Need for choosing the ideal pH value for IVF culture media

Abstract

Purpose

Monitoring the pH of IVF culture media is a good practice, but the required pH levels have been “arbitrarily” set. Assisted reproductive technology centers around the world are spending time and money on pH monitoring without any consensus to date. The objective of this narrative review was to evaluate the importance of pH monitoring during IVF, discover how the oocyte and embryo regulate their intracellular pH and try to determine the optimal pH to be applied.

Methods

A narrative literature review was performed on publications in the PubMed database reporting on the impact of pH on cellular function, oocyte and embryo development, IVF outcomes and pathophysiology, or on physiological pH in the female reproductive tract.

Results

Intracellular pH regulates many cellular processes such as meiotic spindle stability of the oocyte, cell division and differentiation, embryo enzymatic activities, and blastocoel formation. The internal pH of the human embryo is maintained by regulatory mechanisms (mainly Na⁺/H⁺ and HCO₃⁻/Cl⁻ exchangers) that can be exceeded, particularly in the oocyte and early-stage embryos. The opinion that the optimal pH for embryo culture is physiological pH is not correct since several physicochemical parameters specific to IVF culture conditions (temperature, medium composition, duration of culture, or implication of CO₂) can modify the intracellular pH of the embryo and change its needs and adaptability.

Conclusions

Because correct and stable extracellular pH is essential to embryo health and development, monitoring pH is imperative. However, there is a lack of clinical data on choosing the ideal pH for human IVF culture media.

Introduction

Despite progress in assisted reproductive technology (ART), the delivery rate per oocyte aspiration during IVF remains low (14.1 to 37.8%) [1]. As well as being affected by problems of aneuploidy linked to the quality of gametes, embryo development also depends on good culture conditions, since the preimplantation embryo is highly sensitive to its environment. Furthermore, there is growing evidence that embryo culture conditions are critical per se not only for pre-implantation embryo development but also for the postimplantation period and long-term health [2–5].

Numerous data suggest the importance of pH control in the IVF culture medium. These data are derived from animal models or from the study of physiology and pH dynamics in the mammalian female genital tract during conception. The preimplantation embryo maintains its intracellular homeostasis but a variation of extracellular pH can be a cause of cellular stress. Maintaining internal pH (pH_i) is one of the vital roles of homeostasis. Intracellular pH regulates many cellular processes such as enzymatic reactions, cell division and differentiation, calcium concentration, cytoskeleton establishment, and mitochondrial localization [6, 7]. Therefore, variations in pH_i can affect embryonic development. Since most of these data come from animal studies, we need to know which data can be transposed to the human embryo. The human oocyte and embryo appear to have regulation systems, such as membrane ion exchangers, but are these systems effective whatever the preimplantation stage and can they be exceeded? Culture conditions during IVF can be decisive for the good progress of fertilization, then early embryo development and up to the blastocyst stage. It seemed to us essential to present data about the regulatory systems of pHi described at the different stages: oocyte (in particular metaphase II), early embryo, and blastocyst.

Under the conditions of IVF embryo culture, incubator CO₂ dissolves in the medium to form carbonic acid. Primarily, the extracellular pH or outside pH (pH_o) in a culture medium is the result of a balance between the CO₂ concentration in the incubator and the bicarbonate concentration in the culture medium (determined by the medium provider). Therefore, these two settings greatly contribute to the regulation of pH_o. But what are the other physicochemical conditions specific to the IVF process that can also affect pH_o and how do they influence pH_i? In particular, how does the composition of the culture medium used in IVF have an impact on intracellular pH (pH_i) regulation? These are the questions we need to answer to understand the importance of extracellular pH management and to choose its optimal pH.

The objective of this work was to examine the importance of controlling the pH of IVF culture media and to determine if there is an optimal pH value to be applied. We aimed to determine the impact of pH_i on human oocyte and embryo metabolism and the ways in which pH_i is regulated with respect to pH_o. Lastly, what are the available data on the physiological pH of the mammalian female genital tract, and the particular physicochemical conditions during IVF that allow us to choose the optimal pH?

