ART-Laboratory procedures Flashcards
Regarding oocyte assessment two anomalies remain that should be handled with caution:
Regarding oocyte assessment two anomalies remain that should be handled with caution:
- (i) ‘giant’ oocytes because of their likely abnormal` genetic constitution and
- (ii) sER clusters because of their potentially lethal outcomes.
The time for syngamy check is different for ICSI And IVF Insemination
true
The nature of the zona pellucida is important in oocyte grading.
false
Fast Cleaving embryos are considered normal
False
Fast Cleaving embryos are considered at risk of being abnormal
A fragmentation of 25% in a 4cell embryo equates to one blastomere in volume
true
A poor cleaving embryo may have either:…… , …….., …….
- severe fragmentation
- cells out of phase
- multinucleation
A 4AA Grade and a 411 Grade Blastocyst Are the Same?
true
Following aspiration, human oocytes are best held in
- Follicular fluid until fertilization at body temperature
- Bicarbonate buffered media at room temperature
- HEPES or MOPS buffered media at room temperature
- HEPES or MOPS buffered media at 37ºC
4.
If Day 0 is time of ovulation. human embryos would naturally enter the uterus on:
- Day 1
- Day 2
- Day 3
- Day 4
- Day 5
4.
Embryo enter the Uterus d4 of ovulation in natural
Which Statement is NOT true?
- pH strips are useful for checking
- Phenol red is a pH indicator and potentially oestrogenic
- Phenol red turns yellow in media with a pH of 6.5
- pH strips are usually sold non-sterile
- Phenol red is a clear solution that turns red in culture media
5.
The Optimum Embryo Development Stage for Transfer is:
- Always the blastocyst stage as these are the stronger embryos
- What works best in your own clinic
- Cleavage stage embryos since some grading is possible
- Whatever the patient requests
- When the embryonic genome is activated
2.
In ICSI, Immotile Sperm may be used Successfully after:
- positive response following exposure to hypo-osmotic medium
- electroporation
- treatment with calcium ionophore A23187
- showing motility in pentoxifylline containing medium
1, 4
To increase Humidity in Incubator……..
Placing a tray with 1L of water in the incubator will create humidity levels > 75%
advantages of oil overlay in culture media…….
reduce changes in Temp- and PH
Sequential Media was developed because there are changes in………………..
Sequential Media was developed because there are changes in embryo metabolism during cleavage
High O2 Coc- may increase the……….
free radical formation
Mineral Oil and Paraffin Oil
Paraffin oil and mineral oil are chemically slightly different
The polycarbon lipid tail of mineral oil contains more unsaturated bonds than paraffin oil
This makes mineral oil more unstable and prone to attack by free oxygen radicals and photo-oxidation
Paraffin oil is derived from fractionated distillation
Silicone Oil is a polymer
Silicone Oil may be more stable that paraffin oil.
Silicon oil is derived from gas chromatogrphy
High Temperature may be detrimental to the Oil
true
Washing Media Before use is a good idea because….
- Batch To Batch Variability
- It May Reduce Or Remove Water Soluble Toxins
- washing Media Containing EDTA may Reduce or Remove Cations Such as Zinc
*
Some Mineral Oil may Contain Preservatives Such as
Alpha-Tocopherol
Fertilisation Media Not Contain EDTA because:
- EDTA may chelate Ca2++
- Ca2++ is requried for the acrosome reaction
Biopsy Media free of Ca2++ but contain EDTA because….
to perform biopsy ⇒►and reduce blastomere compaction
Ca2++ is required for compaction
If Ca2++ is absent, the blastomeres cannot adhere
► then it can be biopsied
EDTA is a chelating agent so it will remove Ca2++ and facilitate biopsy
The Oocyte has …………..requirement for glucose
little or limited
Glucose is added to fertilization media for…….
sperm metabolism and motility
Penicillamine is sometimes used as a replacement for EDTA
true
Zinc and Cadmium are two potentially toxic cations removed by EDTA
true
EDTA is also used as a Free Radical Scavenger
true
EDTA can …………..Glycolysis in early cleavage stage Embryos
suppress
protein sources supplementation of culture media used in early years….
- Bovine serum albumin
- Human fetal cord serum
- Fertile Human donor serum
- Maternal Human serum
The main problems in their use include:
Occult toxicity
Immense lack of consistency
Variation in the source and grading
Untraceable risk of disease transmission
The development of stage specific media formulations allowed:
Replacement of serum with Human Serum Albumin (HSA)
Development of serum substitutes to augment HSA
The substitutes were mainly serum globulins
Pregnancy has been achieved in protein free media that includes:
- Polyvinvyl pyrrolidone (PVP)
- Polyvinyl alcohol (PVA)
- Hyaluronic acid
true
The requirements for HSA And Supplements such as Globulins and Growth Factors Appear Stage Dependent.
true
Phenol Red is
a guide to media pH
Not toxic to embryos
Not toxic to sperm
Not toxic to eggs
Straw colour at pH 7.2
Yellow at pH 7.0
Red at pH 7.4
Maroon at pH 7.8
The Actual PH Of Culture Medium With 5%-6% CO2 will be influenced by:
- Protein concentration
- The type of protein Supplementation
- Altitude
- Age of the medium
- The number of times a bottle has been opened
- The latitude
- The longitude
1.
A Change in the color of culture media is most likely due to
- The age of the medium
- The number of times the bottle has been opened
- The loss of CO2 from the medium
- Contamination
- Infection
2, 3
The Shelf Life of CM (at 5˚C) is:
- 2 weeks
- 4 weeks
- 6 weeks
- 8 weeks
- 10 weeks
- 12 weeks
5.
