SDFA KIT brochure (download) License to make SDFA KIT (download) General information Application: routine analysis of semen is the first method in evaluating men's fertility status. This method provides useful information about the number, movement, morphology, sperm viability and gonad function. However, this evaluation does not provide any information about one of the most important parameters, namely the integrity of the DNA molecule, which is essential for sperm fertility and embryo development. The amount of sperms with healthy DNA in semen can be considered as a predictive factor in evaluating male fertility and the possibility of pregnancy. In fact, the presence of DNA breaks is associated with a decrease in male fertility, infertility and abortion.
Sperm DNA Breakage Assessment Kit (SDFA) by evaluating the integrity of the sperm DNA molecule and calculating the DNA Breakage Index (DFI) provides valuable information regarding the status of patients referred to the infertility treatment center to choose the appropriate treatment protocol.
Conditions and duration of storage: After receiving and every use, the components of the kit should be kept at a temperature of 2-8 degrees Celsius.
Indications: Oligostenotratozoospermia, male infertility with unknown cause, history of repeated abortion, varicocele, exposure to toxins and pollutants, checking the effectiveness of drug treatments, fetal growth arrest, unfavorable embryo quality, fertility evaluation after cancer treatments.
Considering that performing assisted reproductive cycles (ART) are expensive and time-consuming, it is recommended to evaluate the amount of sperm DNA breakdown before starting treatment cycles for all couples.
Principles of the test: This test is based on the expansion of sperm chromatin. In this method, after placing the sperms in a bed of microgel on special slides, acid is used for denaturation. Next, using lysis solution, sperm proteins are removed. Finally, by the staining solution, the expansion of the sperm DNA molecule can be seen in the form of a halo of different sizes (based on the integrity of the DNA) around the nucleus.
Kit contents: Each kit contains the contents needed to perform the test for 25 samples.
Kit components include
- 25 coated slides
- Solution A (30 mm)
- Solution B (30 mm)
- Solution C (30 mm)
- Solution D (30 mm)
- Foam for floating microtubes
- Brochure
Other materials and tools needed for the test: - optical microscope
- Chemical hood
- Refrigerator
- Ben Murray
- Sampler and sampler head
- Lamel
- Petri Dish
- 70, 90 and 100 percent ethanol
- Distilled water
- Phosphate buffer saline (PBS) with pH 7.4-2.7
Gloves and masks
Sample preparation: First, a fresh semen sample must be collected in a sterile container (melted semen sample can also be used). After liquefaction and evaluation of sperm concentration, wash twice with PBS at 300-400 g for 5 minutes. Finally, the resulting sediment should be diluted using PBS to the amount of 5-10 million sperm per ml.
Positive and negative controls: - Positive control: In order to prepare a positive control, a sperm sample can be incubated in a boiling water bath for 30 minutes, or it can be exposed to ultraviolet rays (UV) or alkaline sodium hydroxide (NaOH) solution.
- Negative control: In order to prepare a negative control, the processed sperm sample of a person with normal DFI can be used.
instructions The efficacy of the following protocol for the sperm DNA breakage assessment kit (SDFA) by evaluating the state of the integrity of the sperm DNA molecule and calculating the DNA breakage index (DFI) Valuable information regarding the condition of patients referred to the infertility treatment center to choose the appropriate treatment protocol at the disposal of the attending physician and this protocol is presented only as an example:
1- Placing the microtube containing the gel in the foam included in the kit and immersing it in a bain-marie at 85-95 degrees for 5 minutes or until the gel is completely melted.
2- Placing the microtube containing the melted gel at room temperature in order to be at the same temperature as the room (about 2-3 minutes)
3- Add 50 microliters of the prepared sperm sample to the microtube containing the gel and homogenize it.
4- Putting 20 microliters of the above mixture on a special slide and immediately placing the slide on it (avoid creating bubbles)
5- Placing the slide in the refrigerator at 2-8°C for 4 minutes
6- Remove the slide slowly from the slide
7- Placing the slide inside the petri dish and adding solution A and keeping it in the dark for 7 minutes at room temperature
8- Slow draining of solution A and adding solution B and keeping it for 15 minutes at ambient temperature
9- Slowly emptying solution B and adding distilled water and keeping for 3 minutes
10- Slow draining of distilled water and adding 70, 90 and 100% ethanol respectively for 2 minutes each.
11- Completely drying the slide at room temperature
12- Adding solution C to the size (2-3 drops) and immediately solution D to the size (2-3 drops), slowly shaking the slide to mix the two solutions and keeping for 15 minutes
13- Wash the slide with a slow flow of PBS
14- Drying the slide and observing with a light microscope with 1000x magnification
15- Count at least 400 sperm and calculate DFI
Interpretation of results: Healthy sperm without DNA damage 1- Sperm with a large halo: a halo whose width is similar or larger than the diameter of the nucleus (b).
2- Sperm with a medium halo: a halo whose width is between the large and small halo (c).
DNA broken sperm 1- Sperm with a small halo: a halo whose width is smaller or equal to one third of the diameter of the nucleus (d).
2- Sperm without halo (No halo) (e).
3- Sperm without aura with damaged, irregular and weakly stained nucleus (Degraded) (f)
Warnings 1- Strictly avoid contacting the contents of the kit with the skin and eyes, as well as inhaling them.
2- Gloves and mask should be used during the test.
3- The test place should have proper air conditioning.
4- Dispose of waste in accordance with laboratory safety principles.
5- Strictly avoid eating and drinking during the test.
