IVF Equipment and Instrumentation Notes

Incubators

  • Objective:
    • List types of incubators commonly used in IVF labs.
    • Know major features of each type.
    • Know advantages and disadvantages of each type.
    • Explain factors limiting effectiveness of each type.

Types of Incubators Used in IVF Laboratories:

  1. Large Box Incubator
  2. Benchtop Incubator
  3. Time Lapse Incubator

Large Box Incubators

  • Original type used in IVF labs: Borrowed tech from tissue culture labs.
  • Purpose
    • Provide conditions for growth of eggs/embryos in sterile vessel (Petri dish).
    • Humidified chamber at/near body temperature.
    • Gas environment: mix of carbon dioxide, oxygen, and nitrogen.
  • Features
    • Chamber has 3-4 shelves for embryo culture dishes.
    • Shelves have holes for air circulation.
    • Water pan provides near 100% humidity (prevents evaporation).
    • Humidity linked to incubator's ability to sense CO2 level.
    • Heating accomplished by:
      • Electric coils in walls, floor, ceiling to warm air.
      • Electric coils to heat water jacket (more even heating, guards against temp loss).
        • Downside: rust formation if proper water grade not used; water level must be monitored; heavy and requires sturdy bench; relocation requires draining/refilling.
    • Gas concentration:
      • Early goal: 5% CO_2 in air (20% oxygen).
      • Current goal: 5% CO2, 5% O2, 90% N_2 (may vary with media).
    • Large doors cause atmospheric instability: inner door with small, sealed doors added to reduce loss of atmosphere.
  • Advantages
    • Capacity: can accommodate a large number of patients/embryos (if used wisely).
    • Shelf capacity is generous for patients with many embryos cultured in individual drops.
    • Cost: relatively low cost allows purchasing enough incubators.
  • Disadvantages
    • Slow recovery after door opening.
    • Tendency to overload, compromising culture conditions.
  • Optimizing Use
    • Use incubators providing triple gas atmosphere (5% CO2, 5% O2, 90% N_2).
    • Purchase incubators with inner door containing several small doors.
    • Reduce door openings by purchasing enough incubators; limit patients/dishes per incubator.
    • Daily QC checks for temperature, gas concentrations, and humidity.
    • Check pH of media equilibrated in each incubator.
    • Confirm qualified service department and establish routine maintenance.

Benchtop Incubators

  • Purpose: Small growth chamber offers quick recovery to optimal conditions to reduce stress on embryos.
  • Features
    • Small footprint, can be placed in small areas.
    • Chamber has a lid that opens upward; room for 2-3 Petri dishes.
    • Some models heat both lid and floor.
    • Small chamber volume uses small amount of gas.
    • Some require pre-mixed triple gas, others mix from separate CO2 and N2 cylinders.
    • Quick recovery of gas concentration due to faster flow rate, called purge.
    • Humidity accomplished by gas flowing through water before entering chamber.
  • Advantages
    • Faster recovery time than large box incubators.
    • Low gas usage may result in long-term savings.
    • Better use of space in labs with limited bench space.
  • Disadvantages
    • Capacity is smaller than large box incubators.
    • May dictate use of group culture within dishes to save space.
    • Cost of purchasing sufficient incubators can be prohibitive.
  • Optimizing Use
    • Purchase incubators with independent controls for each chamber.
    • Choose an incubator that allows ease of daily QC checks.
    • Confirm qualified service department and establish routine maintenance.

Time-Lapse Incubators

  • Purpose: Benchtop incubator with camera system to record embryo growth.
    • Captures cell division times to determine best quality embryos.
    • Premise: time of key cell divisions provides more information than morphology alone.
  • Features
    • Similar to benchtop incubators: small humidified chambers, heated to 37°C, triple gas.
    • Quick atmosphere recovery.
    • Embryos photographed at regular intervals; images viewed remotely.
    • Some are camera systems placed in large box incubators (take up shelf space, space for only one dish per camera, require computer and monitor).
  • Advantages
    • Increased knowledge of embryos and reduced embryo stress: embryos viewed without removal from incubator.
    • More informed choice of embryos for transfer and cryopreservation.
  • Disadvantages
    • Cost: expensive technology, relatively new.
    • Require special growth dishes (more expensive, not as easy to load/unload).
    • Uncertainty about how best to use the data (differing opinions on critical embryo divisions).
  • Optimizing Use
    • Information should be viewed/analyzed by lab director and senior embryologists.
    • Interpret information after becoming familiar with manufacturer’s suggestions.

