Sperm Selection

During natural conception, sperm are deposited in the vaginal cavity along with other components of the seminal fluid and it is during their passage through the female reproductive system, until they reach the fallopian tubes where the oocyte will be found, that sperm selection and capacitation takes place.

The environment in the vaginal cavity is completely hostile to sperm and will destroy around 90% of them. Only a few will be able to continue on their way and reach the cervix.

In the cervix, more specifically in the cervical canal, there are a series of glands that secrete mucus. This mucus is thick and provides a protective barrier against microorganisms, but also against sperm, for practically the entire menstrual cycle. However, in the period leading up to ovulation, the mucus stops being so thick and a mesh is created that allows the sperm to ascend, selecting those with normal mobility and morphology. The sperm that manage to overcome this barrier will undergo changes that we call capacitation. These changes allow the sperm to acquire hyperactive motility and make them capable of carrying out a reaction essential for fertilization to occur – the acrosome reaction. The acrosome is a structure present on the top of the sperm head that contains enzymes essential for the sperm to penetrate the oocyte. During the acrosome reaction, these enzymes are released.

In cases where ART treatments are necessary, sperm selection and capacitation must be carried out in the laboratory. To this end, there are several protocols and procedures for processing an ejaculate sample that attempt to mimic the natural process.

The most common sperm selection techniques are  Swim-up  and  discontinuous density gradient centrifugation. These techniques allow the selection of sperm with normal morphology and motility, while the media used help the sperm to become more capable of fertilizing an oocyte.

These techniques are used in most cases and with good results, but they still have some limitations, for example, in cases of high sperm DNA fragmentation. DNA fragmentation may not manifest itself in terms of abnormal morphology or motility, but it will have negative effects on fertilization and embryonic development. To overcome some of the limitations of the more conventional techniques, other sperm selection methods have been developed. These are the cases of  IMSI  ( Intracytoplasmic morphologically selected sperm injection ) and  PICSI  (Physiological intracytoplasmic sperm injection), which complement the techniques of centrifugation with density gradients and  swim-up , or  MACS  ( Magnetic-activated cell sorting ) and  microfluidic plates  (such as Zymot or FertileChip) which can replace the most common techniques for processing ejaculate samples.

This technique consists of placing the ejaculate sample in a tube and adding a specific culture medium to the top of the sample. The tube is placed in an incubator with a suitable atmosphere and temperature and at a 45 ^° inclination for 60 minutes. During the incubation period, the sperm with good motility will be able to reach the top of the culture medium. This fraction of the medium is separated from the rest and used for ART treatments.

This procedure uses media with different density gradients that are placed in a tube and overlaid with the ejaculate sample. The tube is placed in a centrifuge. During centrifugation, sperm with good motility and morphology are separated from non-viable sperm and other cell types present in the ejaculate.

Finally, a precipitate is obtained at the bottom of the tube, containing the sperm that managed to pass through the gradient selection mesh. This precipitate is subjected to two more centrifugations with washing media and placed in incubation with culture medium that promotes sperm capacitation until it can be used in MAP techniques.

The density gradient and swim-up techniques are often used simultaneously on the same ejaculate sample in order to further refine sperm selection. Thus, after centrifugation with density gradients and washing, the precipitate obtained is further processed with the swim-up technique, allowing the sperm with better motility to rise.

IMSI is a high-definition variant of the Intracytoplasmic Sperm Microinjection (ICSI) technique, in which sperm are observed under greater magnification under a microscope, allowing the detection of otherwise imperceptible morphological changes.

This technique complements the selection methods already described, but does not guarantee that the selected sperm has a normal genetic composition, nor that it will give rise to normal fertilization and embryonic development.

PICSI is another variation of the Intracytoplasmic Sperm Microinjection (ICSI) technique that uses a special plate containing a substance called hyaluronic acid (HA) at specific points on the plate.

In a natural conception or in the In Vitro Fertilization (IVF) technique, hyaluronic acid, which is present in the cells surrounding the oocyte, plays an important role in selecting the sperm that will fertilize it.

This technique is based on the principle that sperm with fertilizing capacity have receptors for HA, that is, they have completed their maturation and are less likely to have changes in their genetic material.

The sperm are then placed on the PICSI plate after processing by density gradients and/or swim-up. The embryologist selects for microinjection the sperm that remain attached to the spots on the plate containing hyaluronic acid and that are morphologically normal.

Studies conducted to date have not demonstrated significant differences in success rates when compared with conventional ICSI. However, PICSI may be a useful tool for sperm selection in cases with a high percentage of sperm DNA fragmentation and sperm with abnormal morphology.

Apoptosis, or programmed cell death, occurs naturally in order to eliminate abnormal sperm. This process involves marking sperm with a substance, phosphatidylserine (PS), which is available on the outer membrane of sperm. This substance allows other cells in the testis and female tract to recognize the apoptotic sperm and eliminate them.

When MAP techniques are used, some of these barriers are overcome and sperm marked for apoptosis are not eliminated. Conventional sperm selection techniques may not be able to eliminate these sperm since their motility and morphology are not necessarily affected.

In this way, the MACS technique was developed, which involves incubating the ejaculate in a column with magnetic particles bound to a molecule, Annexin V. This molecule binds to the PS present in the membrane of apoptotic sperm, retaining them along the column and separating them from the rest.

This sperm selection method has not yet demonstrated consensual results in the scientific community, but it could be an option for cases of high percentage of sperm DNA fragmentation.

These relatively new plates, commercially available as ZyMot or FertileChip, rely on the ability of sperm to actively swim through membranes or channels. The ejaculate sample is placed in an entrance chamber and 30 minutes later the sperm that have managed to reach the exit chamber are recovered.

This method avoids the centrifugation of more conventional sperm selection methods, reducing the negative effects that reactive oxygen species generated during these centrifugations have on the sperm membrane. It also allows the recovery of a sperm sample with a lower percentage of DNA fragmentation.

Being a relatively recent method, there are still no very conclusive studies on its effectiveness.