Analysis of oocyte physiology to improve cryopreservation procedures

In contrast to the preimplantation mammalian embryo, it has been notoriously difficult to cryopreserve the metaphase II oocyte. The ability to store oocytes successfully at -196 degrees C has numerous practical and financial advantages, together with ethical considerations, and will positively impact animal breeding programs and assisted conception in the human. Differences in membrane permeability and in physiology are two main reasons why successful oocyte cryopreservation has remained elusive. It is proposed, therefore, that rather than relying on technologies already established for the preimplantation embryo, the development of cryopreservation techniques suitable for the mammalian oocyte needs to take into account the idiosyncratic physiology of this cell. Analysis of intracellular calcium, for example, has revealed that exposure to conventional permeating cryoprotectants, such as propanediol, ethylene glycol and DMSO, all independently result in an increase in calcium, which in turn has the potential to initiate oocyte activation, culminating in zona hardening. Quantification of the metabolome and proteome of the oocyte has revealed that whereas slow freezing has a dramatic effect on cell physiology, vitrification appears to have limited effect. This is plausibly achieved by the limited exposure to cryoprotectants. Analysis of meiotic spindle dynamics and embryo development following IVF, also indicate that vitrification is less traumatic than slow freezing, and therefore has the greatest potential for successful oocyte cryopreservation.     

Effect of vitrification on human oocyte maturation rate during in vitro maturation procedure: A systematic review and meta-analysis

Combination of in vitro maturation (IVM) and cryopreservation offers new opportunities for women with contraindication in ovarian stimulation, and females who desire to postpone the childbearing due to different problems. There are still controversies regarding IVM procedure and its impact on oocytes fertilization capability. This systematic review and meta-analysis were conducted to evaluate the impact of vitrification on human oocyte maturation rate during IVM procedure. In this review, we searched Medline, Embase, Scopus and ISI web of science to identify English-language studies. The last search was implemented on 3 February 2018. The original articles which assessed maturation rate after vitrification of MI or GV oocytes were included. Animal trials and the studies that performed cryopreservation using slow-freeze method were excluded. Bias and quality assessments were performed. 2476 articles were screened primarily. After duplication removing and the application of inclusion and exclusion criteria, 14 studies included for the analysis. All studies compared maturation rate between the oocytes that were vitrified at the GV or MI stage before maturation and oocytes which were matured in vitro without vitrification. Meta-analysis showed that oocyte vitrification at GV stage had a significant negative impact on maturation rate (RR = 0.76, 95% CI: 0.66–0.88); I² = 85.2%; P = 0.000). Finally, based on our results, oocyte vitrification decreases the maturation rate by 24%.     

Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos

Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS) and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo's semithin sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of embryos’ and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at stereomicroscopical scoring after controlled slow freezing (P = 0.02), and a significant higher number of grade 3 blastocysts was found at semithin sectioning after OPS vitrification (P = 0.037). The extension of ultrastructural damage, especially of mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification.     

Detection of DNA damage in bovine metaphase II oocytes resulting from cryopreservation

Developmental competence of mammalian oocytes is compromised by currently available oocyte cryopreservation protocols. Experiments were designed to examine the effect of three cryopreservation protocols on the integrity of bovine oocyte DNA. In vitro matured bovine oocytes were cryopreserved either by slow cooling, vitrification in 0.25 ml straws, or in open pulled straws. After thawing/warming, recovered oocytes were immediately subjected to morphological evaluation. Morphologically intact oocytes underwent comet assay to detect cryoinjury at DNA level. All cryopreservation protocols resulted in significant morphological damage as well as DNA damage compared to unfrozen control. Among the morphologically intact oocytes, there was no difference among protocols in the number of oocytes displaying DNA damage. However, oocytes that had been cryopreserved by slow cooling or by vitrification in open pulled straws exhibited more damage than those vitrified in 0.25 ml straws in the extent of DNA damage. If we combine the number of oocytes with morphological damage and oocytes with DNA damage, oocytes cooled by slow cooling resulted in the most damage. This experiment demonstrated that oocyte DNA is a target of cryoinjury and different protocols result in different degrees of damage.     

