OPU, Insemination and Fertilisation Flashcards
When does OPU occur
36 hours after trigger
At OPU what is the role of an embryologist once the clinician has collected the eggs?
Identify cumulus oocyte complexes (COC’s) and remove them from follicular fluid
(important: cannot identify oocyte maturity or quality)
Why does the oocyte need to be removed from the follicular fluid as soon as possible?
blood and other factors in the follicular fluid cause damage to the oocyte if not removed
Is it possible for the number of oocytes to not match the follicle number?
Yes, in cases with PCOS patients will have a larger amount of follicles without oocytes
What are the essential consideration for an embryologist during an OPU procedure, briefly explain each consideration?
- replicating in vivo conditions
- temp and pH must be controlled and stable for the whole procedure (about 37 degrees)
- oocytes are very sensitive and slight alteration to environmental conditions cause stress - Witnessing
- must have independant witness checks - Speed
- follicular fluid is damaging to COC’s
- follicular fluid should not be placed in culture system
- should be less than 1 minute to locate, wash and place COC in clean media - Accuracy
- supervisor will check and only sign off when zero COC’s are missed
What are the different sperm sources available?
partner or donor
fresh or frozen sperm
- majority are fresh samples
ejaculate or surgical removal
What are the two methods used to prepare a sperm sample
- density gradient centrifugation
- samples are centrifuged for 20 minutes at 1600 rpm
- motile sperm will drop into the lower phase of the centrifuge tube whilst abnormal will remain in the upper phase of the tube just below the seminal plasma - swim up
- sample is incubated for 60 minutes at 37 degrees on an angle
- motile sperm swim up through the medium and found in the top layer of the medium
Once the sperm samples only contains motile sperm it is now considered washed what then occurs for the clinics to determine if the sample is suitable for IUI, IVF or ICSI?
assessment of concentration, motility and morphology using a Makler
Each clinic has their own values to determine which procedure is best suited for the quality of sperm
What occurs after the oocyte retrieval and sperm sample preparation?
Insemination
When does insemination need to occur
38-42 hours post trigger as the oocytes will age quickly
When undergoing conventional IVF what occurs during the insemination step?
- known concentration of motile sperm added with all COC’s (about 80 000 to 120 000 motile sperm)
All clinics have their own protocol regarding:
- sperm concentration
- dish type
- number of COC’s per well/drop
- length of sperm exposure
When undergoing ICSI what is involved during the insemination process?
- oocytes are stripped of cumulus complex and assessed for maturity
- a single sperm is injected into each mature MII oocyte (this does not guarantee fertilisation)
When undergoing ICSI insemination how is oocyte maturity assessed?
- Hyaluronidase breaks down cumulus complex
- very quick exposure as it is toxic to oocytes - narrow pipettes are then used to mechanically loosen the cumulus
- finding oocytes with polar bodies present are mature MII and can be inseminated
Once insemination occurs what is the next step and how long after insemination does this occur?
Fertilisation check which occurs 17 +/- 1 hours post insemination
What are the morphological signs that fertilisation has occurred?
two polar bodies on the oocyte
two dents in the middle of the oocyte
