Capacitation consists of a series of changes in the sperm plasma membrane. These changes are needed for the sperm to fuse with the ovum. This whole process occurs naturally within the female genitalia, but when it comes to assisted reproduction, capacitation should be performed artificially at the laboratory.
There are mainly two techniques for sperm capacitation: the swim-up and the density gradient centrifugation (a.k.a. “Percoll method”). Both techniques have the same purposes:
- To separate the sperms from the seminal fluid
- To obtain spermatozoa with at least 70% of straight moving motility
The choice between these techniques depends on both the quality and the characteristics of the semen being analysed.
It is the most ancient, widely extended technique. It consists of a centrifugation procedure in which the ejaculated cells are centrifuged in a tube for 10 minutes so that the spermatozoa settle down to the bottom of the tube.
The sample is then decanted, discarding thus the supernatant, and the pellet is resuspended. The pellet is combined with 0.3-0.5 mL of culture medium making sure that no air bubbles are formed and it is incubated for 45-60 minutes at 39°C. Over this time the good quality sperm will swim up into the culture medium and then will be pulled in order to use them later in assisted reproductive techniques.
A high percentage of motile sperm as well as morphologically normal cells are recovered thanks to this technique (more than 90%). In addition, separating the spermatozoa from other cells becomes easier. Its efficiency is based upon both the size of the pellet and the initial sperm motility of the ejaculate. The swim-up method requires a quick, short centrifugation in order to bring together every sperm cell into the bottom of the tube.
The main disadvantage is that the spermatozoa in the pellet come in contact with the rest of the sperm cells, which leads to the creation of a greater number of free radicals, with the resulting risk of sperm DNA fragmentation.
Generally, the swim-up technique is used in cases of sperm considered as normal or presenting some mild pathology, in which there is no evidence of free radicals, infections, or inflammations within the man.
Density gradient centrifugation or Percoll method
This method is based upon the combination of, on the one hand, sperm motility and, on the other hand, their capacity to go through different density media.
This method consists of the centrifugation of the semen sample to later pass it through a solution composed of two different density liquids. This solution has different concentration gradients and enables the cells to be separated by means of density gradient centrifugation. Currently, other solutions like PureSperm or SpermGrad are more commonly used due to the presence of endotoxins.
This technique requires a slow, long centrifugation so that the motile sperm are able to pass through the different density gradients from the ejaculate to the bottom of the tube. All cells fall to the bottom of the tube due to the movement; however, only the good quality sperm is able to pass through all layers faster and easier than the immotile cells or the abnormal spermatozoa.
It should be taken into account that density gradient centrifugation is an expensive technique and, furthermore, it is relatively difficult to perform, since the preparation of the gradients must be accurately carried out without mixing its different phases.
This technique is used in cases of moderate to severe sperm-related pathologies, when there is potential evidence of free radicals or when the ejaculate has been subject to enzyme treatment previously.