Vitrification of mouse oocytes results in aneuploid zygotes and malformed fetuses

Vitrification of mouse oocytes adversely affected the subsequent developmental potential of embryos and fetuses derived from the fertilization of such oocytes after thawing. Only 5% of oocytes vitrified formed viable fetuses on the 15th day of gestation as compared to 47% in the controls. The incidence of chromosomally aneuploid zygotes, derived from cryopreserved oocytes, was approximately threefold higher than the controls irrespective of whether the oocytes were cryopreserved by vitrification or DMSO slow-freezing. Malformed fetuses were obtained from oocytes that had been vitrified as well as those that had been exposed to vitrification solutions only, whereas no malformed fetuses were obtained in oocytes slow-frozen by DMSO or fresh controls--thus demonstrating that the exposure of oocytes to the vitrification chemicals was responsible for the fetal malformations. The data in this study suggest that the vitrification technique should be cautiously applied to human oocyte cryopreservation. Furthermore, the data also demonstrate that the exposure of female gametes to carcinogenic and/or teratogenic chemicals may result in malformed embryos when such oocytes are subsequently fertilized.     

Generation of Live Offspring from Vitrified Mouse Oocytes of C57BL/6J Strain

In mammals, unfertilized oocytes are one of the most available stages for cryopreservation because the cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has generally been reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. C57BL/6J mice, an inbred strain, are used extensively for the production of transgenic and knockout mice. If the oocytes from C57BL/6J mice can be successfully cryopreserved, the cryopreservation protocol used will contribute to the high-speed production of not only gene-modified mice but also hybrid mice. Very recently, we succeeded in the vitrification of mouse oocytes derived from ICR (outbred) mice. However, our protocol can be applied to the vitrification of oocytes from an inbred strain. The aim of the present study was to establish the vitrification of oocytes from C57BL/6J mice. First, the effect of cumulus cells on the ability of C57BL/6J mouse oocytes to fertilize and develop in vitro was examined. The fertility and developmental ability of oocyte-removed cumulus cells (i.e., denuded oocytes, or DOs) after IVF were reduced compared to cumulus oocyte complexes (COCs) in both fresh and cryopreserved groups. Vitrified COCs showed significantly (P<0.05) higher fertility and ability to develop into the 2-cell and blastocyst stages compared to the vitrified DOs with cumulus cells and vitrified DOs alone. The vitrified COCs developed to term at a high success rate, equivalent to the rate obtained with IVF using fresh COCs. Taken together, our results demonstrate that we succeeded for the first time in the vitrification of mouse oocytes from C57BL/6J mice. Our findings will also contribute to the improvement of oocyte vitrification not only in animals but also in clinical applications for human infertility.     

Human oocyte morphometry before and after cryopreservation: A prospective cohort study

The cryopreservation of human oocytes is an important strategy to spare fertility in women submitted to gonadotoxic therapy, ovarian surgery, or even to allow gestation by assisted reproduction technology after natural ovarian senescence. Methods to predict oocyte resistance to cryopreservation are still based on qualitative morphological assessment. In this study we evaluated whether morphometric characteristics of mature oocytes before vitrification and after warming are related to successful fertilization by intracytoplasmic sperm injection (ICSI). This was a prospective cohort study including 28 infertile women and 71 oocytes. Morphometric assessments included oocyte diameter, perivitelline space (PS), zona pellucida (ZP) and first polar body (PB). Out of 49 warmed oocytes, 27 (55%) survived cryopreservation and their pre-vitrification measures were similar to those of the 22 oocytes that perished. However, the oocytes that eventually failed to be fertilized had undergone more enlargement of the total diameter (p = 0.029) and shrinking of the PS (p = 0.033) after cryopreservation, compared to oocytes that were successfully fertilized. These findings suggest that the morphometric characteristics of fresh oocytes do not predict their survival to vitrification, while fertilization failure is associated with oocyte enlargement and PS shrinking after cryopreservation.     

