Dr. Alex Varghese:

“The jury is still out regarding the optimal pH of human embryo culture. For safety purpose we may suggest that pH <7.2 and >7.4 is detrimental for the molecular health of the denuded oocytes and embryos based on the animal studies. It would be interesting to see the outcome of the human clinical trial currently underway by Fawsy et al- Fawzy M, Emad M, Wilkinson J, et al. Triple-arm trial of pH (Tri-pH) effect on live birth after ICSI in Egyptian IVF facilities: protocol of a randomised controlled trial. BMJ Open. 2020;10(2):e034194.”

Dr. George Liperis:

“As demonstrated in hamster embryos, early embryos have very little ability to regulate their intracellular pH (pHi) and deviations of the scale of 0.2 in the pH scale can have a detrimental effect on developmental competence (Zander-Fox et al., 2010). A 0.2 change in external pH (pHe) will inflict a 60% change in H+ concentration on the logarithmic scale. It is also suggested from mouse studies that cleavage stage embryos don’t have the same capacity to regulate their pHi in comparison with morula and blastocyst stage embryos (Edwards, et al., 1998).”

Dr. Ralf Böhm:

“Actually, nobody knows. I think the pH should be as stable as possible within the range given by the manufacturer of the culture medium. The cell’s work is done by enzymes. Enzymes have the highest value of for example activity at a pH optimum. But the optimum also has a range and is not a strict value of, for example, 7.32. It has perhaps an optimum in the range of 7.22 to 7.40. So slight variations in pH should not be harmful to embryos.”

Dr. Alex Varghese:

“Setting 5 or 6% CO ₂for the culture incubators may not fix the right and optimal pH for your culture media. One should check the pH of each new lot of the media and after changing the gas bottles too. pH related studies by Dr. Pool noted that while during the simultaneous monitoring of the CO₂ and pH, drifts in CO₂ values does not corresponds to change in pH of the media for some time. That also warrants the importance of the continuous monitoring of the pH in incubators where embryos reside.”

Dr. George Liperis:

“Even if the CO₂ is tracked appropriately with calibration and validation of the measuring device by readings of a standard gas, it is still very important to track pH at frequent intervals. This is of particular importance having in mind that certain lots of culture media might have altered their pH due to changes in manufacturing, transportation, storage and handling conditions and will need to be validated. Similarly, when a new pre mixed gas cylinder is in place, pH tracking is recommended to verify gas quality and pressure control in the gassing system.”

Dr. Ralf Böhm:

“A CO₂-Meter is a good and fast tool to make a quick check of your incubators. If your pH is too low or too high a check of the CO₂ concentration can give you a quick information whether your incubator is in the correct range, If it is outside, you can change it and correct by this the pH. This is much faster than do this via pH. A CO₂ meter is also good to harmonize the your incucators regarding CO₂ concentration and pH respectively”

Dr. Alex Varghese:

“Very recent studies indicate that microdroplet continuous culture in a non-humidified environment for 5/7 days might drift the osmolality and pH from the optimal range. Hence continuous pH monitoring systems serves in providing critical information on actual culture conditions, so that embryologists can take informed decisions to modify their culture systems.”

Dr. George Liperis:

“Minimal differences in pH stability are anticipated for the two types of incubators if osmolality is accurately controlled. Incubator humidity is crucial when not using oil overlay. Osmolality can be affected by temperature, water and pressure. Water evaporation can lead to increased osmolality that can result in cell shrinkage and developmental blocks (>300 mOsm/kg). Therefore, the process of dish making is crucial in order to avoid evaporation (recommended dish making on laminar-flow hood’s non heated surface with low air-flow setting and media that has been just been taken outside the fridge). Whichever the type of incubator used, osmolality levels should be validated to assure that they are stable.”

Dr. Ralf Böhm:

“As far as I can say there isn’t a difference of the pH in media incubated in dry or humid incubators. The pH is a function of Hydrogen ions (Protons) in aqueous solutions and is mainly influenced by the equilibrium of basic and acidic components in the medium and in the surrounding atmosphere. The osmolality can rise over the time of cultivation in dry incubators. Depending on the culture system the evaporation of water can be reduced by a thick layer of mineral oil.”

Dr. Alex Varghese:

“Whichever the pH meter you use in the laboratory, this need to be properly calibrated. Even though bench top pH meters are cheaper, proper training and calibration of the electrodes, measurement at 37 degree Celsius are some key elements for the optimal results. Portable blood gas analysers are available which gives accurate results provided all testing conditions are met properly. We use SAFE Sense continuous monitoring system in our incubators, along with occasional checking with the blood gas analyser to make sure that the system is accurate for the desired pH.”

Dr. George Liperis:

“A blood gas analyser is the most accurate instrument for pH measurement.  After start up calibration (according to manufacturer’s instructions), pre-equilibrated culture medium is loaded into the blood gas analyser and a pH reading is usually obtained together with other blood gas parameters. Alternatively, specialised pH meters can be used. When using a pH meter, the pH needs to be measured using the exact same conditions including volume of media and/or oil overlay which gametes and embryos are exposed to during culture. Before measurements take place, a two-point calibration is required with standard buffers at pH 4 and pH 10 followed by verified reading (6.98 ± 0.02) from a standard at pH 7. If the reading is not within the range, the calibration needs to be repeated. Whichever the selected device, careful validation and calibration is needed.”

