pH in Medically Assisted Reproduction Procedures:
Its Importance, Measurement and Control

Summary

The intracellular pH (pH_i) of gametes and embryos is modulated by a number of systems and, in human oocytes and cleavage embryos, is around 7.1. In in vitro culture, the extracellular pH (pH_e) of culture media is controlled within a typical range of 7.2 – 7.4. Tight control over this range to avoid pH deviation is prudent. An ideal pH_e is, however, difficult to define due to variations between media formulations, culture conditions, as well as gamete specific or embryo stage specific preferences. In the IVF laboratory, pH_e is generally controlled by adjusting the CO₂ concentration of culture incubators, where it works in combination with media components, like bicarbonate, to reach equilibrium. However, due to varying conditions in individual laboratories, including media type, protein supplement and elevation, there is no single % CO₂ concentration that will give the desired pH_e or the best outcomes in all situations. When handling gametes and embryos outside the incubator, buffers such as HEPES or MOPS are typically used to control pH_e.

Testing of pH_e of culture media presents some technical difficulties but is recommended to verify culture conditions and aid in quality control and troubleshooting.

Introduction

The importance of pH in gamete and embryo biology is axiomatic and, in order to optimize embryo viability and reproductive outcomes, it is essential that pH be properly monitored and controlled. This applies equally (or more) to in vitro systems than in vivo. It is therefore essential that we appreciate the role of pH at various stages of the IVF process and understand how we can best control it.

Devised in 1923 by the Danish biochemist, Søren Peter Lauritz Sørensen, pH is a logarithmic scale expressing the ‘potential of hydrogen’ equal to -log₁₀c where c is the concentration of hydrogen ions in moles per liter.

It ordinarily ranges between 1 and 10^-14 hence the scale of 1-14 that we are familiar with. Importantly, as a result of the logarithmic nature of pH measurement, relatively “minor” changes in pH are actually large changes in hydrogen ion concentration.

In biological systems, cells have limited capacity for passive buffering of cytoplasmic pH and thus depend on a number of active transport mechanisms. In IVF systems, control of pH usually utilizes the bicarbonate buffer system (Fig. 1).

pH in reproductive cells

The intracellular pH (pH_i) of human oocytes and embryos is typically believed to be around 7.0-7.1 [Phillips et al, 2000], though a smaller study suggested it was around 7.3 (Dale et al, 1998), and is regulated by various mechanisms (for a review, see Swain 2010).

Interestingly, the capacity of oocytes to regulate pH_i has been shown to be switched off between meiotic progression and fertilization, with the role conferred upon the supporting cumulus cells via gap junctions (FitzHarris and Baltz, 2006).

This exposes denuded metaphase II oocytes to fluctuations of pH_i when pH_e is poorly controlled, for example during procedures such as intracytoplasmic sperm injection (ICSI) or oocyte vitrification. Given that pH_i impacts the cytoskeleton and meiotic spindle in rodents (Squirrel et al, 2001; Swearman et al, 2018), and similar impacts may be present in other mammalian oocytes, poorly controlled pH_e has potential implications for rates of aneuploidy (Swain, 2019).

In preimplantation embryos, pH controls various cellular functions that influence embryo development. Squirrel and colleagues (Squirrel et al, 2001) reported disrupted localization of mitochondrial and actin filaments in mouse embryos when pH_i was raised or lowered compared to controls at pH 7.2. Metabolism is also affected with increased glycolysis and reduced oxidative metabolism in response to rising pH_i in hamster embryos (Lane et al, 2000). This is especially important in early cleavage embryo culture: in 1-cell mouse embryos, culture at an inappropriately low pH_i resulted in reduced cell number in blastocysts, higher levels of apoptosis and reduced fetal growth (Zander-Fox et al, 2008). In contrast, later stages of embryos appear to regulate pH_i more efficiently which is linked with the acquisition of tight cell junctions (Spindler et al, 2000). It is vital, however, to recognize that cryopreserved embryos may have a reduced ability to regulate pH_i for 3-4 hours after warming/rehydration (Lane et al, 2000), so extra caution is advisable when handling and/or culturing embryos in these circumstances.

