Gábor Vajta

Open pulled straw (OPS) vitrification: A new way to reduce cryoinjuries of bovine ova and embryos

Although cryopreservation of certain mammalian embryos is now a routine procedure, considerable differences of efficiency exist depending on stage, species and origin (in vivo or in vitro produced). Factors that are suspected to cause most of these differences are the amount of the intracellular lipid droplets and the different microtubular structure leading to chilling injury as well as the volume/surface ratio influencing the penetration of cryoprotectants. A new approach, the Open Pulled Straw (OPS) method, which renders very high cooling and warming rates (over 20,000°C/min) and short contact with concentrated cryoprotective additives (less than 30 sec over −180°C) offers a possibility to circumvent chilling injury and to decrease toxic and osmotic damage. In this paper we report the vitrification by the OPS method of in vitro produced bovine embryos at various stages of development. Embryos cryopreserved from Day 3 to Day 7 (Day 0 = day of fertilization) exhibited development into blastocysts at rates equivalent to those of control embryos; even those cryopreserved on Day 1 or 2 exhibited only somewhat reduced survival. Eighty‐one percent of Day 8 hatched blastocysts also survived the procedure. The method was also successfully used for bovine oocytes; of 184 vitrified oocytes, 25% developed into blastocysts after fertilization and culture for 7 days. Pregnancies were achieved following transfer after vitrification at both the oocyte and blastocyst stage. The OPS vitrification offers a new way to solve basic problems of reproductive cryobiology and may have practical impact on animal biotechnology and human assisted reproduction. Mol. Reprod. Dev. 51:53–58, 1998.

Highly efficient vitrification method for cryopreservation of human oocytes

Two experiments were performed to develop a method to cryopreserve MII human oocytes. In the first experiment, three vitrification methods were compared using bovine MII oocytes with regard to their developmental competence after cryopreservation: (i) vitrification within 0.25-ml plastic straws followed by in-straw dilution after warming (ISD method); (ii) vitrification in open-pulled straws (OPS method); and (iii) vitrification in <0.1 microl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, the Cryotop method, which had yielded the best results, was used to vitrify human oocytes. Out of 64 vitrified oocytes, 58 (91%) exhibited normal morphology after warming. After intracytoplasmic sperm injection, 52 became fertilized, and 32 (50%) developed to the blastocyst stage in vitro. Analysis by fluorescence in-situ hybridization of five blastocysts showed that all were normal diploid embryos. Twenty-nine embryo transfers with a mean number of 2.2 embryos per transfer on days 2 and 5 resulted in 12 initial pregnancies, seven healthy babies and three ongoing pregnancies. The results suggest that vitrification using the Cryotop is the most efficient method for human oocyte cryopreservation.

Comparison of open and closed methods for vitrification of human embryos and the elimination of potential contamination

Survival and development of human embryos was compared following slow cooling versus vitrification involving more than 13,000 vitrified embryos. In addition, the efficacy of an open system, the Cryotop, and a closed vitrification system, the CryoTip(trade mark), were compared using human blastocysts. One hundred percent of vitrified human pronuclear stage embryos survived and 52% developed to blastocysts as compared with 89% survival and 41% blastocyst development after slow cooling. Similar survival rates were seen with vitrification of 4-cell embryos (98%) as compared with slow cooling (91%). Furthermore, 90% of vitrified blastocysts survived and resulted in a 53% pregnancy rate following transfer, as compared with 84% survival and 51% pregnancy rates following slow cooling. All corresponding values were significantly different. When the closed and open vitrification systems were compared, no difference was found with regard to supporting blastocyst survival (93 and 97% for CryoTip and Cryotop respectively), pregnancies (51 versus 59% respectively) and deliveries (48 versus 51% respectively). Vitrification is a simple, efficient and cost-effective way to improve cumulative pregnancy rates per cycle. The use of the closed CryoTip system eliminates the potential for embryo contamination during cryopreservation and storage without compromising survival and developmental rates in vitro and in vivo.

