Divergent national strategies define the maximum number of embryos that can be transferred in any one cycle, and elective single embryo transfer (eSET) is recommended for selected patient populations, in order to decrease the incidence of multiple births associated with ART.
In routine practice, embryo grading for transfer continues to be based primarily on morphological assessment, often with multiple observations over the course of the embryo’s development. Disturbance to the embryo culture system should be minimized, with assessment no more than once per day; some laboratories skip assessment on Day 2 or Day 4. Embryo development should also be checked within a period that is appropriate to IVF laboratory workload.
Routine morphological assessment is highly subjective, with both inter- and intra-observer variation repeatedly documented. In addition, embryo development is affected by differences in culture media and environment, as well as by handling procedures. These two factors make it difficult to compare embryo scores/success rates between different units, and embryo assessment procedures should be validated both within laboratories and for each individual carrying out the assessment. Online external quality control schemes provide an extremely useful tool for training and validation procedures.
For cleavage-stage embryos, assessment criteria include:
Rate of division judged by the number of blastomeres,
Size, shape, symmetry and cytoplasmic appearance of the blastomeres presence of a nucleate cytoplasmic fragments,
Appearance of the zona pellucida.
Criteria are frequently combined to produce composite scoring systems which may incorporate pronuclear scoring of zygotes. Although morphological assessment is recognized to be highly subjective, arbitrary and unsatisfactory, it is quick, noninvasive, easy to carry out in routine practice, and does help to eliminate those embryos with the poorest prognosis. Evaluation of blastomere shape, size and number will reflect synchronous cleavage of the blastomeres, and embryos with asynchrony in either the timing of cleavage or the process of blastomere division will be given lower scores.
Unfortunately, embryo cleavage in vitro rarely follows the postulated theoretical timing of early development, and computer-assisted morphometric analyses confirm that large variations in blastomere size and fragmentation are frequently observed. Large variations in blastomere size have been linked to increased chromosomal errors.
The timing of the assessment is crucial, as pronuclear development is a dynamic process, and therefore zygote assessment should be used with caution and only in combination with other assessment methods.
Timing of the first cell division of the embryo has been investigated as a predictor of developmental competence, with the suggestion that “early cleavage” is associated with higher pregnancy rates. However, there is only a certain window where true early cleavage can be seen, and this extra assessment may be difficult to fit into the normal routine of the IVF laboratory. Once the embryos have undergone the second division to the four-cell stage, those that might have cleaved a few hours earlier will have similar morphology to those that did not, and it is not possible to differentiate between them.
Multinucleated blastomeres can sometimes be observed at early cleavage stages, most easily on Day 2. Studies have revealed that blastocysts developing from embryos showing multinucleation at early cleavage stages have similar aneuploidy rates to blastocysts that resulted from embryos that did not have multinucleation. Transfer of binucleated and multinucleated frozen-thawed embryos does not apparently increase the incidence of congenital anomalies and chromosomal defects in newborns. Aneuploidy activates a spindle-apparatus checkpoint in different types of cells, and it is possible that blastomere multinucleation may represent the activation of a cell cycle checkpoint that can convert a mosaic embryo to one that is euploid.
Cumulative Scoring Systems
Multi-day scoring systems can enhance embryo selection by combining both developmental rate and morphological assessment. These should provide a more accurate picture of developmental progression than can be obtained from a single observation. However, the ultimate combination of morphological features required for optimal evaluation of developmental competence has yet to be resolved. The optimal timing of embryo transfer will be more accurately determined when agreement on this is reached. In addition, multi-day assessment requires repeated embryo handling and exposure to potentially hazardous conditions outside of the incubator.
Fragmentation in the human embryo is very common, affecting up to 75% of all embryos developed in vitro. It is not clear whether this is an effect of culture conditions and follicular stimulation, or a characteristic of human development.
Extensive fragmentation is known to be associated with implantation failure, but the relationship between the degree of fragmentation and the developmental potential of the embryo is far from clear. Researchers have found that when embryos with more than 15% fragmentation were cultured to the blastocyst stage, they formed fewer morulae, fewer cavities and fewer blastocysts compared to those embryos with less than 15% fragmentation. When fragmentation was greater than 35%, all processes were compromised. Retrospective analysis of embryo transfer data revealed that nearly 90% of embryos selected for transfer were developed from embryos with less than 15% fragmentation observed on Day 3.
They have used an analysis of patterns of cell fragmentation in the human embryo as a means of determining the relationship between cell fragmentation and implantation potential, with the conclusion that not only the degree, but also the pattern of embryo fragmentation determine its implantation potential. Five distinct patterns of fragmentation which can be seen by Day 3 were identified:
Type I: <5% of the volume of the perivitelline space (PVS) occupied by fragments.
Type II: Small localized fragments associated with one or two cells.
Type III: Small, scattered fragments associated with multiple cells.
Type IV: Large, scattered fragments associated with several unevenly sized cells and scattered throughout the PVS.
Type V: Fragments throughout the PVS, appearing degenerate such that cell boundaries are invisible, associated with contracted and granular cytoplasm.
