The effects of vitrification on oocyte quality.

Vitrification, is an ultra-rapid, manual cooling process that produces glass-like (ice crystal-free) solidification. Water is prevented from forming intercellular and intracellular ice crystals during cooling as a result of oocyte dehydration and the use of highly concentrated cryoprotectant. Though oocytes can be cryopreserved without ice crystal formation through vitrification, it is still not clear whether the process of vitrification causes any negative impact (temperature change/chilling effect, osmotic stress, cryoprotectant toxicity, and/or phase transitions) on oocyte quality, which translates to diminished embryo developmental potential or subsequent clinical outcomes. In this review, we attempt to assess the technique's potential effects and the consequence of these effects on outcomes.

Mangosteen Concentrate Drink Supplementation Promotes Antioxidant Status and Lactate Clearance in Rats after Exercise.

High-strength or long-duration exercise can lead to significant fatigue, oxidative stress, and muscle damage. The purpose of this study was to examine the effect of mangosteen concentrate drink (MCD) supplementation on antioxidant capacity and lactate clearance in rats after running exercise. Forty rats were divided into five groups: N, non-treatment; C, control; or supplemented with MCD, including M1, M5, and M10 (0.9, 4.5, and 9 mL/day) for 6 weeks. The rats were subjected to 30 min running and exhaustive-running tests using a treadmill. The blood lactate; triglyceride; cholesterol and glucose levels; hepatic and muscular malonaldehyde (MDA) levels; and antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT), were analyzed. The results of this study demonstrated that MCD supplementation can increase GPx and CAT activities, alleviate oxidative stress in muscle, and increase lactate clearance, and is thereby beneficial to reduced muscle fatigue after exercise.

Vitrification of the human embryo: a more efficient and safer in vitro fertilization treatment.

Cryopreservation has become a central pillar in assisted reproduction, reflected in the exponential increase of "freeze all" cycles in the past few years. Vitrification makes it possible to cool and warm human eggs and embryos with far less cryo-damage than 'slow-freeze' and allows nearly intact survival of embryos with very high survival rates for eggs as well. This has resulted in a complete transformation how we manage treatment for in vitro fertilization patients. Fresh transfers can be avoided without compromising outcomes, and in fact, cumulative pregnancy/delivery rates may be improved by performing sequential elective "frozen" single embryo transfers. Some recent evidence suggests that previously vitrified embryos give better perinatal outcomes than fresh embryo transfers. Frozen embryo transfer, especially when coupled with preimplantation genetic testing allows for highly efficient single embryo transfers that translate to more singleton and therefore safer pregnancies, as well as healthier babies. Additionally, vitrification has also opened new options for patients, most notably fertility preservation (through oocyte cryopreservation), and donor egg banking.

Production of Live Offspring from Vitrified-Warmed Oocytes Collected at Metaphase I Stage.

Vitrification of matured oocytes is widely adopted in human clinics and animal research laboratories. Cryopreservation of immature oocytes, particularly those at metaphase I (MI), remains a challenge. In the present work, mouse MI oocytes denuded of cumulus cells were vitrified and warmed (V/W) either prior to (V/W-BEFORE-IVM, n = 562) or after (V/W-AFTER-IVM, n = 664) in vitro maturation (IVM). Derivative metaphase II (MII) oocytes were then used for intracytoplasmic sperm injection (ICSI). In the control groups, in vivo matured MII oocytes were used freshly (FRESH-MII, n = 517) or after V/W (MII-V/W, n = 617). In vitro and in vivo developmental competencies were compared among groups. Satisfactory blastocyst rates were achieved in V/W-BEFORE-IVM (27.5%) and V/W-AFTER-IVM (32.4%) groups, albeit as expected still lower than those from fresh-MII (56.1%) or MII-V/W (45.6%) oocytes. Similarly, the term development rates from V/W-BEFORE-IVM and V/W-AFTER-IVM were 12.4% and 16.7% respectively, acceptable but lower than those of the fresh-MII (41.2%) and MII-V/W (23.3%) groups. These data demonstrate that oocytes collected at MI stage are amenable to V/W, which can be performed before or after IVM with acceptable development rates including production of healthy pups. These findings provide useful knowledge to researchers and clinical practitioners for preservation and use of the otherwise discarded MI oocytes.

