evaluation of the embryo culture media using targeted spectroscopic analysis and bioinformatics. DESIGN: A prospective multi-center study was conducted in 2 academic, and one private ART centers. MATERIALS AND METHODS: Embryo culture medium of 108 embryos from ART cycles using fresh donor or nondonor oocytes were evaluated. Media (G1.3; VitroLife, Sweden) were individually collected after embryo transfer on day 3, and analyzed using both Raman, and Near Infrared Spectroscopy (NIR). The spectra obtained from each instrument were separately analyzed using a wavelength selective genetic algorithm (GA) to determine regions predictive of pregnancy outcome as determined by a logistic regression of the light attenuation from the wavelength included. To avoid random correlations, a leave-one out cross-validation was used. Sensitivity and specificity of predicting pregnancy (defined as presence of fetal cardiac activity) were calculated. RESULTS: Spectral profile describing differences in -CH, -NH and -OH concentrations showed distinct differences between culture media of embryos that resulted in pregnancy and those that did not. The ratio of the -CH to ROH content in the media that is reflective of oxidative stress was also different between the two groups. Using GA with Raman, four spectral regions associated with these molecular species were identified as predictive of pregnancy outcome. Compiled outcomes from the leave-one-out crossvalidation of the logistic regression using the Raman measurements resulted in a specificity of 80% and a sensitivity of 95%. Analysis of the NIR required two wavelength regions, and provided a specificity of 83% and a sensitivity of 73%. CONCLUSION: Our findings suggest that a detectable difference exists in the metabolomic profiles found in culture media obtained from embryos that cause pregnancy compared to those that do not. The reported metabolomic parameters were established using two different forms of spectroscopic analysis, with samples from 3 different centers, and achieved high sensitivity and specificity. Confirmation of these initial observations is planned through a larger prospective study involving additional ART centers. Metabolomic profiling may serve as a useful methodology for rapid, non-invasive embryo assessment and selection. Metabolomic evaluation may make it possible to decrease the number of embryos transferred, lead to a decrease in multiple-infant live birth rates, and possibly also improve the pregnancy rates. Supported by: None.

Wednesday, October 25, 2006 3:45 pm O-271

Wednesday, October 25, 2006 3:30 pm

EVALUATION OF METAPHASE II SPINDLE LENGTH, RETARDANCE AND ITS RELATIONSHIP TO EMBRYO QUALITY ON DAY 3 AND DAY 5. T. H. Taylor, T. Elliott, S. A. Gitlin, S. Jones-Colon, H. I. Kort, Z. P. Nagy. Reproductive Biology Associates, Atlanta, GA; Eastern VIrginia Medical School, Norfolk, VA.

NON-INVASIVE METABOLOMIC PROFILING OF HUMAN EMBRYO CULTURE MEDIA CORRELATES WITH PREGNANCY OUTCOME. INITIAL RESULTS OF THE METABOLOMICS STUDY GROUP FOR ART. E. Seli, D. Sakkas, B. Behr, P. Nagy, J. S. Kwok, D. H. Burns. Yale Univ School of Medicine, New Haven, CT; Stanford Univ, Stanford, CA; Reproductive Biology Associates, Atlanta, GA; McGill Univ, Montreal, PQ, Canada.

OBJECTIVE: Recent studies suggest a relationship between meiotic spindle retardance and embryo development. The aim of this study is to determine if meiotic spindle retardance in metaphase II human oocytes, as viewed by the Oosight, are effective predictors of embryo development. DESIGN: This study was conducted in a prospective randomized way. This study was approved by Sterling Institutional Review Board (Atlanta, GA). MATERIALS AND METHODS: Twenty-six patients who elected for intracytoplasmic sperm injection (ICSI) insemination were also consented

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FERTILITY & STERILITY威

S115