- Sperm Preparation
An ideal isolation technique should be rapid , inexpensive & isolate all the sperm without damaging them. IUI offers a simple and cost effective method of isolating the most functional spermatozoa from the ejaculate. It helps obtain an enriched fraction of motile spermatozoa and also removes of seminal plasma and other cellular components from ejaculate. This is important because the cellular debris, prostaglandins and any micro organisms if present must be removed so that the sample can be used for IUI procedure. However, there is no technique, which is superior to all other techniques. Ideally, at the end of a sperm wash, we should aim to be able to attain minimum criteria of sperm quality in order to enhance the chances of pregnancy.
- 1. Simple Washing
Washing is oldest method described. The aim is to remove only the seminal plasma, and not the cellular debris, bacteria or non-motile spermatozoa.
After liquefaction, the semen is mixed with culture medium (1:1) and centrifuged at 300-400 g for 10 minutes. The supernatant is discarded and pellet is suspended in 2 ml of culture medium. This is centrifuged again at 300-400 g for 5-10 minutes and supernatant against discarded. The final pellet is suspended in 0.4-0.5 ml of medium and immediately used for insemination. This is especially useful when sperm density is very low and you are doing an IUI.2. Gradient Separation
The layers are made with 1-2 ml of gradient solution (80% lower and 40% upper layer) 2 ml of semen is then carefully layered on top of 40% and centrifuged at 400-600 g for 15-20 min. cell debris, immobile an abnormal sperm all accumulate at interfaces and a soft pellet is formed at the bottom of the tube. This pellet is aspirated and suspended in 2 ml of culture medium (Depending on sperm density either simple washing or wash with swim-up technique is used). This is centrifuged at 300 g for 5-10 min. the pellet is now suspended in 0.3-0.5 ml of culture medium and used for insemination. The final wash with culture medium to remove the gradient is drawback of this method. Sedimentation Method / Layering under paraffin.3. Swim up
- Swim – up form Pellet
This is the most widely used technique for separation of most sperms from non-motile sperms and cellular debris. It is used with normal semen samples and is based on principle active self-migration.
After liquefaction, the ejaculate is mixed with culture medium and then centrifuged at 300-400 g for 10 minutes. The supernatant is discarded and the sperm pellet is gently over lay with 1 ml of culture medium and stored inside incubator at 37°C for 30-45 minutes. The 0.4-0.5 ml of the supernatant is then gently aspirated and used for insemination.Swim-up from Ejaculate
This is the ideal method because it obviates the needs for centrifugation . There are some workers who still prefer to centrifuge the recovered sperms.