Cryopreservation of eggs.

Oocyte cryopreservation is playing an increasingly important role in the field of human infertility treatment. The ability to store viable oocytes for later use has given many women the option to delay childbearing in order to pursue other ventures in life, without the concern of losing the opportunity to have a family. Furthermore, oocyte cryopreservation is very valuable for diseased patients who have to undergo treatments that may compromise fertility. Also, infertility patients who produce large numbers of oocytes during a retrieval cycle now have the option of storing some eggs prior to fertilization, thereby reducing the number of embryos that have to be managed. Lastly, oocyte cryopreservation enables egg donation programs that are independent of fresh donations, which makes it possible for numerous recipients to benefit from a single donor.Traditionally, slow freezing was the only method available for oocyte cryopreservation. However, recent years have shown that ultrarapid cooling of oocytes results in higher survival and developmental rates. Thus, vitrification is today's preferred method of oocyte cryopreservation and therefore the only technique described.In this chapter, we present two reliable methods of oocyte vitrification that have been in use for several years and that have been experimentally validated. Since no single vitrification method is clearly superior to the rest, other systems are also briefly described to give the reader options when deciding which methods to utilize in their practice.

Prospective controlled study to evaluate laboratory and clinical outcomes of oocyte vitrification obtained in in vitro fertilization patients aged 30 to 39 years.

OBJECTIVE: To determine whether the process of oocyte vitrification affects oocyte viability in in vitro fertilization (IVF) patients between 30 and 39 years of age. DESIGN: Prospective controlled study. SETTING: Private IVF practice. PATIENT(S): A total of 30 women assigned and 22 qualified. INTERVENTION(S): Denudation of oocytes, cryopreservation of oocytes using vitrification method in a medium with 15% ethylene glycol (EG), 15% dimethylsulfoxide (DMSO), and 0.5 M sucrose. MAIN OUTCOME MEASURE(S): Oocyte survival, fertilization, day-3 embryo quality, blastocyst formation, clinical pregnancy, implantation, and live-birth rates. RESULT(S): After denudation of oocytes, mature sibling oocytes were randomly allocated to the fresh and vitrified groups. The survival rate was 79.6% after vitrification/warming. Overall, no statistically significant differences were found in fertilization, day-3 embryo quality, or blastocyst formation rates between the fresh and vitrified groups. The positive ß-human chorionic gonadotropin, clinical pregnancy rate, and implantation rate were 13 (59.0%) of 22, 10 (45.4%) of 22, and 16 (30.1%) of 53 for the vitrified group. The overall efficiency in achieving a live birth was 11 (5.9%) of 186 per vitrified oocyte. CONCLUSION(S): The impact of vitrification can be reduced to a minimal level, making it possible to achieve high pregnancy and implantation rates in this age group of IVF patients.

Oocyte cryopreservation for donor egg banking.

