basic embryology

Question 1

What happens after the trigger

Answer 1

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

Question 2

The oocyte

Answer 2

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)

Question 3

stages of development - immature oocytes

Answer 3

Germinal Vesicle (GV)-immature - 46 chromosomes (maternal)
metaphase 1 (M1) - progressed but still immature - still 46 chromosomes (maternal)

Question 4

Mature oocyte: M2

Answer 4

Polar body extruded-> 23 chromosomes​

Now can be fertilized..​

Must be M2 to be frozen or fertilized

Question 5

oocyte crypotreservation

Answer 5

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

Question 6

inseminatio

Answer 6

ICSI, conventional insemination (IVF)

Question 7

Embryo Development

Answer 7

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

Question 8

watch the progression

Answer 8

ature Egg (M2)  Fertilized Embryo (2PN), Cleavage (Day 2) Blastocyst (Day 5/6)

Question 9

A fertilized oocyte—2 pronuclei (2PN)

Answer 9

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.

Question 10

Assisted Hatching (AH)

Answer 10

When is it typically completed? ​

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For embryos undergoing biopsy for PGT, it is completed on Day 3​

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For embryos being frozen without biopsy, it will be completed after thaw, pre-FET​

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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

Question 11

cleavage stage of embryos

Answer 11

number of cells, symmetry, fragmentation

Question 12

Blastocyst

Answer 12

Day 5 (120 hrs.) or ​

Day 6 (148 hrs.) of development
ICM-Inner cell mass (fetus)
TE-Trophectoderm (placenta)​

Question 13

day 4 and early Blastocyst

Answer 13

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.

Question 14

Embryo Biopsy for PGT

Answer 14

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…

Question 15

day 7 embryos

Answer 15

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

Question 16

Blastocyst stage: Grading

Answer 16

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..

Question 17

Embryo Cryopreservation (Vitrification)

Answer 17

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..

Question 18

Preimplantation Genetic Testing

Answer 18

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. ​

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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.​

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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.

Question 19

How does the embryologist select the best to transfer?

Answer 19

All AA, AB and BA have similar results, if they are Euploid​

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D5 or D6 Bx doesn’t seem to factor in​

Selection of D5 Blast over D6 is more by habit than by superior outcome​

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Cryopreservation only done on those graded BB and above.

Question 20

Expected Attrition – Basic Stats

Answer 20

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

Question 21

Summary

Answer 21

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

Question 22

we’ll hold until day 7

Answer 22

but if it’s not a blastocyst at this point, we have to discard

Question 23

fluid cavity size - 3 - what happens at this point?

Answer 23

we can grade it A, B, C