биолог извършва специализирани процедури за обработка на сперматозоиди в болница "Надежда"


Specialised procedures

Sometimes, in specific indications and cases of impaired sperm parameters, additional techniques for selecting spermatozoa are employed in the Andrology lab.


Before fertilization can happen, spermatozoa need to undergo a process of biochemical and structural changes known as capacitation. In normal conditions, sperm capacitation occurs in the female reproductive tract, while in assisted reproduction, it is achieved in Andrology and Embryology lab immediately before fertilization.

Typically ejaculate is produced by masturbation, collected in a sterile container and handed over to the biologists in the Andrology lab for processing or analysis.

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.

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.

If you are having your sperm frozen, you will sign a contract for cryopreservation and storage, which specifies all the conditions of freezing and the period of storage of part or all of the viable genetic material, the rights and obligations of the parties, the methods of payment and termination of the contract. After the expiry of the contract, if you so wish, it may be extended by signing an annex. If you require any assistance or further information, please contact tel. 0887 701 927.




In accordance with Regulation No. H-2 of 12 July 2023 on assisted reproduction activities of the Ministry of Health

Art. 71. Good reasons for the withdrawal and destruction of ova, spermatozoa and zygotes are:


  1. the written certified wish of the patients;
  1. the expiry of the maximum storage period requested by the patients when signing the declaration of informed consent prior to cryopreservation;
  1. the risk of cross-contamination or transmission of bacterial or mycotic infection;
  1. the risk of cross-contamination or transmission of a transmissible disease (HIV, hepatitis B and C, syphilis, etc.);
  1. the expiry of more than 6 months after the requested storage period for unclaimed sperm, ova or zygotes;
  1. the death of the spouse/partner, irrespective of the existence of a declared wish during his/her lifetime, in respect of spermatozoa or zygotes, except in the cases referred to in paragraph 5.9 of Section IV of Annex 1 to Art. 1, т. 1;
  1. the death of the spouse/partner, notwithstanding the existence of a declared wish during lifetime, in respect of oocytes or zygotes;
  1. the establishment of a disease in an anonymous donor which can be transmitted to the offspring or the recipient;
  1. the impossibility of securely identifying donors or tracing the path of genetic material due to incorrect or missing labelling or confusion during processing.

See also

Semen analysis
Specialised tests
Fertility preservation
Reproductive surgery for the male partner