To date, conventional morphological indicators of embryos are still used as the primary rating parameters or criteria. However, standardized and easy-to-use morphological evaluation criteria for human cleavage-stage embryos and blastocysts remain lacking. According to the Istanbul Consensus, morphological scoring systems for cleavage-stage embryos, trophectoderm (TE) cells, and inner cell mass (ICM) include three ratings, namely good, fair, and poor. However, the criteria are straightforward and less detailed, which increases the subjectivity of the embryo evaluation procedure. In this consensus, we provide a scoring system for cleavage-stage embryos and blastocysts in which the evaluative indicators are explained using illustrative images. In addition, this consensus quantifies a variety of evaluative indicators, such as the number of cells in cleavage-stage embryos and the number of TE and ICM cells in blastocysts, thereby improving the operability in practice. Simultaneously, it refines the grade of embryo assessment, defines high-quality and usable embryos, and displays abnormal morphological characteristics of embryos, which facilitates the selection of embryos for transfer. This consensus was initiated by the Chinese Association of Reproductive Medicine and compiled jointly by European and Oceanian embryologists in an effort to further optimize the quality management systems of assisted reproduction laboratories and enhance the performance of assisted reproduction technology.
Morphological indicators for cleavage-stage embryos
The number of blastomeres, degree of fragmentation, cell size, and multinucleation profile of the embryo are the main morphological indicators for scoring cleavage-stage embryos.
The number of blastomeres signifies the developmental rate of the embryo and is considered the most important indicator of embryo scoring. Generally, high-quality cleavage-stage embryos exhibit the appropriate developmental kinetics and synchrony of division.
Embryo fragmentation is strongly associated with pregnancy outcomes. The degree of fragmentation is usually expressed as the percentage of fragmented blastomeres in the total embryo volume, with <10% being preferable. When larger fragments are indistinguishable from blastomeres, a round cytoplasm diameter of <45 μm on day 2 or <40 μm on day 3 is the recommended definition of a fragment [Supplementary Figure 1, https://links.lww.com/CM9/B448].
Theoretically, embryonic mitosis produces daughter cells of equal sizes. Embryos with even cleavage have shown lower rates of multinucleation and aneuploidy, as well as a significantly elevated implantation rate. However, the symmetry and size of all blastomeres also depend on the developmental pattern and specific stage of the embryo, namely the stage-specific cleavage pattern [Supplementary Table 1, https://links.lww.com/CM9/B449].
An embryo is considered multinucleated if more than one nucleus is present in one or more blastomeres. However, the smaller size, larger number, and overlapping of embryonic blastomeres on day 3 would considerably increase the difficulty of observation. Therefore, for day-3 embryos, where multinucleation is difficult to observe, incorporating the observation results of multinucleation on day 2 for embryo scoring is possible.
Scoring criteria for cleavage-stage embryos
The scoring criteria for cleavage-stage embryos on day 2 are presented in Supplementary Table 2, https://links.lww.com/CM9/B449. The superiority hierarchy of grades I, IIa, IIb, III, and IV embryos descends sequentially.
The scoring criteria for cleavage-stage embryos on day 3 are presented in Supplementary Table 3, https://links.lww.com/CM9/B449 and Supplementary Figure 2, https://links.lww.com/CM9/B448. The following points should be noted: (i) Grade I embryos should be 8-cell embryos (day 3) developing from 4-cell embryos (day 2), and these 4-cell embryos have no multinucleation phenomenon. Grade I embryos should also be characterized by clear homogeneous cytoplasm (no vacuolation), tight intercellular junctions, and normal zona pellucida (no darkening, excessive thickness/thinness, abnormal shape, etc). However, the impacts of several abnormal morphological features on embryo development lack robust data support; therefore, these are not included as main scoring indicators. Embryos with any of the abnormal morphological features cannot be assessed as grade I or considered as the preferred choice [Supplementary Figure 3, https://links.lww.com/CM9/B448]; (ii) Relatively even cells are represented by the following equation: (large cell diameter–small cell diameter) / large cell diameter × 100% < 20%; (iii) The superiority hierarchy of grade I, IIa, IIb, IIIa, IIIb, and IV embryos descends sequentially.
Scoring criteria for blastocysts
Blastocyst culture is an effective means for enabling embryo preference and selective single-embryo transfer. Therefore, refining the morphological scoring system for blastocysts is particularly important for accurately assessing their developmental potential. This consensus is based on the Gardner and Schoolcraft blastocyst scoring system, combined with the Consensus on Human in Vitro Fertilization and Embryo Transfer Laboratory Manipulation (2016) developed by Chinese experts. It divides the grading of embryos into six stages according to the expansion and hatching status of the blastocyst and adds a grade D to evaluate ICM cells [Supplementary Tables 4–6, https://links.lww.com/CM9/B449, Supplementary Figure 4, https://links.lww.com/CM9/B448].