Analysis of the Phospholipid Profile of Metaphase II Mouse Oocytes Undergoing Vitrification

Oocyte freezing confers thermal and chemical stress upon the oolemma and various other intracellular structures due to the formation of ice crystals. The lipid profiles of oocytes and embryos are closely associated with both, the degrees of their membrane fluidity, as well as the degree of chilling and freezing injuries that may occur during cryopreservation. In spite of the importance of lipids in the process of cryopreservation, the phospholipid status in oocytes and embryos before and after freezing has not been investigated. In this study, we employed mass spectrometric analysis to examine if vitrification has an effect on the phospholipid profiles of mouse oocytes. Freshly prepared metaphase II mouse oocytes were vitrified using copper grids and stored in liquid nitrogen for 2 weeks. Fresh and vitrified-warmed oocytes were subjected to phospholipid extraction procedure. Mass spectrometric analyses revealed that multiple species of phospholipids are reduced in vitrified-warmed oocytes. LIFT analyses identified 31 underexpressed and 5 overexpressed phospholipids in vitrified mouse oocytes. The intensities of phosphatidylinositol (PI) {18∶2/16∶0} [M−H]− and phosphatidylglycerol (PG) {14∶0/18∶2} [M−H]− were decreased the most with fold changes of 30.5 and 19.1 in negative ion mode, respectively. Several sphingomyelins (SM) including SM {d38∶3} [M+H]+ and SM {d34∶0} [M+K]+ were decreased significantly in positive ion mode. Overall, the declining trend of multiple phospholipids demonstrates that vitrification has a marked effect on phospholipid profiles of oocytes. These results show that the identified phospholipids can be used as potential biomarkers of oocyte undergoing vitrification and will allow for the development of strategies to preserve phospholipids during oocyte cryopreservation.     

Safety and efficiency of oocyte vitrification

As the oocyte is the starting point for a new life, artificial reproductive technology (ART) techniques should not affect the (ultra) structural and functional integrity, or the developmental competence. Oocyte vitrification -one of the most significant achievements in human ART during the past decade- should therefore be a safe and efficient technique. This review discusses the principles and developments of the existing and future techniques, applications possibilities and safety concerns.The broad range of vitrification media and devices that are currently available, show differences in their effects on the oocyte ultrastructure and preimplantation development. It is not yet fully decided whether this has an influence on the obstetric and neonatal outcome, since only limited information is available with different media and devices. For autologous oocytes, the obstetric and neonatal outcomes appear promising and comparable to pregnancies obtained with fresh oocytes. This however, is not the case for heterologous fresh or vitrified oocytes, where the immunological foreign foetus induces adverse obstetric and neonatal outcomes. Besides the oocyte vitrification process itself, the effect of multiple stimulations (for oocyte banking or for oocyte donors), seems to influence the possibility to develop gynaecological cancers further in life.Automated vitrification/warming should offer a consistent, cross-contamination free process that offers the highest safety level for the users. They should also produce more consistent results in survival, development and clinical pregnancies between different IVF clinics.     

Effects of reproductive aging and postovulatory aging on the maintenance of biological competence after oocyte vitrification: insights from the mouse model

Cryopreservation of female reproductive cells allows preservation of fertility and provides materials for research. Although freezing protocols have been optimized, and there is a high survival rate after thawing, the in vitro fertilization (IVF) pregnancy rate is still lower in cycles with cryopreserved oocytes, thus highlighting the importance of identifying intrinsic limiting factors characterizing the cells at time of freezing. The aim of the present study is to investigate in the mouse model the impact of reproductive aging and postovulatory aging on oocyte biological competence after vitrification. Metaphase II oocytes were vitrified soon after retrieval from young and reproductively old mice. Part of the oocytes from young animals was vitrified after 6 h incubation (in vitro aged oocytes). All classes of oocytes showed similar survival rate after vitrification. Moreover, vitrification did not alter chromosomal organization in young cells, whereas in vitro aged and old oocytes presented an increase of slightly aberrant metaphase configurations. Compared to fresh young oocytes, in vitro aged and old oocytes showed increased ROS levels which remained unchanged after vitrification. By contrast, cryopreservation significantly increased ROS production in young oocytes. Both the aging processes negatively impacted oocyte ability to undergo pronucleus formation and first cleavage after vitrification by stimulating cellular fragmentation. These results could be helpful for establishing the correct time table for cryopreservation in the laboratory routine and improving its application in reproductively old females. Moreover, our observations highlight the importance of oxidative stress protection during vitrification procedures.     

