Background: Many variations in oocyte and embryo grading make inter-laboratory comparisons extremely difficult. This paper reports the proceedings of an international consensus meeting on oocyte and embryo morphology assessment.
Methods: Background presentations about current practice were given.
Results: The expert panel developed a set of consensus points to define the minimumcriteria for oocyte and embryomorphology assessment.
Conclusions: It is expected that the definition of common terminology and standardization of laboratory practice related to embryo morphology assessment will result in more effective comparisons of treatment outcomes. This document is intended to be referenced as a global consensus to allow standardized reporting of the minimum data set required for the accurate description of embryo development.

The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting

Transcriptional regulation of nanog by OCT4 and SOX2.

Oxygen tension was measured using flexible polarographic microelectrodes within the oviductal and uterine lumen in rhesus monkeys (n = 9), golden hamsters (n = 21) and rabbits (n = 6), during the reproductive cycle (monkey), during oestrus and pseudopregnancy (hamsters, rabbits) and during pregnancy (hamsters). In general, oxygen tensions in each species were much less than half of atmospheric O2, ranging from high values of about 60 mm Hg (8.7% O2) in the rabbit oviduct, rabbit and hamster uterus, to as low as 11 mm Hg (1.5% O2) in the monkey uterus. Oxygen tensions did not vary significantly between left and right sides of the reproductive tracts (all species), nor between pregnant and pseudopregnant states nor between oviduct and uterus (hamsters). Differences owing to reproductive stage were found in the monkey oviduct, hamster oviduct and uterus, and rabbit uterus. Oxygen tensions were consistently very low (11-14 mm Hg) in the monkey uterus throughout the menstrual cycle. In hamsters and rabbits, intrauterine O2 decreased significantly at about the normal time of blastocyst formation and implantation, to 37 mm Hg (5.3% O2) and 24 mm Hg (3.5% O2), respectively. This study indicates that embryos develop in vivo under low oxygen concentrations, especially during the peri-implantation period. The data have implications for investigations of embryo metabolism and for improving embryo development in vitro.

/pdf/oxygen-tension-in-the-oviduct-and-uterus-of-rhesus-monkeys-2jdlhsv14m.pdf

Oxygen tension in the oviduct and uterus of rhesus monkeys, hamsters and rabbits

The objective of the research described was to devise an efficient procedure to cryopreserve in vitro-matured bovine oocytes, using in vitro fertilization (IVF) and development of resultant zygotes into blastocysts as criteria of oocyte survival. Oocytes at metaphase II were found to be extremely sensitive to chilling. Cooling them to O degrees C for as little as 5 sec significantly decreased their capability to cleave and develop further after IVF; after 80 sec at 0 degrees C, only approximately 10% of chilled oocytes developed into blastocysts. Oocytes were also adversely affected by brief exposures to 4 M and 5.5 M ethylene glycol (EG) solutions supplemented with sucrose; after being suspended in either of these EG solutions in plastic straws and plunged directly into liquid nitrogen (LN2), few of the oocytes were fertilized and developed. To "outrace" chilling injury, oocytes contained in < 1 microliter of EG solution were placed onto electron microscope grids and plunged directly into N2 slush or LN2. After such ultra-rapidly cooled oocytes were warmed, 30% of them cleaved after IVF, and half of these developed into blastocysts-- survival rates equivalent to those for oocytes that had been exposed to EG without any cooling. This method offers promise as a novel way to cryopreserve bovine oocytes.

/pdf/development-into-blastocysts-of-bovine-oocytes-cryopreserved-2w1ugynzlg.pdf

Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling.

Type 1 diabetes (T1D) is the result of an autoimmune destruction of pancreatic β cells. The cellular and molecular defects that cause the disease remain unknown. Pluripotent cells generated from patients with T1D would be useful for disease modeling. We show here that induced pluripotent stem (iPS) cells can be generated from patients with T1D by reprogramming their adult fibroblasts with three transcription factors (OCT4, SOX2, KLF4). T1D-specific iPS cells, termed DiPS cells, have the hallmarks of pluripotency and can be differentiated into insulin-producing cells. These results are a step toward using DiPS cells in T1D disease modeling, as well as for cell replacement therapy.

/pdf/generation-of-pluripotent-stem-cells-from-patients-with-type-4t1as51n7o.pdf

Generation of pluripotent stem cells from patients with type 1 diabetes.

