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Role of IVF Cabinets in IVF Labs

By Esco Medical, 07 May, 2015

External Factors Affecting Oocytes and Embryos in Vitro

 

During all handling of oocytes/embryos they must be protected from physical/chemical changes, specifically:

 

  • Temperature changes
  • Culture medium osmolarity and pH
  • Air quality

 

A. Temperature

 

  • The oocyte (female germ cell in the process of development) in particular is extremely sensitive to alterations in temperature.
  • Tempeature shifts can affect trans-membrane transport and many intracellular metabolic processes.
  • Human oocytes and embryos must be held as closely as possible at a stable 37 degree C.
  • Temperature also affects the pH value of the sample.
  • Evaporation affects the pH value of the sample.
  • A significant, but poorly-recognized, confounding aspect of temperature control during the microscopic observation of oocytes and embryos in dishes is that the design of all disposable plastic dishes does not allow the base of the dish to come into direct contact with the microscope stage, there is always an air gap (see Figure). Because air is a poor conductor of heat, this air gap greatly reduces the efficacy of heated stages (either those attached to or built into the microscope, or the heated work surfaces of the cabinet, allowing the medium in dishes to cool below the temperature at which the heated surface is set.

 

B. Culture Medium Osmolarity and pH

 

The composition of culture media used for human IVF have evolved markedly over recent years with the increasingly widespread use of "sequential culture media" suites that comprise a range of media and buffers designed to provide optimized support to each stage of the process from oocyte retrieval through fertilization and embryo cleavage to the blastocyst.

 

Correct use of all culture media requires the use of special atmospheres that are, at a minimum, enriched in carbon dioxide.

Additionally it is now recognized that the human embryo shows better development, and implantation potential, if the oxygen tension is reduced from that in normal air. However, there is great confusion between CO2 and O2 concentrations (expressed in volumetric percentage terms) and their partial pressure. Firstly, at higher elevations, not only does atmospheric pressure decrease, but the relative proportions of oxygen and nitrogen (and the other minor gases) also change: for example, whereas there is 20.95% O2 at sea level, at an elevation of 1000 m there is only 18.55% O2 and at 1600 m (e.g. Denver) there is just 17.2%. The same changes affect CO2, so that if one wishes to achieve a pH of 7.3 in a solution of 25mM bicarbonate at sea level, 6.0% CO2 is required according to the Henderson-Hasselbach equation otherwise the pH will shift and bicarbonate ions will be lost until the solution reaches a new equilibrium. But at 1600 m above sea level, almost 7.5% CO2 is required to achieve the correct partial pressure (ppCO2) to maintain the bicarbonate ions in solution and the pH at 7.3.

 

A major problem with bicarbonate-buffered media is that they take a long time to reach equilibrium, but out-gas very quickly. Recent studies [4] have shown that a 50 ul droplet of medium under oil will out-gas after removal from a CO2 incubator so that the pH has shifted above 7.45 within 2 minutes and that after replacing the dish in the CO2 incubator it will take 35 minutes to re-equilibrate the pH (only about 15 min for Petri dishes containing 5ml of medium). These differences are due to the relative magnitudes of the differential CO2 contents between the equilibrated medium and air and between the incubator's atmosphere and the partially out-gassed medium.

 

Finally in this regard, if culture medium is exposed to air that is not well-saturated with water vapor then there will be evaporative loss from the medium with concomitant increases in medium osmolarity. Evaporation is also higher at warmer temperatures. Whereas culture under oil helps combat this problem, during oocyte retrievals the dishes are typically open without an oil overlay (which would greatly complicate the procedure).

 

C. Air Quality

 

Because of their high metabolic and cell division rates, embryos are highly sensitive to toxic chemicals, thereby making IVF/ART laboratories and procedure rooms especially high risk areas for volatile organic compounds (VOCs) and other air pollutants.

 

It is a common misconception that HEPA (high efficiency particulate air) filtration removes gaseous organic and inorganic molecules. HEPA, as its name implies, is a highly efficient system for removing particulates from the air (the standard requires a 99.97% efficiency for a 0.3 um particle), not low molecular weight gaseous molecules.

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