basic embryology Flashcards
What happens after the trigger - how many mm do we want the follice?
If no egg collection, ovulation would occur ~ 36 hours post trigger
Egg collection scheduled 34-36 hours after trigger
At time of egg collection->Embryologist looks for eggs—under microscope (oocyte covered w/cumulus cells (i.e.. Fluffy cloud..)
1 hour later embryologist->Denude(strip) the egg from the cumulus cells ->able to assess egg maturity
Goal-> Metaphase 2 (M2) –these normally come from those follicles >16mm
~3 hours later (if creating embryos) the eggs will be fertilized (6:30am-noon embryologist very busy)
16-18 hours later, embryologist checks for fertilization—2PN
The oocyte
a. An image of an oocyte initially after retrieval, surrounded by the cumulus cells
b. An image of oocytes after the “stripping” of the cumulus cells in preparation for ICSI or cryopreservation. The embryologist will look for a polar body to confirm if the egg is mature (m2)
stages of development - immature oocytes
(the gv germ is 46)
Germinal Vesicle (GV)-immature - 46 chromosomes (maternal)
metaphase 1 (M1) - progressed but still immature - still 46 chromosomes (maternal)
Mature oocyte: M2
Polar body extruded-> 23 chromosomes
Now can be fertilized..
Must be M2 to be frozen or fertilized
oocyte crypotreservation
During the vitrification process it is essential to first reduce the intracellular water of the oocyte by partial dehydration using brief exposure to high concentration of cryoprotectants. This helps to avoid ice formation (crystallization) during freezing process that would be destructive to the egg
inseminatio
ICSI, conventional insemination (IVF)
Embryo Development
Day 0: egg
collection—if oocyte cryo, they are frozen today
Day 1: Fertilization check
Day 3: cleavage stage: 6-8 cells (goal: 8 cells)
Day 4: Morula (compacting ball of cells)
day 5: blastocyst
day 6: blastocysts/crypotreservation
watch the progression
ature Egg (M2) Fertilized Embryo (2PN), Cleavage (Day 2) Blastocyst (Day 5/6)
A fertilized oocyte—2 pronuclei (2PN)
Fertilization check (day following egg retrieval)
Looking for 2PN
Genetic material from both gamete providers
2PN is only visible for a few hours..
Could see 0PN
No fertilization –or-
PN already disappeared
These are kept and watched for signs of development
Could see 3PN
Extra genetic material likely present….
2 sperm penetrated the egg or the egg never extruded the second polar body……….
If PGT planned, these embryos will be watched and biopsied if they become blasts.
Recent data has shown a possibility for 3PN (tripronuclear embryo) to be euploid.
Assisted Hatching (AH)
When is it typically completed?
For embryos undergoing biopsy for PGT, it is completed on Day 3
For embryos being frozen without biopsy, it will be completed after thaw, pre-FET
For embryos being transferred fresh it likely will not be completed for blastocysts unless indicated due to thick zona
or history of implantation failure. For cleavage stage embryos it will be completed pre-transfer
cleavage stage of embryos - what 3 things to look for -
(cleavage has chronic fatigue syndrome)
number of cells, symmetry, fragmentation
Blastocyst
Day 5 (120 hrs.) or
Day 6 (148 hrs.) of development
ICM-Inner cell mass (fetus)
TE-Trophectoderm (placenta)
day 4 and early Blastocyst
Day 4 Embryos are called Morula (“Mor”). There is no grading mechanism for this stage. They are in a transitional phase between cleavage and early blastocyst. During this stage compacting of the cells will begin to take place (starts as early as late Day 3).
Between Morula and Blastocyst are two stages of Early Blastocyst (EB1 and EB2).
These are also transitional phases and have no grading mechanism.
Embryo Biopsy for PGT
Embryo must become a blastocyst to have a biopsy for PGT
Cells from the TE (placenta) are what are removed and sent to the lab for analysis
Once biopsied, embryo is frozen/labeled to match with results once available
Results will advise on embryo sex and which are Euploid (Chromosomally normal)…
No call-> the lab unable to analyze the cells sent. Would need to thaw/rebiopsy/refreeze to get results…
day 7 embryos
In embryology, everything is about timing. This is true in every phase including blastocyst. A viable embryo should typically reach the blastocyst in 5 to 6 days. However, in some cases with specific circumstances, we will culture embryos to Day 7. These embryos have a significantly lower chance of success than Day 5 or 6 embryos.
The criteria for going to Day 7 is as follows:
The patient has a very low number of embryos (typically only one or none frozen on Day 5/6)
The embryo has shown continued progress between the Day 5 and Day 6 check. Example: D5 Mor D6 EB or D5 EB1 EB2
Blastocyst stage: Grading
Blastocyst fluid cavity size:
1-cavity is small (EB1)
2-cavity up to 50% (EB2)
3*-cavity the diameter of the embryo
4*-Shell is thin around the outside
5-Embryo is leaving shell (hatching)
6-Embryo has left the shell
Quality of ICM(inner cell mass - the baby) & TE (trifectoderm)
A=Good
B=Okay
C=Not great..
A=Good
B=Okay
C=Not great..
Embryo Cryopreservation (Vitrification)
Embryologist place the embryo in a solution to “dehydrate” the embryo
Freeze w/cryoprotectant
No crystals to harm embryo upon freeze/thaw..
Thaw rate—is very high
Will only freeze embryos that have a high chance of surviving this process..
Preimplantation Genetic Testing
PGT is the process of screening cells from embryos for genetic diseases and chromosomal disorders
PGT-A (formally known as PGS): A stands for “Aneuploidy”
Embryo screening that identifies embryos with extra or missing chromosomes, also called aneuploidy.
PGT-M (formally known as PGD): M stands for “Monogenic” or Single Gene Conditions
Embryo screening for inherited single gene conditions such as cystic fibrosis, SMA, or Tay Sachs disease. In many cases, couples are “carriers” of a gene mutation that does not affect their health but puts their children at risk of inheriting the condition.
PGT-M will identify embryos that are un-affected, carriers, and affected.
PGT-SR: SR stands for “Structural Rearrangements” such as translocations and inversions
Couples in which one partner carries a chromosomal rearrangement (such as a translocation or inversion) are at increased risk, beyond maternal age-related risk, to have embryos with chromosomal abnormalities.
Translocation/inversion PGT-SR can identify embryos that have inherited extra and/or missing material as a result of the paternal chromosomal rearrangement.
How does the embryologist select the best to transfer?
All AA, AB and BA have similar results, if they are Euploid
D5 or D6 Bx doesn’t seem to factor in
Selection of D5 Blast over D6 is more by habit than by superior outcome
Cryopreservation only done on those graded BB and above.
Expected Attrition – Basic Stats
75-80% of mature eggs will fertilize, 85-95% of 2PNs will progress to the cleavage phase, 40% (this is where the big drop is) of cleavage stage embryos will make it to blastocyst
Summary
20% of mature eggs will reach blastocyst stage, Of those genetically testing the euploid rate will be dependent on the age of the egg. additionally, frozen eggs have an 80-90% survival rate upon thaw
we’ll hold until day 7
but if it’s not a blastocyst at this point, we have to discard
fluid cavity size - 3 - what happens at this point?
we can grade it A, B, C
