*6.2.1 Open-pulled straws*

Open pulled straw (OPS) is a specially designed carrier for ultra-rapid vitrification. The tool was introduced in 1998 by Professor Gábor Vajta and it is considered to be highly efficient for single sperm cryopreservation. At considerably low risk of microbial contamination while in LN2, OPS could be incorporated for cryopreservation of very small volumes (1–5 μl), without the use of cryoprotectants. The reproductive cells are loaded in the end of the OPS by spontaneous capillary action when the straw is submerged into microdroplets of sperm suspension. The loaded straws, inserted into 90 mm straws, are hermetically closed and submerged in LN2 [53]. Preselection and loading of the sperm cells into the OPS could be performed via polar body biopsy (PBB) pipette [54]. In order to thaw the sperm cells, the outer straw is cut and the open pulled straw is drawn out. The tip should be immediately plunged into microcentrifuge tube containing media.

The OPS tool is of comparable efficiency to other systems and methods when rare sperm cells are cryopreserved. It is relatively easy to use and allows selection of sperm by its morphokinetic parameters prior cryopreservation.

## *6.2.2 Cryoloops*

Cryoloops have been explored as a rare sperm freezing tool by Schuster et al. In 2002 [55]. Further investigation on nylon cryoloops with aspect to successful loading of preselected spermatozoa and cryopreserving oligozoospermic samples and surgically retrieved epididymal or testicular spermatozoa was conducted [56]. The open cryoloop should be dipped in small droplet of sperm suspension and placed into cryovial. Ultra-rapid freezing by either direct submersion in LN2, or 5 min of exposure to liquid nitrogen vapor prior submersion was performed. Standard slow freezing protocol could also be applied. Vitrification in cryoloops without additional cryoprotectants was also investigated and higher sperm motility compared to control group with cryoprotectants was reported (89.5 ± 7.1% vs. 77.5 ± 8.9%) [36].

Sperm samples were thawed by resuspendation in media immediately after the cryovial was taken out of the liquid nitrogen.

Sperm motility, viability, plasma membrane and acrosome integrity were assessed. Certain concerns according to acrosome damage and sperm cryo-capacitation after thawing have been arisen [57]. Cryo-injury is common consequence to different freezing protocols and cryoprotective media. When such small numbers of spermatozoa are frozen any structural or functional damage to the cell could be crucial to the overall treatment outcome.

This method would be convenient for sporadic sperm freezing due to its simplicity. It also provides a set of samples that could be used in multiple ART cycles for ICSI procedures. Cross-contamination should be considered as the cryoloop system is opened and allows liquid nitrogen flow.

## *6.2.3 Cryopreservation in microdroplets*

Ultra-rapid cryopreservation of rare sperm cells in microdroplets results in high post thaw total motility and progressive motility rates compared to slow freezing technique. Sperm DNA fragmentation index (DFI) was also comparable to stated values in the fresh sample. On contrary, when standard slow freezing was conducted, DFI values increased significantly after cryopreservation in the post-thawed samples [58]. This method was adopted and resembles oocyte and embryo vitrification.

Suspension, obtained by the mixture of proceeded sperm and cryoprotective media (1: 1) added by drops in every 30 seconds, was prepared. Modified French mini straws, cut in half from the center to the end by its length, were loaded with 2 μl droplets of the suspension after it was equilibrated at room temperature for 10 minutes. Each hemi-straw (HS) was loaded by the extents of the open gutter with 10–15 droplets placed at equal distance of 2 mm in-between. The HS was placed in 0,5 ml straw and was secured through the two-stage stoppers which it has. After short exposure to LN2 vapor the straws were plunged into liquid nitrogen. Droplets carrying sperm cells were derived by gentle struck of the HS against the bottom of 1.5 ml Eppendorf tube. The samples were incubated at 37°C for 15 minutes. No additional centrifugation for enrichment of the sample was necessary. Washing the cryoprotective media was performed for the selected spermatozoa in droplets of the ICSI dish before sperm injection. This seems to be quite convenient when poor semen samples are frozen, as reduction of numerous centrifugation and washing steps increases the chance for higher number of sperm cells retrieved after thawing.