Effect of oocyte vitrification on DNA damage in metaphase II oocytes and the resulting preimplantation embryos

As an assisted reproduction technology, vitrification has been widely used for oocyte and embryo cryopreservation. Many studies have indicated that vitrification affects ultrastructure, gene expression, and epigenetic status. However, it is still controversial whether oocyte vitrification could induce DNA damage in metaphase II (MII) oocytes and the resulting early embryos. This study determined whether mouse oocytes vitrification induce DNA damage in MII oocytes and the resulting preimplantation embryos, and causes for vitrification‐induced DNA damage. The effects of oocyte vitrification on reactive oxygen species (ROS) levels, γ‐H2AX accumulation, apoptosis, early embryonic development, and the expression of DNA damage‐related genes in early embryos derived by in vitro fertilization were examined. The results indicated that vitrification significantly increased the number of γ‐H2AX foci in zygotes and two‐cell embryos. Trp53bp1 was upregulated in zygotes, two‐cell embryos and four‐cell embryos in the vitrified group, and Brca1 was increased in two‐cell embryos after vitrification. Vitrification also increased the ROS levels in MII oocytes, zygotes, and two‐cell embryos and the apoptotic rate in blastocysts. Resveratrol (3,5,4′‐trihydroxystilbene) treatment decreased the ROS levels and the accumulation of γ‐H2AX foci in zygotes and two‐cell embryos and the apoptotic rate in blastocysts after vitrification. Overall, vitrification‐induced abnormal ROS generation, γ‐H2AX accumulation, an increase in the apoptotic rate and the disruption of early embryonic development. Resveratrol treatment could decrease ROS levels, γ‐H2AX accumulation, and the apoptotic rate and improve early embryonic development. Vitrification‐associated γ‐H2AX accumulation is at least partially due to abnormal ROS generation.     

Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes

Considerable attention has been focused on the cryopreservation of mammalian oocytes, as a consequence of poor development of cryopreserved bovine oocytes in vitro, in order to enhance the application of genetic engineering. Experiments were carried out to evaluate the viability and ultra-structural changes of bovine oocytes cryopreserved by ultra rapid cooling methods. Oocytes that had been allowed to mature for 22 hr were exposed to a mixture of cryoprotectants (3.2 M ethylene glycol, 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose), and were cryopreserved by very rapid cooling either within glass capillaries or as droplets on copper electron microscope grids. After being warmed, the oocytes were cultured in in vitro maturation (IVM) medium for an additional 2 hr. Viability was assessed by determining the development rate after fertilization with frozen-thawed semen from which motile sperm had been recovered using a Percoll density gradient, and by immunochemical evaluation of microtubule and mitochondrial morphology. Cleavage and development rates were significantly (P < 0.05) lower in oocytes cryopreserved by vitrification than in in vitro fertilization (IVF) control group, but did not differ in the open-pulled glass (OPG) or copper grid (CG) groups. In most oocytes cryopreserved by vitrification, the microtubules were partially or completely broken. Similarly mitochondria appeared to be abnormal compared to that of unfrozen oocytes. Oocytes cultured in IVM medium supplemented with both cytochalasin B (a protein synthesis inhibitor) and 2-mercaptoethanol (an antioxidant) showed less damage to microtubules, but not to mitochondria after cryopreservation. In conclusion, this study showed that bovine oocytes can be cryopreserved by vitrification within small droplets using CGs. While damage to microtubules and mitochondria may be involved in reduced viability, supplementation of IVM medium with cytochalasin B appears to enhance stabilization of microtubules during oocyte cryopreservation.     

Effect of different cryopreservation protocols on cytoskeleton and gap junction mediated communication integrity in feline germinal vesicle stage oocytes

Oocyte cryopreservation in carnivores can significantly improve assisted reproductive technologies in animal breeding and preservation programs for endangered species. However, the cooling process severely affects the integrity and the survival of the oocyte after thawing and may irreversibly compromise its subsequent developmental capability.In the present study, two different methods of oocyte cryopreservation, slow freezing and vitrification, were evaluated in order to assess which of them proved more suitable for preserving the functional coupling with cumulus cells as well as nuclear and cytoplasmic competence after warming of immature feline oocytes.From a total of 422 cumulus enclosed oocytes (COCs) obtained from queens after ovariectomy, 137 were stored by vitrification in open pulled straws, 147 by slow freezing and 138 untreated oocytes were used as controls. Immediately after collection and then after warming, functional coupling was assessed by lucifer yellow injection and groups of fresh and cryopreserved oocytes were fixed to analyze tubulin and actin distribution, and chromatin organization. Finally, COCs cryopreserved with both treatments were matured in vitro after warming. In most cases, oocytes cryopreserved by slow freezing showed a cytoskeletal distribution similar to control oocytes, while the process of vitrification induced a loss of organization of cytoskeletal elements. The slow freezing protocol ensured a significantly higher percentage of COCs with functionally open and partially open communications (37.2 vs. 19.0) and higher maturational capability (32.5 vs. 14.1) compared to vitrified oocytes. We conclude that although both protocols impaired intercellular junctions, slow freezing represents a suitable method of GV stage cat oocytes banking since it more efficiently preserves the functional coupling with cumulus cells after thawing as well as nuclear and cytoplasmic competence. Further studies are needed to technically overcome the damage induced by the cryopreservation procedures on immature mammalian oocytes.     

