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. A closed system whereby the embryo does not come in direct contact with liquid nitrogen is preferred by many clinics and is a regulatory requirement in some countries. STUDY DESIGN, SIZE, DURATION: The Gavi system, an automation instrument with a novel closed system device, was used to equilibrate embryos prior to vitrification. Outcomes for embryos automatically processed with the Gavi system were compared with those processed with the manual Cryotop method and with fresh (non-vitrified) controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: The efficacy of the Gavi system (Alpha model) was assessed for mouse (Quackenbush Swiss and F1 C57BL/6J x CBA) zygotes, cleavage stage embryos and blastocysts, and for donated human vitrified-warmed blastocysts. The main outcomes assessed included recovery, survival and in vitro embryo development after vitrification-warming. Cooling and warming rates were measured using a thermocouple probe. MAIN RESULTS AND THE ROLE OF CHANCE: Mouse embryos vitrified after processing with the automated Gavi system achieved equivalent in vitro outcomes to that of Cryotop controls. For example, for mouse blastocysts both the Gavi system (n = 176) and manual Cryotop method (n = 172) gave a 99% recovery rate, of which 54 and 50%, respectively, progressed to fully hatched blastocysts 48 h after warming. The outcomes for human blastocysts processed with the Gavi system (n = 23) were also equivalent to Cryotop controls (n = 13) including 100% recovery for both groups, of which 17 and 15%, respectively, progressed to fully hatched blastocysts 48 h after warming. The cooling and warming rates achieved with the Gavi system were 14 136°C/min and 11 239°C/min, respectively. LIMITATIONS, REASONS FOR CAUTION: Testing of the Gavi system described here was limited to in vitro development of embryos from two mouse strains and a limited number of human embryos. Validation of Gavi system advanced production models is now required to confirm the success of semi-automated vitrification, including clinical evaluation of pregnancy outcomes from the transfer of Gavi vitrified-warmed human embryos. WIDER IMPLICATIONS OF THE FINDINGS: The Gavi system has the potential to revolutionize and standardize vitrification of embryos and oocytes. The success of the Gavi system shows that it is possible to semi-automate complicated labour-intensive ART methods and processes, and opens up the possibility for further improvements in clinical outcomes and efficiencies in the ART clinic. STUDY FUNDING/COMPETING INTERESTS: This study was funded by Genea Ltd. S.B., N.M.T., T.T.P., S.J.M., M.C.B. and T.S. are shareholders of Genea Ltd. E.V., C.H., C.L., S.R.L. and S.M.D. are shareholders of Planet Innovation Pty Ltd. The remaining authors are employees of either Genea Ltd. or Planet Innovation Pty Ltd.

Cell lines from morphologically abnormal discarded IVF embryos are typically euploid and unaccompanied by intrachromosomal aberrations.

Routine IVF practices result in the discarding of a significant proportion of embryos due to their unsuitability for transfer or cryopreservation. The present study plated clinically unusable human blastocysts to derive cellular outgrowths for aneuploidy studies and genome-wide analysis of DNA copy number variations, and to evaluate their potential as a source for pluripotent stem cells. Just 79 cellular outgrowths were obtained from 1026 abnormal blastocysts (7.7%), reflecting their low developmental potential. Of these, 13 (16.5%) were karyotypically abnormal and included trisomies frequently detected in miscarriages, each of which was uniform (nonmosaic) and the result of meiotic nondisjunction. Evaluation of submicroscopic DNA gains and losses in 10 diploid cellular outgrowths did not identify increased rates of copy number variations. Five of these outgrowths were shown to express pluripotency markers and could be developed into cell lineages representative of the three germ layers. These data suggest that embryos with chromosomal abnormalities resist cell-line derivation, and mosaic aneuploidy produced from mitotic nondisjunction, common in preimplantation embryos, is likely to be diminished or lost under conditions of diploid cell competition. Furthermore, this work demonstrated that abnormal embryos discarded in IVF programmes can provide a valuable source for pluripotent stem cell lines. During IVF, a large proportion of embryos are clinically unsuitable due to abnormal development and these embryos only have a small chance of achieving a pregnancy. Here we used these abnormal embryos to create cell lines for genetic testing and to determine their potential as stem cells. Of the 1026 abnormal embryos used, 79 (7.7%) created cell lines, reflecting their low developmental potential. Of those, only 16.5% had chromosomal anomalies, a much lower number than expected. This included chromosome abnormalities frequently observed in miscarriages, all of which were found in each cell tested (nonmosaic) and originated from the egg or the sperm as opposed to cell division. In-depth testing of 10 normal cell lines for small DNA gains and losses did not reveal an increased frequency of mutations. Furthermore, five of the cell lines were examined for stem cell properties and found to exhibit the hallmark features of stem cells including their ability to make mature cells from different parts of the body. Our data suggest that embryos with abnormal chromosomes resist making cell lines and that abnormalities that arise during cell division are likely to be lost due to competition with normal cells. We also demonstrated that abnormal embryos usually discarded in IVF programmes can provide a valuable source for stem cell lines.

