International Conference and Exhibition on Tissue preservation & Bio-banking July 20-22, 2015 Barcelona, Spain

Koichi Kyono received his MD from Fukushima Medical College in 1978 and his PhD in obstetrics and gynecology (reproductive biology) from Tohoku University in 1984. He was a member of the Tohoku University team which achieved the first IVF pregnancy and delivery in Japan in 1983. He is currently the president of Kyono ART Clinic in Sendai and Tokyo. His long-term research interests include studies on cryopreservation (oocytes, ovarian tissue and testicular spermatozoa), in vitro culture, andrology (Micro-TESE, IMSI) and endocrinology (ovarian stimulation).

Overview: Cryopreservation of ovarian tissue (after transport on ice 4°C for 18 hours) performed at reproductive medicine centers is very promising alternative to preserve fertility for women who undergo chemotherapy. Introduction: It’s been reported that measuring the oxygen consumption rate (OCR) using a scanning electrochemical microscope (SECM) may be effective as a non-invasive evaluation of oocyte/early embryo quality of various mammalian species, including human oocyte/early embryo. The purpose of this study was to evaluate the effect of transportation at prolonged low temperatures on the survival of pre-antral follicles. Methods: Ovarian tissue was removed from six women with gender identity disorder. Tissues were stored in an icebox at 4°C for 6 or 18 hours prior to vitrification. After warming, those were cultured for 24 hours and follicle survival was assessed via a viability/cytotoxicity kit. Morphological features and OCR were evaluated by SECM. Results: Survival rate of isolated primordial follicles was 95.7% and 100% and that of primary follicles was 91.7% and 81.8% in the 6 hour and 18 hour groups respectively. There was no difference in morphology between the 6 hour and 18 hour storage groups. After being vitrified and warmed and then cultivated for 24 hours, the OCR in primordial follicles was significantly higher than prior to cultivation in both 6 hour (0.02±0.02 vs. 0.07±0.04, P<0.05) and 18 hour groups (0.02±0.02 vs. 0.11±0.10, P<0.05). Conclusions: This strongly suggests that prolonged transportation of ovarian tissue at low temperatures is useful when there are no available local systems for fertility preservation.

Background
Embryo cryopreservation is considered a vital part of successful assisted reproduction technology (ART) treatment and improves the cumulative pregnancy rate per IVF (In Vitro Fertilization) cycle allowing additional chances of pregnancy without reexposure to exogenous gonadotropins and subsequent oocyte retrieval procedure (1,2,3). Slow freezing was the dominant method of cryopreservation in human assisted reproduction laboratory for many years. Recent studies have reported increasingly successful clinical results with vitrification (4). Vitrification is an ultra-rapid method of cooling cells into a glass-like state. In contrast to slow-freezing techniques, vitrification procedure requires an extremely high cooling rate and much higher concentrations of cryoprotectant. Compared with the traditional slow freezing method, embryo vitrification is relatively simple, inexpensive and potentially faster as the ultrarapid cooling technique requires no expensive programmable controlled-rate freezing equipment and avoiding damage to the cells or tissues(5).

Objective
To compare vitrification versus slow freezing cryopreservation outcome for cleavage stage day 2-3 embryos.

Materials and Methods
A retrospective study of 121 patients under IVF treatment with 323 cryopreserved embryos. Two basic techniques have been employed for the cryopreservation of human day 2-3 embryos: slow-freezing and vitrification. The patients parameters evaluated were age, pregnancy and abortion rate. The embryos parameter evaluated was survival rate according to embrionary quality. In our center we scored embryos quality according to Veeck (1999):
Grade 1 - embryo with blastomeres of equal size, no cytoplasmic fragments;
Grade 2 - embryo with blastomeres of equal size, cytoplasmic fragmentation less than 20%;
Grade 3 - embryo with blastomeres of distinctly unequal size; cytoplasmic fragmentation between 20-50%;
Grade 4 - embryo with blastomeres of equal or unequal size; cytoplasmic fragmentation above 50%.

Results

The patients average age of the slow freeze group was 36.2 years and the vitrification group was 37.1 years. The embryo survival rate was stratified according to the embryo quality. The survival rate of slow freeze group was: Grade 1= 97%, Grade 2= 76%, Grade 3= 70%, Grade 4= 50% and the vitrification group was: Grade 1= 96,1%, Grade 2= 92%, Grade 3= 86%, Grade 4= 100%.

The pregnancy rate in the slow freeze group was 35,1% and the vitrification group was 36,2%. Abortion rate was 11,5% and 11,7% respectively.