Methods

We conducted a narrative review of the relevant literature. The PubMed database was used to retrieve works published between January 1970 and November 2019 using the following search terms: “pH and (preimplantation embryo or cleavage stage embryo or 2-cell embryo or blastocyst or embryo culture or oocyte or IVF)” in all mammalian species. The search was restricted to English-language articles. The titles, abstracts, and reference lists were reviewed and only relevant publications (i.e., those reporting on the impact of intracellular/extracellular pH on cellular function, oocyte and embryo development, IVF outcomes and pathophysiology, or on physiological pH in the female reproductive tract) were selected (Fig. 1). This review examined, compared and discussed study methodologies and results, including experimental data and patients’ characteristics. As this was a narrative review, no review protocol or registration number was required. No specific funding was received.

Fig. 1.

Flow chart for selection of relevant publications

Impact of pH_i on mammalian cell function

General data

Maintaining intracellular pH (pH_i) is one of the vital roles of cellular homeostasis. For example, it has been shown in various types of somatic cells that ionic membrane permeability into Na⁺, K⁺ or Cl⁻ ions, as well as membrane conductance, are modified according to pH_i values [7]. Ion channel activity may vary according to pH_i values. In addition, the H⁺ ions bind to the membranes, attract the anions and thus modify the conductance. The mitochondrial redox chain requires a proton gradient across the mitochondrial membrane, so its function is partly related to pH_i [7]. The same is true for lysosomal enzymes [7]. At the nuclear level, even small changes in pH_i (0.1 pH units) can alter DNA synthesis [8].

Oocyte

The meiotic spindle stability of the mouse oocyte is significantly impacted by pH changes [9]. Using polarized light microscopy, Swearman et al. have shown a reversible increase in the density of the meiotic spindle (actin filament polymerization) after incubation of denuded mouse oocytes in a medium at pH 7.4–7.5 instead of pH 7.33 [9]. Disturbance of the meiotic spindle may be a cause of aneuploidy and maturation arrest. Other authors emphasize the importance of pH_i in the occurrence of oocyte aneuploidy: Cheng et al. have shown that mouse oocyte pH_i increases with advancing age, which may be a result of decreases in HCO₃⁻/Cl⁻ exchanger activity [10]. In aged oocytes, the increase in pH_i may damage the cohesion complex structure, resulting in aneuploidy [10]. Some authors have demonstrated oscillations in pH_i during bovine oocyte maturation and in connection with the activity of the M-phase promoting factor (MPF) [11]. In humans, some data suggest that a slight increase in pH_o (7.5) may be beneficial for fertilization [12]. These authors found significantly lower fertilization and cleavage rate at pH 7.0 compared with pH 7.5.

Early-stage embryo

In the cytoskeleton, alkalinization, and acidification of pH_i alter the actin network (microfilaments). These cytoskeletal disturbances also result in a disruption of mitochondrial distribution in the cytoplasm. In the study of Squirrell et al., changes in the pH_i of embryonic hamster cells were induced either by a weak trimethylamine (TMA) base or a weak 5.5-dimethyl-2.4-oxazolinedione (DMO) acid [6]. The normal pH_i of these 2-cell embryos is 7.4, and it was modified by these acidic or alkaline treatments to range from 6.85 to 7.68. Both treatments displaced mitochondrial organization from a perinuclear localization to a more homogeneous localization throughout the cytoplasm (with a faster effect after alkalinization by TMA). pH_i changes by TMA and DMO also had an effect on the cytoskeleton (on microfilaments but not microtubules). With an alkaline pH_i, microfilaments formed aggregates while with an acidic pH_i, the microfilament network was established but weakened [6]. These cellular effects of pH_i changes were possibly caused by indirect mechanisms via cellular pathways involving Ca²⁺ signaling. Development up to the hamster morula and blastocyst stage was reduced by both acidic and alkaline treatments in a dose-dependent response. This effect was partially reversible when exposure did not exceed 12 h and was irreversible if it exceeded 12 h. This suggests that the embryo has limited ability to recover from cellular stress induced by pH_i variations.

At the metabolic level, the pH_i value can have an impact on enzymatic activities: for example, a pH_i above 7.2 stimulates phosphofructokinase (PFK) activity, which can be altered even by very moderate pH variations of 0.2. The increase of PFK activity can prematurely activate the glycolysis process and may be responsible for stopping development in culture [13]. The presence of weak acids in the extracellular media can decrease the pH_i of the mouse early-stage embryo and cause a reduction in glycolytic activity [13]. This effect is not found at the mouse morula stage, which seems to have more efficient regulation systems.