Generation of human embryonic stem cells.

This unit describes generation of human embryonic stem cell lines from early human embryos. The focus is on actual handling of embryos and early embryonic outgrowths, omitting steps required for actual generation, freezing, and thawing of embryos, as well as further culture and characterization of newly derived stem cells. Hence, the initial culture of embryos to a blastocyst stage is described, followed by removal of the protective zona pellucida layer, isolation of the inner cell mass (ICM), subsequent plating of ICM or whole embryo and, finally, the first few passages of an early embryonic outgrowth. A few alternative procedures for some steps such as zona removal and inner cell mass isolation are described, to allow procedures to be modified according to circumstances.

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. All 5 hESC lines had a normal karyotype, expressed pluripotency markers including Oct4, SSEA3, and Tra-1-81, and could be maintained in culture for multiple (>40) passages. Teratoma studies revealed that the hESC lines were capable of differentiating into cells representative of the 3 germ layers. Furthermore, in vitro neuronal differentiation experiments have confirmed that the hESC lines were able to generate MAP2-positive neuronal cells that express the Htt protein. Combined, these experiments confirm that the cell lines represent pluripotent stem cell lines. These HD-affected hESC lines will be made available to biomedical research laboratories and will provide a valuable tool to investigate the mechanisms and potential treatments for HD.

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. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines.

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. We describe here methods required to derive new embryonic stem cell lines starting from the initial cryopreservation of an embryo and finishing with a new cell line. We cover embryo cryopreservation and warming using a highly efficient vitrification method, the production of feeder cells and feeder plates, as well as embryo handling, plating and critical early passages, including earliest possible cryopreservation of putative stem cells using vitrification.

Improved membrane proteomics coverage of human embryonic stem cells by peptide IPG-IEF.

Protein biomarkers are fundamental tools for the characterization of stem cells and for tracking their differentiation and maturation down developmental lineages. Technology development allowing increased coverage of difficult cellular proteomes should allow for the discovery of new and novel membrane protein biomarkers for use by the stem cell research community. The amphipathic and highly hydrophobic nature and relative low abundance of many membrane proteins present significant analytical challenges. These difficulties are amplified when the source material (tissue or cells) is only available in limited quantities (e.g., embryonic stem cells). Recent advances in enrichment for purer membrane fractions, the enzymatic and chemical digestion of membrane proteins in the presence of solvents or chaotropes, and the use of "shotgun" proteomics methodologies have gradually resulted in increased membrane proteome coverage with numbers of predicted integral membrane proteins now in excess of 1000 being routinely reported. We have recently demonstrated the advantages of using peptide isoelectric focusing in the first dimension on immobilized pH gradients (peptide IPG-IEF) followed by reversed phase chromatography and tandem MS to increase membrane proteome coverage. This study looked at achieving a similar level of membrane proteome coverage using modifications to reported methodologies while restricting the number of characterized human embryonic stem cells to 10(7) cells. Two-thousand two-hundred and ninety-two (2292) nonredundant proteins were identified with two or more high accuracy peptide matches from 260 mug of a human embryonic stem cell membrane enriched fraction with a false discovery rate of 0.32%. Gene Ontology (GO) mapping predicted 1279 (44.9%) of this list to be membrane proteins of which 395 proteins were predicted to be derived from the plasma membrane compartment. The TMHMM algorithm predicted 904 integral membrane proteins with up to 16 transmembrane helices. Collectively, we assert that the substantial membrane proteome coverage achieved using these procedures will enable rapid advances in the identification and quantitation of novel membrane proteins as markers of differentiation status and/or genetic mutation from relatively low numbers of cultured embryonic stem cells.

Karyotypically normal and abnormal human embryonic stem cell lines derived from PGD-analyzed embryos.