Timing of observation
Referring to the Istanbul Consensus, the time points for embryo observation were 44 ± 1 h and 68 ± 1 h after insemination for cleavage-stage embryos on days 2 and 3, respectively, and 116 ± 2 h, 140 ± 2 h, and 164 ± 2 h after insemination for blastocysts on days 5, 6, and 7, respectively. Each center may rationally modulate the timetable for embryo observation according to the characteristics of embryo development and working schedules; however, they should ensure consistency in the daily observation time.
The following is the suggested format for writing cleavage-stage embryo grading: cell number and grade (eg, I/8 or 8I). Since the cell number is the most important indicator for embryo scoring, it is integrated into the grading criteria of this consensus and is also reflected in the writing format.
Notably, not all embryos have visible blastomere nuclei (either mono- or multinucleated) on day 3. The clear presence of mononucleated blastomeres in the embryo and the proportion of mononucleated blastomeres to total blastomeres may be positively correlated with the developmental potential of the embryo and live birth rates. Therefore, this phenomenon can be recorded specifically. To report this, N indicates mononucleated blastomeres, MN indicates multinucleated blastomeres, and a number indicates the number of mono- or multinucleated blastomeres observed. Furthermore, blastomeres without nuclei are not recorded. For example, an 8-cell grade I embryo with six blastomeres observed as mononucleated is recorded as I/86N. Moreover, a 6-cell grade III embryo with one blastomere observed as multinucleated is recorded as III/61MN. This labeling approach may help improve the preferential selection of embryos. However, the presence and number of visible mononucleated blastomeres are not the main reference indicators for embryo assessment.
The writing format for blastocyst grading is relatively consistent, and the scoring system can be further refined by adding a “+” or “−” to the TE and ICM grades, for example, 4B+A and 4BB−. This annotation approach may facilitate the preferential selection of embryos at additional levels; however, it is not used as an inter-laboratory embryo quality control criterion.
Definition of high-quality embryos and blastocysts
Based on the Vienna Consensus combined with the practical experience of multiple centers, this consensus recommends redefining high-quality embryos and usable embryos at the cleavage stage, as shown in Supplementary Table 7, https://links.lww.com/CM9/B449.
All usable embryos can be used for embryo transfer, cryopreservation, or blastocyst culture. The grade I/8 (Day 3) embryos without fragments, developed from I/4 (Day 2) embryos, are defined as optimal embryos and are preferred for transfer. The early compaction of embryos on day 3 is a common phenomenon [Supplementary Figure 5, https://links.lww.com/CM9/B448]. With reference to relevant research combined with the practical experience of multiple centers, this consensus recommends the early compaction of embryos with ≥7 cells and the percentage of fragments <10% to be summarized as high-quality embryos. Additionally, blastocyst culture is recommended for grade IIIb embryos (Day 3), while grade-IV embryos should be discarded and are not recommended for embryo transfer or cryopreservation.
Based on the Gardner and Schoolcraft blastocyst scoring system combined with the practical experience of multiple centers, this consensus recommends redefining high-quality blastocysts and usable blastocysts, as shown in Supplementary Table 8, https://links.lww.com/CM9/B449.
All usable blastocysts can be used for embryo transfer or cryopreservation. The following points are noteworthy: (i) Stage 3 and above low-quality blastocysts graded as CC on day 5/6 could be transferred, cryopreserved, or discarded, depending on the patient's condition and the low-quality blastocyst application strategy of each center; (ii) Early blastocysts that remain at stages 1 or 2 on day 6 are recommended to be cultured until day 7. By this time, if the cells exhibit good refractive properties, no large degenerative foci, and can reach stage 4 and above with a grade of AA, AB, BA, or BB, they can be used for embryo transfer or cryopreservation; (iii) This consensus adds a grade D for ICM to the conventional Gardner blastocyst scoring system, which aims to refine the conventional grade C for ICM. Blastocysts with an ICM grade D, as defined in this consensus, should be discarded to reduce the unnecessary financial burden on patients and the workload of laboratory personnel.
In this consensus, we provide a scoring system for cleavage-stage embryos and blastocysts in wherein the evaluative indicators are explained using illustrative images. It is our hope that these “consensus values” will form a common language for embryologists worldwide to further optimize the quality management systems of assisted reproduction laboratories and improve the performance of assisted reproduction technology.
Conflicts of interest
These guidelines are a reference for reproductive medicine specialists in clinical practice. The Chinese Association of Reproductive Medicine assumes no responsibility for results involving the inappropriate application of these guidelines and reserves the right to interpret and revise the guidelines.
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