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The modern successes of reproductive medicine are based on the achievements in the fields of artificial fertilization and cryobiology over the last 50years. Cryopreservation of oocytes makes it possible to preserve their reproductive potential after surgical interventions, treatment of cancer, for delayed pregnancy and to use cells for donation. Cryopreservation of embryos allows not only to reduce the multiple pregnancies rate and to increase the cumulative pregnancy rate as a result of embryo transfer in the following favorable cycles of the patient, but is also a necessary procedure in case of genetic diagnosis or in the case of contraindications for embryo transfer in the stimulated cycle due to possible complications. However, the viability of cryopreserved oocytes and embryos depends on the degree of their cryo damage during the process of freeze-warming. In this regard, it is very important to develop such freezing protocols that minimize the damages caused by the intra- and extracellular ice crystal formation, toxic effect of high concentrations of cryoprotectants and osmotic stresses. The effectiveness of cryopreservation of gametes and embryos is assessed on the basis of morphological, functional and genetic changes in the cells after warming. Special attention should be paid to the ethical issues of assisted reproductive technology, including cryobiotech technologies, which in many countries remain open and in need of settlement.

We analyzed the influence of initial properties of biopolymer fi lm extracellular matrices made of chitosan on adhesion of swine embryonic kidney epithelium-like cells, their morphology, growth, and proliferation. It was found that adhesion index of cells on the studied matrices was similar, but cell morphology had significant qualitative differences. Round non-flattened cells were described in endothelial cell cultures on chitosan-based matrices. Local variations in charge density on matrices induced by the presence of chitin nanofibers led to the appearance of flattened spindle-shaped and non-flattened sphericalal cells. Long-term culturing on biopolymer extracellular fi lm matrices was associated by the formation of spatial conglomerates of sphericalal cells.

cryopreservation of spermatozoa is widely used in the treatment of infertility by assisted reproductive technologies. however, the cryopreservation causes an oxidative stress which can induce pathological changes in the male gametes. The aim of the research was to evaluate lipid peroxidation (lPO) and DNa fragmentation as well as correlation between these parameters in the fresh and cryopreserved spermatozoa of men with normozoospermia or oligoasthenoteratozoospermia (OaT) and spermatozoa derived from the epididymis of men with azoospermia. The level of malondialdehyde (MDa) in a TBa test, superoxide dismutase (SOD) activity and total antioxidant activity (aOa) were assessed. DNa integrity was estimated by acridine orange staining technique. It was shown that MDA level and SOD activity were significantly higher in the fresh spermatozoa of oligoasthenoteratozoospermic group compared with the fresh spermatozoa of normozoospermic group. after cryopreservation the MDa level increased in all groups and was the highest in OaT group where the greatest aOa decline was detected. DNa fragmentation frequency was 2.6 and 4.1 times higher in the fresh and cryopreserved OaT spermatozoa respectively as compared with the fresh normozoospermic ones (7.2%). DNa fragmentation was found to be the lowest in the fresh (6.2%) and cryopreserved (5.8%) epididymal spermatozoa. after cryopreservation SOD activity in epididymal spermatozoa was lower than in normozoaspermic. In spermatozoa of the studied groups the MDa level positively correlated with DNA fragmentation (0.79 Pearson's correlation coefficient) both before and after cryopreservation. It is suggested that due to the detected low DNa fragmentation and lPO level in epididymal spermatozoa their use could be an alternative approach for infertility treatment by assisted reproductive technologies.

SummaryThe complexity of predicting embryo development potential at the cleavage stages and the emergence of epigenetic risks during prolonged in vitro culture of pre-implantation embryos made it more advantageous to transfer embryos at the morula stage to the uterine cavity. The criteria for estimating embryos at this stage that allow prediction of cryopreservation outcomes have been poorly described. All day 4 embryos (n = 224) were graded 1, 2, 3, 4 or 5 according to blastomere compaction degree (BCD = 100, 75, 50, 25 or 0%, respectively) and the survival and blastocyst formation rate of these morulae were studied after cryopreservation. An inverse dependence was found between survival rate and BCD. Excluded fragments were characterized by low osmotic reaction during exposure to cryoprotective medium and, after freeze-thawing, they were destroyed. As damaged necrotic areas of the embryo can affect their further development rate we proposed blastomeres and biopsy fragments of incomplete compacted morula be removed before embryo cryopreservation. This step led to significant increase in the post-thawing survival rate up to 93.1 ± 4.1%, 75 ± 8.8% and blastocyst formation rate up to 85.2 ± 10.4%, 59.4 ± 5.2% in grade 2 and grade 3 embryos, respectively. There was no significant difference in grade 4 embryos. Therefore the removal of blastomeres and biopsy fragments in incomplete compacted morulae can improve cryopreservation outcomes of grade 2 and grade 3 embryos with BCD.

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Agricultural and Wild Animals Реферат: В представленном обзоре описывается текущее состояние проблемы криоконсервирования сперматозоидов, ооцитов и эмбрионов животных. Проанализированы методические подходы для эффективного низкотемпературного сохранения генетических ресурсов животного мира, описаны этапы криоконсервирования гамет и эмбрионов животных. Показана результативность применения технологии криоконсервирования репродуктивных клеток и эмбрионов животных. Отмечено, что на данный момент успешно криоконсервированы половые клетки и эмбрионы многих видов лабораторных, сельскохозяйственных и диких животных, однако остаются нерешенными вопросы криоконсервирования ооцитов и эмбрионов некоторых видов птиц, рыб и млекопитающих. В работе обсуждаются подходы к разработке методов сохранения генетического материала тех видов, которые на сегодняшний день криоконсервировать не удалось. Ключевые слова: криоконсервирование, сперматозоиды, ооциты, эмбрионы, витрификация. Реферат: У представленому огляді описується поточний стан проблеми кріоконсервування сперматозоїдів, ооцитів і ембріонів тварин. Проаналізовано методичні підходи для ефективного низькотемпературного збереження генетичних ресурсів тваринного світу, описано етапи кріоконсервування гамет і ембріонів тварин. Показана результативність застосування технології кріоконсервування репродуктивних клітин і ембріонів тварин. Відзначено, що на даний момент успішно кріоконсервовані статеві клітини і ембріони багатьох видів лабораторних, сільськогосподарських і диких тварин, проте залишаються невирішеними питання кріоконсервування ооцитів і ембріонів деяких видів птахів, риб і ссавців. У роботі обговорюються підходи до розробки методів збереження генетичного матеріалу тих видів, які на сьогоднішній день кріоконсервувати не вдалося.