To the Editor:
In their article “Differential pH in Embryo Culture” (1), Hentemann et al. attempt to demonstrate differential requirements of media pH during stages of mouse embryo development. Unfortunately, in its current state, it is not clear that any conclusion regarding pH can be drawn from the publication. In fact, results should be interpreted with caution.
Authors varied sodium bicarbonate levels of their commercial media to adjust pH, similar to the approach utilized previously by Quinn & Cooke (2). Using this method, it is impossible to attribute any subsequent impact on embryo development to pH alone. A title of “Differential Bicarbonate Levels in Embryo Culture” may be just as appropriate. Bicarbonate concentration may impact embryo development independent of pH, as it is used by a variety of cellular transport mechanisms and could impact metabolism. This same limitation of confounding variables exists with prior attempts at determining an optimal culture media pH by adjusting CO2 levels (3). Carbon from CO2 is fixed differentially by embryos for incorporation into various molecules (4) and may impact development independent of pH .
The cause for concern lies in the fact that readers of the current article who may wish to adopt a similar differential pH paradigm for embryo culture would presumably accomplish this by altering incubator CO2, as bicarbonate levels are set by the media manufacturer. However, doing so may result in entirely different outcomes than observed in the current study.
Furthermore, the apparent lack of statistics or statistical differences in the article precludes any definite conclusions regarding the potential impact of bicarbonate/pH on the embryo. What is known is that the authors have begun to optimize their own commercial culture medium formulation and culture environment for mouse embryos. This is a necessary step in the pursuit of improving culture conditions and embryo development in vitro, and authors should be commended. However, similar results may not be apparent when using other media, as differing media composition, such as lactate or pyruvate concentration or amino acid composition, can differentially impact internal pH of the embryo (5,6). Therefore, to determine an optimal media pH for embryo development, it is advisable for each laboratory to do their own direct comparisons in their respective culture system.
Jason Swain, Ph.D.
University of Michigan
Ann Arbor, Michigan
1. Hentemann M, Mousavi K, Bertheussen K.Differential pH in embryo culture. Fertil Steril. 2011 Mar 15;95(4):1291-4.
2. Quinn P, Cooke S. Equivalency of culture media for human in vitro fertilization formulated to have the same pH under an atmosphere containing 5% or 6% carbon dioxide. Fertil Steril. 2004 ;81(6):1502-6.
3. Carney EW, Bavister BD.Regulation of hamster embryo development in vitro by carbon dioxide. Biol Reprod. 1987 ;36(5):1155-63.
4. Quinn P, Wales RG.Fixation of carbon dioxide by pre-implantation mouse embryos in vitro and the activities of enzymes involved in the process. Aust J Biol Sci. 1971 ;24(6):1277-90.
5. Edwards LJ, Williams DA, Gardner DK.Intracellular pH of the preimplantation mouse embryo: effects of extracellular pH and weak acids. Mol Reprod Dev. 1998 ;50(4):434-42.
6. Edwards LJ, Williams DA, Gardner DK.Intracellular pH of the mouse preimplantation embryo: amino acids act as buffers of intracellular pH. Hum Reprod. 1998 ;13(12):3441-8.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.04.024
The Authors Respond:
We appreciate the comments from Dr. Swain regarding out article on “Differential pH in Embryo Culture” in the March 2011 issue of Fertility and Sterility. Embryo culture media have evolved from simple balanced salt solutions to highly complex composition of diverse media components that interact dynamically with each other, with the cultured embryo and with the physical environments at different stages of embryo growth, thus contributing to optimal embryo development.
Successful outcome of any culture system is highly dependent upon the minimizing in vitro stress to the cultured embryos, which can manifest in altered metabolism, embryo quality, viability, implantation potential and thereby the final pregnancy outcome. Stabilization of the environmental components like pH and osmolarity are therefore crucial to minimize physico-environmental stress during culture. Carbon dioxide and bicarbonate levels in culture media, through their interactions under aqueous conditions, are main contributors to generating the optimal pH levels necessary for optimal culture conditions, but are not necessarily the only conjugate acid-base pairs present in the media (1,2).
The authors agree that bicarbonate and CO2 levels, along with other media components like lactate, pyruvate and amino acids can independently impact embryo development by variety of cellular mechanisms. However, pH levels are also a manifestation of complex interactions between media components, especially bicarbonate and CO2 and their influence on in vitro culture environments, and perhaps one of the most reliable laboratory variables available to the clinical embryologist to regulate to optimize culture conditions.
Our studies corroborate other studies and underline the importance of pH regulation in any culture medium, because culture media react rapidly and directly to differences in environmental conditions, like temperature, humidity and CO2. Since bicarbonate levels in commercially available media are fixed, the only practical possibility to fine tune pH is through altering the CO2 levels. The ability to adjust CO2 concentrations in the incubator offers the ability to provide the reliable control over pH (2). Whether changing CO2 levels alone without altering bicarbonate composition, could have any impact on the culture medium is speculative and should be reason for further studies.
My coauthor, Prof Kjell Bertheussen, who did some pioneering work in embryo culture media formulation for human IVF, died unexpectedly in February this year. His comments would have been very relevant in this discussion.
Martha Agnes Hentemann, M.D.
Department of Obstetrics and Gynecology
University Hospital of Northern Norway
1. Pool TB. Optimizing pH in clinical embryology. Journal of Clinical Embryology 2004; 7(3):1-17.
2. Swain JE. Optimizing the culture environment in the IVF laboratory: impact of pH and buffer capacity on gamete and embryo quality. Reprod Biomed Online 2010; 21:6-16.
Published online in Fertility and Sterility doi:10.1016/j.fertnstert.2011.04.023