Oocyte donation is an efficient alternative to using own oocytes in IVF treatment for different indications. Unfortunately, 'traditional' (fresh) egg donations are challenged with inefficiency, difficulties of synchronization, very long waiting periods and lack of quarantine measures. Given the recent improvements in the efficiency of oocyte cryopreservation, it is reasonable to examine if egg donation through oocyte cryopreservation has merits. The objective of the current manuscript is to review existing literature on this topic and to report on the most recent outcomes from two established donor cryobank centres. Reports on egg donation using slow freezing are scarce and though results are encouraging, outcomes are not yet comparable to a fresh egg donation treatment. Vitrification on the other hand appears to provide high survival rates (90%) of donor oocytes and comparable fertilization, embryo development, implantation and pregnancy rates to traditional (fresh) egg donation. Besides the excellent outcomes, the ease of use for both donors and recipients, higher efficiency, lower cost and avoiding the problem of synchronization are all features associated with the benefit of a donor egg cryobank and makes it likely that this approach becomes the future standard of care. Oocyte donation is one of the last resorts in IVF treatment for couples challenged with infertility problems. However, traditional (fresh) egg donation, as it is performed today, is not very efficient, as typically all eggs from one donor are given to only one recipient, it is arduous as it requires an excellent synchronization between the donor and recipient and there are months or years of waiting time. Because of the development of an efficient oocyte cryopreservation technique, it is now possible to cryo-store donor (as well as non-donor) eggs, maintaining their viability and allowing their use whenever there is demand. Therefore, creating a donor oocyte cryobank would carry many advantages. In the present manuscript, the current experience with oocyte donation using cryopreservation technology is reviewed. The outcomes of two recently established donor egg cryobanks at Instituto Valenciano de Infertilidad in Spain and Reproductive Biology Associates in the USA (involving a large number of cases) demonstrate that egg cryo-survival is high and that fertilization, embryo development, implantation and pregnancy rates are similar to those reported after fresh egg donation. It also provides additional advantages of being more efficient, more economical, easier for both donors and recipients and potentially also safer, because eggs can now be quarantined for 6 months (or longer) to retest for infectious diseases in the donors. It is the opinion of the authors, based on several advantages associated with the use of donor egg cryobanking, that in the future there will be fewer traditional egg donations and increasingly more cryo-egg donations.

Cryopreservation of oocytes in experimental models.

Until recently, success in oocyte cryopreservation has been very limited mainly due to poor understanding of the complex physiological processes that lead to cell damage during cryopreservation. In the past three decades, however, a wealth of information has been collected using various different animal models, which has led to development of new technologies and optimization of existing ones. The use of these models has provided the opportunity for research that may not have been possible with human material. Today, results of these studies still continue to form the basis of oocyte cryobiology. This review discusses these studies, especially the physiological impacts of cryopreservation on oocyte biology. It will also focus on the role that animal models have played in improvement strategies, validation before translating new techniques into the human model and the advances made in the human in IVF because of these animal models. Finally, existing investigations and their potential impact in other areas of research will be discussed. Until recently, success in oocyte cryopreservation has been very limited mainly due to poor understanding of the complex physiological processes that lead to cell damage during cryopreservation. In the past three decades, however, a wealth of information has been collected using various different animal models, which has led to development of new technologies and optimization of existing ones. The use of these models provided the opportunity for research that may not have been possible with human material. Today, animal models still continuously provide imperative data that facilitate further advancements in oocyte cryobiology. This review will focus on the physiological impacts, current improvement strategies and future applications of oocyte cryopreservation using animal models as they benefit not only human oocyte cryopreservation procedures, but also the human species through their usefulness in agriculture, medicine and conservation.

Impact of phase transition on the mouse oocyte spindle during vitrification.

During vitrification, the glass-like solidification is the phase-transition process from liquid to solid. Phase transition is one of the major factors suspected to affect the physiology of the oocyte, such as the structure of the meiotic spindle. Therefore, it is very important to investigate the systematic and morphological alterations of the metaphase-II spindle and chromosome arrangement during complete course of a vitrification and warming process. B6D2F1 (C57BL/6 X DBA/2) mouse oocytes were cryopreserved by minimum volume cooling (MVC) method of vitrification in a solution with 15% ethylene glycol, 15% dimethylsulphoxide and 0.5 mol/l sucrose. To examine the spindle, oocytes were fixed before, during and after vitrification and were analysed by immunocytochemistry and confocal microscopy. It was shown that spindles in all oocytes could be maintained through the vitrification and warming process, even though they were exposed to extreme temperature and two rounds of phase transition. According to the sequential observations, chromosome alignment was maintained throughout the complete course of vitrification, warming and post-warming stage. The impact of phase transition was barely detectable when the oocyte was exposed to the vitrification and warming process. The oocyte spindle was able to recover immediately after warming.

Efficient derivation of embryonic stem cells from nuclear transfer and parthenogenetic embryos derived from cryopreserved oocytes.

