Methods of sperm selection and separation
Having explained the importance of sperm collection, selection and separation, it is now time to look at the various techniques employed to identify the quality of sperm and for isolation of the best spermatozoa.
Sperm Wash: This basic method uses a solution containing antibiotics and protein supplements which is added to the ejaculate. After centrifugation, the concentrated sperm cells are separated from the seminal fluid and isolated.
Disadvantage: It is a low yield method, for ejaculates with high sperm quantity and motility. ROS can damage sperm.
Swim-up: is another basic method of sperm selection. Motility is used as an indicator of sperm quality. Hence, the sperm are allowed to swim upwards and those that reach the higher medium (upper fraction) are considered as displaying higher motility, increased velocity, good morphology and with improved fertilization rates.
Disadvantage: Reduced number of recovered sperm, low efficiency and can be used only with samples having high volume and motility.
MACS (Magnetic activity cell sorting): It is a technique to choose a live sperm cell, from an apoptotic one, which has DNA damage or sperm DNA fragmentation. When the sperm cells are collected and before separation, the damaged sperm is labelled with magnetic nanoparticles and they are put through a column where the apoptotic sperm is detained. The live sperm flow through the column and are collected.
Disadvantage: No recognition of the types of motility. Insufficient literature on the percentage of live births, additional cost factor.
Density Gradient Centrifugation: This procedure uses the cells’ density to separate them. Different cells have varying densities. Hence a normal sperm cell (morphologically) will have a different density from an abnormal spermatazoon. At the end of centrifugation, each cell is located at the gradient matching the density. The normal sperm with high motility and viability move quickly to the bottom of the gradient. However, the morphologically abnormal sperm are seen at the gradients towards the top. So it is easy to discard such sperm with the cell debris. Density Gradients can be continuous or discontinuous. With the former, there is a gradual increase in density from the top to the bottom of the gradient. However, with discontinuous gradients, there are clear boundaries among them. During the procedure when the semen ejaculate is placed on top of the density media, those with high motility move toward the sedimentation gradient and penetrate the boundary quicker than the poor motility or immobile cells.
Disadvantage: High ROS production, inefficiency when sperm has high viscosity. Overloading the sample can cause a cluster of sperm cells with other cells.
Microfluidics: This method is chemical-free, using a disposable chip. It has an inlet for the sample and is linked by a microfluidic duct to the outlet opening. The sperm sample is pipet dropped into the inlet, where it moves towards the outlet against the movement of the fluid and the micro-barriers. Healthy motile sperm will be collected from the outlet whereas the low quality sperm will be trapped in the duct.
Disadvantage: Additional cost factor and low yield, doesn’t help when the majority of sperm are immotile.
MTT (Mechanical Touch Technique): This process is used to identify the viability of sperm, by evaluating those cells with an intact plasma membrane. If, after contact with an ICSI injection needle, the tail remains flexible and recovers its original position, it is considered viable. Those that remain rigid and are unable to recover the initial position are considered non-viable.
Disadvantage: The success depends on the expertise of the embryologist conducting the test.
HOS Test (Hypo-osmotic swelling test): When the sperm is in a hypoosmotic (having a lower osmotic pressure than the surrounding fluid) environment, if the tails of spermatozoa swell and bend, they are considered viable. HOS Test is used to measure the functional integrity of the sperm, using seven distinct patterns of swelling.
Disadvantage: It is not efficient for small volumes of semen. The fertilization rate is lower when incubation in hypoosmotic solution is for more than 30 minutes.
PICSI (Preselective Intracytoplasmic Sperm Injection): is a technique to select the highest quality sperm for the process of IVF. The fertilisation of an oocyte (egg cell) in the ovary can take place only by a mature sperm which can bind to hyaluronan (a natural polymeric substance found in the reproductive tract and other parts of the body) on the surface of the oocyte. The PICSI method adds tiny droplets of hyaluronic acid to the hydrogel containing the sperm sample. The selection of the best mature sperm is done by ascertaining how well they bind to the hyaluronan. Sperms selected by this method have lower levels of DNA damage and aneuploidy (missing chromosomes resulting in an unbalanced chromosome complement).
Disadvantages: It involves more control of the individual sperm cells and the culture may damage the performance of some cells. Motile sperm are required, so it cannot be practised on TESA samples (removal of sperm cells and tissue from the testicle through a needle and syringe) where the sperm samples are immotile.
Conclusion: These are just some of the methods used for sperm selection and separation. With modern technology emerging, there are new techniques which give a higher percentage of detail and accuracy in sperm selection for ART procedures.
