118 Micro magnetic resonance spectroscopy for noninvasive metabolic assessment of individual bovine embryos

Noninvasive selection of optimal embryos for transfer in ART is challenging, but magnetic resonance spectroscopy (MRS) offers a promising solution owing to its chemical sensitivity and noninvasive nature. Although MRS has been effective for larger organisms, it faces sensitivity challenges with embryos. Our study aims to overcome these obstacles by using innovative microchip-based MRS sensors. We conducted multiple micro-MRS measurement on in vitro-produced IVP bovine embryos produced by our collaborator in Zurich at Paladis GmbH. Culture procedures were performed at 38.5°C under 5%–6% O₂ in humidified air, all reagents for IVP and in vitro culture (IVC) were purchased from IVF-Bioscience. Bovine ovaries were recovered from a local abattoir and transported to the laboratory. Follicles were aspirated, and oocytes with cumulus cells were isolated and cultured for IVM for 24 h. Post-IVM, oocytes were fertilized with cryopreserved bull semen. Embryo development was monitored with a stereomicroscope until blastocyst formation (Day 6+). All embryos for the MRS analysis were cryopreserved, and both vitrification and thawing were performed with Kitazato kits. Before MRS analysis, embryos were thawed and cultured for 3 h to allow equilibration. Samples were loaded in our 1-nL micro-MRS probe in pre-equilibrated BO-IVC medium and subjected to a 50-min MRS measurement at 38.5°C. For statistical analysis, normality was assessed using the Lilliefors test. When normality was confirmed, a two-tailed unpaired t-test with Welch’s correction was performed, with statistical significance set at P < 0.05. If normality was not met, the Mann-Whitney U-test was used. In Experiment 1, in vitro naturally arrested embryos at early or late stages were cryopreserved according to their developmental stage. Two predefined cohorts were studied: Cohort 1 included embryos arrested at early stage (8-cell-early morula), and Cohort 2 included embryos arrested at late stage (morula-blastocyst) (n = 32 embryos). This experiment aimed to establish the presence of MRS markers in well-defined categories of preimplantation embryos. Up to nine lipid markers were identified as valuable targets. Late arrested embryos showed higher levels of lipid biomarkers compared with early arrested embryos. Late arrested embryos showed elevated levels of mono-unsaturated lipids, as well as saturated lipids, with high statistical significance (P < 0.05). The poly-unsaturated lipid marker was equally present in both cohorts (P > 0.05). In Experiment 2, IVP embryos were cultured up to Day 2 post-fertilization (8-cell stage) and cryopreserved. On the day of MRS analysis, embryos were assigned unique IDs (n = 61 embryos). After MRS, the embryos were cultured in single 50-µL drops of medium overlaid with oil. Embryos were imaged on Days 8 and 10 (6 and 8 days after MRS) to assess development. After verifying the presence of the lipid markers identified in Experiment 1, machine learning models were applied to evaluate the effectiveness of Day 2 (8-cell) stage MRS biomarkers in predicting subsequent embryo development. Despite no significant P-values, the biomarkers formed distinct clusters. The biomarkers were then collected into a features matrix, normalized, and reduced with PCA. Machine learning combined with Monte Carlo simulation was used to project the performance of a classifier based on these data. The obtained performance indicators were accuracy (85%–88%), precision (65%–82%), and sensitivity (93%–94%). This analysis revealed up to 80% precision in predicting the embryo development to the blastocyst stage solely based on the metabolic profile obtained at Day 2. These findings suggest analysis of embryos using micro-MRS can effectively uncover the significance of embryonic metabolic status during preimplantation in vitro development.