Source: HAWAI'I INNOVATION LABORATORY, INC. submitted to
NON-INVASIVE BOVINE EMBRYO ASSESSMENT USING AI AND IMPEDANCE SPECTROSCOPY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1028469
Grant No.
2022-33530-37152
Cumulative Award Amt.
$175,000.00
Proposal No.
2022-00897
Multistate No.
(N/A)
Project Start Date
Sep 1, 2022
Project End Date
Apr 30, 2024
Grant Year
2022
Program Code
[8.3]- Animal Production & Protection
Project Director
Rahman, A.
Recipient Organization
HAWAI'I INNOVATION LABORATORY, INC.
1919 CITRON ST APT 308
HONOLULU,HI 968262628
Performing Department
(N/A)
Non Technical 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 super ovulatory 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 novel embryo 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 as blastocyst expansion rate, blastomere symmetry, and percentage of the intact embryonic cells represented by the extruded material in the perivitelline space. The embryo assessment takes place in 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 Artemia cysts 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. 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. 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. If successful, this effort will be the path forward to a commercial tool for the ET/IVF practitioner and embryo producers. The tool will help determine bovine embryo viability quantitatively before 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 fostering an innovative research strategy that may be used to develop safer and more effective means of regulating, preserving, or achieving animal reproduction.
Animal Health Component
30%
Research Effort Categories
Basic
50%
Applied
30%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3013999202050%
3013999100030%
3013999104020%
Knowledge Area
301 - Reproductive Performance of Animals;

Subject Of Investigation
3999 - Animal research, general;

Field Of Science
2020 - Engineering; 1040 - Molecular biology; 1000 - Biochemistry and biophysics;
Goals / Objectives
The overall goal of this project is to develop a novel, non-invasive, quantifiable bovine embryo assessment technique free from human subjectivity. Being able to grade the embryo according to its potential to result in pregnancy is critical for successful pregnancy in ET and IVF procedures. Current methods of embryo assessment are morphological scoring, which is based on the morphological observation of an embryo. Still, it does not provide inside health information of an embryo and suffers from human subjectivity (Mapletoft and Hasler 2005). Thisproject will develop an embryo assessment technique that is non-invasive, unlike PGD, and provide quantifiable health information of an embryo related to its internal biological growth, unlike morphological scoring.Specific Aim 1: Design and fabricate a microfluidic chip that will facilitate precise manipulation of single embryos suspended in culture medium for impedance measurement: The dimensions and the position of the electrode in the chip will be simulated in COMSOL multiphysics for optimum performance. The microfluidic chip will be fabricated using photolithography and soft lithography.Specific Aim 2: Measure the Impedance of 20 bovine embryos using the microfluidic chip designed in aim 1 and record time-lapse images during the measurement. Model the equivalent circuit and fit the impedance data to calculate the membrane capacitance and cytoplasmic conductivity.Specific Aim 3: Train a neural network using manually annotated embryo images and process the images recorded in aim 2 to identify the expansion rate, blastomere symmetry, and percentage of the intact embryonic cells represented by the extruded material in the perivitelline space. Also, measure the metabolic activities of the embryos (in aim 2) by measuring the depletion of glucose & pyruvate and the production of lactate in the embryo medium using the ultra-microfluorometric technique. Finally, correlate the membrane capacitance and cytoplasmic conductivity with the morphological observation, morphokinetics (expansion rate), hatchability, and metabolic rate.
