Cryopreservation of reproductive entities is gaining a constantly growing importance. What used to be a means for storage of semen and embryos now has a much wider application. On top of being part of ongoing subfertility treatment, it now also plays an important role in extension and preservation of fertility.We see an increase in cumulative pregnancy rate, a possibility of single embryo transfer, a less troublesome second treatment, a possibility to deal with legal restrictions (e.g. Italy), and a number of practical issues.We have the possibility of extending and preserving fertility of health reasons, e.g. cancer and premature menopause, and of social and personal reasons varying from country to country.With more or less success, we are now able to preserve all types of reproductive structures – from gonadal tissue and immature oocytes to hatched blastocysts. Still there are only two main methods – slow freezing and VITRIFICATION.This lecture will touch on slow freezing, but mainly look at various aspects of VITRIFICATION: What is VITRIFICATION, which tools are used, how is it done, and how successful is it?

In this research, cryopreservation was performed by VITRIFICATION method with two types of solution protecting PVS2 and PVS3 in 7 time levels (20, 40, 60, 80, 100, 120 and 140 minutes). This factorial experiment was conducted in completely randomized design (CRD) with two factors of protective solution (two level) and time levels (seven level) and three replications. The results of analysis of variance for treatment time and type of solution, in three traits, germination percentage, root and shoot length showed significant differences at the level of 1% (P<0. 01). Interaction effect of time treatment in solution type for the length of root and shoot length were significant at the level of 1% (P<0. 01) and for germination percentage was insignificant at the level of 5% (P<0. 05). Overall, analysis of variance showed that the protective solution type and levels of treatment time in the process of VITRIFICATION and the cryopreservation sesame plant is effective in assessment indicators.

In this research the potential development of vitrified early preantral of neonate mouse follicle was studied. Early Preantral follicles with the diameter of 100-130 mm were isolated mechanically from 14 days old NMRY mice ovaries.Follicles were vitrified using PBI containing 30% WN Ficoll, 0.5 M sucrose, 105 mg BSA, 10.7 WN Acetamide and 40% V/V ethylene Glycol (EGFA40) as a cryopreservation solution and then plunged into liquid nitrogen, Stored for two weeks. Thawing was performed in one step manner using 0.5 M Sucrose in BPI and then washed with PBI, transferred to the drops of DMEM/Hams F12 containing 5% FCS. 253 intact vitrified preantral follicle were cultured in drops in 20 ml of culture medium containing 5 mg/ml insulin, 10 mg/ml transferrin and 100 mlU/ml rFSH. two day after culturing, survive follicles evaluated under invert microscope. preantral follicar development followed up to day 12 and culture media renewed every two days. At day 12, preovolatory follicles were stimulated with 1.5 IU/ml hCG. After 24 hours, mature, ovulated oocytes inseminated with the capacitated epidydimal sperms as the same strain of mice. Germinal vesicle (GV), germinal vesicle breakdown (GVB), extrusion of first polar body, fertilization and development of two cells embryos in vitrified were evaluated by using inverte microscope. results showed that after 48h, 91.2% of vitrified follicles were survived and at the 6th day of culturing, follicular follicles changes to outgrowth follicle and finally 15.9% of AFF follicles were ovulated and 42.1% of oocytes were showen to be 2-cell embryos. There fore it can be conclued that preantral follicle of neonate as a vitrified can develop in DMEM-Hams F12 toward germinal vesicle breakdown successfully but their further progression need to be investigated.

Cryopreservation of reproductive entities is gaining a constantly growing importance. What used to be a means for storage of semen and embryos now has a much wider application. On top of being part of ongoing subfertility treatment, it now also plays an important role in extension and preservation of fertility.We see an increase in cumulative pregnancy rate, a possibility of single embryo transfer, a less troublesome second treatment, a possibility to deal with legal restrictions (e.g. Italy), and a number of practical issues. We have the possibility of extending and preserving fertility of health reasons, e.g. cancer and premature menopause, and of social and personal reasons varying from country to country.With more or less success, the embryologists are now able to preserve all types of reproductive structures – from gonadal tissue and immature oocytes to hatched blastocysts. Still there are only two main methods – slow freezing and VITRIFICATION.Royan VITRIFICATION Workshop was held on August 26th, in cooperation with Medicult Company. This workshop and was aimed to touch on slow freezing, but mainly looked at various aspects of VITRIFICATION: What is VITRIFICATION, which tools are used, how is it done, and how successful is it?

Background: The survival of cells after the difficult process of cryopreservation and warming is possible only by using appropriate methods of cryopreservation and the use of appropriate cryoprotective agents (CPAs). This article is a review of some of the research that has been done on cryoprotectants in ovarian VITRIFICATION. Materials and Methods: To conduct this review study, first Pubmed, Scopus, and Embase databases, were researched, with the keywords of cryoprotectant and VITRIFICATION. In this study, in addition to introducing some common methods of ovarian cryopreservation, the combination of protective factors against intracellular, extracellular, polymeric, and natural cryopreservation was investigated. Results: For VITRIFICATION of ovarian tissue from intracellular types of CPAs such as ethylene glycol (EG), dimethyl sulfoxide (DMSO), glycerol, propylene glycol, acetamide, and formamide and non-cellular CPAs such as serum proteins, sugar, Polymers are used. Each of these materials has a special efficiency and performance and is generally used in frozen solutions to minimize the damaging effects of the formation of ice crystals both at the intracellular and intra-tissue level. Conclusion: It seems that more comprehensive research is needed on the best protective compound against cryopreservation so that in addition to maintaining the morphological parameters of the ovary, a variety of functional parameters of this tissue are also preserved after cryopreservation.