Some embryos have no distinct pattern of fragmentation.
No definite cause of fragmentation has been identified, although speculations include high spermatozoa numbers and consequently high levels of free radicals, temperature or pH shock, and stimulation protocols. Apoptosis has been suggested as a possible cause, and a progressive shortening of telomere length, which induces apoptosis, has been linked with fragmentation. However, this study was not definitive. Mitotically inactive cells do not exhibit fragmentation, and it has been suggested that aberrant cytokinesis in the presence of the spindle and cyto-skeletal abnormalities may be associated with fragmentation.
Observed via the scanning electron microscope, the surface of fragments is made up of irregularly shaped blebs and protrusions, very different from the regular surface of blastomeres, which is organized into short, regular microvilli.
Interestingly, programmed cell death in somatic cells also starts with surface blabbing, and is caused, in part, by a calcium-induced disorganization of the cytoskeleton. We can speculate that similar mechanisms operate within human embryos, but there is so far no scientific evidence that this is the case.
There does appear to be an element of programming in this partial embryonic auto-destruction, as embryos from certain patients, irrespective of the types of procedure applied in successive IVF attempts, are always prone to fragmentation. Surprisingly, fragmented embryos, whether reconstructed or not, make implants and often come to their senses. Time-lapse photography technology has clearly demonstrated that an individual embryo can radically change its morphological appearance in a short period of time: fragments that are apparent at a particular moment can be subsequently absorbed with no evidence of their prior existence. This demonstrates the highly regulated nature of the human embryo, as it can apparently lose over half of its cellular mass and still recover, and also confirms the consensus that the mature oocyte contains much more material than it needs for development.
The reasons why part and only part of the early embryo should become disorganized and degenerate remain a mystery. Different degrees of fragmentation argue against the idea that the embryo is purposely casting off excess cytoplasm, somewhat analogous to the situation in annelids and marsupials that shed cytoplasmic lobes rich in yolk, and favor the idea of partial degeneration.
Perhaps it involves cell polarization, where organelles gather to one side of the cell. It is certain that pH, calcium and trans-cellular currents trigger cell polarization, which may in certain cases lead to an abnormal polarization, and therefore to fragmentation. Describing fragmentation as a degenerative process may not be justified, but more research is needed to elucidate whether the implantation of embryos with extensive fragmentation at the cleavage stages has any long-term effects.
Embryo Grading: Cleavage Stages
Despite the introduction of extended culture and sophisticated monitoring and genetic screening technologies, a significant number of apparently high-quality and chromosomally normal embryos still do not implant, and selecting embryos for transfer remains an ongoing challenge in routine clinical IVF.
After more than 40 years, and the birth of close to 10 million babies, the basic selection criteria in a routine IVF laboratory without research facilities continue to be based on assessment of morphology, despite numerous attempts to find new biomarkers and variables. Preimplantation genetic screening has shown a surprising discrepancy between gross morphology and genetic normality of embryos.
Even the most “beautiful” Grade 1 embryos may have numerical chromosomal anomalies, whilst those judged to be of “poorer” quality, with uneven blastomeres and fragments, may have a normal chromosome complement. The variables that continue to prove consistently useful as predictors of viability remain: numbers of cells and fragmentation, cell symmetry and nucleation.
Even, regular spherical blastomeres,
Moderate refractivity (i.e., not very dark),
No, or very few, fragments (less than 10%).
Allowance should be made for the appearance of blastomeres that are in division or that have divided asynchronously with their sisters, e.g., three-, five-, six- or seven-cell embryos, which may be uneven. As always, individual judgment is important, and this is a highly subjective assessment.
Uneven or irregularly shaped blastomeres,
Mild variation in refractivity,
No more than 10% fragmentation of blastomeres.
Fragmentation of no more than 50% of blastomeres,
Remaining blastomeres must be at least in reasonable (Grade 2) condition,
Refractility associated with cell viability,
Intact zona pellucida.
More than 50% of the blastomeres are fragmented,
Gross variation in refractivity,
Remaining blastomeres appears viable.
Zygotes with two pronuclei on Day 2 (delayed fertilization or re-insemination on Day 1).
Nonviable: fragmented, lysed, contracted or dark blastomeres,
No viable cells.
It is important to bear in mind that the time during which the assessment and judgment are made represents only a tiny instant of a rapidly evolving process of development. Embryos can be judged quite differently in two different time periods, which can be seen if a comparison is made between estimates made in the morning and later on the same day, immediately before the transfer.
Individual judgment should be exercised in determining which embryos are selected. In general, those embryos at later stages and of higher grades are preferred, but the choice is often not clear-cut. The Grade 2 category covers a wide range of morphological states but, provided the blastomeres are not grossly abnormal, a later stage Grade 2 embryos may be selected in preference to an earlier stage Grade 1 embryo. Attention should also be paid to the appearance of the zona pellucida and to the pattern of fragmentation. Embryos of Grade 3 or 4 are transferred only where no better embryos are available. If only pronucleate embryos are available on Day 2, they should be cultured further and transferred only if cleavage occurs.