In order to be able to describe the processes of preimplantation development in vitro, embryologists need to conduct a detailed assessment of the gametes and embryos they work with. According to international criteria and consensus decisions, specialists do not only choose the optimal gamete fertilization protocol, but also the most promising embryos for transfer or cryopreservation.
For each IVF attempt, an embryological protocol is filled out, in which the fate of each embryo is reflected from the moment of oocytes receiving to the moment of transfer or freezing. The main criterion for quality assessment is the morphological one – a description of the embryo appearance.
Day 0 is the day of fertilization. As a result of the ovarian puncture, it is possible to obtain mature, immature, as well as degenerative and destroyed cells. An accurate assessment of the oocyte maturity state is possible only after its denudation before the ICSI procedure. In mature, ready-to-fertilize oocytes, the first polar body is determined. In the embryological protocol, a mature oocyte is specified as MII. If the oocyte maturation process in the follicle is disrupted for internal or external reasons, then there is a high probability of immature cells obtaining – MI and GV. Complete oocyte atresia (Atr) is also possible.
Day 1. 16-20 hours after the addition of spermatozoa in the IVF protocol or after the ICSI procedure, during normal fertilization, two pronuclei are formed (areas of the cytoplasm containing the parents’ DNA, which are visible in the microscope). These are the precursors of the future cells’ nuclei (blastomeres). Pronuclei contain genetic material – half of the chromosomes from the maternal genome, and half from the paternal one, so there should be two pronuclei, ideally of equal size. With normal fertilization, both pronuclei are clearly distinguishable. In this case, they are assigned with a score of 2pN. If the pronuclei are not visible, which is usually due to the fertilization absence, 0pN is recorded in the protocol. With abnormal fertilization, several pronuclei may appear, for example, 3pN, 4pN, etc. Such zygotes are not suitable for further work and are discarded because they contain insufficient or excessive amounts of DNA that cannot ensure normal development of the embryo.
Day 2-5. Over the next few days, further development of the embryo (cleavage) occurs. There is a large number of different cleavage stage classifications, but not all of them have clinical significance. According to the generally accepted international criteria of ASEBIR&ESHRE, embryo cleavage should be symmetrical (resulting in blastomeres of the same size) and uniform (all blastomeres undergo division). For purposes of visual expression, embryologists use a numerical – letter quality assessment system, where the number means the number of blastomeres, and the letter means their quality. To label an embryo of good quality, the letter “A” is used, for fair quality there will be letter “B”, and for poor quality the letter “C” is used.
For the second day of culture, embryos with four blastomeres (4A, 4B) are considered promising. For the third day they should be eight-cell (8A, 8B). Such embryos are the most promising for further development and transfer. Embryos of poor quality (C grade), usually are not used for embryo transfer. The embryo evaluation is influenced by various parameters of its morphology, such as stadium–specific cleavage and fragmentation. It is believed that for the cleavage stage, presence of fragmentation of less than 25 % does not affect the further embryo development being a feature of human preimplantation development. Normally, each blastomer carries one nucleus, if more than one nucleus is visualized in at least one blastomer, this is called multi-nucleation and indicates a significant probability of chromosomal pathology of this embryo.
By the end of the third and fourth days of culture, the embryo begins to compact (the boundaries of its cells become indistinguishable) and prepares to formate a blastocyst.
On the fifth day, approximately 120 hours after fertilization, the embryo forms a blastocyst (Bl). The blastocyst quality assessment is carried out in accordance with Gardner’s criteria, which implies a numerical expression of its size (in numbers from 1 to 6); a letter description of the inner cell mass (ICM) (from “A” to “C”) and the surrounding trophoblast cells (from “A” to “C”). The best for transfer will be blastocysts of size from 3 to 5, having a multicellular ICM and trophoblast – Bl4AA, Bl4AB, for example. Blastocysts of fair quality with rating below Bl3BB have reduced chance of implantation.
As a conclusion, it should be noted that the described morphological criteria for embryo quality assessment are basic, necessary, but not always sufficient for choosing an embryo for transfer. So, often after the transfer of good quality embryos, implantation does not occur and, conversely, there are cases of pregnancy onset and successful development after the transfer of embryos of poor quality. In most cases, this situation is caused by genetic status of the embryo, which we can determine with help of PGT-A.