Application of different in-situ hybridization detection methods for human sperm analysis

Area of Science:

• Reproductive Biology
• Genetics
• Cytogenetics

Background:

• Fluorescence in-situ hybridization (FISH) is used for aneuploidy detection in human spermatozoa.
• Current FISH methods face challenges in sperm decondensation, leading to distorted morphology and loss of tail structures.
• Distinguishing between disomic, diploid, and abnormal spermatozoa, as well as somatic cells, remains difficult with existing techniques.

Purpose of the Study:

• To adapt and improve decondensation and visualization steps for single sperm cells in FISH analysis.
• To enhance the discrimination capabilities for various sperm cell types and potential contaminants.
• To develop a more robust and accurate method for sperm aneuploidy detection.

Main Methods:

• Adapted DNA decondensation using 25 mM dithiothreitol in 1 M Tris at pH 9.5, preserving sperm head and tail morphology.
• Applied a novel detection method utilizing enzyme immunocytochemical reactions with colored precipitation products.
• Combined the improved in-situ hybridization (ISH) procedure with Diff-Quik staining and bright-field microscopy.

Main Results:

• Achieved intact sperm cell morphology (head and tail) post-decondensation, enabling efficient single-, double-, and triple-target ISH.
• The enzyme-based detection method provided a permanent signal with enhanced visualization of the sperm plasma membrane outline.
• Successfully discriminated between disomic, diploid, abnormal spermatozoa, and somatic cells due to preserved cell morphology and intact tails.

Conclusions:

• The adapted ISH procedure significantly improves the accuracy of aneuploidy detection in human spermatozoa.
• Preservation of sperm morphology and tail visualization allows for reliable differentiation of cell types.
• This enhanced method offers a valuable tool for genetic analysis of sperm and reproductive research.