Introduction: Slow-cooling (SC) cryopreservation of supernumerary pronuclear stage oocytes during IVF/ICSI is well established and routinely implemented in the clinical IVF-programme. Recently, worldwide high survival and pregnancy rates with Cryo-Embryotransfer by verification using minimum volume cooling method have been reported. The radical strategy of verification is to result in a total elimination of ice crystal formation, both within the cells being vitrified (intracellular) and the surrounding solution (extracellular). In the present study, we examined the survival rate of vitrified and rewarmed human pronuclear stage oocytes that were cultured for additional 24 h before Cryo-ET as well as to evaluate the pregnancy rate. The results were compared to survival- and pregnancy rate using the slow-cooling cryopreservation method retrospectively.Materials and Methods: Between January 2000 and November 2005 a total of 752 patients had 3616 supernumary zygotes during IVF/ICSI treatment. These zygotes were cryopreserved using the slow-cooling method. A total of 1005 supernumary zygotes from 211 other patients were vitrified between April 2004 and January 2008 using the Cryotop (Kuwayama, RBM-online, 2005, pp 608-615). For VITRIFICATION, zygotes were placed into equilibration solution (7.5% Ethylenglycol; 7, 5% DMSO) and incubated for 8 min. at room temperature (RT). Hereafter zygotes were incubated in VITRIFICATION solution (15% Ethylenglycol; 15% DMSO; 0,5M Saccharose) for 45-60 sec. at RT and placed on the Cryotopstrip and were plunged directly into the liquid nitrogen. After Vitrifiction a hard plastic cover is attached to protect the strip during storage in liquid nitrogen. In total 1438 zygotes were thawed according to the conventional Slow-cooling-protocol. 107 zygotes were rewarmed after being vitrified: the hard plastic cover was removed in liquid nitrogen and the Cryotop was plunged in thawing solution (1M Saccharose) at 37 C for 1 min. Zygotes were placed in diluent solution (0,5M and 0.25M Saccharose) at RT each for 3 min. Washing was done many times before culture. After both procedures, vitality of zygotes was evaluated under dissecting microscope one hour after rewarming. Embryo transfer was done 24 hours after culture in programed cycles. Clinical pregnancies per Cryo-ET were evaluated and compared for both methods. Results: In total 1438 zygotes were thawed after being cryopreserved with the slow-cooling method. 848 zygotes seemed to be vital after thawing with a survival rate of 59%, while 381 zygotes were rewarmed after being vitrified corresponding to a survival rate of 96.3%. 583 patients underwent Cryo-ET after Slow-cooling procedure of zygotes. The clinical pregnacy rate per Cryo- ET was 10.2% (n=111). In contrast 115 patients underwent Cryo-ET after VITRIFICATION of zygotes. Pregnacy rate was 33.3% (n=69). Out of these 39 healthy babies were born.Conclusion: These retrospective comparative results clearly demonestrate, that the Cryotop VITRIFICATION method of supernumary zygotes showed a high postthaw survival and pregnancy rates suggesting that the VITRIFICATION-protocol may be preferable because of its simplicity, cost-effectiveness and time saving in a busy laboratory daily-work.

Background: The cryopreservation of human oocyte would make a significant contribution to infertility treatment, such as using it for oocyte donation and for patients a bout to lose ovarian function due to surgery or chemotherapy. Despite of using standard freezing straws and cryovials or even open pulled straws, only a few successful pregnancies have been arisen from cryopreserved human oocytes. This situation has been primarily attributed to poor survival, fertilization and development of cryopreserved oocytes.Objective: The aim of this study was to evaluate the novel cryoloop VITRIFICATION method for cryopreservation of human oocytes.Materials and Methods: Nine infertile couples participated in this study. In all women proper regulation and desensitization was done using GnRH agonist during luteal phase. Mature oocytes allocated into two groups randomly. In group I, 34 oocytes were vitrified in conventional straws, while in group II, 33 oocytes were vitrified in cryoloop. After a store time of 1-6 months the oocytes were thawed, incubated for 2 hours and subsequently the ICSI was done on survived oocytes. To verify normal fertilization of vitrified oocytes the number of pronuclei in the cytoplasm was counted 16-18 hours after ICSI and good morphological quality embryos were transferred on day 2 or 3 after sperm injection. Pregnancy was identified by the serum ک HCG level, checked 14 days after embryo transfer.Results: The present study shows that the rate of survival of vitrified human oocytes in two groups has no significant difference (52.94% in group I versus 63.63% in group II) but the fertilization rate of vitrified oocytes by cryoloop was greater than vitrified oocytes by conventional straws (73.7% versus 55.55% respectively). One of the embryo transfers achieved clinical pregnancy and resulted in the delivery of healthy baby.Conclusion: VITRIFICATION by using cryoloop can improved the fertilization rate and developmental capacity of vitrified thawed oocyte.