Laminar Flow Hoods

  • Objectives
    • Explain the purpose of laminar flow hoods in the IVF laboratory.
    • Understand the importance of working in a laminar flow hood with culture media and patient’s cells.
    • Learn how to work in a laminar flow hood without contaminating biological specimens and their media.
  • Purpose
    • Prevent contamination of cells (sperm, eggs, embryos) being prepared for placement into patient.
    • All media/chemicals coming into contact with cells must be handled within hood.
    • Culture dishes prepared for retrieving eggs and growing embryos must be kept within hood.
    • Bacterial contamination kills sperm, eggs, and embryos.
  • Design
    • Bench-style cabinets with stainless-steel work surface.
    • Air pulled through HEPA filter to remove contaminants before flowing over work surface.
    • Airflow continues toward lab worker and out into room.
    • Some hoods re-capture air and re-sterilize it.
  • Method
    • Assume air in room is contaminated with bacteria and mold spores.
    • Assume technician’s hands are contaminated.
    • Arrange hood so no objects are between origin of sterile air and specimen.
  • Maintenance
    • Keep clean at all times.
    • Wipe spills immediately and decontaminate with 6% hydrogen peroxide.
    • Do not use alcohol for decontamination.
    • Clean and decontaminate between each patient’s specimens and at end of workday.
    • Check and change prefilters on regularly scheduled intervals.
  • Additional Features
    • Stereomicroscopes mounted directly to heated work surface.
    • Triple gas airflow for culture dishes.
    • Camera for projecting image of specimen.

Microscopes

  • Objectives
    • Explain the function of each part of the light microscope.
    • Illustrate the basic design of the inverted microscope.
    • Explain the basic structure, operation and importance of the stereomicroscope.
    • Demonstrate the basic principles of microscopic techniques used for improving specimen contrast or imaging (darkfield, polarized, phase contrast and Hoffman Modulation Contrast).

Types of Microscopes Used in the ART Laboratories

  1. Light Microscope
  2. Inverted Microscope
  3. Stereomicroscope

Light Microscope

  • Purpose
    • View stained or living cells placed on glass slides.
    • Used in Andrology lab for semen analyses and other diagnostic tests.
    • Used in viewing sperm samples being prepped for IUI and IVF.
  • Components
    • Illumination Source
      • Incandescent tungsten halogen bulb in a protective/reflective housing.
      • Light projected by collector lens through condenser aperture diaphragm into sub-stage condenser.
    • Condenser Aperture Diaphragm
      • Light intensity adjusted by varying the opening of the aperture.
    • Sub-Stage Condenser
      • Receives light from collector lens and concentrates it in a cone of light.
      • Illuminates specimen from below and directs its image to the lens of the objective.
      • Rotating turret with varying number of apertures (each projects a different size cone of light).
      • Sub-stage condenser must be rotated to project the corresponding cone of light to optimize image quality.
    • Specimen Stage
      • Opening in middle for viewing the specimen.
      • Slide or counting chamber secured with clips or holder.
      • Mechanical stages equipped with control arm.
      • May have heating element to maintain body temperature.
    • Objectives
      • Most important components of an optical microscope.
      • Located above the stage on a rotating nosepiece.
      • Quality ones correct image aberrations.
      • Markings on housing explain aberration corrections, magnification, and type of contrast enhancement.
    • Eyepieces (Oculars)
      • Further magnify the image of the specimen.
      • Adjusted for viewer’s pupillary distance.
      • Individually focused to account for focusing distance of each eye (diopter adjustment).
        *Designed so the observer may see a single, three- dimensional image while using both eyes to view the specimen.
    • Adjusting the microscope for parfocality
      • Turn the rotating nosepiece to the highest objective (ex. 40X).
      • Using the right eye only (close the left eye), focus on a sharp edge of the specimen by adjusting the fine focus of the microscope.
      • Turn the rotating nosepiece to the lowest objective (ex. 10X).
      • Using the left eye only (close the right eye), focus on the same sharp edge of the specimen by adjusting the left eyepiece.
      • Using both eyes, the image should be in focus at each magnification (parfocal).