Dr. Ralf Böhm:

“The most used pH devices use glass electrode sensors. They are easy to use and, when of high quality, can deliver precise values. They offer a precision measurement of +- 0.002 or even better. The problem is the size of the sensors but in the meantime one can buy sensors with a diameter of 6 or even 3,5 mm that allow measurements in small tubes.

Another principle is the FET technology. There are some devices on the market (RI and Horiba). They are easy to use but not that precise (+- 0.1). I had the chance to use the RI device some years ago, but it made a lot of problems. May be the newer versions now are better. Blood gas devices are known to deliver precise pH values. But they are expensive in purchase and use.

There are some other systems available, but I do not have any experience with them. The SAFE Sense system I’ve tested for a while in my former lab. It is a nice way to monitor the pH. I cannot say anything regarding precision. I think to get reliable pH values it is not just a matter of the device it is also a matter of the way you do the measurement. You have pay attention on the temperature and the calibration. It is the best to do the calibration and the measurement of the media at 37 ° C. This means that you have to keep the sensor and all solutions at this temperature.”

Dr. Alex Varghese:

“One should make sure that the certificate analysis (CoA) is readily available for each lot of media that they receive. This will give an overview of the QC check done by the manufacturer. However the end user must adjust the pH as per their requirement. We recently had a single step media where the CoA results showed a pH of 7.5. We adjusted the CO₂ so that we got a pH of 7.3 for our condition.”

Dr. George Liperis:

“Commonly, media manufacturers will have the pH range between 7.2 to 7.4, allowing the embryo to maintain a pHi of 7.1 to 7.2. Theoretically, the pH is affected by altitude, as the amount of CO₂in the incubator atmosphere changes. In practical terms however, the affect is minimal. Increasing CO₂ levels might be considered if the laboratory is set in a high altitude. An altitude of 2 km will change the pH by 0.13.”

Dr. Ralf Böhm:

“The manufacturers always give a range of the optimal pH that the culture medium should have during culture. The CO₂ concentration must be adapted to the geographical altitude. This can be done by stepwise increasing or decreasing the CO₂ concentration. The pH in the Certificate of analysis just gives the value of the finished product. With the CO₂ concentration one should adjust the incubator to get a pH on the middle of the pH range given by the manufacturer. If the range is from 7.2 to 7.4 you should adjust the CO₂ concentration to get a pH of 7.3 at the end.”

Dr. Alex Varghese:

“If the pH system is working fine under proper calibrated conditions there is no need to use multiple systems at the same time that might give rise to many confusions. An once in 3/6 months checking with portable BGA is more than enough If someone is using bench top or continuous monitoring systems. Nowadays some culture media companies offer the add on service of BGA which one can utilize without investing in the costly device.”

Dr. George Liperis:

“Measuring pH effectively can be difficult. If measuring devices are calibrated and validated prior to use and preventative maintenance is implemented at frequent intervals, use of a single monitoring device should be sufficient. Personnel’s training in the correct use of equipment is of highest importance regardless of the type of device. If more than one type of pH monitoring device is available, they can be used as a source of additional validation.”

Dr. Ralf Böhm:

“No. If you use three different types it will confuse you as there will be deviations between them. When you use a high class standard pH meter and do thoroughly calibrations before every measurement session with fresh calibration solutions it is sufficient.”

Dr. Alex Varghese:

“Each model of the incubator varies how it process the gas supplied to it and the  actual gas concentration inside the chamber varies depending on various factors. If one connects multiple (say 2-4) MINC/Planer/C-top kind of bench top incubators to a single line of incoming gas mixture, the last one in the series may not get enough gas pressure. One should make sure that each incubator gets the optimal chamber CO₂.”

Dr. George Liperis:

“At first, the measuring device error should be examined. It is very important that the pH meter in use has been carefully calibrated and validated. Gametes and embryos are particularly vulnerable to extracellular pH changes, but media manufacturers have validated their buffer stability at 37°C. It is well known that pH measurements are highly temperature dependent. Therefore, sample measurements should be performed at the same temperature. Temperature mapping for the 3 incubators is required as it can reveal locations in the incubators that fall outside the desired temperature range. For this purpose, a thermocouple device with a micro-probe or surface probe validated to an accuracy of ±0.2°C is required and with the dish/tube to be set-up under the exact same conditions as the gametes/embryos are held for all 3 incubators (including type of dish, volume of media and oil overlay, position of droplets). ”

Dr. Ralf Böhm:

“Good question. Are these incubators all the same type and do they all close tightly? If the doors are not closing completely, air from outside could get in influencing the inner milieu. This also could happen via cable bushings.

Can the differences also be seen in the early morning after undisturbed overnight operation? The measurements of pH always should be done in the morning with the first opening of the incubator.”