The Lab Flashcards
Units of TSH
mIU/mL
Units of Estrogen
pg/mL
Units of Testosterone
ng/dL
Normal range 20-80
Liquid chromatography and mass spec
What do day 3 embryo media need?
lactate, pyruvate, non essential AA, Ca chelator (EDTA), no glucose
What do blast media need?
non essential and essential AA, glucose
Benefits of extended blastocyst culture?
1) Blastocyst transfer is associated with a shorter time to pregnancy - emotional value and reduced cost to patients
2) LBR was significantly improved in day 5 / 6 (39.1 vs 30.3)
3) Embryonic genome activates on day 3 - so those that dont activate dont make it to day 5-6 ; aka Fewer embryos in cryo that would not end up being viable / end in a viable pregnancy → better quality blasts are the ones making it to day 5
4) Better uterine receptivity and environment with day 5 transfers
Cons of day 5 include obstetric outcomes as above and potential increase in MZT
If a patient has poor quality blasts that are not going to make it to day 5 - better to put back in the uterus with appropriate counseling of lower success rates than to discard / die off in the lab?
Units of progesterone
ng/dL
Normal volume of SA
1.5-5.5 mL
What is hypospermia?
Low volume < 1.5
Normal pH of semen
7.2-7.8
What are round cells in an SA?
Either immature germ cells or WBC
Purpose of eosin staining?
Differentiate dead cells from living non motile cells eg. necrozoospermia versus motile dyskinesis from immotile cilia syndrome (Eg. Kartageners)
Consider doing vital staining if motility is less than 40%
Asthenozoospermia
Decreased motility
Polyzoospermia
> 250 x 10^6
Azoospermia
No sperm
Post centrifugation (1000 g x 15 min) wet prep
Crypotozoospermia
so few sperm only noted in the post centrifugation sample
Aspermia
no ejaculate volume
Evaluation post vasectomy
2 months post op and after 10 ejaculations
Also perform centrifugation
Patient considered sterile if no sperm in SA/centrifugation sample 1 month apart
Normal vaginal pH
3-5
Purpose of cervical mucus
Vaginal mucus protects sperm from hostile, acidic vaginal environment
Provide energy
Filters sperm
Acts as a reservoir to store sperm in the crypts
Initiate sperm capacitation
**Estradiol stimulates cervical mucus production that is copious, thin, acellular, and alkaline
Clinical exam of cervical mucus
Volume (peaks at 500 uL), viscosity (watery at ovulation versus high viscosity premenstrual), ferning (want teriatry or quaternary ferns), spinnbarkeit (stretchiness of mucus and measuring length before it breaks), cellularity
Score > 10 favorable for sperm penetration
pH not measured, but ideal is 7-8.5
Post coital test
Evaluate cervical mucus 9-24 hours after intercourse
Unlubricated speculum and sample discharge in posterior fornix
Evaluate number and motility of sperm
Cause of antisperm antibodies in men?
Autoimmunization to sperm antigens should not happen because of the blood testes barrier and epididymis from tight junctions, but anything that disrupts this can result in ASAB
Vasectomy, congenital anomaly, CBAVD, CF, trauma
50-70% of men with vasectomy have ASAB
Theoretical effects of ASAB?
Cause agglutination and clumping that would prevent sperm motility and cervical mucus penetration
Hypo-osmotic swelling test
Differentiates dead sperm from living sperm with an intact membrane that are immotile
If living sperm is placed in a hypo-osmotic liquid, the sperm will swell to maintain an osmotic gradient resulting in swelling of the membrane and curling of the tail
Normal if > 60% sperm swell in the sample
Also useful during TESE because sperm become motile after passing through the epididymis
What is an acrosome?
Modified golgi apparatus covering upper 75% of the sperm head between the inner and outer acrosomal membranes
Contains hydrolases (most common acrosin)
At time of acrosome reaction the plasma membrane and outer acrosomal membrane fuse (exocytic process)
Globospermia
Genetic disease leading to absence of the acrosome
Standard cryoprecipitant for sperm?
