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 minimum criteria for oocyte and embryo morphology 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.

Three-dimensional cytoskeletal organization of detergent-treated epithelial African green monkey kidney cells (BSC-1) and chick embryo fibroblasts was studied in whole-mount preparations visualized in a high voltage electron microscope. Stereo images are generated at both low and high magnification to reveal both overall cytoskeletal morphology and details of the structural continuity of different filament types. By the use of an improved extraction procedure in combination with heavy meromyosin subfragment 1 decoration of actin filaments, several new features of filament organization are revealed that suggest that the cytoskeleton is a highly interconnected structural unit. In addition to actin filaments, intermediate filaments, and microtubules, a new class of filaments of 2- to 3-nm diameter and 30- to 300-nm length that do not bind heavy merymyosin is demonstrated. They form end-to-side contacts with other cytoskeletal filaments, thereby acting as linkers between various fibers, both like (e.g., actin- actin) and unlike (e.g., actin-intermediate filament, intermediate filament-microtubule). Their nature is unknown. In addition to 2- to 3-nm filaments, actin filaments are demonstrated to form end-to-side contacts with other filaments. Y-shaped actin filament "branches" are observed both in the cell periphery close to ruffles and in more central cell areas also populated by abundant intermediate filaments and microtubules. Arrowhead complexes formed by subfragment 1 decoration of actin filaments point towards the contact site. Actin filaments also form end-to-side contacts with microtubules and intermediate filaments. Careful inspection of numerous actin-microtubule contacts shows that microtubules frequently change their course at sites of contact. A variety of experimentally induced modifications of the frequency of actin-microtubule contacts can be shown to influence the course of microtubules. We conclude that bends in microtubules are imposed by structural interactions with other cytoskeletal elements. A structural and biochemical comparison of whole cells and cytoskeletons demonstrates that the former show a more inticate three-dimensional network and a more complex biochemical composition than the latter. An analysis of the time course of detergent extraction strongly suggests that the cytoskeleton forms a structural backbone with which a large number of proteins of the cytoplasmic ground substance associate in an ordered fashion to form the characteristic image of the "microtrabecular network" (J.J. Wolosewick and K.R. Porter. 1979. J. Cell Biol. 82: 114-139).

The relationship between the ATP content of mature human oocytes and developmental potential after uterine transfer of sibling embryos was examined in 20 non-male factor in-vitro fertilization (IVF) patients matched for age, fertility history, ovarian stimulation protocol, oocyte quality and number, stage and morphology of embryos at uterine transfer. ATP content was determined for uninseminated and unfertilized oocytes for each cohort, and for embryos that developed after dispermic fertilization or which showed significant fragmentation. The effect of reduced ATP content on meiotic maturation, fertilization and preimplantation development was examined in cultured mouse oocytes treated with uncouplers of mitochondrial oxidative phosphorylation. The results demonstrated that meiotic maturation occurs in both mouse and human oocytes over a wide range of ATP contents, and that the ATP content of normal-appearing, metaphase II human oocytes can differ significantly between cohorts; however, a higher potential for continued embryogenesis and implantation in the human is associated with embryos that develop from cohorts of oocytes with ATP contents ≥2 pmol/oocyte. The findings are discussed with respect to possible aetiologies and developmental consequences for embryonic development of different oocyte ATP contents, and the extent to which mitochondrial function may determine or influence the continued developmental capacity of embryos which appear normal and developmentally viable at the early cleavage stages.

Regardless of whether fertilization occurs in vivo or in vitro, a large proportion of human embryos do not develop progressively through the pre-implantation stages or arrest development after implantation. This study examined the association between the chromosomal/spindle normality of the mature human oocyte and the dissolved oxygen content, vascular endothelial growth factor concentration (VEGF) and perifollicular blood flow characteristics of the corresponding ovarian follicles. Findings from >1000 samples of follicular fluid show that developmentally significant differences in dissolved oxygen content occur in follicular fluids aspirated from follicles of equivalent size and ultrasonographic appearance. Oocytes from severely hypoxic follicles were associated with high frequencies of abnormalities in the organization of the chromosomes on the metaphase spindle that could lead to segregation disorders and catastrophic mosaicisms in the early embryo. Oocytes with cytoplasmic defects and cleavage stage embryos with multinucleated blastomeres are derived predominantly from severely hypoxic follicles. VEGF measurements of follicular fluid and colour pulsed Doppler ultrasonographic analysis of follicle-specific blood flow characteristics indicated a potentially important role for this factor both in perifollicular angiogenesis and in the regulation of intrafollicular oxygen levels. The results are discussed with respect to how severe intrafollicular hypoxia may influence the normality of chromosomal organization and segregation in the oocyte, and whether detailed pulsed Doppler analysis of individual pre-ovulatory follicles may provide an indirect indication of the 'health' of the follicle and possibly the developmental competence of the corresponding oocyte.

Mitochondria are the most abundant organelles in the mammalian oocyte and early embryo. While their role in ATP production has long been known, only recently has their contribution to oocyte and embryo competence been investigated in the human. This review considers whether such factors as mitochondrial complement size, mitochondrial DNA copy numbers and defects, levels of respiration, and stage-specific spatial distribution, influence the developmental normality and viability of human oocytes and preimplantation-stage embryos. The finding that mitochondrial polarity can differ within and between oocytes and embryos and that these organelles may participate in the regulation of intracellular Ca2+homeostasis are discussed in the context of how focal domains of differential respiration and intracellular-free Ca2+regulation may arise in early development and what functional implications this may have for preimplantation embryogenesis and developmental competence after implantation.