Project Methods
Approach for Specific Aim 1: A microfluidic device will be designed that can precisely manipulate individual cow embryos. The device will have two metal conductors embedded in it. The dimensions of the channels, electrodeposition, and overall dimensions will be optimized using COMSOL simulation. The tested embryo will be positioned between a pair of electrodes (Figure 2) connected to an impedance analyzer. The impedance analyzer will pass a small amount of current through the embryo and measure the electrical impedance and phase. The impedance analyzer operates in CV (constant voltage) mode applying a small electric field. The maximum electric field applied will be 6.7·103 V/m, whereas up to 2·105 V/m electric field has been reported as safe for various mammalian cells, so this amount of electric field should not affect the health of the. Embryo. However, we will investigate the optimum electric field and frequency, which presumably should be lower than the above. Also, the electric field applied to Artemia cysts while measuring their impedance did not affect their viability. The microfluidics channel will be designed with gradual width varies from 100-150 μm to fit the embryo expansion as it grows. Also, the impedance measuring electrodes is designed for long-term monitoring of the cultured embryo in a controlled environment. Besides the impedance-measuring electrodes function near the embryo, the electrode material will be chemically and physically stable across the experiment. The material of choice is gold, platinum, palladium, and titanium. Overall the design will be optimized for minimizing the background noise due to the conductive medium.Approach for Specific Aim 2: Measure the Impedance of 20 bovine embryos using the microfluidic chip designed in aim one and record time-lapse images during the measurement. Model the equivalent circuit and fit the impedance data to calculate the membrane capacitance and cytoplasmic conductivity.Approximately 20-25 batches of experiments will be carried out using the microfluidic device fabricated in aim 1. Frozen bovine embryos produced by ET (morula/ blastocyst) or IVF (blastocyst) will be used for the experiment. Different grades (1-4) of the embryo will be used. Hoofstock genetics will donate 300 embryos for this research. Once thawed, the embryo will be moved through 2-3 drops of holding or wash media to remove all freeze media. Then the embryo will be transferred to the microfluidic chip in culture media cultured until they hatch in the microincubator at 37°C under the microscope. During culturing, impedance data will be measured and documented. The culture media will be equilibrated with tri-gas (CO2, O2, and N2). Usually, 5-6% CO2, 5-6% O2, and 85-90% N2 are maintained in the microincubator. Under optical microscopy, a micro-syringe pump will manipulate the embryo inside the microfluidic chip. Fig. 5 shows the schematic of the experimental setup and the zoomed-in schematic (not-to-scale) of the microfluidic device working area. Unlike most single-cell impedance measuring device, which uses a planar electrode, we plan to use a cuboid or cylindrical gold/ platinum electrode. In the case of a cuboid electrode, a square face, and the case of a cylindrical electrode, a circular face of the electrodes will be in contact with the embryo for optimum sensitive measurement. However, the electrode dimension and position will be finalized through COMSOL modeling. The top view of the preliminary design (Fig. 5 inset) shows the electrode configuration. The chip will have a lateral inlet port through which fluid will be withdrawn to trap two embryos entering paths A and B (Fig. 5 inset) using a hydrodynamic trapping mechanism(Tan and Takeuchi 2008). Embryo entering through path A will be trapped between two electrodes for impedance measurement, and another embryo will be trapped nearby as the control for comparing biological growth. Also, there will be a pair of control electrodes adjacent to the traps to measure the impedance of the media only. The Electrodes (insulated, 100-120 μm-dia Ti/ Au) face will be flattened by a microelectrode polisher before inserting it into the microfluidic chip. The device will be placed inside a moist microincubator, where the gas (5% CO2, 5% O2, 90% N2) and temperature (37°C) are controlled for strict pH maintenance of the media. An inverted microscope records the time-lapse images, and a high precision impedance analyzer (Hioki IM 3570) measures the impedance spectra (Fig. 5). After measurement, the data will be fitted to equations 1-5 in Fig. 3 through non-linear regression to calculate the membrane capacitance and cell mass/ cytoplasm conductivity. In addition, the blastocyst time-lapse images recorded during the experiment will be analyzed using AI-based CNN (convolutional neural network) tools to measure the blastocyst expansion rate and prepare expansion maps. At the same time, the morphology grading of the test embryo and control embryo will be recorded, along with hatching.Approach for Specific Aim 3:To achieve aim 3, the morphological, morphokinetic, and metabolic activities of the embryo under test will be determined using the ultra-microfluorometric technique and correlate with the impedance data. First, the time-lapse images will be sent to Agrimark genetics and Whitaker embryo transfer service for visual grading. In addition, an AI-based image segmentation tool developed by the proposer will be used to measure biological data such as blastocyst expansion rate, cell mass volume, ZP & ICM intactness. The accurate segmentation of the embryo's inner cell mass (ICM) and trophectoderm epithelium (TE) is essential, as these parameters can help predict the embryo viability and live birth potential. However, the ICM and