Abstract
Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.
Highlights
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis
The chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21
Add freshly prepared Carnoy’s fixative (3:1 methanol:acetic acid) drop by drop while mixing on a vortex to obtain a final volume of 8 ml. 8
Summary
7. After resuspending the pellet, add freshly prepared Carnoy’s fixative (3:1 methanol:acetic acid) drop by drop while mixing on a vortex to obtain a final volume of 8 ml. 9. Add freshly prepared methanol:acetic acid (3:1) drop by drop to adjust the final volume to the cellular concentration required for obtaining good cell spreading (in those cases with a small pellet, few drops will be enough whereas samples with a high amount of sperm recovered, the final volume can reach [1-2] ml). A test slide can be made by dropping the resuspended sample onto an ungreased slide and examining it under a phase contrast microscope This will let to check the cellular dispersion obtained and would allow correcting it, if it is necessary, in further slides.
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