Vitrification of the oocytes and embryos of domestic animals

After the first successful application of vitrification for embryo cryopreservation 15 years ago, a rapid application of the method in domestic animal embryology was presumed. However, although the advantages of vitrification (simplicity, cost efficiency, speed of the procedure) were widely acknowledged, its use has been mainly restricted to experimental studies. For commercial embryo transfer purposes, the traditional slow-rate or equilibrium freezing has been used. This review attempts to explain the reasons for this phenomenon and discusses the theoretical and practical differences between the two technologies as well as their commercial prospects. Recent developments that improve the efficiency of vitrification and applications to other reproductive technologies are also summarized. These advances may result in considerable advantage and could lead to widespread application of vitrification in certain areas of domestic animal embryology.

Consistent and predictable delivery rates after oocyte vitrification: an observational longitudinal cohort multicentric study

BACKGROUND An efficient method for cryopreservation of human oocytes may offer solutions to legal and ethical problems in routine infertility programs and may also be used for fertility preservation for medical and social reasons. METHODS We conducted an observational longitudinal cohort multicentric study to investigate the efficacy and reproducibility of oocyte cryopreservation outcomes in IVF/ICSI cycles. Moreover, the effects of patient and cycle characteristics on the delivery rate (DR) were analyzed. RESULTS In 486 cycles performed in 450 couples, 2721 oocytes were warmed and 2304 of them survived cryopreservation (84.7%). Of the 2182 oocytes subjected to ICSI, the rates of fertilization and development to top-quality embryos were 75.2 and 48.1%, respectively. A total of 128 deliveries were obtained (26.3% per cycle and 29.4% per transfer) for 450 patients (28.4%) and 147 babies were live born from 929 embryos transferred (15.8%). The forward logistic regression analysis on a per patient basis showed that female age [odds ratio (OR): 0.93, 95% confidence interval (CI): 0.88-0.98], number of vitrified oocytes (OR: 1.08, 95% CI: 1.01-1.17) and the day of transfer (OR: 1.97, 95% CI: 1.14-3.42) influenced DR. By recursive partitioning analysis, it can be estimated that more than eight oocytes vitrified are required to improve the outcome (22.6 versus 46.4% DR, respectively). When fewer oocytes are available in women aged >38 years, results are dramatically reduced (12.6 versus 27.5% DR, respectively). Conversely, when >8 oocytes are available, blastocyst culture represents the most efficient policy (62.1% DR; data from one center only). CONCLUSIONS Oocyte vitrification is an efficient and reliable approach, with consistent results between centers and predictable DRs. It should be applied routinely for various indications. A predictive model is proposed to help patient counselling and selection.

Somatic cell cloning without micromanipulators.

Until now, micromanipulators have been regarded as indispensable for somatic cell nuclear transfer. This paper describes an improved zona-free nuclear transfer procedure with manual bisection of oocytes, selection of cytoplasts by Hoechst staining, and two-step fusion of somatic cells from primary granulosa cell cultures with two cytoplasts. Blastocyst rates in the three systems tested for zona-free embryo culture were 0%, 18%, and 36% for microdrops, well of the wells (WOW system), and microcapillaries (GO system), respectively. This simple, rapid, and inexpensive procedure may become a useful alternative to the existing techniques for somatic cell nuclear transfer for large-scale application of the technology.

Predictive value of oocyte morphology in human IVF: a systematic review of the literature

BACKGROUND Non-invasive selection of developmentally competent human oocytes may increase the overall efficiency of human assisted reproduction and is regarded as crucial in countries where legal, social or religious factors restrict the production of supernumerary embryos. The purpose of this study was to summarize the predictive value for IVF success of morphological features of the oocyte that can be obtained by light or polarized microscopic investigations. METHODS Studies about oocyte morphology and IVF/ICSI outcomes were identified by using a systematic literature search. RESULTS Fifty relevant articles were identified: 33 analysed a single feature, 9 observed multiple features and investigated the effect of these features individually, 8 summarized the effect of individual features. Investigated structures were the following: meiotic spindle (15 papers), zona pellucida (15 papers), vacuoles or refractile bodies (14 papers), polar body shape (12 papers), oocyte shape (10 papers), dark cytoplasm or diffuse granulation (12 papers), perivitelline space (11 papers), central cytoplasmic granulation (8 papers), cumulus–oocyte complex (6 papers) and cytoplasm viscosity and membrane resistance characteristics (2 papers). None of these features were unanimously evaluated to have prognostic value for further developmental competence of oocytes. CONCLUSIONS No clear tendency in recent publications to a general increase in predictive value of morphological features was found. These contradicting data underline the importance of more intensive and coordinated research to reach a consensus and fully exploit the predictive potential of morphological examination of human oocytes.