While oocytes and embryos favor mildly alkaline conditions, spermatozoa will function in the same conditions but thrive at higher pH. The pH_i follows pH_e in a quasi-linear relationship in human spermatozoa (Hamameh et al, 1996) but capacitation may rely on the selective activation of one major pH_i regulator in the mouse at least (Zeng et al, 1996). Achikanu and colleagues (Achikanu et al, 2018) reported that higher pH_e led to increased pH_i and intracellular calcium concentration which resulted in hyperactivated motility; higher bicarbonate (HCO₃¯) also drove increased sperm motility. Comparing various formulations of sperm washing media, de Rosa et al (2015) demonstrated how the composition impacted sperm function, again showing high pH_e, as result of high HCO₃¯ was beneficial. This begins to also demonstrate the difficulty in isolating an ideal pH_e due to the independent actions of both CO₂ and HCO₃¯ on various cellular processes (Quinn & Wales 1974, Swain 2012, Swain 2011, Hentemann et al, 2011).

Control of pH in IVF systems

For routine culture, the pH_e is determined by the equilibrium between the HCO₃¯ in the medium and the level of CO₂ in the incubator (as fig 1). Since most labs have moved away from making their own culture media, the concentration of HCO₃¯ is effectively pre-set by the manufacturer, leaving the % CO₂ as the parameter that can be manipulated by the laboratory to set pH_e. Commercial media preparation is performed under tight controls and regulatory oversight, giving a high level of batch-to-batch consistency, and manufacturers test media for pH against a standard % CO₂ which is then reported in the certificate of analysis provided with each new batch. Whilst a useful general guide to show that the medium composition is within specification, there are a number of factors to consider that might lead to variations in pH_e in each user laboratory.

One factor affecting pH_e is altitude: simply, the higher above sea level a laboratory is located, the higher the % CO₂ in the gas mix to achieve the same pH_e. For example, at sea level the standard barometric pressure is typically 101kPa (or 760mm Hg) but at 500, 1000m and 2000m above sea level, this drops to 96kPa, 90kPa and 81kPa respectively. Put another way, only 94%, 89% and 80% of the CO₂ available at sea level is available at these altitudes even though the gas mix (by percentage) is the same (see https://baillielab.net/critical_care/air_pressure/).

It should be noted that the pH_e will also depend on temperature, with pH_e decreasing with increasing temperature, especially in zwitterionic buffered solutions, so stable control of this parameter is equally important.

Time for complete equilibration will depend on volume of medium, surface area (influenced by drop size and/or well shape), oil overlay and type of dish used, as well as starting and ending pH_e.

Sufficient time should be given for pH to reach equilibrium before exposing gametes or embryos. Steel and Conaghan (2008) suggested a minimum of 10 hours, whether using 50µl or 500µl under oil, though the ideal situation would be to validate equilibration times in each laboratory.

Set point is influenced primarily, but not exclusively, by CO₂ and HCO₃¯ with other components of the medium also having an impact. As already discussed, the pH_i of oocytes and embryos is around 7.1. In embryo culture, it is crucial to avoid inducing acidosis and driving pH_i below this figure and so the generally accepted lower limit for pH_e is 7.2 to avoid acidification by intracellular metabolic processes. The upper limit is typically 7.4 since some reports (see Gatimel et al, 2020) suggest poorer outcomes at pHe >7.4. There may be some value in targeting pHe nearer 7.2 but it is impossible to give an ideal pHe as media composition also affects pH_i (Swain 2010; Swain, 2012; Gatimel et al, 2020). Bearing in mind that a move from pH 7.2 to pH 7.4 represents a very significant 60% increase in hydrogen ions, it makes sense to attempt to stabilize pH_e in our culture systems within a much narrower band. Various methods can be employed to aid in this stabilization.