TE segmentation are complex due to variations in their shape and similarities in their textures, both with each other and their surroundings. A deep neural network (DNN) based segmentation approach is implemented to tackle this problem. The neural network will be trained using manually annotated bovine embryo images and a U-Net-based DNN to extract local (texture of the ICM and TE) and contextual information (spatial arrangement of the ICM and TE) to segment the ICM and TE regions. The tool will measure the embryo expansion rate, blastomere symmetry, and percentage of the intact embryonic cells. This parameterprovides qualitative embryo viability data based on their physical parameters. For example, the averaged euploid blastocyst expansion rate is significantly (52.8% average) faster than in aneuploid blastocysts (Thomas TF Huang et al. 2019), so a higher expansion rate indicates a healthier embryo. Similarly, the IETS manual mentions that an embryo with 85% intact cell material is code-1, 50% code-2, and 25% code-3, which will be calculated by the image segmentation tool. Also, the embryo impedance will be measured until it hatches, so hatchability is an additional health parameter.Also, the metabolic activity of the embryo under experiment will be measured through molecular testing of the medium fluid adjacent to the incubated embryo (control and ). Here, the consumption of pyruvate and glucose and the release of lactate by single bovine embryos will be measured by analyzing the sample using a fluorescent spectrophotometer plate reader. The assays for this test will be performed, collecting 1μl sample medium. For pyruvate test, 1μl sample is added to 10 μl of assay mixture containing 0.1 mM NADH and 40 IU lactate dehydrogenase ml−1 in 4.6 mM EPPS buffer, pH 8.0 incubated at 25°C for 3 minutes. Finally, the morphological, morpho-kinetical, hatching, and metabolic activity data will be analyzed correlated with impedance data to determine the feasibility of the proposed method.

Progress 09/01/22 to 04/30/24

Outputs
Target Audience: The primary target audience for the project spans several key groups within the bovine reproduction industry, particularly those involved in embryo transfer (ET) and in vitro fertilization (IVF). These audiences are crucial stakeholders whose work directly benefits from the advancements made in this project. Bovine Embryologists and Veterinarians: Role and Importance: Bovine embryologists and veterinarians are the frontline professionals responsible for the handling, assessment, and transfer of cattle embryos. Their expertise in selecting viable embryos is critical to the success of ET and IVF procedures. Traditionally, embryo viability has been assessed through subjective morphological grading, a process that relies heavily on the skill and experience of the embryologist. Project Relevance: The project aimed to develop an objective, non-invasive embryo assessment tool that utilizes impedance spectroscopy and AI-enhanced image processing. This technology provides embryologists with quantifiable data on embryo health, reducing reliance on subjective methods and enhancing the accuracy of embryo selection. By improving the reliability of embryo grading, the technology directly addresses the needs of these professionals, allowing them to offer higher success rates in their reproductive services. Cattle Genetics Laboratories and Research Institutions: Role and Importance: Laboratories specializing in cattle genetics play a vital role in developing breeding strategies and enhancing the genetic diversity of cattle herds. These labs are involved in extensive research and testing to identify genetically superior animals that can improve herd productivity and resilience. Project Relevance: The embryo assessment technology developed through this project is designed to be integrated into the workflows of these laboratories. By providing precise, objective measurements of embryo viability, the tool aids in the selection of embryos that are not only morphologically sound but also exhibit strong internal health markers. This can significantly improve the outcomes of breeding programs and ensure that only the most viable embryos are used in ET and IVF procedures, leading to better genetic outcomes. Animal Genetics and Reproduction Labs for Other Species: Role and Importance: While the initial focus of the project is on bovine embryos, the underlying technology has broader applications across various species. Labs specializing in the genetics and reproduction of other animals could adapt the technology for their specific needs, making it a versatile tool in the field of animal reproduction. Project Relevance: The project's innovation in non-invasive, quantitative embryo assessment can be adapted to different species, providing similar benefits in improving reproductive outcomes. This expands the potential market and applicability of the technology, positioning it as a valuable tool in various sectors of animal genetics and reproduction. Industry Key Opinion Leaders and Potential Customers: Role and Importance: Key opinion leaders (KOLs) in the bovine reproduction industry are influential figures whose endorsement and adoption of new technologies can drive wider acceptance and use. These individuals often serve as early adopters and advocates, helping to establish credibility for new tools and methods. Project Relevance: Throughout the project, significant effort was made to engage with KOLs and potential customers. This included conducting over 160 customer discovery interviews with DVM embryologists, genetics experts, and industry practitioners. The insights gained from these interactions were critical in refining the technology to meet real-world needs. Many of these professionals expressed