Preparing the ejaculated sperm for insemination

The sperm preparation method plays an important role in the outcomes of IUI (intrauterine insemination), IVF (in vitro fertilization) and ICSI (intracytoplasmic sperm injection). Studies have shown that a long waiting period in seminal plasma affects the fertilization capacity of spermatozoa. Therefore, preparation techniques need to be fast, efficient and thorough:

• Microorganisms are discarded by washing the semen sample with antibiotic-containing solutions.
• Seminal plasma, debris and contaminants are also removed.
• Sperm with the best morphology and good motility are selected. Sperm are graded and those with abnormal morphology are excluded.
• After this preparation, sperm are ready to fertilize an oocyte (egg).

The andrology laboratory

Sperm preparation techniques

In Medicana International Hospital ART Center Andrology Laboratory, the following techniques are used, alone or combined:

• Sperm washing
• Swim-up
• Density gradient
• Mini-gradient

Sperm washing

Sperm washing is the first technique applied. The principle of this method is to mix the semen with culture media in defined amounts and to centrifuge it. Although large concentrations of sperm are retrieved, debris and leucocytes cannot be removed by this technique alone. We therefore use this procedure in combination with those described below.

Swim-up

Swim-up is applicable when enough forward moving (progressive) sperm cells are present in the sample. Sperm can be prepared this way for insemination, IVF and microinjection. Semen samples with normal viscosity and containing high motility sperm are divided into several centrifuge tubes and washed by two centrifugations. Sperm washing medium is added onto the pellet and the tubes left at a 45° angle, allowing sperm cells to “swim” to the upper phase.

Combining sperm washing and swim-up

Liquefied semen samples are mixed with sperm washing media in defined ratios, divided between 2 or 4 tubes, and centrifuged. The supernatant lying in the upper part of the tubes is discarded and sperm wash medium is added, yielding a ready-to-use sperm sample. Depending on the properties of the semen sample, this step may be repeated many times. After the washing step, sperm wash media is added to the pellet and the tubes are inclined at a 45° angle and incubated at 37°C with 5% CO2 for 45-60 minutes, allowing sperm cells to swim to the upper layer. At the end of this period, the supernatant, containing viable motile sperm, is collected and directly used for IUI, IVF or ICSI.

Density gradient

Density gradient is used to prepare sperm for microinjection. We use solutions that separate sperm from the rest of the semen sample. Semen is dropped on colloidal silica suspension and centrifuged, forcing cells to fall to the bottom of the tube. Sperm with normal morphology and motility move towards the bottom, and are then collected. With this method, Immotile or abnormal sperm cells, leucocytes and cell debris are thoroughly cleared from the final solution, which contains only sperm with normal morphology. This method, by the elimination in particular of dead cells and leucocytes, also leads to a decreased reactive oxygen species (ROS) leak that can cause DNA damage. Furthermore, since the obtained solution is non-toxic, it will not damage the sperm itself, the endometrium or the oocyte (egg). Viral contamination (Hepatitis B and C, HIV) can be reduced and even eliminated by this procedure.

Mini-gradient

The mini-gradient procedure allows the collection of good quality sperm from a semen sample with a low number of sperm and a high rate of abnormality. Mini-gradient yields a sample that can be used for microinjection directly. This method is applied to testicular sperm rather than ejaculated sperm because only a very low amount of sperm is usually obtained through testes surgery.