Lab procedures Flashcards
IMSI
Intracytoplasmic morphologically selected sperm injection
Examination of individual sperm at high magnification by the invertes microscope equipped with high power optics (x6000+) enhanced by digital imaging.
Assesses - head shape, head size, midpiece and number of vacuoles.
Cochrane review has shown no benefit on LBR compared to conventional ICSI (very low quality data)
Might be beneficial in poor semen parameters in increasing embryo quality and reducing miscarriage.
PIEZO ICSI
Normal icsi needle goes in and push through, suck back and break membrane the cytoplasm goes back into egg.
In piezo - microvibrations in holder allow you to drill a hole in ZP. Pipette can just be slid in, PIEZO pulse then used to break cytoplasmic membrane.
Seems to be assoc with a reduction in lysis. Possibly one extra embryo. Limitation - TGA (therapeutic goods administration) unapproved.
Sperm selection techniques
- Sperm selection techniques base on sperm membrane:
- hyaluronic acid-selected sperm (HA-ICSI)
- Magnetic activated cell sorting (MACS)
- Zeta sperm selection - Sperm selection based on morphology
- normal magnification microscope - embryologist assessment (standard ICSI)
- Intracystoplasmic morphologically selected sperm injection (IMSI) - Sperm viability selection:
- sperm tail flexibility test
- hypo-osmotic swelling test (HOS)
- Laser test
- pentoxyfylline (artificial sperm activation test) - Sperm selection by guidance mechanisms (microfluidic systems)
- Resemblance of guidance within the female genital tract, could be a more physiological mechanism for selection. But no evidence of improved outcomes yet.
- rheotaxis - a flow is created through a series of microchannels towards a well.
- chemotaxis - two wells are connected by a 2mm length and 2.5mm diameter tube. one contains a chemoattactant
- thermotaxis - sperm placed on a drop of medium connected by a capillary to a second drop of free cells. Temperature gradient moves towards warmer temperature
Sperm viability - why is it important and explain tests.
Necessary for fertilisation and embryo development
Testicular sperm samples can sometimes result in only immotile sperm being retrieved
Immotile sperm are not necessarily nonviable
Important to identify which sperm are viable amongst the immotile sperm population
Sperm tail flexibility test
Dead sperm usually display stiff sperm tails.
Sperm gently touched with ICSI needle to establish if sperm are moving or if the tail bends after the capillary touch.
Sperm with flexible and bending tails are more likely to be viable
Method is subjective and not very reliable
Hypo-osmotic swelling (HOS) test
HOS test solution is made by 50% handling media and 50% deionised water.
Mostly used for ejaculated sperm.
Tail of sperm is released into the HOS drop. If the tail curls = viable.
Laser test
Laser focus is placed near sperm tail and using a low pulse length a single shot applied.
Viable sperm demostrate a bend or curl to tail.
Pentoxyfylline/Theophylline
Chemical test that increases sperm motility for a limited duration.
HVAC system
Heating, ventilation, air conditioninng
Separate system for ART lab (separate to other parts of hospital/building)
Controls temp, humidity, concentration or particulates and VOCs.
Must run 24hr/day
VOC
Volatile organic compounds whose composition makes it possible to evaporate under normal indoor atmospheric conditions of temperature and pressure.
Sources:
Off-gassing of lab fabrics and consumables
Gases supplying incubators and workstations
From contaminants which have dissolved in the culture medium or mineral oil
Lab material itself = flooring, benches, paints etc
Not all VOCs are toxic to gametes highly toxic - formaldehyde, acetaldehyde and higher molecular weight aldehydes.
Formaldehyde reacts with albumin and denatures it.
MDF high source of formaldehyde.
Cold sterilisation significant source of high molecular weight aldehydes
Total quality management (TQM) in the laboratory
quality management, which encompasses quality control (QC), quality assurance (QA) and quality improvement (QI);
risk management in all its forms, including risk elimination, risk avoidance, risk minimization, risk transfer, and risk acceptance; and
systems management, incorporating process mapping and systems analysis, which requires Indicators and Benchmarks.
