**4. Zygote and embryo cryopreservation**

Cryopreservation of human embryos is a safe procedure, which has been carried out for more than last 30 years. In development of *in vitro* techniques and together with single embryo transfer becoming greater demand for an efficient and reliable cryopreservation method for surplus embryos. It is possible to cryopreserve the human zygotes immediately after fertili‐ zation, at the pronuclear stage or embryos during early cleavage stages (2–8 cells) or at the expanded blastocyst stage (after 5–7 days in culture).

Embryos are cryopreserved in any embryonic stages. Still there does not exist a common consensus what is the most optimal developmental stage for embryo cryopreservation.

Since morphology of vitrified and thawed embryos is not enough to assess the viability, the possibility of culturing for a few more days before transfer can ensure that embryo is for transfer. In contrary to oocytes, embryos are after cortical reaction, which gives the ooplasmic membrane more stability to cope with the low temperature and osmotic changes.

#### **4.1. Cryopreservation of zygotes**

*3.2.1. Cryopreservation of ovarian tissue*

172 Cryopreservation in Eukaryotes

successes have recently been achieved.

**4. Zygote and embryo cryopreservation**

expanded blastocyst stage (after 5–7 days in culture).

cryopreserved independently of the menstrual phase.

Fertility preservation has a great importance to many young women with cancer [17]. Cryo‐ preservation of ovarian tissue is a safe, simple and effective option for preserving fertility in young patients facing or undergoing gonadotoxic therapy. Oocytes in primordial follicles are very small and tolerate cryopreservation very well. The removal of ovarian tissue is a simple procedure. Ovarian tissue can be obtained using minimally invasive techniques during laparoscopy, with unilateral ovariectomy or partial ovariectomy. Ovarian tissue can be

**Figure 3.** Primary follicle from ovarian cortex before (a) and after (b) cryopreservation with morphological alterations. The oolemma of oocyte after cryopreservation is more undulated and interrupted (E), and the cytoplasm of follicular

In 2004, first live birth after autotransplantation of human ovarian tissue was reported [18]. To date, 60 live births have been reported worldwide following transplantation of cryopreserved ovarian tissue. However, research on the cryopreservation of ovarian tissue as a method of fertility preservation has now been continuing for more than a decade, and considerable

In centres that offer cryopreservation of ovarian tissue, the procedure can be performed one day after the patient's first visit. After the tissue has been removed, it can be processed immediately or transferred in special transportation containers to a centre specializing in the

Cryopreservation of human embryos is a safe procedure, which has been carried out for more than last 30 years. In development of *in vitro* techniques and together with single embryo transfer becoming greater demand for an efficient and reliable cryopreservation method for surplus embryos. It is possible to cryopreserve the human zygotes immediately after fertili‐ zation, at the pronuclear stage or embryos during early cleavage stages (2–8 cells) or at the

cells (F) is vacuolated. N, nucleus; M, mitochondria; L, lipid droplets. Scale bar represents 5 μm [19].

cryopreservation of ovarian tissue, with an associated cryobank (**Figure 3**).

For cryopreservation of human zygotes, it is suitable to use only vitrification method. Slow freezing method has more than threefold worse results than vitrification [20].

The recent reported data for successful pregnancies suggested that the vitrification of human zygotes and early‐stage embryos is a perfect alternative to slow freezing techniques especially in countries where cryopreservation of later stage human embryos is prohibited either by law or due to religious reasons.

#### **4.2. Cryopreservation of cleaved embryos**

Vitrification of early‐stage human embryo is acceptable and better alternative than slow rate freezing because of the higher survival rate and increased rates of pregnancy. Cryopreservation of cleaved embryos is not so effective as cryopreservation of blastocyst.

#### **4.3. Cryopreservation of blastocyst**

Blastocysts are the top embryos, what have successfully passed the critical step of genomic activation and have a high developmental potential. Their advantage is containing numerous small cells; thus, the loss of some cells during freezing and thawing is probably less harmful for future development of the embryo. Furthermore, during extended cultivation, embryos with worse viability are arrested in development and will not be cryopreserved.

**Figure 4.** Laser blastocoel puncture (assisted shrinkage): human expanded blastocyst before puncture; (a) laser pulse open zona pellucida (red circle) and make a small defect in the trophectoderm (b), which resulted to blastocyst shrink‐ ing (c). Scale bars represent 30 μm.

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 (**Figure 4**).