Developmental Competence of Vitrified-Warmed Bovine Oocytes at the Germinal-Vesicle Stage is Improved by Cyclic Adenosine Monophosphate Modulators during In Vitro Maturation

Cryopreservation of mature oocytes and embryos has provided numerous benefits in reproductive medicine. Although successful cryopreservation of germinal-vesicle stage (GV) oocytes holds promise for further advances in reproductive biology and clinical embryology fields, reports regarding cryopreservation of immature oocytes are limited. Oocyte survival and maturation rates have improved since vitrification is being performed at the GV stage, but the subsequent developmental competence of GV oocytes is still low. The purpose of this study was to evaluate the effects of supplementation of the maturation medium with cyclic adenosine monophosphate (cAMP) modulators on the developmental competence of vitrified-warmed GV bovine oocytes. GV oocytes were vitrified-warmed and cultured to allow for oocyte maturation, and then parthenogenetically activated or fertilized in vitro. Our results indicate that addition of a cAMP modulator forskolin (FSK) or 3-isobutyl-1-methylxanthine (IBMX) to the maturation medium significantly improved the developmental competence of vitrified-warmed GV oocytes. We also demonstrated that vitrification of GV oocytes led to a decline in cAMP levels and maturation-promoting factor (MPF) activity in the oocytes during the initial and final phases of maturation, respectively. Nevertheless, the addition of FSK or IBMX to the maturation medium significantly elevated cAMP levels and MPF activity during IVM. Taken together, our results suggest that the cryopreservation-associated meiotic and developmental abnormalities observed in GV oocytes may be ameliorated by an artificial increase in cAMP levels during maturation culture after warming.     

Changes to the meiotic spindle and zona pellucida of mature mouse oocytes following different cryopreservation methods

This study is to investigate the change of morphology of the meiotic spindle and the extent of zona hardening relating to the morphological survival and developmental competence of thawed oocytes. Four- to 8-week-old female mice (C57BL/6) primed with an intraperitoneal injection of pregnant mare's serum gonadotropin and human chorionic gonadotropin. Cryopreserved oocytes using two protocols: vitrificaton using ethylene glycol (EG) and slow freezing using propanediol (PROH). The freezing oocytes were thawed and were fertilized and subsequently cultured in vitro. Spindle/chromosome imagery, dissolution of zona pellucida, and post-thawing survival and development were comparable between two groups. The vitrification cryopreservation method proved to be better than the slow-freezing protocol when comparing the frequency of normal-shaped spindle development post-thawing. The difference in the time required for the dissolution of the zona pellucida under treatment of pronase that was determined to exist between the two cryopreservation methods was statistically significant (P<0.005). The survival rate of post-thawed mature oocytes was significantly greater for the vitrification group than it was for the slow-freezing cryopreservation group (P=0.005). The vitrification cryopreservation of mature murine oocytes would appear to be more satisfactory than the slow controlled-rate freezing method as regards the post-thawing oocyte survival and also the incidence of the normal spindle apparatus in the ooplasm.     

