Embryo Grading, Selection and Preparation for Transfer

Question 1

Which patient characteristics have been associated with favorable prognosis according to ASRM guidleines?

Answer 1

1. young female age
2. expectation of 1 or more high quality embryos available for cryopreservation
3. euploid embryos
4. previous live birth after an IVF cycle
5. FET cycles
6. availability of high quality daty 5/6 blastocysts for transfer
7. patients first FET cycle

Question 2

What characteristics are assessed with pronuclear scoring?

Answer 2

1. alignment of pronuclei and nucleoli
2. number and distribution of nuclei within each pronucleus
3. orientation of pronuclei relative to polar bodies

Question 3

What pronuclei morphology correlates with increased embryo competence?

Answer 3

1. equal number of nucleoli of size size aligned at PN junction (Z1 configuration)

Question 4

How do Z1-Z4 PN grading differ?

Answer 4

Z1- equal sized and number of nucleoli
Z2- unequal number and not aligned
Z3 - unequal number/size ligned up or dispursed
Z4- PNs not touching
Z4- unequal size PNs

Question 5

Describe the SART grading system for cleavage stage embryos

Answer 5

overall grade: good, fair, poor
Cell number : 1-8
fragmentation: 0%, <10%, 11-25%
Symmetry: perfect, moderately asymmetric and severely asymmetric

Question 6

What cell numbers on Days 2 and Day 3 are associated with highest implantation potential?

Answer 6

4 cells on Day 2
7-8 cells on Day 3

Question 7

What is blastomere assymetry associated with?

Answer 7

increased polyploidy
lower implantation rates

Question 8

How is blastomere organization (adhesion) important for implantation potential?

Answer 8

plays role in compaction and differentiation of post-compaction embryo
embryos exhibiting disorganization and reduced cell adhjestion during the cleavage stage have lower implantation potential and dont perform as well in extended culture

spatial arrangement of blastomeres in 4 cell embryos has been associated with developmental prognosis

blastomere organization results from orientation of succcessive cleavage planes which determine what an individual blastomere inherits

Question 9

How does cleavage plane affect blastomeres?

Answer 9

blastomere organization results from orientation of succcessive cleavage planes which determine what an individual blastomere inherits (proteins, cell components, polarized domains etc)

Question 10

what morphological features are assessed on cleavage stage embryos

Answer 10

1. blastomere cell number
2. blastomere assymetry
3. blastomere organization
4. cytopolasmic inclusions
5. multinucleation
6. fragmentation
7. zona pellucida thickness

Question 11

What is the impact of multinucleation on embnryo development and implantation potential?

Answer 11

historically been considered abnormal and associated with poorer implantation rates, a higher incidence of chromosomal aberrations after the first and second cleavage division, mosaicism, and polyploidy. Multinucleated embryos were reported to have a reduced ability to reach the blastocyst stage in extended culture and should not, therefore, be transferred However, in a recent study making use of continual embryo assessment (time-lapse) although the frequency of multinucleation was high in human embryos cultured in vitro, it occurred at an equivalent rate in euploid and aneuploid embryos and most affected embryos were able to self-correct during early cleavage divisions The exact implication of multinucleate blastomeres in cleavage stage embryos should be the topic of further studies to ensure these embryos are categorized appropriately.

Question 12

What is the impact of cleavage stage embryo fragmentation?

Answer 12

blastomeres that limits embryo development in vitro Certain patterns of fragmentation result in partial or total loss of regulatory proteins from affected blastomeres ) and may interfere with the ability of the embryo to compact. Four large studies published on the impact of fragmentation reported low implantation rates after the transfer of cleavage stage embryos with 10%-50% fragmentation on either Day 2 or Day 3 of development established a relationship between the degree and pattern of fragmentation on Day 3 of development, the ability of these embryos to develop to the blastocyst stage, and implantation and pregnancy rates (Figure 3). This study also demonstrated that microsurgical removal of fragments significantly altered the development of some embryos and improved their implantation potential. However, fragment removal may lead to the potential loss of organelles, particularly mitochondria, and the evidence regarding any benefit of this technique to embryo survival has been questioned (Halvaei et al., 2016). The degree and pattern of fragmentation has also been associated with the incidence of chromosomal abnormalities. Based on these data, embryo selection should be based not only on degree, but also on the pattern of fragmentation

Question 13

Which categories of patients would benefit most from assisted zona hatching?

Answer 13

aged 38 years
elevated basal FSH levels
previous IVF failure(s)
excessive fragmentation
slow development of cleavage stage embryos
embryos for transfer were cryopreserved either at their current or an earlier stage of development.

AZH can be accomplished by various techniques including mechanical breach, chemical exposure, acidic medium, laser drilling, or selection of hatching blastocysts through extended culture.

Question 14

What are examples of combined day 3 assessments associated with highest grade embryos and poorest quality embryos?

Answer 14

i. highest grade embryos will possess:
a cell number appropriate for culture time since insemination regular symmetrical blastomeres
, moderate refractility little or no fragmentation
an intact normal width zona (Figure 4).
ii. poorest quality embryos will exhibit:
a cell number inappropriate for culture time since insemination asymmetrical blastomeres gross variations in refractility areas of necrosis fragmentation greater than 25% a thick or thin zona pellucida blastomere multinucleation the presence of various cytoplasmic inclusions (Figure 5).

Question 15

What are the stages of blastocoel expansion grading?

Answer 15

Blastocoel Expansion
1. blastocoel cavity is less than half of the volume of the embryo
2. blastocoel cavity is half or more than half the volume of the embryo
3. blastocoel cavity completely fills the embryo blastocyst cavity
4. greater than the original volume of the embryo, ZP is thinned
5. blastocoel cavity is greater than the original volume of the embryo,
6. TE is herniating through a natural breach in the ZP blastocyst has completely escaped from a natural breach in the ZP

Question 16

What are the ICM and TE grading criteria according to Gardner?

