Letters to the editor |
One-step versus two-step culture of mouse preimplantation embryos
1 Colorado Center for Reproductive Medicine, Englewood, CO, USA and 2 Obstetrics and Gynaecology Department, The Queen Elizabeth Hospital, University of Adelaide, Adelaide, South Australia, Australia
3 To whom correspondence should be addressed at: E-mail: dgardner{at}colocrm.com
Sir,
We would like to offer a different perspective on the study reported by Biggers et al. (2005)
whose findings are contradictory to our own previous studies on these embryo culture media systems.
In our previous publication comparing KSOMAA to sequential media (Gardner and Lane, 2002, 2003; Reed et al., 2003), we were careful to ensure that all conditions used were exactly the same for the medium being tested, e.g. the same volume of medium. This was done because in our extensive experience, the volume of medium has a significant impact on culture outcome if there are problems with the oil source used. Specifically, when suboptimal oil is used, embryo development increases with increasing volumes of medium. In the article by Biggers et al. (2005), two different volumes were used, 50 µl for the KSOMAA and 20 µl for the sequential media. The source of oil used was one not sold for use in human IVF. One therefore has to challenge the validity of this entire study based on differences in the conditions employed to test the different media.
That aside, with regard to the observations and comments made on EDTA, we previously published an extensive analysis on the effects of EDTA on mouse embryo metabolism and viability (Lane and Gardner, 2001). In this work, we analysed the effects of 1, 10 and 100 µM EDTA at two different glucose concentrations, 0.5 and 3.15 mM (i.e. the concentrations present in the sequential media G1 and G2). It was determined that at 0.5 mM glucose, both 10 and 100 µM EDTA significantly reduced glycolytic activity in the 8-cell embryo, but at 3.15 mM glucose, the inhibition of glycolysis by EDTA was diminished. However, subsequent analysis of the glycolytic enzyme 3-phosphoglycerate kinase in 2-cell-, 8-cell- and blastocyst-stage embryos revealed that enzyme activity was significantly reduced by 10 µM EDTA as well as by 100 µM EDTA. Given that Hewitson and Leese (1993) had determined that the inner cell mass uses glycolysis exclusively, we chose to remove EDTA from the second-phase media rather than risk interfering with ICM physiology, which in turn could affect fetal development.
With regard to the renewal of culture medium, the significance of glutamine deamination and the subsequent release of ammonium have been addressed in our laboratory and that of Biggers. Both groups have determined that embryo development is enhanced by the removal and substitution of glutamine with a stable dipeptide form (Biggers et al., 2004), indicating that glutamine is problematic for the embryo. The significance of this is that the ammonium produced by the deamination of amino acids has been shown to induce the neural tube defect, exencephaly (Lane and Gardner, 1994; Biggers et al., 2004). Furthermore, the addition of ammonium chloride to the culture medium can induce the same birth defect (Lane and Gardner, 1994; Sinawat et al., 2003). Therefore, three independent laboratories, including that of Biggers, have demonstrated that ammonium can induce exencephaly in mouse fetuses, following the exposure of the preimplantation embryo. What has been debated is the frequency at which the birth defect is induced. What is not for discussion is that the birth defect is induced by ammonium. As a result, we are obligated to renew media which contain amino acids at 48 h, irrespective of the formulation, to ensure that ammonium levels cannot build up sufficiently to induce the neural tube birth defect. So, irrespective of the article by Biggers et al. (2005), his own previous work (Biggers et al., 2004), and that of others work (Biggers et al., 2004), and that of others such as the recent report by Virant-Klun et al. (2006) that accumulating ammonium in the culture medium can negatively affect human blastocyst development, means that changing embryos to fresh medium every 48 h is necessary.
It is important to acknowledge significant methodology differences between works from different laboratories when comparing data. In the article by Biggers et al. (2005), all of the in vitro blastocyst quality assessments of the media systems were performed after 144 h of culture. This is day 6 of development and 2 days after mouse embryos develop to the blastocyst stage in vivo. Therefore, the significance of this data needs to be discussed in this context, as the majority of all other studies on culture systems for mouse embryos analyse development in a more on-time point, either day 4 or day 5 (96 h of culture), and it is at these time points that there are significant diversions in development rates between a sequential system and KSOMAA. However, in the study by Biggers et al. (2005), at 96 h of culture where from the literature would expect blastocyst rates of >80% in sequential media, these authors report only 30% for the sequential media systems. It is perhaps not in dispute that when embryos are cultured several days past the normal time of blastocyst formation and implantation that there may be no difference in development. However, the significance of this time point is debatable.