The Shelf Life of culture media CM (at 5˚C) is 10 weeks
The Main Source of a buildup of ammonia in culture media is due to
- Glucose
- Pyruvate
- Bicarbonate
- Glutamate
- Glutamate dipeptide
- Non-essential Acids
4.
The ability of an embryo to regulate its metabolism is directly related to its ability to implant and form a viable pregnancy
true
There are suggestions growth that rapid cleavage leads to loss of viability
true
precocious exposure to glucose may cause the early cleavage embryo to divide too fast leading to loss of energy stores and viability
true
The Main Metabolic Pathways Include
Pyruvate and lactate
Glucose
Amino Acids
Fatty acid and b-oxidation
The Oviduct concentration of pyruvate is:
- 0.22 mM
- 0.32 mM
- 0.42 mM
- 0.52 mM
- 0.62 mM
2.
The Uterine concentration of pyruvate is:
- 0.1 mM
- 0.2 mM
- 0.3 mM
- 0.4 mM
- 0.5 mM
1.
The Oviduct Concentration of LACTATE is:
- 6.5 mM
- 7.5 mM
- 8.5 mM
- 9.5 mM
- 10.5 mM
5.
Oviduct natural environment
Uterine
Pyruvate conc-
- 32 mM
- 1 mM
LACTATE conc-
- 5 mM
- 87 mM
Glucose
- 5 mM
- 15 mM
Amino Acids
non-essential amino acids
Mix of ess & non-ess
The Metabolic Quotient of an early-stage cleavage embryo is similar to
- heart tissue
- brain tissue
- muscle tissue
- liver tissue
- tumor tissue
2.
The Metabolic Quotient of a blastocyst is similar to
- heart tissue
- brain tissue
- muscle tissue
- liver tissue
- tumor tissue
5.
remember
Metabolic Quotient of :
early stage embryo ≈►brain tissue
blastocyst is similar to ≈►tumor tissue
Artificial oocyte activation is proposed when
- A few oocytes failed to fertilise
- Less than half the oocytes fail to fertilise
- More than half the oocytes fail to fertilise
- All the oocytes fail to fertilise
- All the mature oocytes fail to fertilise
5
A Common Ionphore used is
- A23185
- A23187
- A13289
- A23191
- A23193
2.
The Common Ionophore Is Known As
- Calcimycin
- Calcomycin
- Calcamycin
- Calcmycin
- Calcemycin
1.
Oocyte Activation
Reinitiates the cell cycle
Induces oscillations in free calcium levels
Controls the timing of subsequent cell divisions
It is assumed to do so by overcoming a failure to up-regulate free calcium levels after sperm entrance into the Ooplasm
The pattern of calcium oscillations (transients)
differs between
normally fertilized and artificial oocyte activation (AOA)
true
the use of Ionophores in IVF
Should be considered experimental
Be restricted to only total fertilisation failure
Has been labelled as experimental by the HFEA
has the potential to be included in all ICSI cases to minimise the risk of failed fertilisation
The HFEA Recommendations:
recommends it use to restricted to selected patients e.g. PLC deficiencies
recommends that patients are provided with its efficacy and potential risks
a post hoc indication
logistic regression to evaluate interdependency
logistic regression to evaluate interdependency.
-Evaluating the interdependency between a variety of factors influencing success rates. Here are some specific examples:
-Evaluate the influence of environmental factors like temperature, pH, and oxygen levels on embryo development
-Compare different culture media formulations by using media composition as predictors and embryo developmental
-Analyze the relationship between sperm parameters like motility, morphology, and concentration with fertilization rates or embryo quality using logistic regression.
What is the laboratory decisions in case of oocyte with large PB?
oocyte with large PB
(this may not be possible for oocytes that are inseminated via IVF, rather than ICSI).
The size of the polar body should only be noted if it is exceptionally large.
It was the consensus that oocytes with an abnormally large polar body should not be inseminated, due to the risk of oocyte aneuploidy.
Oocytes with sER disk ?
oocytes with sER should not be inseminated. In addition, it was noted that the sibling oocytes should also be examined for the presence of sER disks
Large vacuoles
Large vacuoles (>14 mm in diameter) are associated with fertilization failure.
In oocytes that are fertilized, those vacuoles that persist past syngamy can interfere with cleavage planes, resulting in a lower blastocyst rate
pronuclei with no NPBs called?
pronuclei with no NPBs called “ghost pronuclei’
while
a single NPBs (‘bulls-eye pronuclei”)
Multinucleation assessment on Day 2 is more reliable than Day 3 ?
Multinucleation assessment on Day 2 is more reliable than Day 3
due to difficult assess the small size blastomeres on Day 3 compared to D2
early compaction on Day 3 indicates ……?
Early compaction on Day 3 is atypical, of unknown biological significance.
Final Sperm conc after processing for convential IVF?
-After processing by sperm wash or DG,
-Final concentration and motility are reassesed.
Motile sperm are suspended in an amount of media that allows final conc 25*10^3 -50 *10^3 motile sperm per mL.
Main components of the microinjection setupMicroscope for (ICSI)
ICSI setting includes
an inverted microscope capable of 400-600x magnification connected to a video system.
The microscope has a heated stage, two electric motor-driven coarse- and fine-control manipulators, and two microinjectors.
The microscope must be equipped with Nomarski or other phase interference optics
True or false:
PGT-A allowed single ET
Euploid embryo after PGT-a is highly recommended for Single ET
Epigenetics
Changes(reprogramming) in Gene expression by mechanism rather than DNA
-Histon modification
-DNA methylation
women 47 old should be encouraged to ART
women 47 old should discourage to ART
women attende to embryo tansfer but hasband didn’t, and she told that, he already signed the consent yesterday, what’s your decision?
- postpone ET or freez all
- Inseminatin and ET should be performed in presence of the couple.
