understanding lab reports Flashcards
The Oocyte
GV: Germinal Vesicle is pre metaphase and very immature, would not be capable of fertilization
M1: Metaphase 1 is still considered to be immature and not capable of fertilization
M2: Metaphase 2 is full mature and is capable of fertilization
Oocyte CRYOPRESERVATION
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
Gametes orders (this is just how it looks in ideas)
Ovum/Embryo source: Patient or Donor
egg donor eligibility
semen source: donor or partner
semen eligibility
Ovum/Embryo source: Patient or Donor
use of frozen semen
back up sample source
transfer technique
Sperm selection, lab interventions
Physiological ICSI (PICSI)
Mature sperm have a receptor on their head that allows it to bind to hyaluronic acid (HA), which is a main component on the eggs surface.
When completing ICSI the embryologist is picking sperm based only on their general appearance and motility – not this receptor. Therefore with PICSI the sperm are exposed to HA and only the sperm that bind to it are selected from for ICSI.
ZyMot Chip
A device utilized to help with sperm selection
Need to have “some” motile sperm
Sperm is injected into port, fertilization media is placed on top.
Sperm is left to incubate for ~30min. During that time the “best” sperm swim through pores in the media. This is what is selected to use for fertilization.
other interventions - co-culture
(co-cumulus)
Helps to support the egg and therefore the early stages in embryo development, cleavage phase (Day 2 and 3)
Takes the cumulus cells that surround the egg upon extraction and use them in culture media to create a monolayer of cells that the embryo will be placed in after fertilization check. These “helper” cells may improve the embryos development.
the cumulus might help, they don’t know, so they put it back
other interventions -Calcium Ionophore
Helps to improve fertilization rates and potentially embryo progression
When the sperm fertilizes an egg naturally it will activate cortical granules to harden the zona (stop other sperm) this then triggers waves of calcium activation with helps with oocyte activation
To mimic this in the lab, after ICSI is done, the egg/embryo is exposed to concentrations of calcium media for short periods of time (completed twice) and then returned to normal fertilization media
lab orders - how long before baseline to sign orders?
VF lab order must be completed and signed one week before baseine. sections A-D must be completed. Section E will be completed for additional remarks
lab orders
Required Selection Options
IVF vs ICSI
ICSI
No split
Split ICSI (default ICSI)
Split ICSI (default IVF)
Optional add-on Procedures
Zymot
PICSI
IMSI not offered at Spring
CA ionophore
Reason for ICSI
Insemination remarks
Insemination Remarks
Insemination remarks
For ICSI split input the number of eggs for ICSI
Example
ICSI 10 insem rest
Reason for ICSI
Reason for ICSI must be completed
Abnormal sperm parameters
Frozen oocyte
Laboratory Routine
Low oocyte yield
Other
PGS/PGD
Poor Fertilization
Prior Failed Fertilization
Rescue ICSI (not an option)
Section B: Culture
Provider will not select:
time lapse
IVC
Blastocyst culture
If Co-culture selected:
Provider must select granulosa cells
Endometrial cell not offered
PGT indication must be completed:
aneupoloid screening for ARA
aneupoloid screening RIF
aneupoloid determination-balanced translocation
gender selection
gender selection X-linked diseases
HLA typing other
single gene analysis
Other: Will be selected if freezing some oocytes or donating..
i.e. Freeze 10M2, ICSI remaining
ICSI 10 oocytes, donate remaining to _________.
May also see these in Remarks
Assisted hatching: always selected for IVF/PGT & FET
Section C: Transfer
If ET planned transfer section must be completed
Intended blastocyst ET:
Yes, if blastocyst transfer
No, if cleavage embryo (D2 or D3) to be transferred
Day of transfer :
Luteal day 2, 3, 4 or 5
Depends on stage embryo was frozen or type of cycle patient is doing.
See SOP FET 01.
Elective embryo transfer
Number to transfer
Embryo glue—if indicated
Section D: Cryogenic Storage
Freeze must be completed
Most cases will be “ALL”
For rare cases where some oocytes are frozen or donated provider must complete “Some” in this area and “Other” in Culture section (see section B, previous slide)
Reason for freeze all should be entered by provider:
This could be the lab the cells are being sent to:
Natera
Cooper
Igenomix
Other indications:
Donation
Fertility preservation
Gonadotoxic treatment (CA Dx)
NEST
Planned FET
PGT (RGI or other lab not listed)
Egg Freeze
The patient’s report will show the total number retrieved, how many were mature, how many immature (GV & M1) and therefore how many were actually
For patient’s that undergo multiple rounds the report will show the outcome of each cycle and a total
The eggs will be stripped prior to freezing. ICSI will not actually be completed at this time however
Embryo Grading
Embryo grading is a method to assess embryo quality by evaluating and scoring key aspects of their appearance as examined under a high-powered microscope. Can only be completed at specific stages of embryo development.
Helps in selecting best embryo(s) for transfer.
