Ching Chien Chang

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

OBJECTIVEnTo evaluate the efficiency of oocyte donation cycles using egg cryo-banking.nnnDESIGNnStudy 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.nnnSETTINGnPrivate assisted reproductive technology program.nnnPATIENT(S)nTen donors and 20 infertile recipients.nnnINTERVENTION(S)nOocytes 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.nnnMAIN OUTCOME MEASURE(S)nOocyte survival, fertilization, development, clinical pregnancy, and implantation rates.nnnRESULT(S)nA 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.nnnCONCLUSION(S)nOocyte 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.

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.

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.

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.

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

OBJECTIVEnTo determine whether the process of oocyte vitrification affects oocyte viability in inxa0vitro fertilization (IVF) patients between 30 and 39 years of age.nnnDESIGNnProspective controlled study.nnnSETTINGnPrivate IVF practice.nnnPATIENT(S)nA total of 30 women assigned and 22 qualified.nnnINTERVENTION(S)nDenudation of oocytes, cryopreservation of oocytes using vitrification method in a medium with 15% ethylene glycol (EG), 15% dimethylsulfoxide (DMSO), and 0.5 M sucrose.nnnMAIN OUTCOME MEASURE(S)nOocyte survival, fertilization, day-3 embryo quality, blastocyst formation, clinical pregnancy, implantation, and live-birth rates.nnnRESULT(S)nAfter 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 β-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.nnnCONCLUSION(S)nThe 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.

Premature Chromosome Condensation Is Not Essential for Nuclear Reprogramming in Bovine Somatic Cell Nuclear Transfer

Abstract Premature chromosome condensation (PCC) was believed to promote nuclear reprogramming and to facilitate cloning by somatic cell nuclear transfer (NT) in mammalian species. However, it is still uncertain whether PCC is necessary for the successful reprogramming of an introduced donor nucleus in cattle. In the present study, fused NT embryos were subjected to immediate activation (IA, simultaneous fusion and activation), delayed activation (DA, activation applied 4 h postfusion), and IA with aged oocytes (IAA, activation at the same oocyte age as group DA). The morphologic changes, such as nuclear swelling, the occurrence of PCC, and microtubule/aster formation, were analyzed in detail by laser-scanning confocal microscopy. When embryos were subjected to IA in both IA and IAA groups, the introduced nucleus gradually became swollen, and a pronuclear-like structure formed within the oocyte, but PCC was not observed. In contrast, delaying embryo activation resulted in 46.5%–91.2% of NT embryos exhibiting PCC. This PCC was observed beginning at 4 h postcell fusion and was shown as one, two, or multiple chromosomal complexes. Subsequently, a diversity of pronuclear-like structures existed in NT embryos, characterized as single, double, and multiple nuclei. In the oocytes exhibiting PCC, the assembled spindle structure was observed to be an interactive mass, closely associated with condensed chromosomes, but no aster had formed. Regardless of whether they were subjected to IA, IAA, or DA treatments, if the oocytes contained pronuclear-like structures, either one or two asters were observed in proximity to the nuclei. A significantly higher rate of development to blastocysts was achieved in embryos that were immediately activated (IA, 59.1%; IAA, 40.7%) than in those for which activation was delayed (14.2%). The development rate was higher in group IA than in group IAA, but it was not significant (P = 0.089). Following embryo transfer, there was no statistically significant difference in the pregnancy rates (Day 70) between two of the groups (group IA, 11.7%, n = 94 vs. group DA, 12.3%, n = 130; P > 0.05) or live term development (group IA, 4.3% vs. group DA, 4.6%; P > 0.05). Our study has demonstrated that the IA of bovine NT embryos results in embryos with increased competence for preimplantational development. Moreover, PCC was shown to be unnecessary for the reprogramming of a transplanted somatic genome in a cattle oocyte.

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.

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.

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.