Unfertilized mouse oocytes and eggs 1-8 h after fertilization in vitro were examined at the light microscope level for structural changes, distribution of actin (as assessed by both antiactin antibodies and NBD-phallicidin), surface binding of concanavalin A (Con A) and chromosomal distribution and condensation. The influence of cytochalasin D on these events was also assessed. Changes in actin distribution were associated with rotation of the second anaphase spindle, formation of the second polar body, the events following incorporation of the sperm nucleus, and formation and migration of the pronuclei. Cytochalasin D prevented spindle rotation, polar body formation, pronuclear migration and the restoration of Con A binding over the site of sperm entry and the site of polar body formation, but did not affect sperm fusion and entry, the loss of Con A binding at the site of sperm entry and pronuclear formation.

Changes in actin distribution during fertilization of the mouse egg

The effect of cooling on the organization of the microtubule system of the mouse oocyte has been investigated. Cooling to 25, 18 or 4 degrees C for varying periods of time resulted in a progressive disassembly of the spindle and the dispersal of the chromosomes. The extent of the changes observed was greater at lower temperatures and with longer periods of exposure. Transfer of oocytes from either 37 or 4 to 24 degrees C resulted in the rapid and transient appearance of polar asters and of multiple cytoplasmic asters associated with the pericentriolar material present at the spindle poles and in the cytocortex of the oocyte. This transient aster formation appeared to be driven by the elevated levels of free tubulin released during spindle disassembly. An apparent reversal of many of the changes induced by low temperature was observed in many but not all oocytes on their restoration to 37 degrees C for 1 h. These results have implications for our understanding of microtubule organization in the oocyte, and for the handling of oocytes during IVF and GIFT therapeutic procedures and during the cryopreservation of oocytes.

The influence of cooling on the oranization of meiotic spindle of the mouse oocyte

Preimplantation genetic diagnosis (PGD) is an evolving technique that provides a practical alternative to prenatal diagnosis and termination of pregnancy for couples who are at substantial risk of transmitting a serious genetic disorder to their offspring. Samples for genetic testing are obtained from oocytes or cleaving embryos after in vitro fertilization. Only embryos that are shown to be free of the genetic disorders are made available for replacement in the uterus, in the hope of establishing a pregnancy. PGD has provided unique insights into aspects of reproductive genetics and early human development, but has also raised important new ethical issues about assisted human reproduction.

Preimplantation genetic diagnosis.

When mouse ovulated oocytes were exposed to 1.5 M-dimethylsulphoxide (DMSO) the resultant hardening of the zona pellucida was not a direct effect but required the presence of an oocyte. The hardening of the zona pellucida when zonae used were aged in vitro was also dependent upon the presence of the oocyte. Protocols of DMSO exposure that induce zona-hardening also caused depletion of the numbers of cortical granules underlying the oocyte surface, whereas protocols without effect on the zona did not reduce significantly the cortical granule count. It is proposed that the effects of DMSO may be mediated by a release of cortical granule contents.

/pdf/the-hardening-effect-of-dimethylsulphoxide-on-the-mouse-zona-1g19k0ahr4.pdf

The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present.

The effect of dimethylsulphoxide (DMSO) on the organization of the microtubular system of the mouse oocyte has been examined. Exposure to DMSO causes the immediate appearance of multiple, cold-resistant microtubular asters associated with the foci of pericentriolar material (PCM) normally present in the oocyte. More prolonged exposure to DMSO leads to progressive disassembly of the spindle, and as a result dispersal of the chromosomes and polar PCM foci occurs, and tubulin polymerization becomes confined to PCM-organized asters. Those astral microtubules located between the PCM foci and the cortex of the oocyte appear to be particularly stable, resulting in the development of lengthening radial bundles of microtubules between the PCM and the surface and the progressive movement of the PCM foci towards the centre of the cell. In contrast, after activation of the oocyte the microtubules generated in the presence of DMSO remain located in a cortical mesh. The effects of DMSO do not appear to be fully reversible in most oocytes. We discuss the implications of these results both for the cytoplasmic organization of the oocyte and zygote, and for the attempts at cryopreservation of human oocytes for therapeutic use in infertility programmes.

Susan J. Pickering

Papers

Changes in actin distribution during fertilization of the mouse egg

The influence of cooling on the oranization of meiotic spindle of the mouse oocyte

Preimplantation genetic diagnosis.

The hardening effect of dimethylsulphoxide on the mouse zona pellucida requires the presence of an oocyte and is associated with a reduction in the number of cortical granules present.

The effect of dimethylsulphoxide on the microtubular system of the mouse oocyte