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.

https://www.rbmojournal.com/article/S1472-6483(10)60837-1/pdf

Highly efficient vitrification method for cryopreservation of human oocytes.

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. © 1998 Wiley-Liss, Inc.

Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos.

Mouse embryos undergo genome-wide methylation reprogramming by demethylation in early preimplantation development, followed by remethylation thereafter. Here we show that genome-wide reprogramming is conserved in several mammalian species and ask whether it also occurs in embryos cloned with the use of highly methylated somatic donor nuclei. Normal bovine, rat, and pig zygotes showed a demethylated paternal genome, suggesting active demethylation. In bovine embryos methylation was further reduced during cleavage up to the eight-cell stage, and this reduction in methylation was followed by de novo methylation by the 16-cell stage. In cloned one-cell embryos there was a reduction in methylation consistent with active demethylation, but no further demethylation occurred subsequently. Instead, de novo methylation and nuclear reorganization of methylation patterns resembling those of differentiated cells occurred precociously in many cloned embryos. Cloned, but not normal, morulae had highly methylated nuclei in all blastomeres that resembled those of the fibroblast donor cells. Our study shows that epigenetic reprogramming occurs aberrantly in most cloned embryos; incomplete reprogramming may contribute to the low efficiency of cloning.

https://www.pnas.org/content/pnas/98/24/13734.full.pdf

Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos.

Bovine and ovine embryos exposed to a variety of unusual environments prior to the blastocyst stage have resulted in the development of unusually large offspring which can also exhibit a number of organ defects. In these animals, the increased incidence of difficult parturition and of fetal and neonatal losses has limited the large-scale use of in vitro embryo production technologies commonly used in humans and other species. Four different situations have been identified that result in the syndrome: in vitro embryo culture, asynchronous embryo transfer into an advanced uterine environment, nuclear transfer and maternal exposure to excessively high urea diets. However, programming of the syndrome by all of these situations is unpredictable and not all of the symptoms described have been observed universally. Neither the environmental factors inducing the large offspring syndrome nor the mechanisms of perturbation occurring in the early embryo and manifesting themselves in the fetus have been identified.

Large offspring syndrome in cattle and sheep.

The aim of this study is to examine the effect of bovine oocyte maturation, fertilization or culture in vivo or in vitro on the proportion of oocytes reaching the blastocyst stage, and on blastocyst quality as measured by survival following vitrification. In Experiment 1, 4 groups of oocytes were used: (1) immature oocytes from 2-6 mm follicles; (2) immature oocytes from > 6 mm follicles; (3) immature oocytes recovered in vivo just before the LH surge; and (4) in vivo matured oocytes. Significantly more blastocysts developed from oocytes matured in vivo than those recovered just before the LH surge or than oocytes from 2-6 mm follicles. Results from > 6 mm follicles were intermediate. All blastocysts had low survival following vitrification. In Experiment 2, in vivo matured oocytes were either (1) fertilized in vitro or (2) fertilized in vivo by artificial insemination and the resulting presumptive zygotes recovered on day 1. Both groups were then cultured in vitro. In vivo fertilized oocytes had a significantly higher blastocyst yield than those fertilized in vitro. Blastocyst quality was similar between the groups. Both groups had low survival following vitrification. In Experiment 3a, presumptive zygotes produced by in vitro maturation (IVM)/fertilization (IVF) were cultured either in vitro in synthetic oviduct fluid, or in vivo in the ewe oviduct. In Experiment 3b, in vivo matured/in vivo fertilized zygotes were either surgically recovered on day 1 and cultured in vitro in synthetic oviduct fluid, or were nonsurgically recovered on day 7. There was no difference in blastocyst yields between groups of zygotes originating from the same source (in vivo or in vitro fertilization) irrespective of whether culture took place in vivo or in vitro. However, there was a dramatic effect on blastocyst quality with those blastocysts produced following in vivo culture surviving vitrification at significantly higher rates than their in vitro cultured counterparts. Collectively, these results indicate that the intrinsic quality of the oocyte is the main factor affecting blastocyst yields, while the conditions of embryo culture have a crucial role in determining blastocyst quality.

Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality.

High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins.

Culture of mammalian zygotes individually and in small groups results in lower developmental rates than culture of large groups. Zona-free zygotes also have impaired developmental potential in current culture systems. This paper describes a new approach to resolve the problems, the Well of the Well (WOW) system. Small wells (WOWs) were formed in four-well dishes by melting the bottom with heated steel rods. The WOWs were then rinsed, the wells were filled with medium, and the embryos were placed into the WOWs. To test the value of the WOW system a 3 × 3 factorial experiment was performed. Bovine presumptive zygotes were cultured from day 1 to day 7 (day 0: day of insemination) using three modules (single embryos, embryo groups of five, or single zona-digested embryos) and three different culture systems (400 μl medium, 200 μl drops, or WOWs). An additional control group consisted of 40 to 50 embryos cultured in 400 μl medium. The WOW system resulted in higher blastocyst/oocyte rates for all three modules (single: 59%; group of five: 61%; single zona-digested: 53%) than the culture in drops or in wells (P < 0.05 for all). The developmental rate was independent of the number of WOWs per well. The cell number of blastocysts cultured in the WOW system did not differ from that of the controls. Apart from its theoretical value in revealing the role of different factors influencing embryo development in vitro, the WOW system may have immediate practical consequences in certain areas of mammalian embryo production. Mol Reprod Dev 55:256–264, 2000. © 2000 Wiley-Liss, Inc.

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (ops) method

The kinetics of the in vitro development of early embryos from bovine zygotes derived in vitro and in vitro were compared, investigating the effect of serum during in vitro maturation and fertilization (IVM-IVF) and in culture. Zygotes were collected from superovulated heifers or produced in vitro from immature oocytes with or without serum supplementation, and cultured subsequently in defined culture medium (SOFaaci) with or without serum supplementation. Time-lapse images were recorded every 0.5 h throughout the culture period. More in vivo- than in vitro-derived zygotes developed to the compact morula or blastocyst stages (87% versus 47-54%, respectively; P < 0.05). Embryo development was blocked predominantly at the second or fourth cell cycles (28 and 29%). However, blastomeres degenerated at all cleavage stages. Serum supplementation during IVM-IVF resulted in abnormally sized blastomeres at first cleavage (defined serum: 20-22% versus in vivo-derived: 8%, P < 0.05). The duration of the second, third and fifth cell cycles of in vivo-derived zygotes were 1-5 h shorter compared with those of in vitro-derived zygotes cultured under similar conditions (P < 0.05). However, the kinetics of embryo development was affected by serum during IVM-IVF and culture. The first and fourth cell cycles were prolonged by 4-5 h in the absence of serum during IVM-IVF, whereas the presence of serum during culture decreased the duration of the fourth cell cycle and triggered premature blastulation. The results of this study illustrate the differences and similarities between the morphology and developmental kinetics of in vivo- and in vitro-derived zygotes, and show how serum supplementation during IVM-IVF and culture can alter these parameters.

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Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media

Peter Holm

Papers

Open Pulled Straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos.

High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum-proteins.

New method for culture of zona-included or zona-free embryos: the Well of the Well (WOW) system.

Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw (ops) method

Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media