#### *6.2.4 Cryotop*

Cryotop, mainly used as embryo freezing and storage commercialized tool, has been adopted as a carrier for sperm vitrification. Different studies have been conducted in order to establish the most efficient protocol when Cryotop as a carrier is used. The most efficient protocol for the vitrification of rare sperm cells was setting 1 μl droplet of sperm suspension and sucrose as cryoprotectants on the strip. The Cryotop was immediately transferred over LN2 vapor (2 min at −120°C) and then plunged into liquid nitrogen. Reported recovery and motility rates after thawing sperm cells retrieved from the testes were 95% and 42.1%, and for single sperm cryopreservation obtained from ejaculates – 90% and 44.4% respectively [59]. Freezing semen samples on Cryotop and without cryoprotectants show higher viability after thawing and lower damage to DNA integrity compared to samples frozen with sucrose as adjuvant [60].

Cryotop is efficient tool for cryopreservation of embryos and rare sperm samples. It rises no ethical issues as it is of non-biological origin. The tool has been implemented in cryobiology and it has been randomly used resulting in successful pregnancies. In recent years, similar devices, based on knowledge cumulated from Cryotop examination, were developed. Moreover, due to its great performance, Cryotop has been used as a measure tool for newly developed sporadic sperm cells freezing devices and their efficiency [61].

#### *6.2.5 Cell sleeper*

Freezing small number of spermatozoa is still a challenge for modern cryobiology. In order to find not only the most reliable and efficient, but a carrier easy to use and relevantly less time consuming, different devices were fabricated, tested and compared.

Cell sleeper is a novel device with comparatively small use in practice. It is constructed of an inner tray as a sperm sample carrier and an outer vial. The system is closed and when the tray is placed inside the vial they are sealed together by a screw cap [62]. The device could be used as a cryopreservation carrier for preselected spermatozoa derived from ejaculates or from testicular tissue. Sperm cells were transferred from the proceeded samples in a microdroplet of freezing media on the tray of the Cell sleeper through ICSI needle. After placing the tray inside the vial and sealing it through the cup, the vial was positioned over LN2 vapor (2.5 min at −120°C) and then sunken into liquid nitrogen.

#### *Rare Sperm Freezing DOI: http://dx.doi.org/10.5772/intechopen.98388*

For thawing of the sample, the vial was warmed at room temperature for 1 minute. Afterwards the tray was drawn out of the vial and transfered in a petri dish then covered with oil. Following incubation period (37°C for 2 minutes) the recovery and motility rates were observed. Sperm recovery and motility rates of 94% and 56% were stated. Pregnancies were reported for both – ejaculated and retrieved from testicular tissue sperm cells [62, 63].

This device needs further investigation, but the comparatively large volume of the drop placed in the tray inevitably leads to time consuming search for the sperm cells. Since time for micromanipulation of the oocytes is important matter the latter could be considered as great disadvantage of the Cell Sleeper device.

#### *6.2.6 Polydimethylsiloxane (PDMS) chip*

Microfluidic devices for gamete handling have been incorporated in ART more than 20 years ago [64]. Since the initial study of the technology, the microfluidic systems developed specifically for sperm investigation, selection and cryopreservation, have been upgraded, improved and widely used in the clinical practice.

Polydimethylsiloxane (PDMS), silicone elastomer, chips for cryopreservation of single spermatozoa without cryoprotectants have been thoroughly investigated. Microfluidic two-layered chip was fabricated. The upper layer has two separate openings – inlet, transfusing into microchannel, and outlet. The smooth bottom layer is connected through plasma to the upper one. Different height of the microchannel were examined. For ultra-rapid freezing of rare sperm cells without cryoprotective media, best results were obtained when 10 μm height microchannel was tested [65].

Before cryopreservation the ejaculate should be processed by sperm gradient technique. The semen sample was loaded in the microchannel of the sterilized chip through the inlet. Approximately 5 × 10−3 μL medium containing 1000 sperm cells can be injected in the 10 μm channel by needle connected to micro-injector. The whole chip is covered in silver paper, so direct contact with liquid nitrogen could be avoided.

The chip containing sperm cells was thawed at 37°C for 10 min. The content was ejected in a culture petri dish through the outlet by applying pressure of a syringe connected with the input. Sperm viability, motility, DNA and acrosome integrity in post-thawed samples were researched. Comparison for PDMS chips with 10 μm height microchannel, where samples were frozen with ultra-rapid cryoprotectantsfree protocol, and samples frozen through conventional slow freezing protocol was conducted. The compared post-thaw parameters for the investigated parameters were of comparable values. The most essential advantage of the on chip ultra-rapid cryoprotectants-free cryopreservation is the lack of cytotoxic cryoprotective media. The thawing of the sample, as no cryoprotectants were added, is simple and time and consumable saving [66].
