Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Summary

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

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.

Protocol

I. Sample processing and cell fixation

1. Leave the semen sample in sterile containers at room temperature for 20 minutes until liquefaction.
2. Transfer the sample to a centrifuge tube and spin it at 1000g for 5 minutes.
3. Gently remove and discard the supernatant using a Pasteur pipette.
4. Add hypotonic solution (KCl, 0.075M) pre-heated at 37°C, drop by drop, while mixing on a vortex to obtain a final volume of 10 ml.
5. Place the tube in a water bath at 37°C for 30 minutes.
6. Centrifuge at 1000g for 5 minutes. Carefully discard the supernatant by decantation, without disturbing the pellet.
7. After resuspending....

Discussion

This protocol describes how to process human semen samples for obtaining sperm FISH preparations. Using this protocol it is possible to analyze chromosomal abnormalities in male gametes. Spermatozoa aneuploidy screening has applications either in the context of basic research and in the reproductive advice given to infertile males. Although in clinical diagnosis the most widely studied chromosomes are X, Y, 13, 18 and 21, other commercial probes for a wide range of chromosomes and loci are available and can also be used .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,4-Dithiothreitol (DTT)	Material	Roche Group	10197777001
2-Amino-2-(hydroxymethyl)-1,3-propanediol	Material	Roche Group	10708976001
Acetic acid	Material	Merck & Co., Inc.	100.063
AneuVysion® Multicolor DNA Probe Kit 20xSaline-Sodium Citrate 4,6-diamidino-2-phenylindole (DAPI II) CEP 18/X/Y DNA probe NP-40 LSI 13/21 DNA probe	Material	Vysis Inc.	32-161075 30-805850 30-804941 30-171077 30-804820 30-171078
Centrifuge 5804R	Tool	Eppendorf
Centrifuge tube	Material	Nalge Nunc international	347856
Coplin jar (glass)	Material	Barloworld Scientific	Hellendahl (ZCT278)
Coplin jar (plastic)	Material	Deltalab	191087
Cover slips (15x15)	Material	Knittel Glaser	4600115
Cover slips (18x18)	Material	Knittel Glaser	4600118
Diamond pencil	Material	Hammacher	335117010
Ethanol	Material	Merck & Co., Inc.	100.983
Formamide	Material	Roche Group	11814320001
Freezer	Tool	Zanussi
Gloves	Material	Sanyo	101/3300
HYBrite™	Tool	Vysis Inc.
Immersion Oil	Material	Olympus Corporation	35505
Incubator	Tool	Heraeus Instruments
Methanol	Material	Merck & Co., Inc.	106.009
Micropipette	Material	Labsystems	Finnpipette (4500000)
Micropipette replacement tip	Material	Daslab	16-2001
Nail varnish	Material	Quo-cosmetics
Olympus BX60 epifluorescence microscope	Tool	Olympus Corporation		Equipped with a triple-band filter for DAPI/Texas Red/FITC and single-band filters for Aqua, FITC and Texas Red.
Pasteur Pipette	Material	Rubilabor	211.0230
Phase contrast microscope	Tool	Nikon Instruments
Plastic pipette (3ml)	Material	Deltalab	200007
Potassium chloride (KCl)	Material	Fluka	60128
Rubber cement	Material	Best-test
Slides	Material	Knittel Glaser	4520022
Slide Saver Boxes	Material	Deltalab	19276.1
Sterile container	Material	Deltalab	409726
Syringe	Material	PentaFerte	002022300
Thermometer	Material	Comark Instruments, Inc	KM12
Tri-distilled water	Material
Triton X-100	Material	Sigma-Aldrich	9002-93-1
Tweezers	Material	B. Braun Medical	Aesculap (BD224R)
Vertical laminar flow hood	Tool	Burdinola	OR-ST 1500
Vortex mixer	Tool	Fisher Scientific	FB15012
Water bath	Tool	Raypa®