Handmade somatic cell cloning in cattle: analysis of factors contributing to high efficiency in vitro.

Abstract Widespread application of somatic cell cloning has been hampered by biological and technical problems, which include complicated and time-consuming procedures requiring skilled labor. Recently, zona-free techniques have been published with limited or no requirement for micromanipulators. The purpose of the present work was to optimize certain steps of the micromanipulator-free (i.e., handmade) procedure, to analyze the morphology of the developing blastocysts, and to explain factors involved in the high efficiencies observed. Optimization of the procedure included selection of the appropriate medium for enucleation, orientation of pairs at fusion, timing of fusion, and culture conditions. As a result of these improved steps, in vitro efficiency as measured by blastocysts per reconstructed embryo and blastocysts per working hour was among the highest described so far. The cattle serum used in our experiments was superior to other protein sources for in vitro embryo development. One possible explanation of this effect is the considerable mitogenic activity of the cattle serum compared with that of commercially available fetal calf serum. Morphological analysis of blastocysts by inverted microscopy, inner cell mass-trophoblast differential staining, and transmission electron microscopy revealed high average quality. A high initial pregnancy rate was achieved after the transfer of single blastocysts derived by aggregation of two nuclear transfer embryos into recipients. The improved handmade somatic cell nuclear transfer method may become a useful technology as a simple, inexpensive, and efficient alternative to traditional somatic cell nuclear transfer.

Somatic cell nuclear transfer in pigs: recent achievements and future possibilities

During the past 6 years, considerable advancement has been achieved in experimental embryology of pigs. This process was mainly generated by the rapidly increasing need for transgenic pigs for biomedical research purposes, both for future xenotransplantation to replace damaged human organs or tissues, and for creating authentic animal models for human diseases to study aetiology, pathogenesis and possible therapy. Theoretically, among various possibilities, an established somatic cell nuclear transfer system with genetically engineered donor cells seems to be an efficient and reliable approach to achieve this goal. However, as the result of unfortunate coincidence of known and unknown factors, porcine embryology had been a handicapped branch of reproductive research in domestic animals and a very intensive and focused research was required to eliminate or minimise this handicap. This review summarises recent achievements both in the background technologies (maturation, activation, embryo culture) and the actual performance of the nuclear replacement. Recent simplified methods for in vivo development after embryo transfer are also discussed. Finally, several fields of potential application for human medical purposes are discussed. The authors conclude that although in this early phase of research no direct evidence can be provided about the practical use of transgenic pigs produced by somatic cell nuclear transfer as organ donors or disease models, the future chances even in medium term are good, and at least proportional with the efforts and sums that are invested into this research area worldwide.

The Well-of-the-Well system: an efficient approach to improve embryo development

Transfer of human embryos at the blastocyst stage may offer considerable benefits including an increased implantation rate and a decreased risk of multiple pregnancies; however, blastocyst culture requires an efficient and reliable in-vitro embryo culture system. In this study, the effect of the Well-of-the-Well (WOW) system consisting of microwells formed on the bottom of the culture dish was tested in three mammalian species, including humans. The WOW system resulted in significant improvement when comparing the drops for culture of in-vitro-matured and parthenogenetically activated porcine oocytes, and in-vivo-derived mouse zygotes. In human embryos, using a sibling oocyte design, embryos cultured in WOW developed to the blastocyst stage in a significantly higher proportion than did embryos cultured traditionally (55% in WOW and 37% in conventional culture; P < 0.05). In a separate study, also in human, a total of 48 patients with a cumulative 214 unsuccessful previous IVF cycles were selected for the trials. In subsequent intracytoplasmic sperm injection cycles, oocytes/embryos were cultured individually in the WOW system or in microdrops. Transferable quality blastocyst development (48.9% of cultured zygotes) was observed in the WOW system. Ninety-four blastocysts transferred to 45 patients resulted in clinical pregnancy rates of 48.9%, including nine twin pregnancies, seven single pregnancies, five miscarriages and one ectopic pregnancy. The results indicate that the WOW system provides a promising alternative for microdrop culture of mammalian embryos, including human embryos.