a strong desire for an objective, reliable embryo grading tool, and their feedback has guided the development process to ensure the technology aligns with market expectations. Educational and Extension Programs: Role and Importance: Educational institutions and agricultural extension programs that focus on veterinary science and animal husbandry also represent an important target audience. These programs are often involved in training the next generation of professionals and disseminating new technologies and best practices to the broader farming community. Project Relevance: The technology developed in this project can be incorporated into educational curricula and extension programs to teach students and practitioners about advanced methods in embryo assessment and cattle reproduction. By partnering with educational institutions, the project can help ensure that future veterinarians and embryologists are well-versed in the latest tools and techniques, further driving adoption and standardization across the industry. International Markets: Role and Importance: The project also considers the growing demand for advanced reproductive technologies in international markets, particularly in regions where cattle reproduction is a key economic activity. Countries in Asia-Pacific and Latin America are experiencing rapid growth in the veterinary artificial reproduction market. Project Relevance: The commercialization strategy for the project includes targeting these international markets, where there is a significant opportunity for growth. By addressing the specific needs of these regions, such as cost-effectiveness and ease of use in varying environmental conditions, the technology developed can help improve cattle reproduction outcomes on a global scale. Broader Impact: The project's focus on these diverse audiences is crucial for ensuring that the developed technology is not only scientifically sound but also practically useful and commercially viable. By directly engaging with the end-users--those who will implement the technology in their day-to-day operations--the project has been able to tailor its objectives and outcomes to meet the actual demands of the market. This strategic alignment with the needs of various stakeholders ensures that the technology has a clear pathway to adoption and can make a meaningful impact on the cattle reproduction industry. Moreover, the involvement of industry experts, KOLs, and potential customers throughout the project has provided valuable validation and feedback, helping to refine the product and positioning it for successful commercialization. The emphasis on customer discovery and market research demonstrates a commitment to understanding and addressing the real challenges faced by the industry, ensuring that the final product is both innovative and relevant. Changes/Problems:During Phase I, several critical changes and optimizations were implemented to ensure the success and repeatability of the impedance spectroscopy (IS) experiments on bovine embryos. Initially, the project focused on optimizing the percentage of current passing through the embryo rather than leaking through the surrounding media. This optimization, which was not originally proposed, became essential for achieving reliable IS measurements and was accomplished through extensive COMSOL simulations. This step was crucial in minimizing noise and enhancing signal accuracy, ensuring that the IS data accurately reflected the biological properties of the embryos. Using a micro incubator for real-time embryo imaging and incubation under a microscope also presented unique challenges. Unlike standard incubation chambers, this setup required the development of a new, standardized protocol due to the absence of existing guidelines. Over 200 frozen embryos were tested across various microfluidic devices and incubation conditions within this microincubator. This extensive experimentation was necessary to establish an optimized and controlled environment for IS measurements, minimizing environmental variation and focusing on capturing biological development alone. Additionally, the original Phase I plan included developing a microfluidic channel for embryo manipulation. However, consultations with bovine embryologists revealed that industry professionals need to be more typically trained to manipulate embryos in channels, which could introduce complexity and resistance to adoption. Consequently, the design was modified to incorporate a hybrid microfluidic channel that integrated conventional reservoirs--familiar to embryologists--with precise channel positioning for accurate IS measurement. This hybrid design allowed for reliable and accurate measurements while ensuring ease of use and compatibility with existing industry practices. A significant deviation from the original plan involved the measurement of biomarkers, such as glucose consumption, and comparing the IS data with morphological and biomarker data. To maintain a robust and uniform incubation environment and minimize environmental variation during IS measurement and embryo growth, embryos were incubated in 50-80 µl droplets under an oil layer. However, due to the largevolume of media (~20 ul is suitable for highly sensitive measurement) and the relatively short incubation period (5-7 hours), the changes in biomarkers, specifically glucose, pyruvate consumption, and lactate production, were not substantial enough to be sensitively measured using the ultrafluorometric cuvette reader. The small changes in these biomarkers under these conditions made it challenging to obtain meaningful comparative data, leading to a focus on optimizing the IS measurements as the primary indicator of embryo viability and comparing them with the morphological growth markers. Though