Blastocyst

In addition to previous data on the importance of pH_i in cell metabolism, pH regulation systems (in particular ion exchanger function) are important for mammalian blastocyst development. For example, Na⁺/H⁺ exchanger activity contributes to the trans-trophectodermal Na⁺ flux that is required for blastocoel expansion [14, 15]. Blastocoel formation is driven by an osmotic gradient with Na⁺ accumulation in the cavity. We will see below how the blastocyst regulates its pH_i.

How do the mammalian oocyte and the embryo regulate their pH_i?

Data on the pH_i values of the human oocyte and embryo are rather sparse: Phillips et al. found a pH_i of 6.98 ± 0.02 for the metaphase II oocyte (n = 9) and 7.04 ± 0.07 for the germinal vesicle (n = 4) [16]. Concerning the early-stage human embryo, the same authors found a pH_i of 7.12 ± 0.01 in cleavage stage human embryos in the largest published series (n = 199) [16]. In a smaller series (n = 13 to 32), Dale et al. reported pH_i values ranging from 7.3 ± 0.3 to 7.4 ± 0.1 from the germinal vesicle stage to the zygote stage [12]. These differences between the findings of the two teams cannot be explained except as a result of different media compositions that may affect pH_i. Moreover, we must consider the oocyte source (the woman’s characteristics such as age or ovarian function, immature or failed fertilized oocyte) when discussing pH_i regulation. Unfortunately, these data are not detailed in most studies. Another limitation of these data is that pH_i was measured in embryos developing outside their natural environment, and it is possible that the incubation conditions change the pH_i, especially the ambient bicarbonate-CO₂ complex. It is currently impossible to measure the pH_i of the embryo in vivo. It has been shown that the pH_i of mammalian embryos (mouse and hamster) is not equivalent to pH_o. pH_i can be maintained for pH_o values of 7.0 to 7.4 but beyond that, a change in pH_i occurs and impairs the developmental capacities of the embryo [17, 18]. Intracellular cytoplasmic buffer systems exist that are short-term regulatory systems, active in seconds and immediately correcting the pHi. This immediate buffering capacity is measured in mM protons. In hamsters, this fast regulatory buffer system is significantly greater in the oocyte at 51.4 ± 2.4 mM/pH compared with 1-cell embryo post-activation at 17.9 ± 0.5 mM/pH (p < 0.05) [19]. This is probably related to the fact that ion exchangers are not very active in the hamster oocyte.

In addition to the short-term regulation systems, which are rapidly exceeded, there are more efficient regulation systems by ion channels. The classic regulatory mechanisms are the Na⁺/H⁺ exchanger and the Na⁺ dependent HCO₃⁻/Cl⁻ exchanger against acidosis and the HCO₃⁻/Cl⁻ exchanger against alkalosis. The Na⁺/H⁺ antiporter is active below pH 6.8 in humans and the HCO₃⁻/Cl⁻ exchanger is active at approximately pH 7.2 to 7.3 (again in humans) requiring intracellular HCO₃⁻ [16] (Fig. 2). There are several isoforms of Na⁺/H⁺ antiporters and NHE (Na⁺/H⁺ exchanger) 1 and 3 are the most common isoforms in oocytes and embryos [14]. In mice, the Na⁺/H⁺ antiporter appears to be activated at below pH_i 7.1 [20]. Also in mice, it seems that NHE1 alone plays an active role in recovery from acidosis [21]. The HCO₃⁻/Cl⁻ exchanger exists as four anion exchanger (AE) isoforms, AE1–AE4, belonging to solute carrier family 4 (SLC4) [22]). Mice embryos express AE2 and AE4 at each stage but not AE3 [23]. AE2 must be produced from both the maternal and embryonic genomes and is the most likely to be responsible for HCO₃⁻/Cl⁻ activity at the one-cell stage [18].

Fig. 2.