Conclusions

The vitrification method was more efficient than slow freezing for cryopreservation of human cleavage stage embryos in terms of post-warming survival rate. No significant difference in the pregnancy rate was observed between the cryopreservation methods. In conclusion, our study provides evidence that vitrification is more efficient than slow freezing cryopreservation in terms of survival rates of human cleavage embryos, specially when we have poor quality embryos. Regardless of cryoprotectants and their concentration, vitrification gives better embryo survival rate compared to slow-freezing. Even if no significant differences in pregnancy and abortation rate were observed between the cryopreservation methods studied, through a higher survival rate and quality of the embryos at warming, vitrification may improve the clinical outcome of IVF by maximizing the cumulative efficiency of the cycle.

High-quality randomized controlled trials should be pursued to find out which vitrification method is the best and when will be the time to completely abandon slow freeze embryo method.

Estefania Paredes holds a degree in Ciencias del Mar (2008) at Universidade de Vigo-Spain with a specialization in Marine environment conservation and marine pollution. She obtained her PhD in 2014 at the Universidade de Vigo with the tesis entitled “Cryopreservation of marine invertebrate early‐life stages: applications in marine water quality assessment and aquaculture” work which was awarded by Universidade de Vigo as outstanding Thesis 2014. Estefania spent some time working in several international research centers during her doctoral studies and currently holds a postdoctoral research associate position in Dr. Peter Mazur’s lab at the University of Tennessee-Knoxville working in Vitrification.

Vitrification is now a main route to the cryopreservation of human and animal oocytes and implantation of embryos. It is strongly believed that for success, the cells must be placed in very high concentrations of permeating cryoprotective solutes and must be cooled extremely rapidly. These facts may limit the applications of vitrification to CPA-sensitive cell types. In our research group we have shown that several of these beliefs are incorrect. Our new research suggest that the key to vitrification survival does not rely only in achieving a fast cooling rate but in achieving a fast warming rate that prevents the recrystallization of the vitrified water inside of the cell. Mouse oocytes and embryos survive being cooled slowly even in diluted solutions provided that they are warmed ultra-rapidly by a laser pulse. Research carried out in Mazur’s lab stated that not only vitrification is possible without high concentration of solutes; it is possible without permeating solutes because the degree of vitrification success seems to be in the prior dehydration of the cell not in the amount of permeating solute equilibrated, provided that the cells are being warmed at an ultra-rapid rate to avoid recrystallization. This new point of view about vitrification will potentially widespread its use to a wide amount of cells, even those which were very sensitive to the high concentration of solutes used in the past.

Haritz Gurruchaga has completed his bachelor degree in Pharmacy in 2013. The past three years, he has being working in the department of Pharmacy and Food Sciences at the University of the Basque Country (UPV/EHU). In the meanwhile, he has completed a M.Sc. degree in Pharmacology and completed a research work entitled “Cryopreservation of microencapsulated murine mesenchymal stem cells genetically engineered to secrete erythropoietin” that has being published recently in the International Journal of Pharmaceutics. Currently, he is enrolled as a PhD student in the University of the Basque Country (EHU/UPV) and he is working in the project entitled “Optimization of cryopreservation of microencapsulated cells by slow cooling and vitrification protocols”. Nowadays, we are testing the optimal combination of CPAs for the vitrification of microcapsules.

Cell microencapsulation permits the immobilization of desired cell and its protection from the immune system. This technology is being studied as drug and cell delivery system to treat different diseases. However, different aspects need to be improved to reach the clinics, as the preservation process, which would facilitate the “on demand” access of patients to the treatment in a clinic far from the manufacturer and reduce the costs of this technology. Several parameters should be considered on the encapsulated cell-cryopreservation such as the time and temperature during the process, or the cryoprotectant solutions (CPAs) used. Two different freezing methods are being used with specific freezing rates and CPAs. On the one hand, the slow freezing protocol uses slow cooling rates and low concentration of CPAs, minimizing the creation of intracellular ice as well as the toxic and osmotic damage of cells when exposed to CPAs. We have compared several penetrating and non-penetrating cryoprotectants at different concentrations to elucidate the optimal cryoprotectant solution with slow cooling protocol, concluding that DMSO 10% is the most suitable cryoprotectant solution, among the assessed ones. On the other hand, in vitrification protocols, the sample is rapidly cooled down with high concentrations of CPAs achieving an ice-free/ vitreous state, not only on cooling but also on thawing. Interestingly, vitrification could be an alternative to slow freezing if it can promote better integrity of the capsules while maintaining a good cell viability, being then, the future perspective of microencapsulated cells preservation.