How to secure your culture system when introducing a new type of culture media

Temperature, pH and osmolality are some of the most important parameters to take into account during In Vitro embryo culture. In any good quality management system these parameters, amongst others, should be monitored and regulated on a regular basis to ensure optimal culture conditions are maintained. In this blog post I will specifically highlight the importance of pH and its relation to CO₂ measurements for successful results in IVF.

What is pH?

pH is a figure expressing the acidity or alkalinity of a solution on a logarithmic scale on which 7 is neutral, lower values are more acid and higher values more alkaline. pH is the negative of the logarithm to the base 10 of the activity of the hydrogen ion (-log [H⁺]).  H⁺ ions are needed for metabolic pathways and the effect of a pH-difference of 1 unit implies a 10-fold increase/decrease of [H⁺].  Therefore the pH-difference of 0.2 units from 7.4 to 7.2 ,which is the acceptable range with which we work in the IVF lab, is actually a 60% change in [H⁺] ^1.

The relationship between pH and CO₂

The bicarbonate buffering system is mostly used in culture media as a means to buffer the acid-base equilibrium. In this system, carbon dioxide (CO₂) combines with water (H₂O) to form carbonic acid (H₂CO₃), which in turn rapidly dissociates to form hydrogen ions (H⁺) and bicarbonate (HCO₃⁻) as shown in the following reactions:

CO₂ + H₂O <=> H₂CO₃ <=> H⁺ + HCO₃^-

By increasing the CO₂ concentration, the equation shifts towards the right, releasing more H⁺ ions and the medium becomes more acidic, resulting in a lower pH.

The effect of pH on the embryo

Many of the intracellular processes that take place in the embryo are regulated by pH. In mice, the raising (about pH 7.4) and lowering (about pH 6.8) of pH for only 3 hours disrupts localisation of mitochondrial and actin filaments compared with controls (pH 7.2)².  The regulation of various enzymes, embryo glycolysis and oxidative metabolism are a few other intracellular processes that can have an impact on the developmental competence of an embryo¹.

Embryos show stage-specific sensitivity to stress

Intracellular pH is much more difficult to control by the oocyte than by the later stage embryos and it is during the oocyte and early stages of embryo development that sub optimal handling in the lab have the most significant impact. This is a reason why embryos show a stage-specific sensitivity to stress³. There are certain intracellular mechanisms in place enabling the human embryo to regulate its internal pH but it is still the responsibility of the embryologist to minimise fluctuations of pH during embryo culture.

_{Illustration from Wale and Gardner 2015³.}

A correct measurment of pH can be complicated 

Measuring pH is a very accurate way of monitoring the conditions of the culture environment. However, pH measurements are very easy to get wrong due to the complexity of the measuring process and the amount of variables that will influence the outcome. Most pH measuring devices are designed to measure larger volumes and not micro droplets of media that are often used in the IVF labs.  

Very few laboratories have access to equipment that can perform accurate measurements in a controlled environment. Blood gas analysers are often used in larger hospitals where this type of equipment is used for patients. IVF labs associated with such hospitals and in close proximity of the blood gas analyser may use this as a method to very accurately measure the pH of the culture media. There is usually a distance to cover with the sample from the laboratory to the analyser and therefore important to make sure that samples are tightly capped to avoid any gas phase exchange. More recently portable blood gas analysers have become more readily available on the market, but considering the cost of the device and consumables this is not the obvious choice for many labs.

A correct measurment of CO₂ is much easier

Measuring the concentration of CO₂ on the other hand is less complicated and has much less variables that can affect the final reading. Your media manufacturer should provide a certificate of analysis for each product, indicating the pH that will be achieved in the media at a specified setting of CO₂ at sea level. Digital electronic CO₂ analysers are the preferred device when it comes to measuring CO₂ and far more accurate than a liquid-based system such as Fyrite. CO₂ analysers should be calibrated regularly with a traceable standard and known sample gas to assure its accuracy.

Culture media to fit your everyday routines

When it comes to embryo culture, there are several different media types to choose from but the preferred choice would be to maintain the pH of the culture media at a level that reflects physiological pH as close as possible. The pH of every batch of Vitrolife culture media is meticulously measured and validated during the production process with a tried and tested method. Due to the relationship between CO₂ concentration and pH, we can then recommend the correct concentration of CO₂ to be used inside the incubator to maintain the correct pH for that media type at sea level. If a lab is situated in a location significantly higher than that of sea level, the altitude will affect the partial pressure of the CO₂ in the medium. Be sure to accurately measure the pH first and then adjust the CO₂ output of the culture incubator accordingly³.

Follow recommendations from manufacturers

It is common practice that a lab may change the type of culture media they use at some time or another. It is very important that the CO₂ concentration recommended by the media manufacturer is used when changing from one commercial media to another. If the pH is already at the edge of the physiological range due to an incorrect CO₂ setting of the incubator, something as simple as incubator door openings will cause a prolonged exposure of the embryos to a sub-optimal culture environment with detrimental effects on the eventual outcome. 

Minimise exposure to sub optimal CO₂ levels

Be meticulous in the way you work and handle the embryos to minimise exposure of the embryos and culture dishes to sub optimal CO₂ concentrations. The use of IVF chambers or isolettes is one way of minimising the changes in CO₂ concentrations when embryos have to be handled outside of the incubator. Most commonly, a good handling media containing either a MOPS or HEPES buffer, is used during the handling steps of oocytes and embryos outside of the incubator such as oocyte collection, denudation, ICSI, assisted hatching and biopsy.

Time-lapse culture can reduce pH fluctuations

It is very important to minimise the number of incubator door openings on a daily basis. Too often labs do not have enough incubators for the number of patients they are treating. As a clinic grows and cycle numbers increase, pay attention to the ratio of cases per incubator. The proper use of time-lapse culture systems in recent years is one way of circumventing the problem of door openings and the associated problems regarding fluctuations in pH in the culture dishes.

_References

_{1. Swain J.E. Optimizing the culture environment in the IVF laboratory: impact of pH and buffer capacity on gamete and embryo quality. RBM Online (2010) 21, 6-16}

_{2. Squirrell, J.M., Lane, M., et al., 2001. Altering intracellular pH disrupts development and cellular organisation in preimplantation hamster embryos. Biol. Reprod. 64, 1845-1854}

_{3. Wale, P.L., Gardner, D.K., 2015. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Reprod. Update. In press.}