The embryologist’s daily bread, or a few words about the new standards of cell and embryo assessment recommended by the Polish Society of Reproductive Medicine and Embryology for assisted reproduction laboratories..
A part of the daily work of embryologists is the assessment of the development of embryos at individual stages of their incubation. The assessment criteria take into account the size and shape of cells inside an embryo as well as the degree of fragmentation (anuclear cytoplasmic structures, forming in the process of cell division). The comprehensive assessment is most often carried out on culture day 1, 3 and 5 in the morning hours. This aims to minimize the external intervention into the conditions prevailing in the embryos’ environment.
Depending on the day of culture, various stages of embryo development are assessed:
- Day 1: egg fertilization, approx. 17 hours from the event
- Day 3: approx. 68 hours, expected 8-blastomere embryo
- Day 4: approx. 92 hours, expected morula-stage embryo
- Day 5: approx. 116 hours, expected blastocyst
Oocyte assessment
The assessment of an egg cell is carried out already at the time of its isolation from follicular fluids. According to the new standards recommended by the Polish Society of Reproductive Medicine and Embryology, the assessment of the cumulus-oocyte complex (COC) has been introduced. The analysis in the following form is propounded:
COC- 1: good quality cumulus with numerous, well developed and evenly distributed radiating corona cells. It can be presumed with high probability that the complexes will contain a mature egg cell.
COC -0: all others which do not meet the above criteria.
The detailed and precise assessment of cells is carried out after a cell has been cleaned of the surrounding granulosa layer (so-called denudation). An embryologist assesses the cell for its morphology, and every parameter which deviates from average values is recorded. The analysis includes, but is not limited to the presence of vacuoles, size of the first polar body.
Normal, mature oocyte (MII stage)
Oocyte with vacuoles
Oval-shaped oocyte with the larger than average perivitelline space
Fertilization assessment
On day one, after about 17 hours from performing the fertilization procedure the embryologist assesses its effectiveness by observing the fertilization markers:
- 1PN
- 2PN
- 3PN
- >3PN
PN is an abbreviation of the term pronucleus, and the digit denotes their number. On day one, a properly fertilized oocyte should have two pronuclei = 2PN
1PN
2PN
3PN
In addition, the most recent guidelines take into account also the type of distribution of pronuclei:
Type 1 – symmetrical distribution of 2PN
Type 2 – asymmetrically distributed pronuclei
Type 3 – pronuclei with one or no precursor body
Evaluation of embryo development
During day two and three of culture, the embryo’s cell divisions are analyzed. The assessment concerns the number of blastomeres and the degree of fragmentation. The assessment is carried out by the blastomere’s size.
Degrees of fragmentation are divided into three groups:
- 1: mild <10% of the embryo volume
- 2: moderate 10-25% of the embryo volume
- 3: severe >25% of the embryo volume
How does it look like in practice? For example:
Cl. 4.1: the embryo has four blastomeres, fragmentation is below 10%, the size of blastomeres is adequate to the number of cells, no features of multinuclearity (according to the former nomenclature: 4A)
Cl. 2.2: the embryo has two blastomeres, moderate degree of fragmentation, the size of most blastomeres is optimal for a given stage, no features of multinuclearity (according to the former nomenclature: 2B)
Cl.8.3: the embryo has 8 blastomeres, fragmentation exceeds 25% of the embryo’s volume, cell size highly differentiated, blastomeres with features of multinuclearity (according to the former nomenclature: 8C)
| embryo | old | new |
|---|---|---|
| 2 blastomeres | 2A | CI.2.1 |
| 2B | CI.2.2 | |
| 2C i 2D | CI.2.3 | |
| 4 blastomeres | 4A | CI.4.1 |
| 4B | CI.4.2 | |
| 4C i 4D | CI.4.3 | |
| 6 blastomeres | 6A | CI.6.1 |
| 6B | CI.6.2 | |
| 6C i 6D | CI.6.3 | |
| 8 blastomeres | 8A | CI.8.1 |
| 8B | CI.8.2 | |
| 8C i 8D | CI.8.3 |
On day 4, the embryo should reach the stage of morula (blastomeres begin to compact). The quality of embryos is assessed according to a 3-grade scale:
M.1- good: full compaction of all blastomeres (former nomenclature: P8A)
M.2- fair: most embryo blastomeres are compacted (former nomenclature: P8B)
M.3- słaby: kompaktacja niepełna, ograniczająca się do mniejszej niż połowa objętości zarodka, obecne dwa, trzy wolne blastomery lub brak kompaktacji (poprzednia nomenklatura: P8C, P8D)
Prawidłowo rozwijające się zarodki w 5. i 6. dniu hodowli powinny osiągnąć stadium blastocysty. Ich ocena prowadzona jest według nomenklatury Gardnera (Gardner and Schoolcraft, 1999). Zgodnie z wytycznymi Polskiego Towarzystwa Medycyny Rozrodu i Embriologii morfologia trofoektodermy i węzła zarodkowego została zastąpiona wartością liczbową (1 – odpowiednik klasy A, 2 – klasy B, 3 – klasy C).
| embryo | old | new |
|---|---|---|
| blastocyst | 1AA | BI.1.1.1 |
| 1AB | BI.1.1.2 | |
| 1CC | BI.1.3.3 | |
| 2AA | BI.2.1.1 | |
| 2BA | BI.2.2.1 | |
| 2CB | BI.2.3.2 | |
| 3AC | BI.3.1.3 | |
| 3BB | BI.3.2.2 | |
| 3CC | BI.3.3.3 | |
| 4CA | BI.4.3.1 | |
| 4BA | BI.4.2.1 | |
| 4AC | BI.4.1.3 |
Bl.1.2.3
Bl.2.1.1
Bl.3.1.2
Bl.4.1.1
Bl.5.2.2
Kiedy zarodek jest TQ, a kiedy NTQ?
Zarodki TQ to zarodki wykazujące prawidłowe tempo rozwoju, których morfologia w 2., 3., 4. dniu hodowli dostała klasę 1. lub 2.
W 5. dobie zarodkiem TQ jest blastocysta na poziomie rozwojowym minimum 3 z dodatkową klasą trofoektodermy i węzła zarodkowego minumium 1 lub 2 (dawniej A lub B).
TQ- top quality (spełniający dobre kryteria morfologiczne).
Cl.8.1 w dobie 3.
Bl.4.1.2 w dobie 5.
NTQ- non- top quality ( non-top quality)
Cl.4.3 w dobie 2.
C l.12.2 w dobie 4.
Bl.2.3.3 w dobie 5.
Na koniec zarodek- smutas (prawidłowy zarodek w pierwszej dobie hodowli. Widoczne dwa przedjądrza).