Glycerol
Ovarian tissue cryopreservation
~130 live births
Primordial follicles/primary oocytes in the ovarian cortical tissue are frozen
Thin layer of cortex is frozen (1 mm)
What stage is MII arrested in
Metaphase of meiosis II with chromosomes arranged on bipolar spindle
Polymerase chain reaction
Denaturation -> Annealing -> Extension/Elongation
Denaturation: heat DNA (20-30sec) yields 2
ss DNA molecules @ 92 degrees
Annealing: lower temp to 50–65 °C for 20–40 seconds,
allowing annealing of the primers to each of the single-stranded DNA templates. One for each of the two single-stranded complements containing the target region.
Extension/elongation: Raise temp @ 72 degrees, DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free nucleotides (dNTP)
PCR pros and cons
Advantages: Rapid, fast, the sequencing of unknown etiologies of many diseases are being figured out
by the PCR. Allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail (e.g. Down syndrome for only a few fetal cells)
Disadvantages: One major limitation of PCR is that prior information about the target sequence is
necessary in order to generate the primers that will allow its selective amplification.
Real time PCR
faster than conventional PCR
Uses fluorescent probes to measure the amount of DNA produced while PCR is occurring
Provides a quantitative comparison between samples by limiting the analysis to the stage where the reaction shows linear amplification
Used in the diagnosis of infectious disease or to measure gene expression (when combined with reverse transcription)
Competitive immunoassay
Excess labelled hormone + unknown amount in sample and limited antibody
Competition between labeled hormone and that in sample. Less bound labeled hormone means more in the sample
Noncompetitive immunoassay/ELISA
Accuracy
extent to which a measurement agrees with the actual value
QC: standard assay curve
Precision
variability encountered when measuring same sample multiple times
Expressed as coefficient of variation = SD/mean of replicate determinations x100 (%)
AMH
homodimeric glycoprotein linked by disulfide bonds
TGF-B superfamily
Typically measured by ELISA
Trouble shoot low level HCG from cross-reacting heterophile aBs
switch from sandwich (ELISA assay) to competitive RIA
Postmenopausal low level HCG
consider using elevated E2/P4 to suppress
Chromatography
The physical separation of components in a mixture via two phases
Mobile phase: mixture is dissolved in fluid
Stationary phase: substance is fixed in place
Works because constituents of a mixture travel at different speeds, causing separation.
Differential retention occurs in the stationary phase due to differences in a compound’s partition coefficient.
Can be used to purify components in a mixture as well as to analyze for the presence of (or measure proportions) of analytes in a mixture.
Mass Spectometry
Measures the masses within a sample via substance ionization and subsequent sorting of ions into a spectrum based on their mass-to-charge ratio
Plot of the relative abundance of detected ions as a function of the mass-to-charge ratio is generated so that atoms or molecules can be identified via:
Correlating known masses to identified masses
Detection of a characteristic fragmentation pattern
Often used in combination with chromatography to detect components of the mixture being separated The physical separation of components in a mixture via two phases
Mobile phase: mixture is dissolved in fluid
Stationary phase: substance is fixed in place
Works because constituents of a mixture travel at different speeds, causing separation. Differential retention occurs in the stationary phase due to differences in a compound’s partition coefficient.
Can be used to purify components in a mixture as well as to analyze for the presence of (or measure proportions) of analytes in a mixture.
What do you need for PCR?
1) Free nucleotides
2) Primers
3) DNA polymerase Taq = Heat-stable
4) DNA template (target)
5) Thermal cycler
Reverse transcription PCR
Amplifies a sequence of RNA after it is converted to its complementary DNA by reverse transcriptase (originally discovered in retroviruses)
cDNA is then processed by standard PCR procedure to yield large quantities of DNA corresponding to the RNA of interest
DNA sequencing
Set of approaches that allow the nucleotides in a particular portion of the genome to be identified in order
Most common approach for the past 40 years was Sanger sequencing (also known as chain-termination sequencing)
An in vitro DNA replication is carried out using DNA polymerase at sites directed by DNA primers
Nucleotides include a subset that terminated DNA strand elongation- called dideoxynucleotides or ddNTPs
ddNTPs are typically fluorescently labeled to allow automated detection with the fluorescent wavelength matched to the length of the fragment
Rate of monozygotic twins in blastocyst culture
2-3%
Maximum number of recommended oocyte donor cycles
6
NNT with PGT-A to have additional live birth among > 37
21 cycles and 35 ET
Motility stimulants
caffeine or pentoxifylline can also be used where sperm are alive but not swimming and it works on sperm that has been frozen/thawed