Deriving histocompatible embryonic stem (ES) cells by somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA) requires fresh oocytes, which prevents their applications in humans. Here, we evaluated the efficiency of deriving ES cells from mature metaphase II (MII) and immature metaphase I (MI) vitrified oocytes, by PA or SCNT, in a mouse model. We successfully generated ES cell lines from PA (MII and MI) and SCNT (MII and MI) blastocysts. These cell lines expressed genes and antigens characteristic of pluripotent ES cells and produced full-term pups upon tetraploid embryo complementation. This study established an animal model for efficient generation of patient-specific ES cell lines using cryopreserved oocytes. This is a major step forward in the application of therapeutic cloning and parthenogenetic technology in human regenerative medicine and will serve as an important alternative to the iPS cell technology in countries/regions where these technologies are permitted.

Rapid elimination of the histone variant MacroH2A from somatic cell heterochromatin after nuclear transfer.

Oocytes contain a maternal store of the histone variant MacroH2A, which is eliminated from zygotes shortly after fertilization. Preimplantation embryos then execute three cell divisions without MacroH2A before the onset of embryonic MacroH2A expression at the 16-cell stage. During subsequent development, MacroH2A is expressed in most cells, where it is assembled into facultative heterochromatin. Because differentiated cells contain heterochromatin rich in MacroH2A, we investigated the fate of MacroH2A during somatic cell nuclear transfer (SCNT). The results show that MacroH2A is rapidly eliminated from the chromosomes of transplanted somatic cell nuclei by a process in which MacroH2A is first stripped from chromosomes, and then degraded. Furthermore, MacroH2A is eliminated from transplanted nuclei by a mechanism requiring intact microtubules and nuclear envelope break down. Preimplantation SCNT embryos express endogenous MacroH2A once they reach the morula stage, similar to the timing observed in embryos produced by natural fertilization. We also show that the ability to reprogram somatic cell heterochromatin by SCNT is tied to the developmental stage of recipient cell cytoplasm because enucleated zygotes fail to support depletion of MacroH2A from transplanted somatic nuclei. Together, the results indicate that nuclear reprogramming by SCNT utilizes the same chromatin remodeling mechanisms that act upon the genome immediately after fertilization.

The oocyte spindle is preserved by 1,2-propanediol during slow freezing.

OBJECTIVE: To investigate the specific changes in oocyte spindle subjected to severe challenges of low temperature, as well as to examine the effect of cryoprotectants in preserving oocyte spindle during cryopreservation. DESIGN: In vitro experimental study. SETTING: Academic research laboratory. ANIMAL(S): B6D2F1 (C57BL/6 X DBA/2) mice. INTERVENTION(S): Mouse oocytes were cryopreserved using a slow freezing method in a sodium-depleted medium with 1.5 mol/l 1,2-propanediol (PROH) and 0.3 M sucrose. To examine the spindle, oocytes were fixed before, during, and after cryopreservation, and oocytes were analyzed by immunocytochemistry and confocal microscopy. RESULT(S): The MII spindle was preserved during the slow freezing, because the cryoprotectant PROH was found to support the organization of MII spindle in resisting the subzero temperature. In contrast, the MII spindle was disassembled gradually during the thawing process with or without PROH. Most of the oocytes were able to recover the MII spindle after thawing, but a portion of thawed oocytes could not sustain the meiotic spindle because of parthenogenetic activation. CONCLUSION(S): 1,2-Propanediol can support the organization of MII spindle to defy the subphysiologic temperature; however, the PROH cannot sustain oocyte spindle structure after the subsequent thawing process.

The efficacy and safety of human oocyte vitrification.

Vitrification is now a widely applied and highly successful approach for cryopreservation in reproductive biology. Rapidly increasing data prove that it is also a highly efficient technique for low-temperature storage of human oocytes. The latest approaches with appropriately selected cryoprotectants, tools and techniques, and properly adjusted parameters allow close to 100% morphological survival rates, and in vitro embryo development, as well pregnancy and implantation rates, comparable with those achieved with fresh oocytes. With standardization of the technique and elimination of biosafety problems by preserving all the positive features, vitrification may become a common part of the everyday routine in a human embryo laboratory, and it may offer a solution for various medical and social situations as well as for simple logistic problems commonly occurring in assisted reproduction.

Nuclear transfer and oocyte cryopreservation.