In conclusion, sequential media were in fact based on available data not only on the composition of the female reproductive tract, but also on studies on the physiology of the embryo as it develops and differentiates. The development of such media that were designed to account for the known effects of medium components such as EDTA and the breakdown product ammonium has resulted in increased efficacy of human IVF. This is supported by a Cochrane review by Blake et al. (2003) who reported that ‘The subgroup of trials employing sequential media, did however demonstrate a substantial improvement in implantation rates and similar pregnancy rates, despite the transfer of less embryos’ while inclusion of one-step culture systems in this analysis eliminated this benefit. Therefore, this evidence appears to validate our decision to keep EDTA out of the second-phase medium for fear of compromising ICM metabolism and for renewing the medium after 48 h to ensure that we minimize the exposure of embryos to ammonium which has teratogenic properties. There can be no discussion on the changing physiology and metabolism of the embryo during the preimplantation period and that the zygote and blastocyst are as different as skin and heart muscle. Biggers and colleagues wanted to let the embryo adapt to a single-medium formulation; we simply chose to facilitate embryo development and differentiation by using the physiological data we had at hand and make the culture environment free of teratogens. Whatever the outcome of this debate, there can be no question that sequential media formulations used by Biggers et al. (2005) [which have now been superceded (Balaban and Urman, 2005)] have been shown to support excellent human blastocyst development (around 60%) and result in excellent implantation rates (65% fetal heart beat) (Schoolcraft and Gardner, 2000). In this case, the data are from an oocyte donation program (Schoolcraft and Gardner, 2000), where the average age of the donors is 27 and where one cannot be accused of patient selection, as the oocytes do indeed come from younger women. Furthermore, when compared to in vivo developed mouse embryos on the morning of implantation, those cultured in sequential media have the same number of cells and the same cell allocation to the inner cell mass (Gardner and Lane, 2002). We have not found this to be the case using KSOMAA.
References
Balaban B and Urman B. (2005) Comparison of two sequential media for culturing cleavage stage embryos and blastocysts: embryo characteristics and clinical outcome. Reprod Biomed Online 10:485–491.[Medline]
Biggers JD, McGinnis LK, Lawitts JA. (2004) Enhanced effect of glycyl-L-glutamine on mouse preimplantation embryos in vitro. Reprod Biomed Online 9:59–69.[Web of Science][Medline]
Biggers JD, McGinnis LK, Lawitts JA. (2005) One-step versus two-step culture of mouse preimplantation embryos: is there a difference? Hum Reprod 20:3376–3384.
Blake D, Proctor M, Johnson N, et al. (2003) Cleavage stage versus blastocyst stage embryo transfer in assisted conception. Cochrane Database Syst Rev 1:1–92.
Gardner DK and Lane M. (2002) Development of viable mammalian embryos in vitro: evolution of sequential media. In Cibelli J, Lanza R, Campbell K, West MD (Eds.). Principles of Cloning (Academic Press, New York) pp. 187–213.
Gardner DK and Lane M. (2003) Towards a single embryo transfer. Rep Biomed Online 6:470–481.
Hewitson LC and Leese HJ. (1993) Energy metabolism of the trophectoderm and inner cell mass of the mouse blastocyst. J Exp Zool 267:337–343.[CrossRef][Web of Science][Medline]
Lane M and Gardner DK. (1994) Increase in postimplantation development of cultured mouse embryos by amino acids and induction of fetal retardation and exencephaly by ammonium ions. J Reprod Fert 102:305–312.
Lane M and Gardner DK. (2001) Inhibiting 3-phosphoglycerate kinase by EDTA stimulates the development of the cleavage stage mouse embryo. Mol Reprod Dev 60:233–240.[CrossRef][Web of Science][Medline]
Reed LC, Lane M, Gardner DK. (2003) In vivo rates of mouse embryo development can be attained in vitro. Theriogenology 59:349.[CrossRef]
Schoolcraft WB and Gardner DK. (2000) Blastocyst culture and transfer increases the efficiency of oocyte donation. Fertil Steril 74:482–486.[CrossRef][Web of Science][Medline]
Sinawat S, Hsaio WC, Flockhart JH, Kaufman MH, Keith J, West JD. (2003) Fetal abnormalities produced after preimplantation exposure of mouse embryos to ammonium chloride. Hum Reprod 18:2157–2165.
Virant-Klun I, Tomazevic T, Vrtacnik-Bokal E, Vogler A, Krsnik M, Meden-Vrtovec H. (2006) Increased ammonium in culture medium reduces the development of human embryos to the blastocyst stage. Fertil Steril 85:526–528.[Medline]
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