Lab Stuff Flashcards
What is the effect of FSH on corona radiata?
What is the effect of LH on corona radiata?
FSH»_space; granulosa cell proliferation»_space; MORE granulosa cell layers in corona radiata
LH/HCG»_space; expansion of cumulus cells»_space; less dense/tightly packed
What do oocytes undergoing premature luteinization look like?
Premature luteinization due to early elevated progesterone
Granulosa cells become dark/less refractile
What do follicles responding poorly to FSH indicate?
Scanty corona radiata
What is the percentage of IVF cases involving contamination?
0.7%
Which type of contamination may still allow for ET?
Fungal
Even if contamination cannot overtly be observed on IVF dishes, what percentage contain bacteria when tested?
1/2 !
Why is inside air dirtier than outside air?
Human skin debris!
Which bacteria can pass through filters?
Mycoplasma (small, lack cell wall)
What is likely the largest source of IVF culture contamination?
Follicular aspirates, Sperm
What is gentamicin active against?
Gram negative bacteria
First source to evaluate for contaminants?
Media
Pre-compaction (D1-3) v Post-compaction (D4-6)
Embryo genome activates between day 2-3 (8-cell stage)
Day 1-3:
Pyruvate
Lactate
Non-essential AAs
Low O2 consumption
Day 4-6:
Glucose
Essential AAs
High O2 consumption
Blastocyst stage transfer has a higher ___ rate than day 3 transfer
implantation/pregnancy
Pros/Cons of blastocyst transfer
Pros:
- Higher implantation/pregnancy rate than with Day 3 transfers
- More viable option for eSET (opportunity for more embryo development allowing for better embryo selection)
- Decrease potential for embryo trauma with trophectoderm biopsy (compared to cleavage stage biopsy)
Cons:
- Longer culture time, longer exposure to in vitro environment
- More labor intensive for lab
- Higher chance of lacking embryo development –> no embryos for transfer
- High rates of multiple gestation
- Increased chance of monozygotic pregnancy
- Possible inadequate endometrial receptivity with early stage blastocyst
Describe the Gardner grading scale
- Progression score
- ICM grade
- TE grade
Gardner scale allows for counseling of likelihood of success based on individual available embryos.
It also provides quality control metric for the lab.
Progression score: degree of expansion and hatching status
- Early blastocyst - blastocoel <50% of embryo volume, ZP thick
- Expanding blastocyst - blastocoel >50% of embryo volume, ZP starting to thin
- Full blastocyst - blastocoel fills entire embryo, ZP not completely thin
- Expanded blastocyst - blastocoel volume now greater than the early embryo (fully expanded embryo), ZP very thin
- Hatching blastocyst - trophectoderm starting to herniate through ZP
- Hatching blastocyst - blastocyst completely escaped from ZP
ICM grade:
A- tightly packed, many cells
B- loosely packed, several cells
C- very few cells
TE grade:
A- cohesive epithelium, many cells
B- loose epithelium, few cells
C- very few, large cells
*3AB or better considered good quality
Gardner scale correlates with increased ___
Pregnancy/delivery rates
Blastocyst quality score (BQS) calculation
Expansion score x ICM score (3-A, 2-B, 1-C) x TE score
Score of at least 36 or above (correlating to 4AA) associated with highest clinical pregnancy rates compared to <9 (correlating to <1AA)
Blastocyst ___ score equally important to blastocyst quality score for implantation and pregnancy in fresh transfers
Blastocyst PROGRESSION score equally important
Blastocysts transferred on D5 vs D6 have ___ implantation, pregnancy, and multiple gestation rates
Higher (by about 2x)
In both fresh/frozen transfers
(Though D6 embryos still have good outcome)
Trophectoderm score is/is not a good indicator of outcome
IS
Increased pregnancy rates with increased/decreased:
- Blastocyst:ICM ratio
- ICM area
- ICM diameter
- Embryo area
- Embryo diameter
- Zona thickness
- INCREASED:
- Blastocyst:ICM ratio
- ICM area
- ICM diameter
- Embryo area
- Embryo diameter
- DECREASED:
Zona thickness
Day 3 cell progression (# of cells) can be predictive of ___
Blastocyst progression
Euploid embryos develop ___ than aneuploid embryos
Faster
A ___ euploid rate is seen in D5 vs D6 blastocysts
Greater
What were early vitrification solutions composed of?
High molar
High viscosity
- Ethylene glycol
- DMSO
Vitrification survival is dependent on the cooling/warming rate?
warming
Open/closed system devices have exceptionally high cooling rates due to low/high thermal mass of large/small volumes
Open
Low
Small
What is considered an ultra-rapid cooling rate?
Ultra-rapid warming rate?
How is this ultra-rapid warming rate achieved?
- 50,000 degrees Celsius/minute
+100,000 degrees Celsius/minute
Direct placement of device into sucrose solution
What is a universal warming solution for vitrified and slow-freeze embryos/oocytes?
1.0 M sucrose > transfer in multi-step decreasing dilutions at 3 min intervals
What is the survival rate of vitrified blastocysts?
95%
Number of eggs needed to approach fresh oocyte transfer pregnancy rates among vitrified oocytes is:
12 oocytes if <36 yo
33 if >36
What is meant by an orthotopic site for ovarian auto-transplantation?
Pelvic auto-transplantation (as opposed to heterotopic)
Which is currently the standard method of freezing of ovarian tissue?
Slow-freezing, though vitrification studies are promising
What is important to leave attached when removing a whole ovary for cryopreservation?
Large vascular pedicle - to introduce cryoprotectant to all cells and to subsequently remove it upon re-warming