Answer 16

Inner Cell Mass
1. many cells that are tightly packed
2. several cells that are loosely grouped
3. very few cells that are loosely bound
Trophectoderm
1. many cells that form a cohesive epithelium
2. few cells forming a loose epithelium
3. very few large cells that struggle to form a cohesive epithelium

Question 17

WHat are the functions of TE vs ICM cells?

Answer 17

Blastocysts with an expansion grade of 3 or more show a clear distinction between two cell populations; the outer cells of the blastocyst, forming the blastocyst structure (trophectoderm [TE] cells) and the cells located inside the blastocoel (Inner Cell Mass (ICM) cells). The ICM cells will develop into the embryo proper while the TE cells give rise to the fetal extra-embryonic membranes as well as the placenta. The TE cells are also actively involved in breaking free of the ZP as well as the production of several molecular factors that aid in the implantation process. ICM and TE cells are scored according to their number and cohesiveness (Table 3).

Question 18

Describe the SART Grading System for Morula and Blastocyst Stage embryos

Answer 18

Growth Phase: Morula
Good/Fair/Poor
Stage: Compaction (incomplete or complete)
Fragmentation 0, 10 11-25, >25%

Blastocyst
Good/Fair/Poor
Expansion: Early, expanding, expanded, hatched
ICM: Good/Fair/Poor
TE:Good/Fair/Poor

Question 19

What is morphokinetics?

Answer 19

a) Morphokinetics: Embryo development is monitored continuously by the acquisition of images at a defined frequency until the time of transfer (Time-lapse Analysis; TLA). Continual, cumulative data collection builds on the daily scoring system used by most laboratories, with the aim to provide an effective embryo selection tool. Thus, analysis of the embryo’s cumulative pattern of development (kinetics), combined with its morphological characteristics can be used to select a single embryo portraying viability and implantation potential, according to pre-determined biomarkers and algorithms (Chavez et al., 2012; Herrero et al., 2013; Chen et al., 2013; Conaghan et al., 2013).

The non-invasive nature of this approach is appealing and it also eliminates the need to remove embryos from optimized culture conditions. Several positive reports regarding the use of time-lapse monitoring in the IVF laboratory setting have been published, including using observation of dynamic blastomere behavior as a method with which to diagnose ploidy in early cleavage-stage embryos (Chavez et al., 2012). However, the data reported from TLA lack a consistent use of time points and nomenclature, as well as a consensus on defined biomarkers and algorithms between users. This is likely due to differences in embryo health and quality between patient populations, use of different methods of insemination, culture conditions and other variables that impact embryo development and viability. TLA
has been supported commercially with the production of stand-alone units for purchase. Three systems have been developed that share some common features, but differ markedly in others, which may also have contributed to the difficulty in reaching an agreement on biomarkers (Racowsky et al., 2015). Together, these data indicate the need for additional, well-designed studies before time-lapse analysis can be applied as a standardized embryo selection method (Racowsky et al., 2015).

Question 20

What is proteomics?

Answer 20

b) Proteomics: including analysis of the embryonic secretome (proteins produced and secreted by the embryo during development) is another non-invasive approach to obtain information about the embryo’s physiological status (well-being) (Katz-Jaffe et al., 2013). Assessment of molecules involved in implantation potential and pregnancy, such as HLA-G production by human embryos, may also provide non-invasive markers of embryo competence (Kotze et al., 2013). These data can be used for selection purposes in combination with an assessment of the embryos morphological characteristics and may additionally have the potential to develop non-invasive methods of aneuploidy assessment (Katz-Jaffe and McReynolds, 2013). Analysis of the embryo’s metabolism (metabolomics) has also been investigated for use as a method of assessing embryo viability (Gardner & Wale, 2013).
These newer approaches aimed at adding to and improving available methods for embryo selection are, on the one hand, supported by evidence of their potential to make selective measurements, however their broad application in the clinical IVF laboratory is currently restricted by the following: the need for specialized expertise equipment that can handle large analytical volumes daily lack of standardized selection parameters cost.

Question 21

What is metabolomics?

Answer 21

Analysis of the embryo’s metabolism (metabolomics) has also been investigated for use as a method of assessing embryo viability (Gardner & Wale, 2013).

Question 22

What are the limitations to newer approaches of embryo selection?

Answer 22

Analysis of the embryo’s metabolism (metabolomics) has also been investigated for use as a method of assessing embryo viability (Gardner & Wale, 2013).
These newer approaches aimed at adding to and improving available methods for embryo selection are, on the one hand, supported by evidence of their potential to make selective measurements, however their broad application in the clinical IVF laboratory is currently restricted by the following: the need for specialized expertise equipment that can handle large analytical volumes daily lack of standardized selection parameters cost.

Question 23

What is a normal kinetic timeline of development of the human embryo?

Answer 23

To that end, normally fertilized zygotes demonstrating early division to 2-cells at 27 hours post-insemination (hpi), 4-cells at 42 hpi, 6- to 8-cells at 66 to 72 hpi, compacted morula on day 4 and expanded blastocoel with a good number of tightly packet ICM cells and a cohesive TE have met the accepted benchmarks for cell number and timing of blastocoel formation

Embryos are transferred at the cleavage stage for some women. Priority should be given to embryos with 6 to 8 cells on day 3 of development (66-72 hours postinsemination), with little or no evidence of fragmentation, good blastomeres symmetry and organization. Good quality embryos with more than 8 cells may be selected if no fragment-free or minimally fragmented embryos with an appropriate cell number are available.