Chapter 20 Gavi-Automated Vitrification Instrument.

Gavi is intended for use in a laboratory or clinic environment for the preparation and vitrification of oocytes, cleavage stage embryos and blastocysts. Gavi is designed to automate the equilibration steps in the vitrification process to minimize the variability that occurs during cryopreservation. This automated process reduces the potential for errors and ensures a standardized, repeatable procedure for vitrification in a controlled, closed-system environment.

Embryo vitrification using a novel semi-automated closed system yields in vitro outcomes equivalent to the manual Cryotop method.

Study Question: Can the equilibration steps prior to embryo vitrification be automated?

Summary Answer: We have developed the 'Gavi' system which automatically performs equilibration steps before closed system vitrification on up to four embryos at a time and gives in vitro outcomes equivalent to the manual Cryotop method.

What Is Known Already: Embryo cryopreservation is an essential component of a successful assisted reproduction clinic, with vitrification providing excellent embryo survival and pregnancy outcomes. However, vitrification is a manual, labour-intensive and highly skilled procedure, and results can vary between embryologists and clinics.

Derivation of Human Embryonic Stem Cell Lines from Vitrified Human Blastocysts.

Human embryonic stem cells are pluripotent cells typically derived from blastulating embryos that have become excess to clinical needs in assisted reproduction programs. They provide cellular models for embryonic development and disease, and are thought to be useful for future cell replacement therapies and regenerative medicine. Here we describe methods to derive human embryonic stem cell lines.

Derivation of Huntington's disease-affected human embryonic stem cell lines.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by an expansion of cytosine-adenine-guanine (CAG) repeats in the Huntingtin gene Htt. To facilitate research into HD, we have derived 4 human embryonic stem cell (hESC) lines containing ≥ 40 CAG repeats in exon 1 of Htt: SIVF017-HD (CAG₄₀), SIVF018-HD (CAG₄₆), SIVF020-HD (CAG₄₈), and SIVF046-HD (CAG₄₅). Additionally, we have derived a normal sibling-matched control for SIVF020-HD, cell line SIVF019.

Derivation of three new human embryonic stem cell lines.

Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal.

Derivation of human embryonic stem cell lines from vitrified human embryos.

Human embryonic stem cell lines are usually derived from human embryos that have become excess to clinical needs in assisted reproduction programs, whether because the couple in question has completed their family or because the embryo was found to be clinically unsuitable for transfer due to severe genetic condition (in case of pre-implantation genetic diagnosis, PGD). Culturing embryos to a blastocyst stage (5-6 days after IVF) before embryo transfer or cryopreservation instead of earlier commonly used 8-cell stage (3 days after IVF) calls for new methods for embryo cryopreservation and allows higher efficiencies for the actual stem cell derivation. Despite the vast advances in other fields of embryonic stem cell research, methods for derivation of new lines have not changed much over the years, mainly due to scarcity of embryos limiting experimentation.

Phenotypes of the ovarian follicular basal lamina predict developmental competence of oocytes.

Background: The ovarian follicular basal lamina underlies the epithelial membrana granulosa and maintains the avascular intra-follicular compartment. Additional layers of basal lamina occur in a number of pathologies, including pili annulati and diabetes. We previously found additional layers of follicular basal lamina in a significant percentage of healthy bovine follicles.

Telomere length status of somatic cell sheep clones and their offspring.

This study was carried out to determine the telomere length status of sheep clones and their offspring, and to examine telomere dynamics and chromosomal abnormalities in culture propagated donor cells. Skin samples were collected from somatic cell nuclear transfer-derived sheep clones, and three of their progeny generated by natural mating. Samples were collected from control animals (n = 35), spanning in age from 1 month to 36 months of age.

Derivation of human embryonic stem cell lines.

Human embryonic stem cells (hESC) are undifferentiated cells derived from an early embryo that can grow in vitro indefinitely, while retaining their capability to differentiate into specialized somatic cell types. Over the last decade there has been great interest in derivation and culture of these cells, as they can potentially provide a supply of readily available differentiated cells and tissues of all types to be used for therapeutic purposes in cell transplantation in humans, as well as for other medical uses such as drug discovery. The source of hESC lines is usually excess human embryos from in vitro fertilization treatments, although novel ways of producing hESCs have been suggested recently.

Highly efficient and reliable chemically assisted enucleation method for handmade cloning in cattle.

The purpose of the present study was to find an efficient and reliable chemically assisted procedure for enucleation related to the handmade cloning (HMC) technique. After in vitro maturation oocytes were incubated in 0.5 microg mL(-1) demecolcine for 2 h.

Ovine ooplasm directs initial nucleolar assembly in embryos cloned from ovine, bovine, and porcine cells.

Here we present ultrastructural and immunocytochemical evidence that ovine ooplasm is directing the initial assembly of the nucleolus independent of the species of the nuclear donor. Intergeneric porcine-ovine somatic cell nuclear transfer (SCNT) and intrageneric ovine-ovine SCNT embryos were constructed and the nucleolus ultrastructure and nucleolus associated rRNA synthesis examined in 1-, 2-, 4-, early 8-, late 8-, and 16-cell embryos using transmission electron microscopy (TEM) and light microscopical autoradiography. In addition, immunocytochemical localization by confocal microscopy of nucleolin, a key protein involved in processing rRNA transcripts, was performed on early 8-, late 8-, and 16-cell embryos for both groups of SCNT embryos.

A comparison of established and new approaches in ovine and bovine nuclear transfer.

Several breakthroughs in nuclear transfer research were first achieved in sheep, although cattle soon became the main livestock species of interest. However, sheep still offer significant advantages both in basic and applied research. With increased interest in cloning of livestock, new approaches have been developed for both sheep and cattle nuclear transfer technology.

Effect of nutrition of oocyte donor on the outcomes of somatic cell nuclear transfer in the sheep.

The purpose of this study was to determine if the nutrition of the oocyte donor ewe influenced the success of somatic cell cloning. Merino ewes were fed at either a high- or a low-nutrition level for 3-5 mo before superovulation treatments. Freshly ovulated oocytes were enucleated and fused with serum-starved adult granulosa cells, and resulting reconstructed embryos were cultured for 6 days in modified synthetic oviduct fluid.