Milken Institute School of Public Health Poster Presentations (Marvin Center & Video)
Semi-Automated Scoring of Triple-Probe FISH in Human Sperm Using Confocal Microscopy
Poster Number
42
Document Type
Poster
Publication Date
3-2016
Abstract
Aneuploidy, or an abnormal number of chromosomes, is found in human gametes and is caused by errors during meiosis. Although most human fetuses conceived from aneuploid gametes do not survive to term, some aneuploid conceptuses do result in offspring with compromised phenotypes. Kleinfelters, Turners, and XYY syndromes are examples of well-characterized chromosomal disorders resulting from aneuploid conceptuses. To study sperm aneuploidy and its impacts on fertility and reproduction, fluorescence in situ hybridization (FISH) is used to quantify the frequency of aneuploidy in sperm and requires the scoring of thousands of nuclei. Traditional manual scoring of FISH sperm can be time consuming, which can lead to scorer fatigue and increased error. Semi-automated methods that rely on computer software to objectively count fluorescence signals using specified criteria are needed.
In this validation study, we used a Zeiss LSM 710 confocal microscope combined with the Zen software (Zeiss, Jena, Germany) for image acquisition. Application of online spectral linear unmixing allowed for effective separation of the four fluorochromes used to identify chromosomes X, Y, 18 and the nuclei. Image processing, segmentation, classification, and scoring were performed using custom analysis software developed in MATLAB®.
The semi-automated results were compared with manual scoring results in 10 slides. In comparing percent disomy calculated by each scoring method for each slide, a significant difference was found on one slide for XX18 (0.62% automated vs 0.05% manual). For the other 9 slides, XX18 estimates were comparable by method, as were the YY18 and XY18 estimates across all 10 slides. These results demonstrate that semi-automated methods using spectral imaging on a confocal microscope are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods.
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Open Access
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Semi-Automated Scoring of Triple-Probe FISH in Human Sperm Using Confocal Microscopy
Aneuploidy, or an abnormal number of chromosomes, is found in human gametes and is caused by errors during meiosis. Although most human fetuses conceived from aneuploid gametes do not survive to term, some aneuploid conceptuses do result in offspring with compromised phenotypes. Kleinfelters, Turners, and XYY syndromes are examples of well-characterized chromosomal disorders resulting from aneuploid conceptuses. To study sperm aneuploidy and its impacts on fertility and reproduction, fluorescence in situ hybridization (FISH) is used to quantify the frequency of aneuploidy in sperm and requires the scoring of thousands of nuclei. Traditional manual scoring of FISH sperm can be time consuming, which can lead to scorer fatigue and increased error. Semi-automated methods that rely on computer software to objectively count fluorescence signals using specified criteria are needed.
In this validation study, we used a Zeiss LSM 710 confocal microscope combined with the Zen software (Zeiss, Jena, Germany) for image acquisition. Application of online spectral linear unmixing allowed for effective separation of the four fluorochromes used to identify chromosomes X, Y, 18 and the nuclei. Image processing, segmentation, classification, and scoring were performed using custom analysis software developed in MATLAB®.
The semi-automated results were compared with manual scoring results in 10 slides. In comparing percent disomy calculated by each scoring method for each slide, a significant difference was found on one slide for XX18 (0.62% automated vs 0.05% manual). For the other 9 slides, XX18 estimates were comparable by method, as were the YY18 and XY18 estimates across all 10 slides. These results demonstrate that semi-automated methods using spectral imaging on a confocal microscope are a feasible approach for analyzing numerical chromosomal aberrations in sperm, and are comparable to manual methods.
Comments
Presented at: GW Research Days 2016