Intracellular pH (pH_i) regulation by ion channels in mammalian cleavage stage embryos. The ion channels are the Na⁺/H⁺ antiporter (active at a pH below 6.8 in humans) and the Na⁺ dependent HCO₃⁻/Cl⁻ exchanger against acidosis, and HCO3⁻/Cl⁻ exchanger against alkalosis (activated at a pH higher than 7.2/7.3 in humans). NHE: Na⁺/H⁺ exchanger, NHE 1 and 3 isoforms (described by Barr et al. 1997 (mRNA) in mice [14]), Lane et al. 1998 in hamsters [20], and Phillips et al. 2000 in humans [16]). AE HCO₃⁻/Cl⁻: anion exchanger (solute carrier family 4, SLC4) AE2 and AE4 isoforms (described by Dagilgan et al. 2015 in mice [23] and Romero et al. 2013 in humans [22]). NBC: Na⁺-driven HCO₃⁻/Cl⁻ exchanger: in mouse cleavage-stage embryos, NBC is less active than NHE against acidosis (Erdogan et al. 2011 [30]), Siyanov and Baltz, 2013 [21]). The outer grey circle is the zona pellucida. The circles inside the zona pellucida are blastomeres of an early-stage embryo

Thus, in mammalian embryos, pH_i is maintained between 7 and 7.3 and this requires the presence of HCO₃⁻, Na⁺, Cl⁻, and H⁺. The function of the HCO₃⁻/Cl⁻ exchanger and the Na⁺/H⁺ antiporter has been demonstrated in mice, cows, hamsters, and humans [11].

The regulatory mechanisms do not activate above or below a specific pH_i threshold, so there may be a lag time during which pH_i follows pH_o [24]. Moreover, if pH_o variations are too abrupt or too marked, the regulation mechanisms may be exceeded. Moreover, in humans, Phillips et al. have shown that oocytes and early embryos are unable to regulate mild acidosis (pH 7.0) at any stage of development up to the human blastocyst, but can regulate against alkalosis (pH 8.0) [16]. We detail below the activity of these regulation systems according to the mammalian oocyte and embryo stages.

Regulation systems: specific data on mammalian oocytes

Activation of the pH regulation mechanisms (Na⁺/H⁺ and HCO₃⁻/Cl⁻ exchangers) was shown in mice at the end of oocyte growth (when the oocyte reaches 80% of its maximum size), leading to a pH_i rise of 0.25 units and coinciding with the acquisition of meiotic skills [25]. Until the mouse oocyte has acquired pH_i regulation systems, it has been shown that in preantral follicles, granulosa cells regulate oocyte pH_i against acidosis via gap junctions and mechanisms including the Na⁺/H⁺ exchanger (NHE) isoforms NHE1 and NHE3 [26]. These regulatory mechanisms are no longer active in the mature oocyte blocked in metaphase II and they become active again after fertilization in hamsters and mice [19, 27]. Some data show that mammalian mature oocytes and zygotes do not have an effective pH_i regulation system by exchangers until 6–10 h after fertilization [11]. These exchangers appear to be active in the mature bovine oocyte [17]. Activation of these exchangers is not related to protein synthesis or cytoskeletal movements but rather is mediated by calcium oscillations and via the protein kinase C pathway [19]. In hamsters, their activity is greatest 8 to 10 h after oocyte activation [19].

In the mouse oocyte, HCO₃⁻/Cl⁻ exchanger activity is high in the germinal vesicle stage and is inhibited in the oocyte during the meiotic maturation process [25, 28]. Inactivation of the HCO₃⁻/Cl⁻ exchanger between the metaphase I (MI) and metaphase II (MII) stages and its activation after fertilization are related to the MEK/MAPK pathway (negative regulation) [27, 29]. Na⁺/H⁺ exchanger activity during oocyte maturation varies in a very similar way to that of the HCO₃⁻/Cl exchanger: present in the GV oocyte, inactivation during oocyte maturation, inactive in the MII stage and active in the 1-cell embryo [19, 25, 30]. Conversely, Na⁺ dependent HCO₃⁻/Cl⁻ exchanger activity is higher in the MII and zygote stages than in the germinal vesicle [30]. Some authors suggest that the inactivation of Na⁺/H⁺ during meiotic maturation allows glycine accumulation that maintains cell volume [31].

Data on human pH_i regulation systems are very sparse. Some authors have found a pH_i of 7.4 in the mature oocyte and during fertilization in women [12]. These authors have shown that fertilization stages (mature oocyte, young zygote) are sensitive to pH variations [12], suggesting ineffective or absent pH_i regulation systems. These data, therefore, suggest that pH_i regulation systems are not active in all stages of human oocyte and embryo development and are sometimes limited.

Regulation systems: specific data on mammalian early-stage embryos

The early-stage human embryo has active regulation systems: the HCO₃⁻/Cl⁻ exchanger counteracts alkalosis, while acidosis is compensated by the Na⁺/H⁺ antiporter [16]. During the human cleavage stage, pH_i appears to be stable [16], but the embryo is less equipped to regulate its pH_i than the blastocyst [32–34]. The Na⁺/H⁺ antiporter against acid overload is not fully functional and HCO₃⁻/Cl⁻ exchanger expression also appears to be reduced [33]. The latter findings have been demonstrated in animal studies; Na⁺/H⁺ antiporter activity in the early stage embryo varies according to the species and may vary within the same species depending on the strain, as has been demonstrated in mice [35].

Na⁺-dependent HCO₃⁻/Cl⁻ exchangers are less active in one-cell stage mouse embryos than in GV and MII oocytes [30]. pH_i in mouse embryos is regulated against acidosis mainly by the Na⁺/H⁺ exchanger NHE1 isoform [21].

In addition, the HCO₃⁻/Cl⁻ exchanger, which is active in mice and hamsters at pH > 7.2, appears to be inactive in bovine embryos (or above pH 7.9) [17]. Bovine embryos appear unlikely to correct a pH rise above 7.2/7.3 [17]. The same authors have shown in bovine embryos that inhibition of these regulation systems, even during very moderate pH_i changes (+ 0.13 or − 0.21), significantly impairs development to the morula and blastocyst stage, as well as blastocyst expansion [17].

Moreover, in mouse embryos, Zander-Fox et al. achieved artificial reduction of the pH_i of the embryo through exposure to a weak acid (DMO) during the preimplantation period, either through “chronic” exposure (from zygote to blastocyst) or through “acute” exposure (2-cell embryo stage or from 2 to 8-cell stage) [34]. They observed consequences on the development of the mouse blastocyst (decrease in the number of cells, increase in apoptosis, decrease in the expression of genes related to cellular stress and involved in apoptosis), irrespective of the periods of exposure. Moreover, exposure to DMO during early embryo cell divisions decreased fetal weight and cranial perimeter (p < 0.05) [34].

Regulation systems: specific data on the mammalian blastocyst stage

Mammalian embryos at the morula and blastocyst stage appear to be more efficient in regulating their pH_i, particularly because of the presence of tight junctions [32, 36]. This is physiologically explained by adaptation of the embryo to the uterine environment which is more acidic than that of the tubes. The impact of embryo cell compaction on the embryo’s ability to regulate its pH_i has been demonstrated by Edwards et al. [32]. These authors showed greater embryo sensitivity to lower pH_i by inducing decompaction of the mouse morula by cytochalasin B and a decrease in this sensitivity by prematurely inducing compaction with wheat germ agglutinin (WGA) [32]. The same team has shown that pH_i increases slightly in mouse blastocysts [32], while others found stable pH_i throughout murine embryonic development [37].

However, it was noteworthy that HCO₃⁻/Cl⁻ exchanger activity was lowest at the blastocyst stage. According to Dagilgan et al., the change is an adaptation to their normal in vivo environment, where the morula and blastocyst are in the acidic uterus (compared with the alkaline oviduct for the early embryo) [23]. However, the same authors showed that despite low HCO₃⁻/Cl⁻ activity at the mouse morula and blastocyst stage, embryos at these stages succeed in complete recovery from alkalosis, probably by other types of mechanism (against ammonium accumulation) [23].

In view of these data on pH regulation systems in mammalian oocytes and embryos, it appears that oocytes and embryos at the early stages of development have limited pH_i regulation mechanisms and thus are highly sensitive to variations in pH_o. The blastocyst seems more competent at regulating its pH_i with more cellular membranes that are less permeable to H⁺ ions [12, 34]. However, there is evidence to suggest that the blastocyst remains sensitive to alkaline environments, which highlights the importance of pH control in IVF culture media during the handling and culture of the in vitro blastocyst.

What is the optimal external pH?

Data on the mammalian female genital tract

The above data highlight not only the importance of pH_i in oocyte and embryo metabolism and development but also the limits of the pH_i regulation systems and therefore the importance of regulating extracellular pH. The question that arises is that of the ideal pH value. To try to answer this question, it is essential to be familiar with the physiology of the female genital tract.

In the female genital tract, the principal pH regulation mechanisms are the HCO₃⁻/Cl⁻ exchanger, which decreases alkalosis, and the Na⁺/H⁺ and Na⁺, HCO₃⁻/Cl⁻ channels, which decrease acidosis [38]. Data on the pH of mammalian tubal and uterine secretions are very sparse and almost non-existent in humans (Table 1). Moreover, these data should be interpreted with caution because of the technical difficulty of pH measurement, and because some authors did not specify the cycle period.

Table 1.

Physiological pH values in tubal and uterine secretions of different species

First author	Year	Localization	Species	pH value
Maas [41]	1984	Tubes	Rabbits	7.94
Nichol [40]	1997		Pigs	7.9 pre-ovulatory 7.3 post-ovulatory
David [42]	1973		Humans	7.3 to 7.7
Mather [44]	1975	Uterus	Cows	7.22 before ovulation 7.35 just after ovulation
Iritani [45]	1971		Rabbits	7.47 to 7.9

Tubes

Human data are very sparse, but data from other mammals (cows, swine, rabbits) suggest that the pH is more alkaline in tubal than in uterine secretions [38–40]. In rabbits, the pH was found to be around 7.94 [41]. This alkalinity is associated with high carbonic anhydrase activity. Moreover, the pH in tubal secretions seems to have some variations during the cycle in some species. Nichol et al. showed pH fluctuations in pigs, with peaks of high amplitude at low frequency and peaks of low amplitude at high frequency (in the peri-ovulatory period) [40]. In women, follicular fluid pH ranged between 7.22 and 7.7 [42, 43] and the pH of tubal secretions between 7.3 and 7.7 [42] (Table 1).

Uterus

Some authors have reported a slightly higher pH in the bovine uterus just after ovulation (7.35) than before ovulation (7.22) [44], while others have reported a pH ranging from 7.47 to 7.9 in rabbits [38, 45] (Table 1).

Particular physicochemical conditions during the IVF process

The opinion that the optimal pH_o for embryo culture is the physiological pH is probably not correct. Several physicochemical parameters specific to culture conditions during IVF can modify the intracellular pH of the embryo, and can probably change its needs and adaptability. The pH in the fallopian tubes or the uterus is therefore probably not the optimal pH in a culture medium. The recommendations of the manufacturers of culture media are to maintain a pH_o slightly higher than the pH_i to compensate for the acidification mechanisms related to cell metabolism (and therefore generally between 7.2 and 7.4). However, as the pH scale is logarithmic, a pH variation of 7.2 to 7.4 corresponds to a decrease in the concentration of H⁺ ions by a factor of 1.6.

Several embryo culture conditions have been identified as being able to affect pH_o and pH_i, such as the temperature of the incubator, the altitude of the laboratory (CO₂ pressure), the composition of the medium and the duration of culture.

Temperature

This can affect pH in two ways: (1) by slightly modifying the conductance of hydrogen ions [7], and (2) by introducing a slight measurement error via a temperature effect on the electrode measuring pH (0.01 to 0.02 pH units/°C) (some devices have a calculation formula to correct this) [46].

Medium composition

High lactate concentrations (5 mM) significantly decrease the pH_i of zygotes by approximately 0.1 units [13]. Lactate acts as a weak acid (as well as pyruvate) in the cytosol of the zygote but this effect is not found in the morula stage which seems insensitive to these variations, probably due to compaction. In addition, the proportions of certain non-essential amino acids such as taurine or glycine have an impact on pH_i by behaving like zwitterions (molecules with a negative and positive electrical charge that can depending on pH, attract anions or protons). Generally, in IVF culture media, they can attach protons and thus buffer the medium and avoid a drop in pH_i [32]. Furthermore, inter-batch pH variations can exist. Tarahomi et al. found that three out of the five media they studied displayed inter-batch pH variations from 0.20 to 0.23 (p < 0.05) [47]. This suggests the necessity of CO₂ adjustment by systematic pH measurement each time the batch of the medium is changed.

Culture time

During 4 days of culture, the pH increases significantly between the first and the fourth day with variations of 0.04 to 0.08 (p < 0.05) depending on the media [47], but still remaining within the tolerated thresholds for clinical practice (7.2 to 7.4). According to these authors, this variation in pH may be due to (1) a change in amino acid protein composition during culture by degradation (during embryo metabolism), (2) ammonium ion release by protein degradation (embryo metabolism), or (3) related to the evaporation of the medium [47].

Differential culture (intentional change in pH_o during culture)

The need to change the pH_o during embryo culture is very little documented. One study has shown a benefit of increasing CO₂ (thus lowering the pH) between fertilization and cleavage time in bovine embryos, but we must note that in this study the fertilization and cleavage media were different and pH_o values were not precisely measured [48]. Hentemann et al. showed in a mouse embryo assay (MEA) model an increase in the rate of good morphology embryos when applying different pH_o: 7.3 to the zygote stage then 7.15 for cleavage stage embryos [49]. The limitation of this study was that pH was modified by altering the concentration of bicarbonates in the medium. But bicarbonates themselves influence pH-independent cell metabolism as they are involved in many cellular transport mechanisms [50].

CO₂

The pH variations induced by different CO₂ levels may need to take into account the impact of CO₂ itself (apart from its effect on pH_o) on embryo metabolism and its action as a weak acid on the embryo’s ability to regulate its pH_i. In hamsters, Carney and Bavister found a benefit of 10% CO₂ content vs 5% on development at the blastocyst stage, but no effect of pH_o or bicarbonate content [51].

Altitude of the laboratory

This can modify the effective CO₂ content in an inversely proportional way. Thus, the higher the altitude, the higher the CO₂ content in the incubator required to obtain a target pH value. Increasing the CO₂ concentration as a percentage of gas composition at a higher altitude merely restores the number of CO₂ molecules to the same number as achieved by lower percentages at a lower elevation.

To conclude this section, we must say that embryologists in IVF laboratories face a real difficulty in choosing the ideal pH_o value or the specific range of values, for the simple reason that we do not have any clinical data (in particular from randomized clinical trials) enabling us to determine the optimal pH value (or range of values) to be applied during embryo culture.

pH measurement methods

Beyond the clinical interest of controlling the pH of the culture medium, we must consider the analytical difficulty of reliably measuring pH in such media. The probes are not always very precise, the frequency of calibration is important (often before each use) and even short exposure of the medium to air must be avoided. From a general point of view, the reference method for measuring pH is hydrogen potentiometry. The silver chloride electrode is considered as a reference electrode in pH meters, although the calomel electrode is an older and robust reference electrode (recommended for solutions with proteins or organic buffers). One constraint of these measurement systems is the necessity of calibration before each use because the electrode does not give reproducible potentials over longer periods of time.

Due to these limitations and because of the need to measure pH in low volumes of the medium, pH measurements by a blood gas apparatus (fixed or portable) after sampling with a needle and syringe can be useful. This method reliably measures pH inside microdroplets of embryo culture medium. A portable blood gas apparatus has been developed and represents a good alternative, avoiding transport of the syringe outside the IVF laboratory.

Continuous pH measurement systems by an optical fluorescence measurement device (suitable for some IVF incubators) are under development or already marketed (OCTAX Log & Guard™ or SAFE Sens™).

Conclusion

Good knowledge of the mechanisms of cellular ion homeostasis is essential to develop cell culture systems that minimize cell stress and improve embryo viability. The mammalian oocyte and embryo stages for which pH_o control is most critical are those whose pH_i regulatory mechanisms are weak, namely the denuded oocyte and the early stages of development. The blastocyst seems more competent at regulating its pH_i with more cellular membranes that are less permeable to H⁺ ions. The importance of pH_i homeostasis for cell metabolism and embryo viability in mammalian embryos has been demonstrated. Because correct and stable pH is essential to embryo health and development, monitoring the pH values of IVF culture media is imperative. However, to date, there are no clinical data enabling us to determine the ideal pH for all specific IVF culture media. Randomized trials are needed to answer this question, although they could only be comparative and not controlled since there is no reference value.

It is clear that the issue of optimal embryo culture conditions is broader than the question of pH alone: the composition of the culture medium (in particular amino acids, ammonium, carbohydrates) [52, 53], the temperature (with the need to maintain 37 °C) [54, 55], the effect of reduced oxygen concentration in the incubator [56, 57], group or single culture [58, 59], the osmolarity and volume of medium [60, 61], incubator performance and oil quality must be taken into account [53]. To date, unfortunately, although we have important data on the impact of pH on embryo development (cf “Early stage embryo” and “Blastocyst”), a limitation is that there are no data demonstrating any influence of pH value (among the various factors relating to culture conditions) on IVF outcomes. Once these culture conditions are optimized, special attention should be paid to selecting the best embryo.