*6.2.7 Sperm vitrification device (sperm VD)*

Sperm vitrification device (Sperm VD) is a novel tool specially designed for cryopreservation of small number of spermatozoa in microdroplets [67]. The device could be exclusively helpful when sperm cells are retrieved by means of TESE. Each Sperm VD plate carrier has three wells for placing sperm cells. Droplets of mixture 1:1 of cryoprotectants and washing media are placed individually for each well of the Sperm VD plate. Sperm VD should be transferred in a petri dish covered with oil. Using the injection needle of the micromanipulator, spermatozoa were transferred from neighboring droplets (PVP/washing media) into the droplets

#### *Infertility and Assisted Reproduction*

of the wells of the Sperm VD. As cryoprotective media has negative influence on sperm cells it is crucial to minimize the time of exposure to the media before freezing the sperm. Substantial freezing should be done within 10 minutes past the first sperm cell transfer [68]. After loading each well of the Sperm VD plate it is transferred in pre-cooled cryotube over liquid nitrogen. The tool should be handled with tweezers and the excess oil should be allowed to drain. The cryotube is sealed with cup.

Immediately after unfreezing (5 min in room temperature) the Sperm VD is placed in a pre-heated petri dish with PVP drops and sperm wash media covered with oil.

Sperm VD is quite convenient for rare sperm freezing and it gives the opportunity for direct sperm washing, selection and injection in the petri dish right after thawing.

#### **7. Rare sperm freezing significance**

Since the first attempts for freezing reproductive cells, a completely new horizon for fertility preservation has been discovered. The opportunity to cryopreserve, thaw and use gametes, embryos and reproductive tissue has given the unique chance to men and women to parent their biological child even when their reproductive functions have been lost.

Modern day society has invented and incorporated countless amenities in day to day life. Unfortunately, some of them have direct negative impact on health: polluted air as the result of factory overproduction, car exhaust gasses, overconsumption and large amount of waste contaminating water and agricultural lands, etc. Others tend to create bad habits: sedentary way of life and lack of physical activity, unhealthy and excessive eating, smoking, and drinking. Mental health, self-esteem and tension should also be considered when certain health issues arise. All of the above mentioned factors, including reproduction postpone and advanced paternal age, could disrupt fertility. Thus cryopreservation has great value in ART.

Cancer, the scourge of modern society, could affect reproduction and leave an indelible mark in one's life. Preserving fertility in cancer patients should be a priority as high as the treatment of the cancer itself – a guarantee for good quality of life after healing. Cancer by itself may affects fertility and alterations in spermatogenesis and low sperm quality could be registered even before treatment [69]. Rare sperm freezing of multiple samples for those patients is mandatory. The unique techniques developed for single sperm freezing and cryopreservation represent the chance for those patients to reproduce in future.

Rare sperm cryopreservation is of high value to patients with severe oligoasthenozoospermia, cryptozoospermia, retrograde ejaculation, and PESA or TESE procedures. All these conditions are associated with extremely low sperm count in the ejaculate and still creates difficulties for laboratory handling. Securing frozen samples through single sperm cryopreservation, inspires calm not only to the patient, but to the clinical and laboratory staff handling the gametes.

Rare sperm cryopreservation could be considered as opportunity beneficial to wildlife and endangered species preservation [70–72]. Breeding and assisted reproduction in animals has long-standing traditions and has been explored in husbandry for centuries. In times when hundreds of animal species are near extinction, rare sperm freezing is a key procedure for stock up of larger amount of samples for future breeding. Genetic diversity should be preserved and on time establishment of cryobanks conserving samples of different representatives of the species on Earth is of high importance [73].