Quality assurance
Quality assurance (QA) is all of the activities undertaken within the quality system, including quality control, that together provide confidence that the service will meet its quality targets.
Quality control
Quality control (QC) is more specific, and is related to the creation of quality specifications for each task or service, the design and application of ways to assess whether these specifications are being achieved, and taking any necessary actions to ensure that they are met.
Quality improvement
Quality improvement (QI) is the part of the quality system that is related to increasing its effectiveness and efficiency. “Continuous Quality Improvement” is an organizational culture of actively seeking QI opportunities to further improve outcomes. The QI process is cyclical, as the effect of any new processes that are implemented to improve outcomes must be monitored and assessed, and new potential solutions devised if necessary
Risk elimination
choosing to not include a process because the associated risk is judged to be too great. An example of risk elimination would be the decision to not offer “rescue ICSI” (the re‐insemination of an oocyte that is apparently unfertilized the day after IVF insemination) because the risk assessment suggests there is a significant risk of polyspermy (i.e. because there was already a spermatozoon in the oocyte, but the oocyte failed to activate)
Risk avoidance:
choosing to avoid an action that would trigger a risk – a real‐world example is deciding not to invest in an opportunity as a consequence of the risk assessment;
Risk reduction / minimization
identifying and modifying processes to reduce the likelihood of the risk, or reducing its effect should it still occur;
Risk transfer:
moving the responsibility for managing the risk, or at least its consequences, to another organization or person. An example would be a warning sign;
Risk acceptance / retention:
accepting the level of risk inherent in a process, because it cannot be reduced any further, at least for the time being.
Media prep
Day before to equilibrate pH, osomlality and temp.
Sequential (D0-3) pyruvate, D3-6 - glucose
Single step (D0-6)
Sperm preparation techniques
Direct swim up
Small volume of liquefied semen is layered under 1ml of warm medium in round bottom tube
Incubated at 37degress for 15-30mins
Top portion removed and centrifuged for 10minutes to remove any residual seminal plasma contamination
Resuspended in fresh media to desired concentration of spermatozoa.
Density gradient centrifugation (DGC)
Works by separating the components of a cell suspension based on each cell’s density.
Two layers of suspension fluid warmed and placed into conical centrifuge tube
2ml liquefied sperm placed on top
centrifuged 20mins
Seminal plasma remains on top = sperm pellet migrates to bottom
Pellet transferred to fresh tube and wash media added
Centrifuged 10mins
Pellet resuspended in fresh medium
Simple washing:
- Dilute with medium to promote removal of seminal plasma and centrifuge for 5-10 minutes, aspirate and discard supernatants and repeat. Gentle pipette so concentration and motility can be determined
- Often used for IUI as has the highest yield of sperm and adequate if good quality sample, won’t be possible for many samples of poorer quality. Does not separate progressive motile sufficiently for IVF
Standard insemination
semen sample processed and diluted to 1million/ml
40-42hours post trigger the lab will place sperm with oocytes.
50 000 sperm added.
Some units will strip off some of the COC
ICSI
39hrs post trigger COC removed (allows identification of where polar body is )
Sperm sample prepared
ICSI is performed 40-42hours post trigger.
Care with
- angle of the ICSI needle and where PB is positioned (underneath polar body is usually where metaphase plate is)
Fertilisation assessment
16-18hours post insemination/ICSI
Visualise pronuclei and polar bodies.
Cleavage embryo development
D2 - 4-6cells
D3 - 7-11cells
Moved into a new media type if sequential media system used.
Can be scored - cell numbe, cell quality, frag present.
Embryo can arrest:
Chromosomal
Egg/sperm abnormaliites
Switch to embryonic genome occurs on day 3.
Considered physiologically premature to expose cleavage stage embryos to the uterine envrinoment.
Day 4
Embryo complaction
Cell division continue and compaction commences
Early blastulation may occur
Sperm genome activates
Failure of compaction may be related to sperm.
D5-6
Blastocoel cavity forms
ICM and Trophectoderm occurs
Embryo transfer
Selection based on morphology
Often placed in hyaluronan specialised media
5-12ul of media used.
If frozen embryo - assessed for survival post warming.