Stabilization is the degree to which the pH_e is unperturbed, taking precautions such as limiting the frequency of incubator doors being opened, using oil overlay or gassed isolettes or zwitterionic buffered media (with temperature considerations). Zwitterionic buffered media are modified media to provide a stable pH_e under ambient atmosphere. These tend to have reduced HCO₃¯ levels and the addition of a buffer, such as HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) or MOPS (3-morpholinopropane-1-sulfonic acid), and allow longer handling times without pH_e changes, which is especially useful for ICSI or embryo biopsy for example, (for review, see Swain & Pool, 2009; Will et al, 2011). A combination of buffers (eg HEPES + MOPS) offers possible benefits in terms of optimizing pKa (the acid dissociation constant equating to the pH where buffering capacity is maximal) and reducing individual concentrations and hence possible risk of toxicity (Will et al, 2011).

An additional consideration is to avoid pH_e perturbation is during dish preparation or culture. Poor technique during preparation of dishes for microdrop culture or extended periods in a nonhumified environment without proper precautions can cause media evaporation (Swain et al, 2012). As a result, the solute concentration of the medium, including bicarbonate, increases. This results in an increase in pH_e.

Testing and internal quality control (IQC)

A recurring question is how and when the pH_e of culture media should be tested. In the first instance, it is worth acknowledging the technical difficulties that many IVF labs encounter with implementing a robust methodology for pH_e testing. These issues generally arise due to limited sample volume or other factors, but with emerging technology, can be addressed.

A reliance on accurate % CO₂ has serious limitations but may have to be adopted as a proxy measure of pH_e and incubator function if the latter cannot be reliably determined using a suitable pH meter. Whilst the use of incubator displays and fyrite should not be heavily relied upon (Pool, 2004), precertified gas mixes or appropriately calibrated external CO₂ probes can provide some reassurances.

That said, provided it is performed well, direct pH testing should remain the gold standard. Accuracy and precision of pH measurement is reliant upon using an appropriate probe which is properly calibrated and used at the correct temperature (Swain, 2010). As well as solution temperature effects discussed above, there may also be electrode temperature effects so appropriate temperature compensation is needed; ideally pH measuring systems should be used and calibrated at the temperature a medium is used, usually 37°C. A very useful alternative is a hand-held blood gas analyzer (Gatimel et al, 2020) since this can test small volumes and has internal calibration, though systems should still be validated (Swain, 2013; Diaz de Pool et al, 2018; Cairo Consensus Group, 2020).

Though each has its pros and cons, none of these systems exactly match the culture system used in treatments and offer only snapshots of the pH. Systems are available, however, that provide real-time monitoring that can lend valuable insight into equipment function and impact of daily workflows or other lab environmental occurrences that may impact pH_e. Systems such as SignipHy™, may offer a more robust approach.

* This article was published in Reprod Biomed Online, 21, Dr Jason E Swain, Optimizing the culture environment in the IVF laboratory: impact of pH and buffer capacity on gamete and embryo quality, 6-16, Copyright Elsevier (2010).

References

Jason E. Swain, PhD, HCLD
Corporate Laboratory Director
CCRM IVF Laboratory Network in North America.

Dr Swain is an Adjunct Clinical Associate Professor in Obstetrics and Gynecology at Rutgers University and the University of Minnesota. He has published numerous peer-reviewed articles, edited and authored several book chapters and contributed to various other publications within the field of assisted reproduction.  Dr. Swain serves on various committees for ASRM, ESHRE, PCRS and other organizations focused on assisted reproduction.

David Morroll, PhD
Director of Clinical Support
CooperSurgical Fertility Solutions

Dr Morroll has worked as a Clinical Embryologist since 1986, training in Manchester, where he also completed his PhD studies. He has managed several laboratories in the UK and was previously Chair of the UK Association of Clinical Embryologists (ACE) and Association of Biomedical Andrologists (ABA). He has also been involved with several working groups, notably the HFEA Working Group on Safety and New Technologies and the Expert Group on Multiple Births after IVF.  He joined ORIGIO a/s as Director of Embryology in November 2011 becoming for CooperSurgical in January 2019.