not initially planned, these adjustmentswere crucial for overcoming technical challenges and advancing the project's objectives. The modifications ensured that the final methodology was robust, repeatable, and aligned with industry standards, thereby paving the way for the successful completion of Phase I and setting a solid foundation for further development in Phase II. What opportunities for training and professional development has the project provided?The project provided significant opportunities for training and professional development, particularly for two engineering interns who worked under the direct supervision of the Principal Investigator (PI). These interns were actively involved in the experimental design, execution, and data analysis, which not only enhanced their technical skills in impedance spectroscopy (IS) and bioengineering but also formed the core of their master's theses. This hands-on experience allowed the interns to gain deep insights into the intricacies of electrical modeling of biological systems, particularly in developing and validating a new equivalent circuit for multicellular bio-objects like bovine embryos. Furthermore, the validation of the new equivalent circuit represents a substantial advancement in the IS science and engineering of multicellular bio-objects. The interns were exposed to cutting-edge research methodologies, including the integration of biological and electrochemical principles into a cohesive analytical model. This experience has prepared them for future careers in biomedical engineering, research, and development, by equipping them with the skills necessary to tackle complex bioengineering challenges. The project also provided opportunities to contribute to scientific publications, enhancing their professional profiles and providing a strong foundation for future academic or industry roles. How have the results been disseminated to communities of interest?The results of this project have been effectively disseminated to relevant communities of interest through the publication and presentation of research findings at two prominent conferences. The engineering processes, experimental results, and innovative methods developed during the project were presented at the 46th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC 2024), held in Orlando, Florida, from July 15-19, 2024. The conference paper, titled "Bovine Embryo Development Monitoring Using Differential Electrical Impedance Spectroscopy," highlighted the use of differential impedance spectroscopy to monitor embryo development, garnering attention from the biomedical engineering community. The research was also submitted at the 2024 AETA-CETA/ACTE Joint Annual Meeting, which will be held in Toronto, Ontario, from October 17-19, 2024. The paper"Non-invasive Bovine Embryo Assessment Using Impedance Spectroscopy and AI-assisted Image Segmentation" will bepresented to professionals and academics in animal embryo technology. This presentation emphasized the integration of AI and impedance spectroscopy as a novel approach to non-invasive embryo assessment, sparking discussions on the future of embryo monitoring technologies. These conferences provided a platform for the project team to engage with experts in bioengineering, veterinary science, and related fields, facilitating knowledge exchange and influencing future research and development in non-invasive embryo assessment technologies. The dissemination of these results through such reputable forums has validated the project's methodologies and findings and contributed to advancing the broader scientific community's understanding of impedance spectroscopy in multicellular bio-object analysis. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1: Design and Fabricate a Microfluidic Chip for Precise Embryo Manipulation and Impedance Measurement Major Activities Completed / Experiments Conducted: The microfluidic chip was designed and fabricated, enabling precise manipulation of bovine embryos for impedance measurements. The chip was tested to ensure accurate electrode placement and minimal damage to embryos during measurements conducted across a frequency range of 100 kHz to 1.5 MHz. Data Collected: Impedance measurements were taken from 20 embryos. For instance, the electrical double-layer capacitance (C1) values ranged from 3.9648E-09 for healthy embryos to 3.9485E-08 for degenerated embryos. The ZP leakage resistance (R5) varied from 1699 ohms in good embryos to 302.7 ohms in degenerated ones. Summary Statistics and Discussion of Results: Healthy embryos consistently showed higher R5 values and lower C1 values, indicating better ZP integrity and more controlled ion transfer. The data supported the hypothesis that impedance measurements can differentiate between healthy and degenerated embryos. Key Outcomes or Other Accomplishments Realized: Change in Knowledge: This work provided new insights into the electrical properties of embryos and demonstrated that impedance spectroscopy could be used as a non-invasive assessment tool. Change in Action: The development of the microfluidic chip and its application in embryo assessment could revolutionize industry practices, moving from subjective grading to objective, quantifiable measurements. Objective 2: Measure the Impedance of 20 Bovine Embryos and Model the Equivalent Circuit In this study, a modified equivalent circuit was developed to accurately model the electrical properties of a bovine embryo using a two-probe electrical impedance spectroscopy (EIS) setup. The circuit accounts for the complex structure of the embryo, including the zona pellucida (ZP), perivitelline space, cell mass, extracellular fluid, and the electrode-fluid interface. Unlike traditional models, this circuit incorporates