From Wired How-To Wiki
About 40 percent of infertility issues in couples are due to male infertility. Even with these compelling statistics, men are still reluctant to go to the doctor to deal with the whole question of fertility for a variety of reasons — awkward conversation in the reception area, waiting sheepishly among other semen-holding guys, the possibility of being caught by familiar faces while stepping out of the clinic, to name a few.
Confirming your virility (or lack there of) from a fertility clinic may also cast shadow of doubt over the veracity of the results, especially if they are not as expected. Was that number really coming from my sample? Were the flasks mislabeled? Was the analyst incompetent? To avoid all this male distress about an issue as intimate as semen quality, there are a few alternative solutions you can explore in the privacy of your own home.
First we'll go through the true DIY bio approach using scientific equipment and bulk chemicals that you could throw a fertility-testing party with the leftovers. Second, the quick-and-dirty home kit procedures.
Cell Counting Technique
What You'll Need
Whether you sneak into a bio laboratory to borrow some stuff or you decide to buy it, here are the basic laboratory items you will need:
- Microscope — You don’t need an electron microscope to peer into the world of sperm. But you won't quite get there if you attempt to use a $15 Toys"R"us plastic microscope. If you are going to buy a microscope for sperm counting, you should observe some minimum specifications regarding optics and materials.
In order to visualize and count individual human sperm, a 400x magnification (10x eyepiece with 40x lens) is needed, as male spermatozoa are about 2.5 to 3.5 microns in width and 5 to 7 microns in length. An additional magnification of 100x (10x eyepiece with 10x lens) must be also available when first observing the sample, to get a wider view of the sample and to assure the presence of sperm. Apart from its power viewing properties, it is desirable to choose one microscope built with metal bodies and metal rack-and-pinion focus, in terms of durability and precise focusing. Another feature to bear in mind in the selection process is the lens material. Glass lenses are preferred rather than plastic toy-like lenses in order to be able to fine focus on thin slides.
All these basic requirements can be found in most standard microscopes for affordable prices. For example, Fisher Science Education Intermediate Compound microscopes are available for approximately $220, while Kokopelli Technologies sells a range of inexpensive microscopes for semen counting purposes starting at $120.
- Collection condom — Collection condoms, which are special, latex-free condoms, are mainly found online from male fertility-related product companies. Kokopelli Technologies offer a $20 pack of two, and similarly, Babyhopes.com sells them for 13 dollars each, or in multi-packs. And (bonus!) they also sell sperm-friendly lube if you need a little... encouragement.
- 1 ml Disposable transfer pipettes — You can find these on any lab supply webpage, or even on eBay at $5 for a 100-pack.
- Hand-held tally counter — While not essential, nothing will make you happier when counting than a hand-held tally counter. You can treat yourself with this essential lab accessory from Fisher Scientific for only $12.
- At least 20mL of Formalin buffer solution — You can purchase this chemical buffer solution from Sigma Aldrich.
Collecting the sample
Prior to collection, the male donor must observe three to five days of abstinence as the sperm needs to build up. (Sorry!)
Sperm can be collected from masturbation or intercourse. Although masturbation seems to be the easiest, some studies have demonstrated that sperm samples extracted from collection condoms after sexual intercourse yields higher total sperm counts, sperm motility, and percentage of normal morphology sperm than those obtained through masturbation. Therefore, the masturbation collection method is believed to give less accurate results when used for semen analysis .
If you decide to collect your sample through intercourse, you can choose to discard the sperm in a collection condom, or for more accurate results (and if you have good aim), directly into the collection containers.
When collecting the sample in a container, it is recommended to discard the first part of the ejaculate as usually the first squirt of semen that gushes forth during the ejaculation is overcrowded. The subject should unload into two adjacent containers so that this first part goes into a waste container, while the rest goes into the sample container.
Then wait for a minimum of fifteen minutes until the sample liquefies.
Diluting the Sample
There is a maximum concentration of cells you can load in a hemacytometer for an accurate counting. Healthy male raw semen is expected to have a very high concentration of sperm cells, so normally the collected sample must be diluted prior to counting.
A common way of diluting biological samples is by using a buffer solution. A buffer solution is basically water mixed with a chemical to provide it with special properties regarding pH. We've listed a pre-mixed solution in the materials list above, which contains formaldehyde that will kill and immobilize the sperm, and therefore, ease their counting.
Note: If you have access to a chem lab, you can mix your own with 100mL distilled water, 1 ml formaldehyde solution and 5 grams sodium bicarbonate
Now you need to perform a 2-part dilution to create a 1:100 semen/buffer solution, so grab your 1mL pipettes. Pipette up 1mL of raw semen, and deposit it into a clean container. Then pipette 9mL of formalin solution (one pipette-full after the other) into the semen vat and gently mix. You now have a 1:10 solution. Pipette 1mL of the 1:10 solution and deposit this in another clean container. Then pipette 9mL of formalin solution into that, and gently mix. You now have a 1:100 semen dilution that is ready to be analyzed with the hemocytometer.