Bovine oocyte vitrification: effect of ethylene glycol concentrations and meiotic stages

Success in oocyte cryopreservation is limited and several factors as cryoprotectant type or concentration and stage of oocyte meiotic maturation are involved. The aim of the present study was to evaluate the effect of maturation stage and ethylene glycol (EG) concentration on survival of bovine oocytes after vitrification. In experiment 1, kinetics of oocyte in vitro maturation (IVM) was evaluated. Germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII) oocytes were found predominantly at 0, 0–10, 10–14, and 18–24 h of IVM, respectively. In experiment 2, in vitro embryo development after in vitro fertilization (IVF) of oocytes exposed to equilibrium (ES) and vitrification solution VS-1 (EG 30%), or VS-2 (EG 40%) at 0, 12 or 18 h of IVM was evaluated. Only blastocyst rate from oocytes vitrified in SV-2 after 18 h of IVM was different from control oocytes. Hatched blastocyst rates from oocytes vitrified in VS-1 after 12 and 18 h, and SV-2 after 18 h of IVM were different from unvitrified oocytes. In experiment 3, embryo development was examined after IVF of oocytes vitrified using VS-1 or VS-2 at 0, 12 or 18 h of IVM. Rates of blastocyst development after vitrification of oocytes in VS-1 at each time interval were similar. However, after vitrification in VS-2, blastocyst rates were less at 18 h than 0 h. Both cleavage rates and blastocyst rates were significantly less in all vitrification groups when compared to control group and only control oocytes hatched. In conclusion, both EG concentration and stage of meiotic maturation affect the developmental potential of oocytes after vitrification.     

A chronologic review of mature oocyte vitrification research in cattle, pigs, and sheep

Vitrification as a means of cryopreservation has become a standard approach for oocytes from livestock. This paradigm shift occurred primarily as a result of the demonstration in 1996 that bovine oocytes are extremely susceptible to chilling injury. Since that early work, numerous devices have been used as supports for oocytes during so-called “ultra-rapid cooling”, and occasionally, trials involving the deposition of small volumes of media containing oocytes directly into liquid nitrogen to facilitate cooling have been reported. Results reporting blastocyst development exceeding 10% are common, but variability remains high, and a standard method for bovine oocytes remains to be established. Oocytes from pigs are particularly difficult to cryopreserve, even with the use of ultrarapid cooling approaches. Few reports have demonstrated blastocyst development exceeding 5%. The application of hydrostatic pressure before vitrification appears to impart stress tolerance to porcine oocytes, as the results of some treatments have shown development to blastocysts at proportions >10%. Work on sheep oocyte vitrification is relatively new, and a few articles have reported blastocyst development at 10% or more. Messenger RNA levels are reportedly altered in sheep oocytes as a result of vitrification, and damage to the cytoskeleton is common across species.     

Cryopreservation of oocyte and ovarian tissue

Cryopreservation of reproductive cells (i.e., oocytes, spermatozoa) and tissues (i.e., ovarian and testicular tissue) is a developing technology that has tremendous implications for rapid advancement of biomedical research in general. Since the early 1980s, advances have been made in establishing optimal conditions for in vitro oocyte maturation, fertilization, and culture of resulting embryos. These in vitro systems have contributed significantly to the utilization of these cells and tissues after thawing and have made it possible to evaluate protocols designed to cryopreserve such biomaterials more effectively. Although cryopreservation of preimplantation embryos from various species including mouse, human, and farm animals has been successful, cryopreservation of oocytes from most mammalian species has been more challenging due to their extreme sensitivity to suboptimal conditions during the cryopreservation process. Cryopreservation on mouse oocytes have been well documented and have resulted in greater success than studies with other mammalian species. Ovarian tissue cryopreservation and transplantation techniques have recently received much scientific and public attention due to their great potential use in human infertility treatment, in safeguarding the reproductive potential of the endangered species, and in genome banking of genetically important lab animal strains. A review of past and current research in the field of oocyte and ovarian tissue cryopreservation and transplantation and discussion of possible strategies for oocyte and ovarian tissue banking are provided.     

Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method

Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this paper is to clarify terms and conditions, to summarize arguments supporting or disapproving the use of vitrification, and to outline its role among assisted reproductive technologies. To provide evidence for the potential significance of vitrification, achievements with the Cryotop technology, an advanced version of the "minimal volume approaches" is analyzed. This technology alone has resulted in more healthy babies after cryopreservation of blastocysts than any other vitrification technique, and more successful human oocyte vitrification resulting in normal births than any other cryopreservation method. The value of this method is also demonstrated by achievements in the field of domestic animal embryology. A modification of the technique using a hermetically sealed container for storage may help to eliminate potential dangers of disease transmission and open the way for widespread application for cryopreservation at all phases of oocyte and preimplantation embryo development in mammals.