Kumi SakuraiSenior Scientist, Irvine Scientific
- Irvine Scientific
- United States of America
In this article, Morbeck et al. showed differences in media composition had varying effects on blastocyst development when murine embryos were cultured under 5% or 20% oxygen conditions. While two simplex optimized KSOM based media showed no difference under varying oxygen concentrations, one of the nutrient-rich media closely reflecting reproductive-tract fluid yielded statistically significant differences in response to oxygen concentration (Ref 1). Notably, Irvine Scientific’s Continuous Single Culture® (CSC) single-step media was also assessed in the study which gave consistently high performance at both oxygen concentrations.We found this study to be highly informative and wished to further contribute to the discussion by providing some of the insights gained throughout our ART media development work at Irvine Scientific.
The importance of protein supplementation to ART culture media has been widely accepted and extensively discussed in the field with a shift observed from the use of poorly characterized whole serum to human serum albumin (HSA) in the culture media (Refs 2-3). It is important to note that each batch of biological protein supplement is distinct even when supplied by the same manufacturer as each lot of protein supplement typically contains undefined components that are lot-specific and potentially harmful to embryos (Refs 1,3). This lot-to-lot variability is generally addressed by screening through multiple lots in order to identify the most optimal one for a desired application. With respects to cell culture media, HSA sourcing and lot selection methodology can vary widely from vendor to vendor and is therefore an important consideration to keep in mind during media selection and comparative studies.In a previous 2014 study, Morbeck et al. elegantly performed a compositional analysis of seven commercially available culture media—two of which were single-step (Global and CSC) and five which were sequential media (Vitrolife, Sage, Cook, In Vitro Care, and Origio)—and evaluated media performance of five protein-free media (Global, CSC, Vitrolife, Sage, and In Vitro Care) (Ref 7). The five protein-free media were supplemented with 5 mg/mL HSA and tested under two different oxygen conditions (5% and 20%). Two of the five media (Sage and In Vitro Care) showed noticeably improved performance with HSA and variable performance at 5% vs 20% oxygen concentration while the other media showed consistent performance throughout all conditions tested. This led to the conclusion that the media + protein and media + oxygen interactions are important parameters to consider for optimizing embryo culture conditions.
In continuation of their 2014 study, the current 2017 study by Morbeck et al. analyzed four commercially available single-step culture media. As two of the media (G-TL and 1-Step) were only available pre-supplemented with HSA, the analysis was performed by obtaining the remaining two media (Global and CSC) with HSA as well (Ref 1). G-TL and 1-Step were shown to contain similar concentrations of energy associated components whereas Global showed fivefold lower concentration of glucose and twofold lower concentrations of pyruvate and lactate. G-TL and 1-Step both were also shown to contain enriched essential amino acids. Interestingly, the embryos cultured in G-TL and 1-Step showed inverse responses with respects to one another with 1-Step showing decreased performance and G-TL showing increased as oxygen concentration is lowered from 20% to 5% while Global and CSC showed consistent performance throughout. Morbeck et al. suggested the difference in performance between Global/CSC versus G-TL/1-Step may reside in species-specific differences (murine vs. human, respectively) in the approaches utilized during the development of each respective media (Ref 1). It is speculated that media performance can be enhanced by better providing nutrient concentrations observed in the human fallopian tube as was done for 1-Step and G-TL. However, the variable and inverse response to oxygen observed in the two media (1-Step and G-TL) suggest that energy source and amino acids may not be the primary contributors to media performance and that other components such as protein source may be playing a role. This is consistent with their 2014 study suggesting that media + protein and media + oxygen interactions are critical parameters for optimizing embryo culture conditions. As mentioned earlier, lot-to-lot HSA variability observed from different suppliers could potentially distort the effects of individual media components and ultimately media performance overall. It would therefore be interesting to see how different sources/lots of HSA affects media performance utilizing Morbeck et al.’s approach under 5% or 20% oxygen conditions, which may help to explain the inconsistency observed between the two studies (Refs 1,7).
Notably, several media including CSC, showed consistent performance throughout the various test conditions utilized in the two studies. Two media components that were not depicted in the studies were EDTA and a dipeptide alanyl-glutamine: EDTA which is added to overcome the 2-cell block (Ref 8) and as an antioxidant in addition to citrate and alanyl-glutamine which is a non-essential amino acid used to reduce ammonium buildup in culture media (Ref 8). These components can help explain CSC’s consistent performance observed under varying oxygen concentrations.
Studies like those undertaken by Morbeck et al. provide a valuable resource, benefitting both the public as well as private sectors within the field. We sincerely appreciate this opportunity to contribute some of the insights gained throughout Irvine Scientific’s development work toward the broader goal of supporting greater crosstalk and dialogue. We welcome any further comments and feedback and look forward to the continued progress and advancement of assisted reproductive technology.
Kumi Sakurai, Ph.D.
Hsiao-Tzu Ni, Ph.D.
Department of Research and Development
Santa Ana, CA USA
1 Morbeck, D. E., Baumann, N. A. & Oglesbee, D. Composition of single-step media used for human embryo culture. Fertil Steril, doi:10.1016/j.fertnstert.2017.01.007 (2017).
2 Lane, M. in In Vitro Fertilization 221-282 (CRC Press, 2006).
3 Meintjes, M. Media composition: macromolecules and embryo growth. Methods Mol Biol 912, 107-127, doi:10.1007/978-1-61779-971-6_8 (2012).
4 Sunde, A. et al. Time to take human embryo culture seriously. Hum Reprod 31, 2174-2182, doi:10.1093/humrep/dew157 (2016).
5 Morbeck, D. E. et al. Composition of protein supplements used for human embryo culture. J Assist Reprod Genet 31, 1703-1711, doi:10.1007/s10815-014-0349-2 (2014).
6 Wale, P. L. & Gardner, D. K. The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction. Hum Reprod Update 22, 2-22, doi:10.1093/humupd/dmv034 (2016).
7 Morbeck, D. E. et al. Composition of commercial media used for human embryo culture. Fertil Steril 102, 759-766 e759, doi:10.1016/j.fertnstert.2014.05.043 (2014).
8 Summers, M. C. & Biggers, J. D. Chemically defined media and the culture of mammalian preimplantation embryos: historical perspective and current issues. Hum Reprod Update 9, 557-582 (2003).