*6.1.1 Evacuated zona pellucida*

Cryopreservation of single sperm cells in empty zona pellucida (ZP) was initially described in 1997 [43]. The biological carrier could be obtained by mouse or hamster pre-fertilization oocytes, human immature oocytes (e.g. germinal vesicle stage) prior fertilization, or embryos with abnormal fertilization and development after ICSI. The cumulus oophorus was removed via hyaluronidase and corona radiate was stripped by micropipettes. The oocyte was fixed with the holding pipette of the ICSI micromanipulator. By applying mechanical breach, chemical reagents (acidified Tyrode's solution) or highly focused laser beam, two small holes in the ZP were perforated. The cytoplasm of the oocyte must be aspirated and fully removed leaving the ZP empty of contents.

Sperm cells were obtained by centrifugation and placed in a droplet of 10% polyvinylpyrrolidone (PVP) solution. Using the ICSI needle each empty zona was injected with one to fifteen sperm cells. Slow freezing protocol and cryoprotective media of 8% glycerol solution in phosphate-buffered saline (PBS) and human serum albumin (3%) were preferred. The empty zonas were transfered in an individual sterile plastic straws of 0.25 ml. For easy location of their position they were situated between two small air bubbles.

Thawing of the biological carrier was implemented through exposure of the plastic straws to 30°C for 30 seconds in water bath. The content between the two air bubbles was extruded into droplets medium.

There are number of studies indicating that empty zona pellucida is an ideal carrier when it comes to freezing extremely small number of spermatozoa [44, 45]. Sperm recovery rate, compared to traditional freezing protocols, was higher, but motility recovery, DNA integrity and fertilization ability of sperm were similar in both methods [46]. From ethical point, it is important also to have an access to donated immature human oocytes and proper informed consent should be obtained (**Figure 2**).

Although this method shows high efficiency it is quite time and cost consuming and requires great precision with micromanipulation techniques and experience of the embryologist.

**Figure 2.** *Spermatozoa freezing in empty zona pellucida.*

#### *6.1.2 Volvox globator algae*

Volvox gobator, a species of green algae of the genus *Volvox***,** was described as a biological carrier for single sperm freezing and storage in 2004 [47]. The technique for loading rare sperm cells into the spherical algae is similar to the one used when freezing in ZP. Spermatozoa were injected in *Volvox globator* algae on a set of petri dish with media microdroplets and ICSI micromanipulator. In the pilot study, each Volvox sphere was loaded with eight male gametes. Cryoprotecting media containing 0.4% human serum albumin was supplemented. Each algae containing sperm was placed in a 0.2 mL plastic straw between two small air bubbles for easier location. Standard slow freezing protocol: 10 minutes at 4°C followed by 10 minutes of LN2 vapor and consequent submerging in LN2, was preferred.

Thawing of the biological carrier was performed through heating the plastic straws in a water bath at 25°C for 20 seconds. The content between the two air bubbles was extruded into droplets of medium. Sperm cells were subtracted through soft suction with the injection pipette at the micromanipulator set. The reported recovery rate (100%) and motility rate (at least 60%) were quite promising, but this method rises certain concerns. According to The United States *Food and Drug Administration (*FDA) and the European Tissue Directive regulations, there is no clear evidence that genetic material from the algae is not transferred and introduced into the oocyte with the injected sperm.

For the time being the use of non-human biological carrier in a clinical setting seems unacceptable. Still, there are countries with strict regulations prohibiting the destructive use of oocytes, and *Volvox globator* algae could be considered as an option for biological carrier when single sperm cryopreservation is nessesary [48].

Recently, non-biological carriers for rare sperm freezing, analogical to ZP and *Volvox globator* algae, were developed. Microcapsules composed of alginic acid, agarose or hollow hyaluronan -phenolic hydroxyl (HA-Ph) were tested with loading, freezing and thawing techniques adopted from the abovementioned biological carriers. First attempts were conducted in 2006, when sperm cells were frozen in polymerized alginic acid drops [49]. Although alginate is a non-toxic polysaccharide, the reported sperm motility after thawing was 20% lower compared to standard protocols. According to the study, decreased motility might be based on the adhesion of alginic acid to sperms surface.

Inactive and biologically sterile empty agarose microspheres of 100-μm in diameter were examined as carrier for the cryopreservation of one to ten sperm cells. The conducted post-thaw results in different studies show high recovery and motility rate (above 90% and above 80% respectively) and preserved membrane integrity of the cells [50, 51]. The hollow-core agarose capsule seems to be a promising substitute to ZP and *Volvox globator* algae.

Enzymatically fabricated hyaluronan (HA) microcapsules with thick membrane (30-μm) and 200 μm in diameter were also tested as carriers for cryopreservation of solitary spermatozoa. No differences according to recovery and motility rates after thawing of spermatozoa loaded into HA-Ph - microcapsules and ZP (95.5 vs. 93.9% and 13.6 vs. 15.1% respectively) were registered [48, 52].

Newly developed spherical analogues for rare sperm freezing would be of great benefit when ethical problems arise, and empty ZP could not be used for clinical or experimental application. No preliminary processing of the carrier is required and the procedure is less time consuming, but still highly qualified and trained embryologist as well as specialized technique is needed.

The inculcation of such promising non-biologically derived carriers in cryobiology needs further investigation and affirmation in regard to their safety and efficiency.

### **6.2 Non- biological carriers for rare sperm cryopreservation**
