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A User’s Guide

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans, cows, and mice. Two basic cryopreservation techniques rule the field--controlled-rate freezing, the first to be developed, and vitrification, which, in recent years, has gained a foothold. While much progress has been achieved in human medicine, the cattle industry, and in laboratory animals, this is far from being the case for most other mammals and even less so for other vertebrates. The major strides and obstacles in human and other vertebrate oocyte and embryo cryopreservation will be reviewed here.

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Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification

The predictable answer to the provocative question of whether programmable freezers are still needed in the embryo laboratory is an even more provocative 'no'. However, such a radical statement needs strong support. Based on the extensive literature of the past 5 years, the authors collected arguments either supporting or contradicting their opinion. After an overview of the causes of cryoinjuries and strategies to eliminate them, the evolution of vitrification methods is discussed. Special attention is paid to the biosafety issues. The authors did not find any circumstance in oocyte or embryo cryopreservation where slow freezing offers considerable advantages compared with vitrification. In contrast, the overwhelming majority of published data prove that the latest vitrification methods are more efficient and reliable than any version of slow freezing. Application of the proper vitrification methods increases the efficiency of long-term storage of stem cells and opens new perspectives in cryopreservation of oocytes, both for IVF and somatic cell nuclear transfer. However, lack of support from regulatory authorities, and conservative approachs regarding novel techniques can slow down the implementation of vitrification. The opinion of the authors is that vitrification is the future of cryopreservation. The public have the final say in whether they want and allow this future to arrive.

https://www.rbmojournal.com/article/S1472-6483(10)61091-7/pdf

Are programmable freezers still needed in the embryo laboratory? Review on vitrification

Improving cryopreservation systems.

The ability to cryopreserve human oocytes and store them indefinitely would be beneficial for cancer patients at risk of becoming sterile after therapy, allow women to delay reproduction, and alleviate religious concerns associated with embryo storage. In 1986, Chen was the first to report a pregnancy originating from a frozen-thawed human oocyte. Although over 100 babies have been born from oocyte storage since then, pregnancy rates remain unacceptably low. Adapting embryo cryopreservation techniques to oocyte storage has had limited success and new reproducible methods are needed. Problem areas other than intracellular ice formation and osmotic effects need to be identified. A broad approach of critical analysis should be conducted regarding the entire cryopreservation process from pre-equilibration and cooling, to thawing and stepout. All established facets deserve reanalysis in order to assess which aspects can be optimized or changed so that cellular demise can be avoided and cellular viability enhanced. New methods, including the use of choline-based media and vitrification have proven useful in increasing survival and pregnancy rates in some clinics. Other methods yet untested, such as injection of complex carbohydrates into the oocyte, deserve further studies. Vitrification research has led to the formulation of new ideas and has demonstrated the flexibility of cells to survive cryopreservation. Although successful, vitrification protocols are potentially harmful and technically challenging, due to elevated cryoprotectant concentrations and rapid cooling rates. Bovine embryo vitrification methods have been used to store human oocytes and embryos, particularly blastocysts with some success. Vitrification solutions containing high molecular weight polymers have also proved beneficial by reducing solution toxicity. In general, further advances are needed to improve human oocyte storage before widespread routine clinical use.

https://www.rbmojournal.com/article/S1472-6483(10)62124-4/pdf

An overview of oocyte cryopreservation.

Calves born after open pulled straw vitrification of immature bovine oocytes.

Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 × 50%); aggregation of two (2 × 50%), three (3 × 50%), or four (4 × 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 × 100%); or aggregation of two clone or parthenote embryos (2 × 100%). Control zona-intac...

Developmental Potential of Bovine Hand-Made Clone Embryos Reconstructed by Aggregation or Fusion with Distinct Cytoplasmic Volumes

Produção In vitro de embriões

Semen deposition by cervical, transcervical and intrauterine route for fixed-time artificial insemination (FTAI) in the ewe.

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and >95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (>90%) for nuclear transfer significantly improved blastocyst yield after cloning.

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Alceu Mezzalira

Papers

Calves born after open pulled straw vitrification of immature bovine oocytes.

Developmental Potential of Bovine Hand-Made Clone Embryos Reconstructed by Aggregation or Fusion with Distinct Cytoplasmic Volumes

Produção In vitro de embriões

Semen deposition by cervical, transcervical and intrauterine route for fixed-time artificial insemination (FTAI) in the ewe.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence.