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Optimizing the culture environment in the IVF laboratory: impact of pH and buffer capacity on gamete and embryo quality

Reproductive BioMedicine Online, 1, 21, pages 6 - 16

Abstract

Supplying and maintaining appropriate culture conditions is critical to minimize stress imposed upon gametes and embryos and to optimize the in-vitro environment. One parameter that requires close scrutiny in this endeavour is pH. Though embryos have a limited ability to regulate their internal pH (pHi), oocytes lack robust mechanisms. Thus, careful attention to external pH (pHe) of culture media is imperative in IVF. Ability to withstand deviations in hydrogen ion concentration varies depending on culture conditions, as well as laboratory procedures. Cryopreserved–thaw–thawed embryos, as well as denuded oocytes, are especially susceptible to perturbations in pHe. Therefore, proper setting, monitoring and stabilizing of pHeduring IVF laboratory procedures is a crucial component of a rigorous quality control programme. Here, importance of both pHiand pHein respect to gamete and embryo quality are discussed. Furthermore, factors influencing selection of pHe, as well as emerging methods to stabilize pHein the IVF laboratory are detailed.

Keywords: embryo, HEPES, hydrogen ion, MOPS, oocyte, pH.

Introduction

One of the primary goals of an embryologist is to improve the quality of embryos developing in the laboratory (which hopefully leads to more babies being taken home). A key factor in this endeavour is minimizing stresses imposed on gametes and embryos during their manipulations within the in-vitro environment. It is readily apparent that improper set-points in growth conditions are stressors and are, therefore, detrimental to embryo development, whether it be improper media energy substrate composition, temperature or osmolality. However, periodic fluctuations in environmental conditions are also harmful stressors, as these are easily transduced into deleterious intracellular perturbations. One such environmental parameter, which not only requires strict attention to its set-point but which is also especially susceptible to these damaging oscillations, is pH.

What is pH?

A detailed review of pH and its derivation can be found elsewhere ( Pool, 2004 ). However, an important concept to note is that pH is dynamic. It depends on the association/disassociation of compounds in solution and any factors which influence this balance, such as temperature. Thus, pH can change and it can do so rapidly. From a practical standpoint, it may be helpful to view pH as occurring in three phases within the laboratory: equilibration, set-point and stabilization ( Figure 1 ). Thinking in these terms helps explain the problematic fluctuations in environmental conditions mentioned above which can be so damaging to the gametes and embryos within the laboratory. Unfortunately, this is often not appreciated because, although the logarithmic pH scale is easier to use, it also gives the false impression of ‘minor’ pH changes. To clarify, because the pH scale is logarithmic, a change in pH of 1 is actually a 10-fold change in H+concentration. Thus, pH being the dynamic entity that it is, these inevitable and seemingly small deviations are actually large changes in H+concentration. As an example, an acceptable pH range for embryo culture media may be set between pH 7.4 and 7.2. However, this is actually a 60% change in H+concentration, which, as will be discussed, can have a dramatic impact on the embryo and likely on the oocyte as well.

gr1

Figure 1 Three phases of pH experienced during in-vitro culture.

How is pH regulated?

Extracellular pH

If pH is so sensitive and important, it would be beneficial to know how to both set and regulate the extracellular pH (pHe) of culture media. Traditionally, pHeis the result of a balance between concentrations of CO2in the cell culture incubator and the amount of bicarbonate in the media. Gaseous CO2dissolves in solution to produce carbonic acid, which reaches equilibrium with the amount of dissolved bicarbonate ( Figure 2 ). Generally the bicarbonate concentration, supplied as sodium bicarbonate, is set by the media manufacturing company. Thus, to regulate the set-point of media pHe, CO2value is controlled on the incubator. This is an inverse relationship, with pHedecreasing as CO2concentration increase.

gr2

Figure 2 pH of culture media is primarily regulated by a balance of CO2concentration, supplied by the incubator, and by concentration of bicarbonate in the media. Because bicarbonate concentrations are set by commercial suppliers of media, it is easiest to adjust CO2concentration in the incubator to adjust pH. Raising CO2lowers media pH, while lowering CO2raises the pH.

This understanding of pHesheds light on the three phases of pH ( Figure 1 ). Equilibration timing then depends on diffusion of CO2into the media and the timing of the above reaction. Thus, volume of media, surface area, use of oil overlay and even the type of lid/dishware can influence this gas exchange and equilibration timing. For the set-point of media pH, although CO2and bicarbonate are the major contributors to pH, they are not the only elements to consider. As an example, protein source and concentration can both affect pHe. Additionally, elevation of the laboratory may also be a factor. Thus, the same basal media may not yield the same equilibration time or set-point pHefrom laboratory to laboratory, even if the same CO2concentrations are supplied. Thus is illustrated the problem with simply setting a specific CO2concentration on the incubator or in the use of pre-mixed cylinders of gas, which do not allow for adjustment. As will be discussed later, the inappropriateness of this practice is further demonstrated by the fact that incubator CO2readings are not always accurate. Regarding stabilization of pH, incubator door openings and closing or other incidents that perturb CO2concentrations can lead to pHefluctuation. Although the use of inner incubator doors can reduce gas escape and the use of smaller incubators can aid in gas recovery, these approaches are not perfect and use of pH buffers within the media may aid in stabilization.

Intracellular pH

Regulation of intracellular pH (pHi) is an important cellular function necessary to maintain intracellular homeostasis. Cells contain various mechanisms to regulate pHi, although specific mechanisms will vary from cell type to cell type. Short-term regulation of pHiis achieved by the limited physiochemical buffering capacity of the cytoplasm and proteins ( Lane and Gardner, 2000 ). Common regulatory systems to combat intracellular acidosis include the sodium-dependentstripin: si1.gif/Clexchanger (HCE) and the Na+/H+antiporter, which add and remove, respectively, bicarbonate and hydrogen ions from within the cell. To combat alkalosis, cells may contain thestripin: si2.gif/Clexchanger. Transcripts and proteins for isoforms of these pHiregulators have been identified in rodent oocytes and embryos (Zhao et al, 1995 and Barr et al, 1998). Additionally, although not a conventional pHiregulator per se, it should be mentioned that the monocarboxylate co-transporter can also influence pHi(Gibb et al, 1997 and Herubel et al, 2002). However, just because these regulators can be identified, it does not confirm that they are active or functioning. Therefore, a very important series of functional studies conducted in the laboratories of Jay Baltz, Michelle Lane and others, have been instrumental in providing insight into pHiand its regulation in oocytes and embryos. These studies have had a tremendous impact on how these sensitive and valuable cells are cultured.

Using cell-permeable fluorophores SNARF and BCECF, these functional studies have demonstrated that embryos from a variety of species possess active pHiregulatory mechanisms, (Barr et al, 1998, Dale et al, 1998, Edwards et al, 1998a, Edwards et al, 1998b, Lane et al, 1998, Lane et al, 1999a, Lane et al, 1999b, Lane and Bavister, 1999, Phillips et al, 2000, Phillips et al, 2002, Zhao and Baltz, 1996, and Zhao et al, 1995). Following calibration, cells are loaded with the fluorophore and a baseline pHiis determined based on fluorescent intensity. Interestingly, this value has been repeatedly shown to be approximately pH 7.1, which is much lower than the pH 7.4 of blood ( Table 1 ). This fact alone has implications on culture practices of embryos. In these studies, cells are subjected to an induced alkalosis or acidosis and composition of the media is then manipulated, or pharmacological inhibitors are used, to determine if cells can recover from the pH change as well as which specific recovery mechanism is present. In human embryos, although the pHiof the embryo initially follows the pHe, thestripin: si3.gif/Clexchanger activates when pHirises above 7.2–7.3 ( Phillips et al., 2000 ). To combat acidosis, the Na+/H+antiporter activates when pHidrops below 6.8 and the sodium-dependentstripin: si4.gif/Clexchanger operates below pH 7.0 ( Phillips et al., 2000 ). Furthermore, morula- and blastocyst-stage embryos appear to have more rigorous control over their pHi, possibly due to formation of tight junctions between cells (Edwards et al, 1998a and Edwards et al, 1998b). This demonstrates the plasticity of embryo development and explains their ability to grow over a variety of pHeconditions ( Kane, 1974 ). Physiologically, this makes sense, as the in-vivo developing embryo appears to adapt to the differing pHeenvironment of the alkaline oviduct and more acidic uterus (Elrod and Butler, 1993, Hugentobler et al, 2004, and Kane et al, 2002). However, as discussed below, just because embryos can form blastocysts over a range of pHeconditions, it does not mean that resulting embryo quality is equivalent.

Table 1 Internal pH (pHi) of human oocytes and embryos source: (adapted from Phillips et al. (2000) ).

Cell stage pHi
Germinal vesicle-intact oocyte 7.04 ± 0.07
Metaphase I oocyte 7.03 ± 0.04
Metaphase II oocyte 6.98 ± 0.02
2–8-cell embryo 7.12 ± 0.01

Although embryos possess functioning pHiregulatory mechanisms, oocyte pHiregulation is a paradoxical event ( Fitzharris and Baltz, 2009 ). Growing mouse oocytes in the follicle lack pHiregulatory capacity ( Erdogan et al., 2005 ). While fully grown immature mouse oocytes can regulate pHiby using thestripin: si5.gif/Clexchanger, these mechanisms are inactivated during meiotic progression through the involvement of the mitogen-activated protein kinase signalling pathway ( Phillips et al., 2002 ). As a result, denuded mature metaphase II oocytes are incapable of actively regulating pHi. This results in a sensitive cell stage, susceptible to even slight deviations in pHe. Interestingly, surrounding cumulus cells convey pHiregulatory capacity to the enclosed oocyte through gap junctions (Fitzharris and Baltz, 2006 and FitzHarris et al, 2007), a factor which as implications in clinical IVF procedures such as intracytoplasmic sperm injection (ICSI) in which cumulus cells are removed. Despite having transcripts for pHiregulatory mechanisms, self-contained pHiregulation in the rodent oocyte does not appear until several hours after oocyte fertilization (Lane et al, 1999b and Phillips and Baltz, 1999). Interestingly, bovine and human oocytes appear to have very limited ability to combat alkalosis (Dale et al, 1998, Lane and Bavister, 1999, and Phillips et al, 2000). Therefore, there may be slight species variations in oocyte pHiregulation or regulatory capacity may be impacted by in-vitro culture conditions ( Lane and Gardner, 2000 ). This possibility raises concerns over procedures such as in-vitro maturation (IVM). One interesting note is that, unlike amphibians, mammalian oocytes do not appear to undergo a rise in pHicoinciding with fertilization ( Phillips and Baltz, 1996 ). Continued work is required to determine exactly how and why oocyte pHiregulatory capacity is turned off during maturation and reinitiated following fertilization. This information will prove valuable in the continued improvement of culture conditions.

Importance of pH

Embryos

Now that the basic groundwork for defining and regulating pH has been established, the tremendous impact it has on cells should be discussed. pH controls several intracellular processes that can impact embryo development. As an example, raising (about pH 7.4) or lowering (about pH 6.8) pHiin mouse embryos for only 3 h disrupts localization of mitochondrial and actin microfilaments compared with controls (about pH 7.2) ( Squirrell et al., 2001 ). Notably, alkaline conditions were worse and effects were not completely reversible. Even minor rises in pH can also dramatically impact embryo metabolism through regulation of various enzymes, such as phosphofructokinase. Raising pHiby about 0.1–0.15 pH units significantly increased embryo glycolysis and lowered oxidative metabolism (Edwards et al, 1998b and Lane et al, 2000), which can dramatically impact developmental competence. Importantly, other common practices in IVF, such as vitrification of embryos, reduces the ability of these already sensitive cells to regulate pHifor about 6 h ( Lane et al., 2000 ). Whether specific cryopreservation (slow or vitrification) protocols are less damaging than others is unknown. Certainly cells are exposed to freezing conditions during slow cooling and are thus subjected to stresses for longer periods of time. Improper pHemay be one of these stresses. Temperature can affect pHe, with decreasing temperatures resulting in a concomitant rise in pHeof certain media ( Swain and Pool, 2009b ). Thus, close monitoring of temperature is also important for maintaining pH stability. Finally, a more recent study suggests that pHecannot only affect embryo development, but resulting fetal development as well. Lowering the pHiof 1-cell mouse embryos from pH 7.25 to pH 7.1 for 19 h resulted in significantly fewer blastocyst cell numbers, higher levels of apoptosis and reduced fetal size/weight compared with controls ( Zander-Fox et al., 2008 ).

Oocytes

As mentioned, denuded mature oocytes lack or have diminished ability to regulate pHiand are therefore dependent upon pHe. Thus, in procedures such as ICSI, where the protective cumulus cells are purposefully removed, cells are created that are extremely susceptible to perturbations in pHeuntil several hours after fertilization occurs. Little work has been done examining the effects of pHeon the mature oocyte. This should be concerning considering the prevalence of oocyte derived aneuploidy and the potential impact pH has on the meiotic spindle. pH is known to affect embryo actin cytoskeletal elements ( Squirrell et al., 2001 ) and the oocyte cytoskeleton is responsible for positioning of the meiotic spindle (Lenart et al, 2005 and Zhu et al, 2003). It is known that alkaline pH affects microtubule assembly/disassembly in bovine brain cells ( Regula et al., 1981 ) and similar actions may be occurring with the meiotic spindle within the oocyte. Additionally, because pH can affect embryo mitochondrial localization ( Squirrell et al., 2001 ), the same may hold true of oocytes. This is concerning because distribution of oocyte mitochondria is correlated to developmental competence (Bavister and Squirrell, 2000 and Nagai et al, 2006). Even small increases in pHiperturb embryo metabolism (Edwards et al, 1998b and Lane et al, 2000), which can profoundly affect subsequent development. Oocyte metabolism is also likely affected by pH and has been correlated with maturational status and developmental competence (Krisher and Bavister, 1999 and Spindler et al, 2000). Additionally, similar to embryo cryopreservation, whether vitrification of oocytes compromises the ability of resulting embryos to regulate pHiis also undetermined. All these factors indicate a strict regulation of pHewhen dealing with oocytes.

An additional consideration in regard to the oocyte and pHeis the use of IVM. The cohort of oocytes collected in these cases have varying amounts of unexpanded cumulus cells, with some cells being denuded, resulting in a wide variability of cumulus cell-media pHiregulation. Therefore, the pHiregulatory capacity of these immature oocytesin vitrois in question. Furthermore, these cells differ from the normally collected mature oocytes, which are protected within their mucified hyaluronan matrix. As a result, it is unknown if maturing oocytes have appropriate pHiregulation or differential pHerequirements than denuded mature oocytes or embryos. Therefore, continued research into oocyte pHiregulation and pHerequirement are prudent and may help improve current inefficient IVM protocols.

Optimal pHe?

The ability of embryos to regulate pHiis shown by various studies that show embryos can develop over a pHerange of pH 7.0–7.4 without any discernable effect on pHior development (John and Kiessling, 1988 and Lane et al, 1998), while excursions of pHeoutside this range have deleterious effects on embryo developmental competence (Lane et al, 1999a, Lane et al, 1999b, Lane and Bavister, 1999, Leclerc et al, 1994, Zhao and Baltz, 1996, and Zhao et al, 1995). However, just because blastocysts can be formed over these pHeranges does not indicate that resulting embryo quality is equivalent. Drifting too far away from the pHiof around 7.1 likely stresses the embryo, as more resources are required to maintain the proper pHi. Future studies examining resulting implantation potential or molecular/genetic profiling of embryos cultured under different pHeconditions will likely reflect this. Conventional wisdom tells us that pHeshould be slightly higher than pHito help offset the acidification that occurs as a result of intracellular metabolic processes. Thus, many laboratories culture their embryos in the range of pH 7.2–7.4. However, as pointed out previously, this is a wide range with more than 60% difference in H+concentration. Unfortunately, it is likely that there is no optimum pHe, as this will vary from media to media based on its ingredients. Anecdotal observations from various laboratories suggest that perhaps culturing cleavage-stage embryos closer to pHe7.2 may give better embryo development. However, the amount of monocarboxylic acids, such as lactate and pyruvate, in culture media can lower pHi(Edwards et al, 1998a, Edwards et al, 1998b, and Gibb et al, 1997). Additionally, certain amino acids, such as glycine, taurine and glutamine, act as zwitterions and help in buffering pHi( Edwards et al., 1998a ). Thus, embryos grown in media with different amounts of these components may have different pHi, although the pHemay be the same. Along with potential species- or strain-specific requirements, this likely explains variations in the literature regarding acceptable and optimal pHe(Brinster, 1965, Hershlag and Feng, 2001, and Summers and Biggers, 2003). Regardless, it would be insightful to see a properly controlled clinical trial to determine if culturing embryos at a pH closer to 7.2 offers any benefit on embryo development, implantation or live birth, compared with culturing embryos in the same media at a pH closer to 7.4. At the moment, there is no ideal pHeat which to culture embryos and this is reflected in the wide ranges of acceptable pHegiven by various commercial media companies ( Table 2 ). Furthermore, despite the growing trend, it remains unknown whether early cleavage-stage embryos prefer a slightly lower pHethan later stages of embryo development (although as mentioned, it is known that later stages of embryos, like the morula and blastocyst, can regulate their pHimore rigorously than early cleavage-stage embryos). That being said, there are data to suggest a slightly more alkaline pHemay benefit fertilization. Dale et al. (1998) found higher rates of sperm binding to empty zona pellucidae at pHe7.5 compared with lower pHs though relevance to subsequent fertilzation events is unknown. This has led to the common practice of fertilizing oocytes in a slightly higher pH, culturing day-1–3 embryos in a slightly lower pH and culturing day-4–6 embryos in a slightly higher pH (high–low–high paradigm).

Table 2 Recommended pH ranges of various commercially available media used in clinical IVF.

Supplier Medium Recommended

pH range
Irvine P1 7.27–7.32
  ECM 7.2–7.25
  Single-step (SSM) 7.28–7.32
  Multi-blast 7.3–7.4
  HTF 7.2–7.3
Sage Fert Media 7.3 ± 0.1
  Cleavage Media 7.2 ± 0.1
  Blastocyst Media 7.3 ± 0.1
  IVM 7.2 ± 0.1
Vitrolife G5 Series Media 7.27 ± 0.07
Life Global Global 7.2–7.4 a
  Global Fert 7.2–7.4 a
  Blastocyst 7.2–7.4 a
  HTF 7.2–7.4 a
  HTFxtra 7.2–7.4 a
Medicult Universal IVF 7.3–7.4
  ISM1 7.2–7.3
  ISM2 7.35–7.45
  EmbryoAssist 7.2–7.3
  BlastAssist 7.35–7.45
Cook Sydney IVF Cleavage 7.3–7.5
  Sydney IVF Blastocyst 7.3–7.5
  Sydney IVF Fert 7.3–7.5

a Company recommends optimal of pH 7.3 and suggests users adjust CO2 readings to reach this after taking into account protein supplementation.

Practically, this changing of media pH for various culture steps means that either specialized media can be used with different bicarbonate concentrations or separate incubators with altered CO2can be used to achieve the differential pHefor fertilization and embryo culture. Thus, while various commercial companies list a wide range of acceptable pHevalues for their media, and some laboratories may argue for a benefit of a slightly lower pHe, there is no argument, that regardless of the final pHe, tight regulation and a narrow acceptable pHerange are critical components of a rigorous quality control programme.

pH buffering

The introduction of more complex culture media formulations has dramatically improved the quest to produce better embryos. Altering energy substrate concentrations, use of sequential culture, incorporation of amino acids and introduction of complex protein sources have all proven beneficial. However, compared with a few short years ago, relatively few advances in culture media formulations have emerged. One area that has especially tended to be overlooked in regard to improving culture media is in the formulation of handling media used outside the laboratory incubator. These media are formulated to resist pH changes due to the change in CO2concentration. Although techniques such as oil overlay can combat this pH rise, dishes of traditional culture media kept out of the incubator for even brief periods can result in the pH rising above 7.4 (Steel and Conaghan, 2008 and Swain and Pool, 2009b) and provide some logistical problems for procedures such as ICSI (multiple dishes, etc). In the past, handling media included phosphate-buffered saline solutions (PBS) and some laboratories continue using this media for oocyte retrieval. However, although the pKavalue of 7.2 and buffering of PBS may be adequate, PBS lacks essential components such as bicarbonate and metabolic substrates. This inadequacy, coupled with elevated concentrations of phosphate, may compromise gamete and embryo metabolic activity via the Crabtree effect, disrupt organelle distribution and interfere with intracellular ionic homeostasis, including pHi(Barnett and Bavister, 1996, Barnett et al, 1997, and Lane et al, 1999c). Indeed, even brief exposure to PBS as a handling media has been shown to compromise hamster and rabbit embryo development (Escriba et al, 2001 and Farrell and Bavister, 1984) and results in aberrant gene expression in bovine embryos when compared with other buffers ( Palasz et al., 2008 ).

Therefore, a better choice for buffer use entails incorporation of handling media that utilize lower concentrations of bicarbonate in conjunction with synthetic organic buffers to maintain media pH within a desired range. (Ferguson et al, 1980, Good and Izawa, 1972, and Good et al, 1966). These buffers, commonly referred to as Good’s buffers, provide supplemental buffering capacity over the physiological pH range of approximately pH 6.1–8.3. Good’s buffers, named for and derived by Norman Good and colleagues, are organic compounds derived largely from zwitterionic amino acids. Thus, these compounds can act as either an acid or base and are excellent pH buffers. The ideal characteristics of effective biological buffers was described by Good et al. (1966) and include: a pKavalue between pH 6.0–8.0; high solubility; no toxicity; limited effect on biochemical reactions; very low absorbance between 240 nm and 700 nm; enzymatic and hydrolytic stability; minimal changes due to temperature and concentration, limited effects due to ionic or salt composition of the solution; limited interaction with mineral cations and limited permeability of biological membranes. However, different cell types display varying sensitivity to individual zwitterionic buffers ( Ferguson et al., 1980 ). Furthermore, no buffer is perfect and each may react differently based on other media components. Thus, determining the suitability of specific buffers for use with mammalian gametes and embryos in IVF is crucial. Two of these commonly used in commercially available handling media for assisted reproduction treatment are 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES) and 3-(N-morpholino)-propanesulphonic acid (MOPS), and are selected based on their pKavalues, an indication of their optimal buffering capacity.

Historically, HEPES at 21 mmol/l has been a standard for IVF handling media. More recently, at least two commercial companies also now include MOPS in their IVF handling media. However, although both buffers are efficient and laboratories using media containing these buffers yield high success rates, there may still be room for improvement when it comes to pH buffering. Although HEPES has been widely used in IVF for years, its suitability for procedures has been questioned (Iwasaki et al, 1999 and Morgia et al, 2006). However, many of the conclusions drawn concerning toxicity of HEPES are often miscited and not fully supported by the studies performed. Early somatic cell studies citing HEPES toxicity stemmed from light exposure and interactions with riboflavin (Lepe-Zuniga et al, 1987 and Zigler et al, 1985). This is not an issue in IVF, as media are void of riboflavin. Several studies actually indicate HEPES is able to support oocyte maturation (Byrd et al, 1997 and Downs and Mastropolo, 1997), fertilization (Behr et al, 1990, Bhattacharyya and Yanagimachi, 1988, and Hagen et al, 1991) and embryo culture (Ali et al, 1993, Hagen et al, 1991, Mahadevan et al, 1986, and Ozawa et al, 2006) at room atmosphere. Those studies indicating lower fertilization rates in the presence of HEPES are likely due to the simultaneous reduction in bicarbonate concentrations ( Lee and Storey, 1986 ). Embryo development is supported in the presence of HEPES when bicarbonate is present, but not when bicarbonate is absent ( Mahadevan et al., 1986 ). Furthermore, when embryos are cultured at room atmosphere and compared with controls cultured in 5% CO2, differences in development cannot be attributed to HEPES alone. Elevated CO2of the laboratory incubator is utilized by embryos for various biochemical processes, such as a carbon source (Graves and Biggers, 1970, Quinn and Wales, 1971, and Quinn and Wales, 1974), and is likely beneficial over culture at room atmosphere. Additionally, it has been reported that HEPES may inhibit mouse oocyte glucose uptake ( Butler et al., 1988 ). However, again, these studies could not attribute this effect to HEPES alone, as HCO3and CO2concentrations were also reduced and both of these molecules can affect transport and utilization of macromolecules such as glucose. Interestingly, inclusion of HEPES at various concentrations from 2.5–25 mmol/l in media used in elevated CO2concentrations has been used to mature oocytes ( Geshi et al., 1999 ), fertilize (Geshi et al, 1999 and Lee and Storey, 1986) or culture embryos successfully (Geshi et al, 1999, Iwasaki et al, 1999, and Liu et al, 1996), yielding rates similar to media with no HEPES present. Only when HEPES exceeded 35 mmol/l was any increased embryo fragmentation observed in pigs ( Iwasaki et al., 1999 ). It has been demonstrated that 25 mmol/l HEPES has no adverse effect on mouse embryo development and that there are no adverse affects of up to 50 mmol/l HEPES when cultured with 25 mmol/l NaHCO3in about 5% CO2(Swain and Pool, 2009a and Swain and Pool, 2009b). Although there may be species specific sensitivities to HEPES, results demonstrate that when adequately controlling for other factors such as osmolality, ionic composition, gas concentrations and pH, HEPES is able to successfully support mammalian embryo development.

An additional organic buffer utilized in commercially available IVF handling medium is MOPS. MOPS was presumably selected due to concerns with HEPES, but also because its pKaof pH 7.2 is the closest of the 20 zwitterionic buffers to the pHiof embryos of 7.12 ( Phillips et al., 2000 ). Thus MOPS would seemingly offer the best pH buffering of available options. However, the pKaof 7.2 for MOPS is at 25 °C. Many laboratories warm their handling media to 37 °C, a temperature at which the pKafor MOPS is actually 7.02. This is low, considering many laboratories target their media pH to 7.3. In general, it is better to utilize a buffer with a pKaslightly above the working pH, as this results in a protonated form of the buffer, which is often less inhibitory to various cellular processes than the non-protonated form ( Good and Izawa, 1972 ). Thus, utilization of MOPS alone may not offer ideal pH buffering for IVF. Furthermore, use of MOPS is not without concern for unexpected cellular actions. Along with other buffers, MOPS can interfere with taurine uptake in tumour cell lines ( Wersinger et al., 2001 ), interact with DNA in cellular preparations ( Stellwagen et al., 2000 ) and interfere with Clconductance in neurons ( Schmidt et al., 1996 ).

Although HEPES and MOPS have been widely used in IVF, there is limited information on other buffers. 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulphonic acid (DIPSO) is a potential candidate because it possessed the next closest pKavalue to the pHiof embryos. Importantly, at 37 °C, the pKafor DIPSO is 7.35. DIPSO was used in a previous study examining oocyte maturation and showed no adverse effects on spontaneous maturation to metaphase II at 20 mmol/l ( Downs and Mastropolo, 1997 ). It was also demonstrated that 20 mmol/l DIPSO can support embryo development in room atmosphere and that a concentration of 25 mmol/l can support embryo development in elevated CO2(Swain and Pool, 2009a and Swain and Pool, 2009b).

As previously mentioned, it is important to note that compounds, such as HEPES, MOPS and DIPSO, may have pharmacological effects other than pH buffering ( Ferguson et al., 1980 ). As an example, both HEPES and MOPS can block a novel chloridechannel inDrosophilaneurons ( Yamamoto and Suzuki, 1987 ). Chloride ion transport is important for blastocoel formation, thus it was suggested perhaps these compounds may be detrimental for embryo development ( Butler et al., 1988 ). However, whether this chloridechannel functions in mammalian embryos is unknown, although it has been shown that blastocyst formation is not affected (Swain and Pool, 2009a and Swain and Pool, 2009b). However, due to the importance of osmolality and embryo development ( Baltz and Tartia, 2009 ), as well as the role of chloridechannels in osmotic regulation, this may be another area in need of future study in regard to use of zwitterionic buffers. It is currently unknown as to whether MOPS or HEPES impacts embryo osmotic regulation. In considering these pharmacological effects, it is essential to note that many of these effects are largely dependent on interactions with other compounds in the media, not due to toxicity of the buffers themselves. Increasing HEPES concentration from 20 to 25 mmol/l did not affect spontaneous oocyte maturation but did suppress ability to induce meiosis in pharmacologically inhibited oocytes with cAMP (but not hypoxanthine; Downs and Mastropolo, 1997 ). Thus, it is important to examine the use of these buffers in the context of specific base media. This may have been a factor in the observations of Butler et al. (1988) . Although no data were presented, authors commented that inclusion of HEPES in the media resulted in increased oocyte degeneration. However, this media consisted of alpha minimum essential medium supplemented with vegetable protein. This suboptimal media contained riboflavin, which, as mentioned previously, is known to interact negatively with HEPES. Furthermore, the source of the buffer is also important. Work in the author’s laboratory, as well as others, demonstrate that different preparations of Good’s buffers can yield varying results and give undesirable side effects (Schmidt et al, 1996 and Swain and Pool, 2009b). Thus, as with any media component, thorough toxicity screening and viability assays should be performed before clinical implementation. Importantly, although much of the focus of zwitterionic buffers is on the negative, there can be beneficial effects on cell growth. HEPES helps preserve the electron transport and phosphorylation capabilities of plant mitochondria ( Good et al., 1966 ). HEPES and DIPSO can also act as chelators of heavy metals such as copper (Mash et al, 2003, Vasconcelos et al, 1996, and Vasconcelos et al, 1998) and have been suggested as a replacement for EDTA to serve this purpose.

Buffers and temperature

Although widely used in assisted reproduction treatment, it is often not appreciated that temperature affects pKaof synthetic organic buffers as well as the actual pH of the media ( Swain and Pool, 2009b ) ( Table 3 ). A previous example regarding the change in pKa of MOPS in response to temperature was given above. Another consideration is how much pKaand pH change in response to temperature. Both MOPS and HEPES display approximately equal changes in pKain response to temperature changes between 25 °C and 37 °C (0.18 and 0.17, respectively) (Sigma–Aldrich). However, other buffers change more significantly. Interestingly, MOPS was reported to be superior to HEPES for vitrification, although the exact reason for this remains unclear and the comparison was not made during the same time period ( El-Danasouri et al., 2004 ). Regardless of the buffer chosen, it is crucial to maintain an appropriate and constant temperature to avoid changes in pH. Due to this relationship, many studies detailing the effects of temperature on cellular structure and function, such as oocyte meiotic spindle organization, cannot rule out a role for pH in regulation of these processes. Therefore, continued research into the role of pH into control of meiotic spindle function is likely prudent.

Table 3 Common Good’s buffers and their effective buffering ranges. Changes in temperature alter buffering capacity of these pH buffers source: (data obtained from Sigma–Aldrich).

Buffer pH buffering range pKa 25 °C pKa 37 °C
MOPS 6.5–7.9 7.20 7.02
TES 6.8–8.2 7.40 7.16
HEPES 6.8–8.2 7.48 7.31
DIPSO 7.0–8.2 7.60 7.35
MOBS 6.9–8.3 7.60 a
TAPSO 7.0–8.2 7.60 7.39

a DIPSO = 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulphonic acid; HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid; MOBS = 4-(N-Morpholino)butanesulfonic acid; MOPS = 3-(N-morpholino)-propanesulphonic acid; TAPSO = 2-Hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid,N-[Tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid; TES = N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid. pKa of MOBS at 37 °C not provided by Sigma-Aldrich.

Combined buffer systems

A concept to address concerns with buffer toxicity, as well as the issue of temperature, is the combination of several organic buffers into a single solution ( Eagle, 1971 ). This approach would allow for the use of lower individual buffer concentrations, thus lowering possible toxicity concerns. Additionally, combining buffers allows for selection of an exact pKavalue at a given temperature, which may be more appropriate and offer better buffering than a single buffer ( Figure 3 ). This approach is a substitute for current mono-buffered handling media. Swain and Pool (2009b) verified that bi- and tri-buffered media with combinations of HEPES, MOPS and DIPSO support mouse embryo development. Preliminary data suggest these buffers may be beneficial for ICSI over mono-buffered media ( Swain et al., 2009 ). Interestingly, these combined buffered media can be used for procedures both inside and outside the laboratory incubator when bicarbonate concentrations and osmolality are adjusted accordingly. With further testing and verification, additional buffers could be explored and added to further refine media. Although developmental studies have proved promising, additional molecular endpoints such as metabolic assessment or transcript profiling would be useful.

gr3

Figure 3 Combination of various Good’s buffers allows for the fine-tuning and shifting of pKavalues, or optimal buffering capacity, while simultaneously reducing individual buffer concentrations and potential toxicity concerns. Data collected at about 25 °C. DIPSO = 3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulphonic acid; HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid; MOPS = 3-(N-morpholino)-propanesulphonic acid.

Measurement of pHe

The importance of pH stability during IVF should now be readily apparent. Thus, measuring pH properly is critical to ensure cells are being exposed to appropriate conditions. Many laboratories use fyrite to measure CO2concentrations in their incubator as a means of quality control by indirectly measuring pHe. However, fyrite is inaccurate at best: fyrite readings often fluctuate and are not overly reliable indicators of pHe( Pool, 2004 ; Figure 4 ). Data show that there is not always agreement between changes in incubator CO2readings, media pH readings and fyrite readings taken at weekly intervals. Furthermore, fyrite readings can fluctuate dramatically over time.

gr4

Figure 4 Demonstration of the fluctuation and inaccuracy of fyrite as an indicator of pH source: (adapted from Pool (2004) ).

A more accurate way of measuring pH is to do so directly. This first requires the appropriate pH probe and calibration. Composition of media can affect probe readings and some pH probes work better than others in solutions with organic buffers or protein content ( Pooler et al., 1998 ). It is often recommend that a double-junction glass calomel electrode be used for media with protein or organic buffers. Cleaning or replacement of probes at set intervals helps ensure rapid and accurate readings. Importantly, it is recommended that the validity of readings be confirmed before implementing a new probe into clinical use, especially if one switches probe technology. Some newer technology probes can vary up to 0.2 pH units from other probes in the same solution (Pooler et al, 1998 and Quinn and Cooke, 2004).

Calibration entails using two standard solutions that bracket your target pH. In the case of IVF, solutions of pH 7 and 10 are recommended. Both these standards should be warmed to 37 °C. Depending on the specific pH probe/meter, the probe should be warmed to 37 °C as well or the working temperature can be manually entered into the pH meter. This is important because, as mentioned, temperature affects pH. As a review, temperature can affect pH in two ways. The first is known as the solution temperature effect. It is the true pH of the solution at the new temperature. However, temperature can also introduce potential error in pH measurements through the pH electrode temperature effect. In this instance, temperature changes induce a change in the electrode’s response to pH. However, at pH 7, the electrode effect is negligible and increases as one moves away from pH 7, approximately 0.003 pH unit/°C. To correct for the electrode effect, one can warm the pH apparatus to the measured temperature or the pH meter can utilize an automatic temperature compensation probe.

Once the probe has been calibrated properly, one should confirm accurate pH readings before taking a measurement. This can be accomplished by placing the probe back into pH 7 solution to verify the appropriate pH is recorded.

Ideally, one would measure pH in the incubator being used, under exact conditions to which the cells are exposed (volume, etc). Although specialized pH meters exist to allow this, some utilize unproven technology and are not overly accurate under testing conditions ( Pooler et al., 1998 ) and most are very expensive. Thus, a bench-top pH meter is sufficient, measuring test tubes of media removed from the incubator. As long as tubes are capped immediately upon removal and quickly measured, accurate readings can be obtained ( Pool, 2004 ). Alternatively, although also expensive, another highly accurate method of determining media pH is through the use of a blood gas analyser.

It should now be clear that because of the dynamic nature of pHeand the effects of protein additives and lot-to-lot variations in media, as well as other variables, the ability to adjust CO2concentrations in the incubator offers the greatest ability to provide the greatest control over pHe. Thus, although the use of pre-mixed gas cylinders in conjunction with desiccator jars, modular chambers or small bench-top incubators may offer advantages in regard to maximizing gas recovery times, they do not offer the same flexibility in fine-tuning pHein response to slight environmental fluctuations. Therefore, it is important verify pHeso that each and every media in use is within an acceptable range when using pre-mixed gases before implementation. Furthermore, regular checks are required to verify that pHeremains with this range throughout the use of an individual tank.

Future directions

Although likely media dependent, there remains a gap in knowledge regarding the ideal pHein which to culture cells during IVF. There may be differential pHeconditions needed for optimal oocyte maturation, fertilization and various stages of developing embryos. Until properly controlled studies are performed, debate will remain as to the proper conditions. What is inarguable is that stabilization of environmental parameters, such as pH, are crucial for optimized culture conditions. Potential for mitigating these damaging oscillations lies in the use of various buffering systems. Use of combined buffers in handling media or within the incubator environment may prove beneficial over current approaches by allowing for selection of a specific pKaand optimal buffering at various temperatures, while lowering individual buffer concentration. Increasing buffering capacity of media, using combination buffers or some other novel approach may also be beneficial during fertilization or IVM, where high concentrations of spermatozoa or cumulus cells could acidify local pHedue to cellular metabolic processes. This would likely be more pronounced in small volumes of media and may be useful for emerging technology such as microfluidics. Finally, examination into the formulation of a temperature-independent buffer, possibly through use of combined buffers, to resist changes in pH due to temperature fluctuation ( Sieracki et al., 2008 ), offers the opportunity of improved pH stability and perhaps benefits during or after cryopreservation. Continued research in this field and examination of molecular endpoints will aid in this endeavour.

Acknowledgements

The author would like to thank Rusty Pool and Charles Bormann for their critical review of this manuscript.

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Footnotes

University of Michigan, OB GYN, Ann Arbor, MI 48108, USA

fx1 Jason Swain, PhD, is an Assistant Professor in Obstetrics and Gynecology and member of the Reproductive Sciences Program at the University of Michigan where he is the Scientific Director of Assisted Reproduction Technology Laboratories. Dr Swain completed his post-doctoral training in the clinical laboratory of Dr Thomas Pool, received his PhD in Molecular and Integrative Physiology from the University of Michigan in the laboratory of Dr Gary Smith, his MSc in Animal Science from Purdue University with Dr Rebecca Krisher and his BSc from Hillsdale College in his native Michigan. Jason’s research interests include elucidating mechanisms involved in oocyte maturation, focusing on the role of reversible phosphorylation, as well as exploring means of improving in-vitro embryo culture conditions.