Somatic cells can be reprogrammed to a totipotent state through nuclear transfer or cloning, because it has been demonstrated that the oocyte has the ability to reprogramme an adult nucleus into an embryonic state that can initiate the development of a new organism. Therapeutic cloning, whereby nuclear transfer is used to derive patient-specific embryonic stem cells, embraces an entire new opportunity for regenerative medicine. However, a key obstacle for human therapeutic cloning is that the source of fresh human oocytes is extremely limited. In the present review, we propose prospective sources of human oocytes by using oocyte cryopreservation, such as an oocyte bank and immature oocytes. We also address some potential issues associated with nuclear transfer when using cryopreserved oocytes. In the future, if the efficacy and efficiency of cryopreserved oocytes are comparable to those of fresh oocytes in human therapeutic cloning, the use of cryopreserved oocytes would be invaluable and generate a great impact to regenerative medicine.

Taurine supplementation improves the utilization of sulfur-containing amino acids in rats continually administrated alcohol.

The main purpose of this study was to evaluate changes in brain sulfur-containing amino acid (SCAA) metabolism to determine whether taurine intervened under continuous alcohol intake. We fed 80 male Sprague-Dawley rats 30% alcohol-containing water for 4 weeks. Eighty animals were divided into two groups (with or without 2 g/kg body weight taurine supplementation), and five were killed every week in each group for monitoring SCAA changes in the brain, liver, kidneys and heart. Results indicated that the plasma alcohol concentration increased from Weeks 1-4; however, animals with taurine supplementation showed a lower plasma concentration of ethanol in Week 2. As to SCAA concentrations, cysteine and taurine were both lower after a week of alcohol ingestion in the brain and plasma; the same declining trend was shown in the liver in Week 2. In contrast, plasma and hepatic concentrations of homocysteine were elevated in Week 2, and the plasma S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) ratio also decreased in Week 1. Furthermore, the key cofactor of transsulfuration, pyridoxal-5'-phosphate, significantly declined in the plasma after a week of the ethanol intervention, whereas an increase was observed in brain tissue. Under taurine supplementation, some recoveries were shown by delaying taurine depletion to Week 2, increasing the SAM/SAH ratio and elevating plasma and brain levels of vitamin B6 in Week 2. In conclusion, daily consumption of 30% alcohol interfered with SCAA metabolism, thus decreasing taurine's role in neurotransmission. The possible mechanism involved might be that ethanol interrupts the production of cysteine, which is the upstream SCAA of taurine, thus decreasing the homocysteine level. Additionally, taurine supplementation delayed this process.

Clinical evaluation of the efficiency of an oocyte donation program using egg cryo-banking.

OBJECTIVE: To evaluate the efficiency of oocyte donation cycles using egg "cryo-banking." DESIGN: Study conditions for vitrified/warmed oocytes for 20 non-autologous recipients (from 10 donors) were set prospectively, and outcomes of it were later compared retrospectively to nine fresh donations cycles. SETTING: Private assisted reproductive technology program. PATIENT(S): Ten donors and 20 infertile recipients. INTERVENTION(S): Oocytes were vitrified 3 to 4 hours after collection and cryo-stored. Intracytoplasmic sperm injection was performed 3 hours after warming, and embryos were in vitro cultured for 5 days. Two or three blastocysts were transferred per patient. MAIN OUTCOME MEASURE(S): Oocyte survival, fertilization, development, clinical pregnancy, and implantation rates. RESULT(S): A total of 153 oocytes were warmed and 134 survived. A total of 117 fertilized and 68% developed to blastocyst stage. A total of 47 embryos were transferred (2.35 embryos per recipient) and 26 implanted. Fifteen patients achieved ongoing pregnancies initially, and two additional pregnancies were obtained after transfer of supernumerary vitrified/warmed embryos. Nine of the 10 donors from the current study had previous fresh donations cycles from where seven clinical pregnancies were established in nine recipients, providing the base for comparison. CONCLUSION(S): Oocyte donation using vitrified/warmed oocytes can provide high pregnancy and implantation rates, and thus can be considered as efficient treatment procedure with additional benefits to recipients.

