IVF – embryo development before transfer

Stages of embryo culture in the ivf procedure

During the first few days following the oocyte fertilization procedure, the resulting cell begins to undergo changes, during which the individual stages of embryo development can be distinguished:

• Zygote (Day 1) – in other words, a fertilized egg cell, which forms 17–20 hours after the sperm is introduced into the gamete. It undergoes numerous divisions (a process known as mitosis), during which the resulting embryo increases its number of cells. It is estimated that after approximately 3 days, it should develop into an 8-blastomere embryo.
• Morula (Day 4) – consists of multiple cells, known as blastomeres (approximately 12–16), which should join together.
• Blastocyst (Day 5/6) – an embryo consisting of more than 100 cells. At this stage, the membrane surrounding it ruptures as a result of the blastocyst's growth; once it breaks, the embryo is released and is capable of implanting in the uterus. Sometimes, to support the blastocyst hatching process (when the membrane is thickened), the AH (Assisted Hatching) procedure is used, which involves making a laser incision in the membrane.

Embryo culture as part of the IVF procedure is usually continued until Day 5–6, although in some cases it may be extended to Day 7 due to difficulties in clearly assessing the embryo’s developmental potential. However, such cases are rare.

IVF and assessment of embryo development

Before the embryos are transferred, embryologists regularly monitor their development and assign them to specific categories that determine their quality and readiness for transfer into the uterine cavity. The assessment usually takes into account aspects such as:

• rate of development,
• size and shape of the blastomeres (i.e. embryo cells),
• degree of fragmentation.

The embryo is assessed during each stage of development, with the exception of Day 4, as this is the compaction stage, during which it is difficult to distinguish individual blastomeres. The following are considered normal:

• Day 2 – the embryo already consists of 4 blastomeres (daughter cells).
• Day 3 – an 8-blastomere embryo is expected; however, each embryo develops individually, and at this stage embryos often have a different number of cells, e.g. 6 or 10, while still remaining within the range of normal development.

The assessment of the morphology of Day 2 and Day 3 embryos takes into account the number of blastomeres and the evaluation of morphological parameters:

• A. – good: <10% fragmentation, blastomere size appropriate for the number of cells, no signs of multinucleation.
• B. – average: 10–25% fragmentation, most embryo cells are of the optimal size for the given stage, no signs of multinucleation.
• C. – poor: >25% fragmentation, considerable variation in cell size, signs of multinucleation present in the blastomeres.

• Day 4 – at the morula stage, a normally developing embryo already consists of more than a dozen cells, which are additionally joined together (the degree of joining, referred to as compaction, is also taken into account during the assessment). At this stage, a three-grade scale is also used.
• Day 5–6 – however, the greatest attention is paid to blastocysts, i.e. embryos on Days 5–6 of development. They are usually assessed using the Gardner method, which involves a six-grade quality classification. A number (from 1 to 6) is used to describe the stage of development they have reached:

• 1 – early blastocyst
• 2 – mature blastocyst
• 3 – expanded blastocyst
• 4 – hatching blastocyst
• 5 – blastocyst undergoing hatching
• 6 – hatched blastocyst

Additionally, they are further classified using letters (A–C), indicating the grade of development and arrangement of the outer cells of the inner cell mass, i.e. the part from which the embryo itself will develop:

• A – many cells tightly packed together
• B – several cells loosely arranged
• C – very few, scattered cells

and the trophectoderm, which will develop into the placenta:

• A – many cells joined together
• B – few cells forming a loose structure
• C – individual cells of relatively large size