both the biological and electrochemical interactions within the embryo, providing a more precise and nuanced representation. By including components such as C1, R5, C5, C7, and R6, the circuit offers a better prediction of embryo viability by accurately reflecting the real conditions and interactions within the embryo during the measurement process. Major Activities Completed / Experiments Conducted: Impedance was measured in 20 embryos, and the data was used to model an equivalent circuit that represents the electrical characteristics of the embryos. Data Collected: Data included C1 values of 3.9648E-09 for good embryos compared to 3.9485E-08 for degenerated ones, and R5 values from 1699 ohms to 302.7 ohms, respectively. Summary Statistics and Discussion of Results: The data showed that impedance spectroscopy could effectively differentiate between healthy and degenerated embryos, providing valuable metrics such as membrane capacitance and cytoplasmic conductivity. Key Outcomes or Other Accomplishments Realized: Change in Knowledge: The equivalent circuit model provided a new framework for understanding the electrical properties of bovine embryos. Change in Condition: This method could improve embryo selection processes, leading to higher success rates in cattle breeding. Objective 3: Train a Neural Network for Embryo Image Processing and Correlate Data with Biological Outcomes Major Activities Completed / Experiments Conducted: A CNN-based image processing platform was developed and trained to segment and analyze bovine embryo images with over 99.5% accuracy. The neural network processed images from over 200 embryos and provided data on expansion rates and blastomere symmetry. Data Collected: The neural network provided quantitative data on cell mass percentage relative to the perivitelline space. The training data was hosted in the cloud and used for cell mass percentage measurement of the embryo for grading as per the IETS manual. The accuracy of the cell mass measurement from the real-time images was over 99.3%. Summary Statistics and Discussion of Results: The neural network's segmentation results correlated strongly with impedance data, demonstrating that embryos with higher cell mass, expansion rates, and better blastomere symmetry had more favorable electrical properties, which are consistently measurable. Key Outcomes or Other Accomplishments Realized: Change in Knowledge: The project showed that AI-driven image processing combined with impedance data could provide a robust method for non-invasive embryo assessment. Change in Action: These findings could lead to adopting more advanced, data-driven methods in reproductive medicine, improving embryo selection, and increasing success rates in fertility treatments. Experimental data analysis: The impedance of embryos was measured before incubation, and the equivalent circuit elements show promise to predict its viability (Good embryos are the ones that grew to expanded blastocyst or hatched and degenerated are the ones that did not grow in the same incubation condition): 1. C1 (Electrical Double Layer Capacitance):C1 values are generally lower in degenerated embryos (2.9894×10?? to 3.7477×10??) and higher in good embryos (3.9648×10?? to 3.2789×10??), suggesting a correlation between higher initial C1 values and healthier embryos. 2. C5 (Perivitelline Space Capacitance):C5 values in degenerated embryos show high variability and tend to be lower or negative, indicating instability, while good embryos have more stable, slightly positive values, reflecting a healthier perivitelline space. 3. R5 (ZP Leakage Resistance):R5 values are lower in degenerated embryos, indicating higher ion leakage, while higher R5 values in good embryos suggest better ZP integrity and lower ion permeability, reflecting healthier embryo conditions. 4. C6 (Perivitelline Space Capacitance):C6 values are lower in degenerated embryos, indicating increased perivitelline space and degeneration, while good embryos show slightly positive or near-zero C6 values, reflecting a stable, healthy perivitelline space 5. R6 (Perivitelline Space Resistance):R6 values are generally lower in degenerated embryos, indicating higher ion transfer and potential degradation, while higher R6 values in good embryos suggest a healthier perivitelline space with less ion transfer. 6. C7 (Cell Mass Capacitance):C7 values are lower and less stable in degenerated embryos, indicating cell mass issues, while good embryos exhibit higher and more stable C7 values, reflecting better cell mass integrity and health. Overall Trends and Hypothesis Validation: Higher C1 and R5 Values Correlate with Good Embryo Health: Embryos that are ultimately classified as "Good" generally show higher initial C1 and R5 values. This suggests that higher electrical double-layer capacitance and better zona pellucida integrity (as indicated by R5) indicate healthier embryos with a higher potential for successful development. Stability in C5, C6, and R6: Good embryos tend to exhibit more stable C5, C6, and R6 values, which reflects better stability in the perivitelline space and less ion transfer. This stability is a positive indicator of embryo health. Variability and Lower Values in Degenerated Embryos: Degenerated embryos tend to show more variability and lower values in key impedance parameters (particularly C1, R5, and C7), suggesting that initial electrical instability correlates with poorer outcomes. Impact Statement:This project has advanced bovine embryo assessment by developing a non-invasive, data-driven method that enhances embryo selection in ET and IVF, improving pregnancy outcomes and breeding efficiency, with broader impacts on sustainable agriculture, food security, and potential applications in human reproductive medicine.