Human oocyte vitrification: in-vivo and in-vitro maturation outcomes.

This study aimed to evaluate oocyte vitrification efficiency using in-vivo matured (IVO) versus rescued in-vitro matured (IVM) oocytes. The results show that oocyte survival (85% versus 81%), fertilization (86% versus 76%) and cleavage rate (98% versus 89%) was not significantly different in IVO oocytes compared with rescued IVM sibling oocytes. The fertilized oocytes from IVO and IVM groups were cultured to blastocyst stage; however, embryo development was significantly reduced in the rescued IVM group (72% versus 15%). Embryo transfer was only performed with the embryos derived from IVO oocytes on day 5; 42 blastocysts were transferred to 18 recipients; 16 of 18 recipients had positive beta-human chorionic gonadotrophin and a total of 26 fetal cardiac activities were detected in 15 recipients (implantation: 26/42, 61.9%). Ten of the 15 recipients have delivered 19 healthy babies, and the other five pregnancies are still ongoing. These data indicate that the combination of oocyte vitrification and rescued IVM not only yield a new strategy to extend the pool of total fertilizable oocytes, but also demonstrate that the efficiency of vitrified/warmed oocytes can be comparable to fresh oocytes with regard to clinical outcomes.

Effect of denuding on polar body position in in-vitro matured oocytes.

The aim of this study was to determine whether the denuding procedure causes the polar body to move within the perivitelline space. Only those patients undergoing IVF who had unused in-vitro matured (IVM) oocytes were included in this study. IVM oocytes were initially viewed under a non-invasive, polarized light microscope. A laser was used to mark the location of the polar body on the zona. Oocytes were subjected to the denuding procedure with a 150 microm, 135 microm and 125 mum diameter pipette. After each pipetting, the oocytes were viewed again to determine whether the polar body had moved. After denuding, the oocyte was left to culture overnight and viewed 24 h later. After denuding with the 150 microm, 135 microm and 125 microm pipettes and after 24 h in culture, the average angle between the spindle and polar body was 15.4 +/- 10.4 degrees , 16.1 +/- 10.1 degrees , 20.9 +/- 11.7 degrees , and 26.7 +/- 18.2 degrees , respectively (P = 0.0021). Slight changes in angles were noted between denuding with the different diameter pipettes. The largest changes in angles were seen after 24 h in culture.

Development of artificial gametes.

Virtually all normal cells can reproduce themselves. The germ cells, however, can initiate reproduction of the entire organism. A special sequence of events, meiosis, is responsible for generating mature germ cells bearing a haploid genome. The basic features of meiosis, i.e. two cell divisions with no intervening DNA replication, resulting in a halving of the chromosome complement, are evident throughout evolution. The idea of generating an artificial gamete was recently put forward to provide an ultimate solution for the treatment of infertility. Currently, there are different strategies to create artificial gametes in vitro, such as converting somatic cells from mitotic division to meiotic division directly (somatic cell haploidization), or dedifferentiating somatic cells into embryonic stem (ES) cells and re-differentiating ES cells into gametes, or extracting adult stem cells and re-differentiating them into gametes.

Two successful pregnancies obtained following oocyte vitrification and embryo re-vitrification.

Recent clinical reports not only show that cryopreserved embryos can be successfully used for human fertility treatment, but also that cryopreserved oocytes may be used successfully as an adjunct to human assisted reproductive technologies. Vitrification is known to establish a glass-like solid state during the cooling process. The high concentration of cryoprotectants and an extremely rapid rate of cooling are responsible for the formation of the solid state, and also prevent formation of intracellular ice crystals. Hence, in theory, vitrification should minimize cryo-injuries, and therefore has great promise for oocyte and embryo cryopreservation. This article describes two pregnancies from vitrified-warmed blastocysts obtained after intracytoplasmic sperm injection fertilization of vitrified-warmed oocytes. Vitrification was employed to cryopreserve the oocytes and the subsequent blastocysts. The results present the intriguing implication that vitrification may serve as an efficient method for clinical oocyte cryopreservation and embryo re-cryopreservation.