Day 3 “Cleavage Stage” Embryos
Day 3 “Cleavage Stage” Embryos
referred to as “cleavage stage” because the cells inside each embryo are dividing or “cleaving” as the genetic material replicates. The embryo itself does not yet increase in size.
The Gardner System
The Gardner System
At Spring, we use a cleavage embryo grading system originally developed and published by pioneer fertility specialist Dr. David Gardner. It has been widely adopted worldwide because of its success rate.
Three separate quality scores help determine the cleavage embryo grade at Day 3:
(grade my CCS)
Three separate quality scores help determine the cleavage embryo grade at Day 3:
Cell Count – How many cells make up the embryo. Post fertilization, we like to see embryo that are at least 2-4 cells by 48 hours (Day 2) and at least 6-8 cells by 72 hours (Day 3).
Cell Fragmentation – Sometimes, as cells divide, small portions can break off. These fragments are usually cytoplasm and do not contain nucleic material but will still like to see little or no fragmentation.
Symmetry Grade – Whether the cells inside the embryo (also known as “blastomeres”) are of equal size, it’s generally better if the individual blastomeres are similar in size versus very different in size.
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
Day 4 and Early Blastocyst
Day 4 Embryos are called Morulas (“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.
Day 5/6/7–Blastocyst Grading
Day 5/6 the embryo should reach to “blastocyst stage.”
Two distinct cell types can be seen, as well as a fluid cavity in the center known as the “blastocoel”:
The inner cell mass (ICM), which will become the fetus
The trophectoderm cells (TE) that will form the placenta
Three separate quality scores help determine the blastocyst embryo grade at Day 5/6:
(IT is first)
Three separate quality scores help determine the blastocyst embryo grade at Day 5/6:
Blastocyst Expansion Stage – The size of the blastocoel relative to the total embryo size, and how far along the blastocyst is towards hating from its outer shell. This is the number on the grading.
Inner Cell Mass (ICM) Quality – The quantity of inner cells and how they are grouped. Generally, it is better to have more and tightly packed cells. This is the FIRST letter on the grading.
Trophectoderm (TE) Quality – The quantity and cohesion of this outermost layer of cells. Generally, it is better to have many cells form a tight, cohesive layer. This is the SECOND letter on the grading
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. If an oocyte was previously frozen, then thawed to form to embryo- culturing to day 7 is expected and you don’t see the same decrease in pregnancy rate.
PGT Results
The PGT results will show up in the Blood/Lab tests once in. To review the full report – click on “Report
The Laboratory details will also have a note with the details of the PGT results in relation to the embryo report
Spectrum Report - pGT
Normal meaning 46 chromosomes
Aneuploidy meaning abnormal number of chromosomes either deletions or duplications
Inconclusive because the biopsied sample provided is not have sufficient DNA
Requirements for Genetic LAbs
We will send cells to the following labs for testing, depending on MD order/patient needs:
Natera
Juno
Igenomix
Cooper
Each lab has its own workflow/requirements that need to be completed PRIOR to the embryologist sending the cells for testing- see CN03 PGT process
It is the PNs responsibility to:
Review SOP to know what is needed to Open the Case
Complete and send the Requisition to the appropriate lab
Consent the patient appropriately
It is the RNs responsibility to:
Double check that all was completed at time of checklist sign-off
Support with any questions/needs
PGT – Next Generation Sequencing
y-axis is the number of copies, ranging from 0 to 4. Normal is 2 (1 from egg & 1 from sperm)
x-axis is individual chromosomes ranging from 1 to 21 and sex (X&Y).
PGT challenges
PGT does not guarantee a successful ET or healthy baby
PGT does not test for all genetic issues
Importance of other genetic tests/evaluation
Prenatal recommendations; POC testing
Providing hope while correcting misconceptions
PGT - mosaic embryos
A mosaic embryo contains some cells with a normal number of chromosomes and others with an abnormal number. This occurs in approximately 15% of embryos.
Mosaic embryos are being reported as aneuploidy (abnormal) on the PGT report.
Low level mosaic embryos can also be reported as “Disomy <80%”
Spring’s policy on Mosaic Embryo Transfer
Expected Attrition – Basic Stats***test question
In Summary ~25% of mature eggs will reach the blastocyst stage, may be as high as 45% depending on age of egg provider
75-80% of mature eggs will fertilize
85-95% of 2 PNs will progress to the cleavage phase
40% of cleavage stages embryos will make it to blastocyst
additionally, frozen eggs have an 80-90% survival rate upon thaw
of those genetically testing the euploid rate will be dependent on the age of the pt
age:
30 75%
35 65%
40 40%
43 15%
Reports to the Patient
Update: As of 8/12/24 we will NO LONGER provide a Day 3 grading update to the patient unless their embryos are being frozen or they are coming in for a fresh transfer.
After the final report, a follow up with the MD should be scheduled to review the outcome of the cycle – this appointment is called a “PIV” (Post IVF). If PGT, the PIV should be scheduled ~3 weeks from trigger at the time of trigger callback.
