**6. Trends and future perspectives**

Many researchers are studying different methods to improve cryopreservation outcome by modification of essential factors (cryoprotectants, freezing rate, warming). Trends and new perspectivities in cryopreservation in human‐assisted reproduction are an important part of this chapter.

#### **6.1. Optimization of current methods**

#### *6.1.1. Inhibition of ROCK kinase*

Several new steps and procedures for optimization of current methods were developed during last years. It is well known that vitrification procedure often increases apoptosis in embryonic cells, and it results in decrease of developmental competence. Specific postvitrification treatment can suppress this effect in somatic cells or animal oocytes. It was reported that inhibition of Rho‐associated coiled‐coil kinase (ROCK) improves developmental competence of vitrified/thawed bovine oocytes [22]. This treatment was also effective in human embryon‐ ic cells [23], or bovine blastocysts [24]. ROCK kinase is involved in regulation of metabolism, apoptosis, growth, cytoskeletal assembly and cell contraction.

#### *6.1.2. New vitrification devices*

Blastocyst presents special challenge to cryopreservation. Excessive water in the blastocoel may lead to ice formation and subsequent damaging of cellular structures. To minimize this risk, removal of some of the blastocoel fluid has been attempted. Removal of blastocoel fluids can be done by perforating the blastocoel and letting the fluid flow passively out [21]. The process called assisted shrinkage can be performed in a variety of ways, including microneedle puncture, repeated micropipetting of the blastocoel or laser‐pulse opening of zona pellucida

**5. Our experiences with cryopreservation of gametes, embryos and tissues**

We have cryopreserved sperm since 1991, and we introduced embryo freezing in 1995. In the beginning, we performed slow freezing by Planer, but from 2007, we prefer vitrification. Well‐ functioning cryopreservation was an essential prerequisite for a donation of gametes and embryos program. Within the centres of assisted reproduction, we had the first and the largest

We have also built a centre for fertility preservation for both male and female oncologic patients. Methods of preserving the fertility in young women can be divided into three cryopreservative methods: embryo‐, mature oocyte‐ and ovarian tissue‐cryopreservations. We have started as the first with ovarian tissue freezing and sperm freezing before gonadotoxic treatment in the Czech Republic. We cryopreserved ovarian tissue of 23 women before gonadotoxic treatment (from January 2006 to December 2015). During October 1995 to December 2015, we cryopreserved the sperm of 1231 men—oncologic patients (587—testicular cancer diagnosis). The testicular cancer survivors have a good chance of fathering a child by using sperm cryopreserved prior to the oncology treatment, even when it contains only limited number of spermatozoa. There are 41 patients in our centre, who returned for infertility treatment underwent 58 treatment cycles with cryopreserved sperm. Totally, 20 pregnancies were achieved, that is 34.5% pregnancy rate. The implementation of all young oncological

sperm bank in the Czech Republic from 1995 (currently, we have 100 donors).

patient sperm cryopreservation has an important place in our laboratory methods.

Many researchers are studying different methods to improve cryopreservation outcome by modification of essential factors (cryoprotectants, freezing rate, warming). Trends and new perspectivities in cryopreservation in human‐assisted reproduction are an important part of

Several new steps and procedures for optimization of current methods were developed during last years. It is well known that vitrification procedure often increases apoptosis in embryonic

**6. Trends and future perspectives**

**6.1. Optimization of current methods**

*6.1.1. Inhibition of ROCK kinase*

this chapter.

(**Figure 4**).

174 Cryopreservation in Eukaryotes

New way to increase the cooling rate is reducing the use of cryoprotectants consist in the reduction of liquid nitrogen temperature. In order to avoid vaporization of liquid nitrogen, the temperature is reduced until −210°C, applying a negative pressure [25]. In this condition, nitrogen partially solidifies and creating nitrogen slush, which is less likely to evaporate on contact with specimen compared to liquid nitrogen. This method was very effective in human blastocyst [26]. Cells immersed into nitrogen slush cool more rapidly because they come into contact with liquid nitrogen sooner than those immersed in normal liquid nitrogen. It can provide very high cooling rate (up to 135,000°C/min. The cooling rate is enhanced mainly in the first part of cooling (from 20 to −10°C).

#### *6.1.3. Hydrostatic pressure*

Survival of cryopreserved oocytes and embryos is affected by many factors, and their role is still unclear. Recent studies also reported promising results after applying of high hydrostatic pressure during pretreatment of oocytes and embryos. Some studies show that cultivation medium has a dramatic effect on efficiency of cryopreservation methods. However, it was tested that short time exposition of high hydrostatic pressure prior to vitrification (probably thought production of HSP proteins [27]) significantly improved the survival and hatching rate in murine blastocyst [28].

#### *6.1.4. Antioxidative treatment*

Oxidative stress has been implicated in many different types of cell injuries, including membrane peroxidation, oxidation of amino acids or nucleic acids, apoptosis and necrosis, which decrease survival rate after cryopreservation. Experiment realized in model animals indicated positive effect of the presence of antioxidant in cultivation medium after thawing of embryos [29]. Indeed, supplementation of α‐tocopherol in recovery culture medium resulted in a significantly higher blastocyst yield from the postwarm bovine oocytes in comparison with control oocytes [30]. Actual methods are capable of achieving proper vitrification attaining high level of viscosity and dehydration and delivering high freezing and warming rates. Recent studies realized on experimental animals bring new applicable knowledge suitable for optimization of current method. In our opinion, further research in vitrification media and devices is important for next development of these methods.

It is well known that type of culture media (where are embryos after thawing) is very important for successful thawing process. This fact is often ignored and we believe that gentle appropriate treatment after thawing can improve the survivability of oocytes and embryos.

#### **6.2. New trends**

#### *6.2.1. Freeze all*

One of the new strategies is also "freeze all". In "freeze all cycle", all embryos are cryopreserved and later used after thawing in another reproductive cycle. This approach is very often used in cooperation with preimplantation genetic screening (PGS) of embryos before their transfer into the uterus. New trend in this approach is genetic screening of blastomere after biopsy at the fifth day of *in vitro* cultivation or later. There is no other way and all embryos must be cryopreserved and stored in liquid nitrogen. Embryos are thawed after final decision about embryo aneuploidy and their suitability for transfer into the uterus.

It was presented that implantation, clinical and ongoing pregnancy rates of ART cycles may be improved by performing cryoembryotransfer compared with fresh embryo transfer [31]. It can be explained by a better embryo endometrium synchrony achieved with endometrium preparation cycles. In frozen embryo transfers, endometrium priming may be achieved with the use of E2 and P, and the endometrial development can be controlled more precisely than in cycles with gonadotropins [32].

#### *6.2.2. Social freezing*

Frozen oocyte replacement is a technique where oocytes are retrieved, frozen, stored and fertilized only after thawing them for transfer. This technique helps women to preserve the future ability of having genetically related children at later point in life. It was first used for cancer patients before chemotherapy or radiotherapy. However, it can be also used for delaying motherhood for any reason, such as an absence of suitable partner or a work career. Large companies like Facebook or Apple have recently included social freezing for female employees as an employment benefit. Indeed, just as for fresh oocyte, the outcome of IVF with vitrified oocytes is highly dependent on maternal age. The most appropriate age for effective cryopre‐ servation is unknown, but ideally, it would be in the early to mid‐30s, before age at which woman's fertility naturally declines. Younger women have higher chance that they will never require these eggs. Elder women can be under risk of insufficient procedure with few amounts of oocytes, aneuploidy oocytes and very low probability of pregnancy [33].