Loading and Imaging the Sample
It is a good practice to place the hemocytometer in the microscope stage and get familiar with the focusing mechanism. This will help you to avoid useless drama in case no sperm is found when counting. Avoid false alarms; sometimes it is just not focused.
To load the sample, pipette about 15 microliters of the sample into the chamber by placing the tip of the pipette in the V-shaped groove of the hemacytometer while firmly holding the cover slip. Make sure the chamber is not overloaded as the result then would be inaccurate. The sample will enter the chamber by capillary action. Next, allow 3 or 4 minutes for the sample to settle before starting the count, but not too long or it will dry out.
The hemacytometer was originally designed for blood cell counting and consists of two chambers, each chamber containing a microscopic grid overlaid with a glass coverslip.
When looking thorough the microscope, you will see that the full grid of the hemocytometer comprises nine squares of 1 mm square each. The central counting area of the hemacytometer has 25 large squares containing 16 smaller squares.
Once you have the central area of the hemocytometer on focus, set the hand-held tally counter to zero and begin counting the sperm heads. To avoid double-counting, count only those on the lines of two sides of the large square.
The volume over the central counting area is known by the geometry of the device, 0.1 mm3 — that is, 0.1 microliter. The average number of sperm over each central counting area multiplied by 10,000 will give you the number of sperm per mL of the diluted sample.
Now you can relax — the math is over. Since you diluted the semen as described in the previous section, no further calculation is needed. The number of sperm that you count in the 25 squares is the total concentration of sperm in millions/mL. For instance, if you count 120 sperm, the total concentration is 120 million/ml.
Note: If you used a different dillution factor than the one we recommended above, multiplying the number of sperm that you count in the 25 squares by 10,000 and by the dilution factor will provide the concentration per milliliter of original sample. Following the previous example, if the dilution factor is 1:20 and you count 120 sperm, the total concentration is 120 x 10,000 x 20 (dilution factor) = 24 million/mL.
Understanding the results
The normal sperm count ranges between 20 million and 250 million sperm per mL of semen. Concentrations of less than 20 million sperm per milliliter of semen are defined as low sperm count according to the World Heath Organization (WHO).
So, what should you do if you get a low sperm count? First, don't panic. Sperm counts tend to fluctuate quite a bit for unclear reasons, so don't jump to quick, tragic conclusions. Simply retest yourself after few weeks and see how you score.
DIY Fertility Tests
A commercial alternative that will rest your eyes and save you from counting hundreds of your little swimmers are male fertility home test kits. They are user-friendly, quite affordable (between 40 and 100 dollars) and quick.
One of the pioneers on the market is FertilMARQ Home Diagnostic Screening Test, which works by staining the cells in the sperm sample to produce a color. The intensity of this color is then compared to a color reference on the FertilMARQ test cassette providing results with an overall accuracy of 78 percent.
Another popular home kit is Spermcheck fertility, known to be 97 percent accurate. This test is based on the detection of SP-10, a protein compound found on the surface of the head of a sperm cell and which concentrations are directly related to the sperm count number.
What is in there?
1 Test device 1 Fertility Reagent Test Solution/ Color Test Solution 1 Package for semen transfer and handling 1 Collection cup 1 Test instructions
First of all, you must carefully read the enclosed instructions. Misreading the directions or personal interpretations can lead to an invalid result, meaning a waste of sperm (and the only test in the package).
Most home tests include a liquefaction cap where the sample must be placed. If using Spermcheck fertility, you should mix the sample with the reagent solution which will release the SP-10 protein from the sperm cells. Once the mixture is ready, you only need to pipette six drops into the sample well of the test cassette.
FertilMARQ test operates slightly different. One drop of liquefied semen must be pipetted directly in the test wells. Then, you should wait some minutes for the drop to soak and then pipette the color solutions in the control and test wells.
Understanding the test results
As with traditional woman pregnancy tests, this male version will give you a positive or negative reading after a few minutes of uncertainty.
A positive test shows the color in the control window as dark as or darker than the reference color, meaning the sperm concentration is equal to or above 20 million/mL. A negative result is obtained when the control window shows a lighter color than the reference one.
Since many additional factors aside from sperm count play a role in male infertility, a positive test is not a guarantee of fertility.
Original article by Pilar Carreras, Wired.com.
This page was last modified 00:41, 8 December 2011 by amyzimmerman. Based on work by howto_admin.