Oocyte and Embryo Grading blastocyst grading.pptx

oocyte grading, zygote scoring, cleavage stage embryo grading, blastocyst grading

1. Oocyte and Embryo Grading R. DEEPTHI IVF LAB DIRECTOR MOHAK IVF

2. STRUCTURE OF OOCYTE

3. OOCYTE

4. OOCYTE GRADING Based on the cytoplasmic and nuclear maturity of oocyte 1. Immature 2. Partially mature 3. Mature 4. Post mature/Luteinized 5. Atretic

5. Immature Dense compact cumulus, very adherent layers of corona cells, Ooplasm if visible with the presence of germinal vesicle, Compact and non aggregated membrana granulosa cells

6. Immature

7. Partially mature Expanded cumulus mass, slightly compact corona radiata, expanded well aggregated membrana granulosa cells. Ooplasm has no germinal vesicle and no extruded polar body

8. Partially mature

9. Mature Expanded cumulus, very radiant corona, distinct zona pellucida, clear ooplasm, expanded well aggregated membrana granulosa cells. Ooplasm extrudes 1st polar body

10. Mature

11. Post Mature Much expanded cumulus with clumps, radiant corona radiata yet often clumped, irregular or incomplete, very visible zona, slightly granular or dark Ooplasm, small and relatively non aggregated membrana granulosa cells. Polar body is still intact or fragmented. Ooplasm may be slightly darkened, mainly granulated.

12. Post Mature

13. Atretic Rarely associated with cumulus mass, clumped and very irregular corona radiata, very visible zona, dark and frequently misshapen ooplasma, membrana granulosa cells with very small clumps of cells.

14. Atretic

15. OOCYTE MORPHOLOGY Based on size and shape of the oocyte 1. Giant oocyte 2. Elongated oocyte 3. Ovoid oocyte 4. Small oocytes

16. Giant oocytes Giant oocyte- improper cytokinesis Giant oocyte with no PB

17. Giant oocytes Tetraploid oocyte- fusion of two oocytes Giant oocyte with two MS spindle under polarized microscope

18. Elongated oocyte

19. Ovoid oocytes

20. Other shapes Two oocytes enclosed in single ZP Small oocytes

21. Oocyte Morphology based on Cytoplasmic Features Normal homogenous oocyte Granular cytoplasm Refractile body Organelle clusters SER Vacuoles

22. NORMAL OOCYTES Steriozome view Polarized microscope view

23. GRANULAR CYTOPLASM GRANULAR CYTOPLASM HIGH GRANULARITY

24. Cytoplasmic Features Refractile body Organelle clustering

25. Cytoplasmic Features SER SER+ GRANULAR CYTOPLASM

26. Cytoplasmic Features MULTIPLE VACUOLES LARGE VACUOLES

27. Extra cytoplasmatic Features Large PVS Large PB Zona Abnormalities

28. Zygote

29. Zygote Zona pellucida-ZP 2 pronucleus-2 PN 2 polar bodies-2 PBs Nucleolar precursor bodies-NPBs ZP 2 PN 2 PBs NPBs

30. Zygote scoring

31. Zygote scoring Zygote scoring system of Scott et al. Z1 zygotes have an equal number of NPBs aligned at the PN junctions (A). Z2 zygotes have an equal number and size of NPBs (3 to 7) that are equally scattered in the 2 PNs(B) Z3 zygotes are characterized by inequality of the NPBs (unequal size, unequal numbers, or unequal alignment at the PN junction)(C and D). Z4 zygotes have PN that are separated or different in size and small NPBs that are partially aligned or scattered.(E and F)

32. Zygote Scoring Z1 Z3 Z2 Z4

33. Cleavage Stage Embryo 2 cell/Day 1 8 cell/Day 3 4 cell/Day 2 Zona pellucida Blastomere Nucleus

34. Cleavage Stage Embryo Cleavage stage embryo grading based on Number of cells/blastomeres Fragmentation Blastomere size Nucleation

35. Cell number Timings schedule Day 1 (26 ± 1 h post-ICSI, 28 ± 1 h post-IVF), 2-cells; Day 2 (44 ± 1 h), 4-cells and Day 3 (68 ± 1 h), 8-cells Early cleavage i.e. the first mitosis occurring before 26±1 h (ICSI) and 28±1 h (IVF) respectively, has been shown to correlate with numbers of good quality embryos, blastocyst development and pregnancy rates ( Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011 ).

36. Fragmentation Small portions of cytoplasm enclosed by a cell membrane but usually not containing DNA are often formed during cell division. Fragmentation is therefore defined as the presence of anucleate structures of blastomeric origin and evaluation of the degree of fragmentation is included in almost every embryo scoring system. The degree of fragmentation is most often expressed as the percentage of the total cytoplasmic volume. The relative degree of fragmentation is defined as mild (<10%) , moderate (10–25%,) and severe (>25%)

37. Fragmentation 10% 40% 20% 30% 50%

38. Blastomere size Diagram illustrating the concept of stage-specific versus non-stage-specific cleavage patterns. The dark green color indicates stage-specific cleavage stage embryos, whereas the light green color indicates non-stage-specific cleavage stage embryo

39. Nucleation

40. Nucleation

41. Cleavage stage Embryo grading Day 2/3 embryo grading: Grade A: Stage specific blastomeres 4 or >4/8 or >8 Fragmentation <10%. No multinucleation Grade B: Stage specific blastomeres 3-4/ 6-8. Fragmentation <25%. No multinucleation Grade C: non-stage specific blastomeres, frag < 25%

42. Day 2/3 embryos Grade A B C

43. Compaction The human embryo appears as an indistinguishable mass of cells on Day 4 of development, the morula. A good quality morula is composed of 16–32 blastomeres and all of the blastomeres should be included in the compaction process. The increase in cell-to-cell adherence should begin at the 8-cell stage and then progress rapidly with time. This process has been linked to activation of the embryonic genome and is therefore considered to be a good sign of the developmental capacity of the embryo.

44. Compaction Good Poor Fair Early cavitating Early cavitating

45. Day 5 blastocyst grading Trophectoderm cells Inner cell mass Blastocoel cavity/ expansion

46. Blastocyst grading The blastocyst grading system introduced by Gardner and Schoolcraft in 1999 was quickly adopted by the majority of IVF laboratories. It has been very useful in classifying the degree of blastocyst expansion as well as the morphological appearance of the inner cell mass (ICM) and the trophectoderm (TE) cells.

47. Selection of Embryo Zygote-Z1, Z2 Cleavage stage embryo- Grade A, B Compaction- Good, Fair Blastocyst- >3 Expansion, TE- A,B, ICM- A,B

48. Thanks