reproductive medicine

Specialised procedures for the male partner

Specialised procedures for the male partner are entirely related to sperm processing, selection, and preparation for fertilisation. Some of the procedures are done at our Andrology lab, like the gradient centrifugation and swim-up techniques, MACS, sperm washing for HIV-positive patients, and sperm cryopreservation. Others are done at the Embryology lab, like zona selection and hyaluronic acid selection. In specific indications, PRP could also be offered to enhance sperm production and quality or to improve sexual function in men with erectile dysfunction.


Sperm preparation techniques

Density gradient centrifugation is a method for separating spermatozoa that aims to isolate the sperm population with the highest motility. At the same time, it reduces the numbers of immotile spermatozoa and cellular debris, as well as cells of the spermatogenesis and the immune system (leukocytes, lymphocytes, erythrocytes).

In this technique, sperm are selected on their motility.

After centrifugation, the sample is overlayed with a special medium. The tube is inclined at a specific angle so that the spermatozoa can have a higher surface contacting the medium. The spermatozoa then swim up to the medium surface, with the most motile getting the closest to the surface.

MACS is a technique for magnetic cell separation. Poor-quality spermatozoa (the ones with DNA fragmentations and low fertilization capacity) are separated by a magnetic field from good quality sperm, which are then collected and used for fertilization. The method is indicated in patients with poor semen parameters, high DFI test results or repeated implantation failure.

In serodiscordant couples where the male is HIV-positive, the only way to prevent the woman and the future child from contracting the infection is to do an IVF procedure with a specific semen processing technique for purifying the ejaculate from viral particles.

The method relies on the fact that the HIV virus is present mainly in the liquid fraction of the ejaculate, the seminal plasma, and cases when viral particles could infect the spermatozoa are highly unlikely. That is why the practice is to process spermatozoa by careful washing and decontamination. This procedure takes up to several hours in contrast to conventional sperm processing for IVF, which takes about 20 to 30 minutes. Interestingly, as opposed to sperm, the viral particles of HIV cannot survive inside the eggs. Relying on the fact that the oocyte destroys the viral particles, there is a negligibly low risk of infection.

Up to the present moment, there have been no cases reported of semen processed by this method resulting in the birth of an infected baby.

Not all andrology labs are qualified to do this kind of processing. 


The Andrology lab at Nadezhda hospital is one of the few in Europe and the only one in Bulgaria that offers this type of sperm washing and the control PCR testing to confirm the absence of viral particles in the processed sample.

Zona selection - method for selecting spermatozoa for ICSI

To select the spermatozoa with the highest quality and fertilization capacity, embryologists employ various sperm selection techniques.

The zona selection method is used at Nadezhda hospital. 

Zona selection is a method for selecting spermatozoa for ICSI, based on the sperm’s functional capacity to discover and bind to the egg’s zona pellucida.

The zona pellucida is one of the egg’s protective layers, which can be penetrated by only the few sperm with the best quality and fertilization potential. This characteristic is the basis of the zona selection technique. The purified spermatozoa are applied over a layer of solubilized zona pellucidae, and the embryologist selects for ICSI only the ones that manage to bind successfully.

Various other methods exist for sperm selection based on functional characteristics. For example, some use hyaluronic molecules, which are part of the outermost egg layer outside the zona pellucida. Those selection methods are not frequently used in clinical practice since research has not shown their undisputed efficacy. However, research on the zona selection technique shows that the percentage of fertilized eggs following its application is significantly higher, and the resulting embryos are of better quality.

Zona selection is different from the PICSI DISH technique in that it uses natural molecules from the outer egg layer of the particular woman in the couple. This contributes to a more detailed and natural communication between the sperm and the selective surface. In contrast, the PICSI DISH method uses modified molecules that are not the natural product of gamete communication, and, consequently, its success rate is very low.

Our research shows that sperm selected by the zona selection technique have close to zero DNA fragmentation. DNA fragmentation directly affects successful fertilization and embryo development, as well as early miscarriage risks. That is why the zona selection method is recommended in patients with high sperm DNA fragmentation.

Very often, the factors behind a couple’s infertility remain unknown: semen parameters are normal, but no pregnancy occurs. In such cases, zona selection can be employed to pick only the best sperm, which significantly improves the chances for successful IVF outcomes.

Zona selection allows the embryologists to pick only the highest quality spermatozoa with the best fertilization potential.

Sperm cryopreservation

The modern freezing technique employed at Nadezhda hospital is called vitrification, or ultra-fast freezing. After washing and purification, spermatozoa a put into a special cryoprotectant media.

The cryoprotectant has to slowly enter the cell and bind all free water molecules inside it. In this way, ice crystal formation is prevented, and the cell structure remains intact. The opposite is true for the reverse process of thawing – the cryoprotectant has to be removed from the cell as quickly as possible to protect its viability.

Sperm are frozen and stored in cryovials placed in special liquid nitrogen vessels under ultralow temperatures of –196°C.

Successful vitrification requires specially trained andrologists. For sperm to preserve their viability, they must be stored under constant monitoring of critical environmental conditions. The cryogenic vessels are equipped with a fully automated system for liquid nitrogen supply and a system for temperature monitoring and control, which guarantees maintaining the required storage temperature for indefinitely long periods.

See also

Specialised procedures for the female partner
Specialised procedures for the embryo
Andrology lab
Reproductive surgery for the male partner