Inverted Microscope

  • Purpose
    • View specimen while contained in a relatively large volume of fluid (Petri dish).
    • Used in IVF lab for viewing eggs and embryos growing in a Petri dish.
  • Components/Design
    • Illumination Source, Condenser Aperture Diaphragm, Condenser
      • Similar to those described for Light Microscopy but positioned above the stage.
    • Objectives
      • Positioned below the stage on a rotating nosepiece.
      • Allow observation of eggs and embryos in detail by positioning objectives very close to underside of Petri dish.
      • Designed to view specimens that are larger than those observed with a light microscope.
      • Require a greater working distance and limits magnification provided by the objective to 40X.
    • Specimen Stage
      • Typically heated to keep Petri dishes containing eggs and embryos near body temperature.
    • Micromanipulators
      • Used for procedures such as ICSI, assisted hatching or embryo biopsy.
      • Translate large movements of the hands of the embryologist to precise, controlled movements of micro tools.

Stereomicroscope

  • Purpose
    • View eggs and embryos in Petri dishes at lower magnification.
    • Allows embryologist to manually move eggs and embryos from one vessel to another with a hand-held pipette.
  • Components/Design
    • accomplished by a single objective which has a fixed distance above the stage.
      • Allows for a range of magnification using a zoom control magnification knob.
      • Magnification generally ranges between 0.5X and 20X.
      • Illumination source in the base, mirror allows adjustment of light shining on specimen.
      • No diaphragm or condenser.
      • Eyepieces similar to light microscopes and inverted microscopes (adjustable for pupillary distance and diopter adjustment).
      • Adjustable for parfocality same as light microscopes and inverted microscopes.

Microscope Contrast Enhancement Techniques

  • Purpose: View living specimens with higher contrast to better see details of morphology.
  • Morphology of embryos influences selection for embryo transfer and cryopreservation.
  • Types of Contrast Enhancement Techniques
    • Darkfield
      • Oblique illumination through the condenser to enhance contrast.
      • Direct light partially blocked by an opaque stop in the condenser.
      • Light transmitted onto the specimen at an oblique angle, specimen partially illuminated on black background.
    • Polarized Light
      • Filter in the condenser blocks all but linearly polarized light rays.
      • Second polarizing filter (analyzer) placed in the condenser.
      • Light exiting analyzer depends on its angle of orientation to the first polarizer.
      • Enhanced contrast achieved by rotating analyzer slowly to an angle in between the two extremes.
    • Phase Contrast
      • Produce high contrast images of transparent living cells.
      • Most used in Andrology laboratory for performing semen analyses and other diagnostic tests.
      • Annulus ring in condenser and phase plate in objective bend light rays.
      • Contrast seen when phase plate in objective is aligned with correct size annulus ring in the condenser.
      • Condenser turret rotated to proper numerical setting with each objective change.
    • Hoffman Modulation Contrast
      • Increase the visibility of living, transparent cells, making their morphology more apparent.
      • Two polarizing light filters (one in objective and one in condenser) alter light intensity.
      • Each objective has a modulator with three zones of polarization: small dark zone (1% light), narrow gray zone (15% light) and larger clear zone (100% light).
      • Rectangular polarizing filter in each condenser setting.
      • Polarizer turned slowly to increase or decrease contrast.
      • Creates sharp-edged images without halo effect; method of choice for observing specimens in plastic containers.