Although a normal karyotype is generally a requirement for stem cell lines, new applications are likely to emerge for stem cells with defined chromosomal aneuploidies. We therefore investigated the use of embryos found to be aneuploid on biopsy followed by preimplantation genetic diagnosis (PGD) with fluorescent in situ hybridization (FISH), and developmentally arrested embryos for stem cell derivation. Eleven stem cell lines were obtained from 41 embryos in 36 cultures, with higher success rate achieved from PGD-analyzed, developmentally advanced embryos (45%) than from clinically unsuitable non-PGD embryos (13%). The resulting stem cell lines were karyotyped, and surprisingly, six of the nine lines from aneuploid embryos as well as both lines from non-PGD embryos were karyotypically normal. Three lines from PGD embryos were aneuploid exhibiting trisomy 5, trisomy 16, and an isochromosome 13, respectively. None of the aneuploid lines presented the same anomally as the original PGD analysis. Our study has three important implications. First, we confirm the ability to produce stem cell lines from PGD-tested embryos as well as developmentally abnormal embryos, offering specialty stem cell lines for research into the clinically important aneuploidies. Second, we observe that stem cell derivation from apparently aneuploid embryos is often thwarted by underlying mosaicism and emerging dominance of the stem cell line by karyotypically normal cells. The corollary, however, is that regular production of normal stem cell lines from developmentally abnormal embryos ordinarity discarded opens a new source of embryos for stem cells, whether for research or for eventual therapeutic use within the donating families.

Variable expressivity of the tumour suppressor protein TRP53 in cryopreserved human blastocysts.

In a mouse model, in vitro fertilization or extended embryo culture leads to the increased expression of TRP53 in susceptible embryos. Ablation of the TRP53 gene improved embryo viability indicating that increased expression of TRP53 is a cause of the reduction of embryo viability resulting from in vitro fertilization or embryo culture. This study investigates the status of TRP53 expression in human embryos produced by intracytoplasmic sperm injection. Following fertilization, embryos were cultured for 96 h and then cryopreserved. Immediately upon thawing they were fixed in formaldehyde and subjected to immunostaining for TRP53. Staining was visualized by confocal microscopy. Negative controls were incubated with isotype control immunoglobulin and showed negligible staining. All embryos showed TRP53 staining above negative controls. TRP53 staining was heterogenous within and between embryos. An embryo that showed retarded development showed high levels of TRP53 expression. A blastocyst that had a collapsed blastocoel also showed high levels of TRP53 compared to morphologically normal blastocysts. Most TRP53 staining was in the region of the nucleus. Morphologically normal blastocysts tended to show little nuclear accumulation of stain. However, some cells within these embryos had high levels of nuclear TRP53 expression. The results show that embryos have varying sensitivity to the stresses of production and culture in vitro, and this resulted in variable expressivity of TRP53.

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. The actual methods of hESC derivation have not changed greatly since the first report by Thomson et al. in 1998 . However, the main emphasis over the last several years has been in finding defined conditions for derivation and culture of hESCs, because to enable the clinical use of hESC for cell transplantation, the use of animal derived biological components is no longer acceptable. For basic research, the aim is to replace even human derived materials with completely defined systems. In this paper we describe methods utilized in our laboratory for hESC derivation and describe two studies conducted in an attempt to improve derivation efficiency and to enable research outcomes to be achieved using fewer embryos.

Flow cytometric sorting of non-human primate sperm nuclei.

Pre-determination of the sex of offspring has implications for management and conservation of captive wildlife species, particularly those with single sex-dominated social structures. Our goal is to adapt flow cytometry technology to sort spermatozoa of non-human primate species for use with assisted reproductive technologies. The objectives of this study were to: (i) determine the difference in DNA content between X- and Y-bearing spermatozoa (ii) sort sperm nuclei into X- and Y-enriched samples; and (iii) assess the accuracy of sorting. Spermatozoa were collected from two common marmosets (Callithrix jacchus), seven hamadryas baboons (Papio hamadryas) and two common chimpanzees (Pan troglodytes). Human spermatozoa from one male were used as a control. Sperm nuclei were stained (Hoechst 33342), incubated and analyzed using a high-speed cell sorter. Flow cytometric reanalysis of sorted samples (sort reanalysis, 10,000 events/sample) and fluorescence in situ hybridization (FISH; 500 sperm nuclei/sample) were used to evaluate accuracy of sorting. Based on fluorescence intensity of X- and Y-bearing sperm nuclei, the difference in DNA content between X and Y populations was 4.09 +/- 0.03, 4.20 +/- 0.03, 3.30 +/- 0.01, and 2.97 +/- 0.05%, for marmoset, baboon, chimpanzee and human, respectively. Sort reanalysis and FISH results were similar; combined data revealed high levels of purity for X- and Y-enriched samples (94 +/- 0.9 and 93 +/- 0.8%, 94 +/- 0.7 and 94 +/- 0.5%, 91 +/- 0.9 and 97 +/- 0.6%, 94 +/- 0.6 and 94 +/- 0.9%, for marmoset, baboon, chimpanzee and human, respectively). These data indicate the potential for high-purity sorting of spermatozoa from non-human primates.