Publications

  • Type: Conference Papers and Presentations Status: Submitted Year Published: 2024 Citation: M. A. Rahman, A. T. Ohta, and M. A. Uzzaman, "Non-invasive bovine embryo assessment using Impedance Spectroscopy and AI-assisted Image Segmentation," AETA-CETA/ACTE Joint Annual Meeting, ON, Canada, October 17-19, 2024.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2024 Citation: M. A. Uzzaman, M. A. Rahman, and A. Ohta, "Bovine Embryo Development Monitoring Using Differential Electrical Impedance Spectroscopy," in Proc. 46th Annu. Int. Conf. IEEE Eng. Med. Biol. Soc., Orlando, FL, USA, Jul. 15-19, 2024.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:1. Bovine embryologist 2. Animal ET/IVFcompanies 3. University animal ET/IVF researchers Changes/Problems:Measuring metabolic activities, especially glucose uptake by embryos incubated in a microfluidic channel, is challenging. The embryo, contained within the channel, is positioned between electrodes using external tubing and fluid flow connected to the microincubator. The excess media and its flow in this setup dilute the glucose and pyruvate concentration near the embryo. As a result, glucose measurements in this media may not accurately represent the actual uptake during incubation. To address this, we aim to reduce the media volume and assess metabolic biomarkers at the start and end of the incubation period. By considering the diffusion of biomarkers throughout the media, we can estimate average concentrations for calculating the consumption or uptake of glucose and pyruvate and subsequently correlate these with impedance data. What opportunities for training and professional development has the project provided?The project recruited an engineering graduate student as an intern and trained him to conduct various experiments related to the projects. How have the results been disseminated to communities of interest?The experiments are still ongoing, and the findings are yet to be conclusively documented. What do you plan to do during the next reporting period to accomplish the goals?We have gathered a comprehensive set of data on bovine embryos, encompassing their physical attributes, morphological details, morphokinetic characteristics, and impedance spectroscopy measurements. Currently, we are in the process of analyzing this data. We'd like to present the findings in our next report, showing the feasibility of the proposed project.

Impacts
What was accomplished under these goals? The following is a brief summary of the goal accomplished: 1.Various microfluidic device designs with embedded electrodes have been developed, with software simulations conducted using the AC/DC module of COMSOL Multiphysics. These simulations evaluated different electrode dimensions, water column sizes, and simulated embryo geometries to optimize the dimensions of electrodes and microchannels. Electrical impedance was measured across the simulated embryo for voltages ranging from 300 mV to 1V, and over a frequency range of 100 Hz to 5 MHz. Our results suggest that aligning the electrode geometry with the embryo's diameter optimizes the signal-to-noise ratio, improves impedance measurement accuracy, and reduces noise, such as current loss in the conductive media. Subsequently, several dozen microfluidic devices were fabricated using photolithography and soft lithography. Following physical experiments using these devices to incubate unfrozen bovine embryos in the microfluidic channels, observations were utilized to refine the designs. This iterative process led to the fabrication of improved devices for further experimentation. 2.We completed a series of critical experiments involving impedance measurement in bovine embryos. Utilizing the microfluidic chip designed in Aim 1 of our project, we successfully measured the impedance of three bovine embryos, accompanied by time-lapse imaging. This process enabled us to model the equivalent circuit accurately and calculate both the membrane capacitance and cytoplasmic conductivity. Notably, this success followed over 100 initial experiments that failed due to unfavorable incubation environmental conditions, which were eventually stabilized using a custom-designed microincubator. Continuing our research, we are now focused on extending these impedance measurements to a more extensive set of bovine embryos. 3. We have developed and trained a neural network using manually annotated embryo images. These images, recorded during embryo incubation, were processed to identify parameters such as the expansion rate, blastomere symmetry, and the percentage of intact embryonic cells, as indicated by extruded material in the perivitelline space. The three previously mentioned embryos' impedance data (membrane capacitance and cytoplasmic conductivity)were correlated with this morphological data. It was observed that cytoplasmic conductivity increases as cell proliferation and total cell mass rise during incubation. We are conducting further experiments with more bovine embryos and planning to explore the correlation between these parameters and metabolic activity soon.

Publications