Methods and systems for assessing and/or quantifying sperm cell subpopulations bearing a specific genetic signature
Inventors
ROTI-ROTI, Elon • CRAY, Nicole • Wagner, Matthias • BOTTS, Michael Reid
Assignees
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Publication Number
US-12258630-B2
Publication Date
2025-03-25
Expiration Date
Abstract
Technologies for assessing, quantifying and isolating sperm cell populations and/or subpopulations having specific genetic signatures are provided, as well as methods and systems to assess the efficacy of chromosomal differentiation processes. Compositions for identification and differentiation of X-chromosomes and Y-chromosomes in DNA are also provided.
Core Innovation
The invention relates to assessing sex-skew in a population of cells by using chromosome-selective oligonucleotide agents. A first oligonucleotide agent selectively binds to an X-chromosome nucleic acid sequence defined by at least one sequence selected from SEQ ID NO: 4-6, 28-35, 222, and 845-847, and a second oligonucleotide agent selectively binds to a Y-chromosome nucleic acid sequence defined by at least one sequence selected from SEQ ID NO: 7-9, 36-55, 767, 850-861, and 864-875. Detection of these bindings is used to quantify sex-skew.
The core approach is implemented as a system and as a method for sperm cells, where the binding agents interact with sperm DNA obtained from a sample. In the system, the combination of X- and Y-chromosome-selective oligonucleotide agents is configured to enable detecting binding of the agents to sperm DNA. In the method, detecting binding is performed on sperm DNA obtained from a semen sample, so that sex-skew is assessed based on the presence and/or binding of the X and Y targets.
The documented implementations further emphasize ddPCR-based measurement of X and Y targets using a housekeeping reference for cell-count normalization. The assay format includes multiplex ddPCR and triplex configurations, and applies gating and Poisson correction as part of ddPCR analysis to support independent quantification of sperm sex-skew.
Claims Coverage
The partial content provides two independent claims: one system claim and one method claim. The independent claim coverage focuses on X- and Y-chromosome-selective oligonucleotide agents defined by specific SEQ ID sets, with the method claim further covering detection on sperm DNA from a semen sample.
X-chromosome-selective oligonucleotide agent
A first oligonucleotide agent that selectively binds to an X-chromosome nucleic acid sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 4-6, 28-35, 222, and 845-847.
Y-chromosome-selective oligonucleotide agent
A second oligonucleotide agent that selectively binds to a Y-chromosome nucleic acid sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 7-9, 36-55, 767, 850-861, and 864-875.
Detecting binding of X- and Y-target oligonucleotide agents to sperm DNA
Detecting binding of a set of oligonucleotide agents to sperm DNA obtained from a sample of sperm cells, wherein the set includes a first oligonucleotide agent that selectively binds to an X-chromosome nucleic acid sequence comprising at least one sequence selected from the group consisting of SEQ ID NO: 4-6, 28-35, 222, and 845-847; and a second oligonucleotide agent that selectively binds to a Y-chromosome nucleic acid sequence comprising at least one sequence selected from the group consisting of SEQ ID: 7-9, 36-55, 767, 850-861, and 864-875.
Sperm DNA isolation from a semen sample
The method further includes isolating sperm DNA from a semen sample containing a population of sperm cells.
Laser-ablated sperm removal prior to DNA isolation
The method further removes laser-ablated sperm cells from a semen sample before isolating sperm DNA.
Multiplex ddPCR detection
The detecting step is performed using multiplex ddPCR.
Across the independent claims, the inventive core is the use of two chromosome-selective oligonucleotide agents defined by specific SEQ ID sets for X and Y sequences, together with detecting their binding to sperm DNA to assess sperm sex-skew. Dependent claim refinements in the provided set further add sperm DNA isolation, optional removal of laser-ablated sperm, and detection performed using multiplex ddPCR.
Stated Advantages
Independent quantification of sperm sex-skew by detecting X- and Y-chromosome-specific oligonucleotide primer/probe targets using ddPCR.
Improved/validated performance metrics reported in the document, including linear correlation with an autosomal housekeeping reference and precision/variance and dynamic range results.
Agreement reported with historical calf sex outcomes using the assay results.
Removal of non-viable (laser-ablated) sperm is described as part of the workflow prior to DNA extraction.
Documented Applications
Sexed semen quality assurance (QA) validation, including assessment of sex-skew in sexed semen products.
Artificial insemination (AI) and in vitro fertilization (IVF) contexts are described as use cases for sexed semen QA.
Assay implementation for Bos taurus, Bos indicus, Bos bubalis, and hybrids is described in the partial claim set.
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