SBIR-STTR Award

Project Summary/In vitro fertilization (IVF) and embryo transfer (ET) are well-established assisted reproductive techniques in the cattle industry. The protocols have matured through decades of improvements yet the conception rate following IVF/ET in cattle is around 35-45%. The major limiting factors for ET and IVF are labor-intensive protocols dependence on practitioner expertise poor superovulatory response and inaccurate embryo assessment. This research effort aims to solve two of the significant challenges mentioned above by (1) improving the labor-intensive embryo dilution process using microfluidics and (2) developing a non-invasive and objective embryo assessment technique. Hawai'i Innovation Laboratory (HIL) in collaboration with Agrimark Genetics Whitaker Embryo Transfer Services and Hawai'i Pacific University plans to develop a novelembryo assessment technique that is non-invasive and objective unlike the subjective IETS- approved morphological grading system. The proposed technique can assess the embryo viability quantifiable by measuring the electrical impedance and observation of morphokinetics such asblastocyst expansion rate blastomere symmetry and percentage of the intact embryonic cellsrepresented by the extruded material in the perivitelline space. The embryo assessment takes placein a microfluidic device that can wash the embryo from the flushing medium easing the embryo dilution process. Also the microfluidic chip allows positioning a single bovine embryo between two micro-electrodes under optical microscopy recording time-lapse images analyzing morphokinetics data and measuring electric impedance circumventing the background noise conductive media. In prior work HIL has measured the electrical impedance of single Artemiacysts at various phases of development in a microfluidic environment. A distinct pattern of impedance changes was observed at different stages of cyst development. The measured impedance changes corresponded to physiological changes as the cyst developed. The change in impedance during the first stage of development provided sufficient quantitative data to predict if the cyst would hatch (Rahman et al. IEEE NANOMED 2018 and Matthews et al. IEEE NEMS2017). Also in a recent work the proposer experimented the morphokinetics observations through time-lapse image segmentation using Deep Neural Network (DNN) which correlates with its genetic health (Huang et al. Reprod Biomed 2019 and Huang st al. Fertil Steril. 2019). We demonstrated genetically normal embryos expand more rapidly than genetically abnormal embryos. Building on the above research HIL combines DNN-assisted morphokinetics and Impedance spectroscopy and proposes developing a non-invasive quantitative embryo assessment technique that can determine viability by measuring internal cell information such as membrane capacitance cytoplasmic conductivity along with the morphokinetic data. During PhaseI of the project the team will design and fabricate a microfluidic device designed for impedancemeasurements on bovine embryos. Also impedance spectra of the embryo will be measured and fitted to the equivalent circuit through non-linear regression to calculate the membrane capacitance and cytoplasm conductivity. In addition the embryo metabolites will be determined by measuring the depletion of glucose and pyruvate and the production of lactate to compare the impedance data to their biological health. This effort will result in a commercial tool for the ET/IVF practitioner and embryo producers if successful. The tool will help to determine bovine embryo viability quantitatively prior to the freezing step. Also the tool can potentially improve the pregnancy rate by 10-15% per transfer by reducing misjudging and discarding of competent embryos and reducing transfer or less competent embryos. This work will help the NIFA achieve its mission of fosteringan i