1 Reproductive Science Center, One Forbes Road, Lexington, MA, 02421 and Department of Obstetrics and Gynecology, University of Massachusetts Medical School, Worcester, MA, 10655, 2 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115 and 3 Beth Israel Deaconess Hospital, Boston, MA 02115, USA
4 To whom correspondence should be addressed. Email: john_biggers{at}hms.harvard.edu
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Abstract |
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Key words: exencephaly/glutamine/glycylglutamine/IVF
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Introduction |
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Serious concern arose in the human IVF community about putative teratogenic effects of ammonium accumulation when Lane and Gardner (1994) reported cases of exencephaly in mice following the transfer into surrogate mothers of blastocysts cultured in medium mMTF containing Gln. This concern was further enhanced by the observation that cases of exencephaly also arose in fetuses that developed from blastocysts produced in the presence of ammonium chloride. The toxic effect of Gln can be avoided by making use of the fact that dipeptides can be directly used by cells in vitro (Eagle, 1955
). Thus, Lane et al. (2001)
replaced Gln with L-alanyl-L-glutamine (AlaGln) for the culture of mouse preimplantation embryos, a practice that has been adopted by several commercial companies that sell media for the culture of human preimplantation embryos (e.g. G-1TM version 3 medium, Vitrolife, Göteborg, Sweden; Global®, LifeGlobal, Canada; Quinn's Advantage®, Cooper Surgical, Trumbull, CT). Recently, we have reported that glycyl-L-glutamine (GlyGln) seems a preferable choice of a dipeptide since it differentially favors the development of the inner cell mass (ICM) (Biggers et al., 2004a
).
The teratological effects of ammonium accumulation described by Lane and Gardner (1994) have not been confirmed by others (Sinawat et al., 2003
; Biggers et al., 2004a
). The reasons for the differences are unknown but may be related to differences in the genetic makeup of the strains of mice used (for review, see Biggers et al., 2004b
). The work done so far has only reported the effects of Gln on zygotes produced in vivo. No comparative studies have been reported in which unfertilized ova were exposed either to Gln or GlyGln before and during the fertilization process. In this paper we describe the results of an experiment in which fertilization occurred in vitro in a medium containing Gln at twice the concentration used routinely in embryo culture media. Parallel studies were perormed using a similar concentration of GlyGln.
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Materials and methods |
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Culture media
All culture media were formulated from KSOM, which contains 1.0 mg/ml BSA (Fraction V; Sigma, Cat. # A9647; lot # 15H0672) (Lawitts and Biggers, 1993). Medium mKSOM is KSOM where the concentration of BSA is raised to 4.0 mg/ml and supplemented with 5.56 mmol/l glucose (Summers et al., 1995
, 2000
). KSOM containing 5.56 mmol/l glucose is designated as KSOMg. KSOM, KSOMg and mKSOM containing AA are designated as KSOMAA KSOMgAA and mKSOMAA, respectively (Table I). KSOMgAA when supplemented with GlyGln at 2 mmol/l is designated as GlyGln-KSOMgAA. KSOMgAA was also supplemented with Gln at 2 mmol/l and is designated as Gln-KSOMgAA; this is in contrast with the concentration of 1 mmol/l Gln normally used in KSOM (Lawitts and Biggers, 1993
). The concentrations of Gln and GlyGln were increased to be consistent with the concentration of GlyGln to exaggerate any effect of ammonium that might result from the chemical breakdown of Gln.
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Sperm, oocyte and zygote collection
Gametes were collected using the methods described elsewhere (Summers et al., 1995, 2000
).
Fertilization in vitro
Fertilization in vitro was done in 1 ml drops of mKSOMAA supplemented with either 2 mmol/l Gln or 2 mmol/l GlyGln under mineral oil as reported in detail previously (Summers et al., 1995, 2000
). The concentration of sperm was
106/ml.
Embryo culture
Ova that had been exposed to sperm from fertilization in vitro and in vivo were cultured in groups of 12 in 50 µ1 droplets of medium consisting of either Gln-KSOMgAA or GlyGln-KSOMgAA overlaid with mineral oil. The ova were allotted at random to the two media. The embryos were incubated at 37 °C in modular incubator chambers (Billups-Rothenberg Inc., Del Mar, CA) which were gassed with a mixture of 5% O2, 6% CO2 and 89% N2 (Lawitts and Biggers, 1993). Culture plates (60 mm suspension, non-tissue cultured-treated; Corning Inc., Corning, NY) were prepared 1 day before embryo collection and equilibrated in the module overnight.
Embryo evaluation
Embryos were observed at x40 to x100 on a warmed (35 °C) microscope stage of a Wild dissecting microscope. The numbers of zygotes that cleaved and developed by 96, 120 and 144 h post-hCG administration into zona-enclosed blastocysts, and partially and completely hatched blastocysts, were counted.
Embryo transfer
Some of the cultured morulae and blastocysts derived 96 h post-hCG in either Gly-KSOMgAA or GlyGln-KSOMgAA were transferred into day 3 pseudopregnant females; six embryos chosen at random to each uterine horn. The embryos produced in each medium were allotted at random to either the left or right uterine horn. The operator performing the embryo transfer experiments was blinded as to the embryo source (i.e. zygotes derived from fertilization in vitro or mated females) and culture conditions used. The embryos were allowed to develop for a further 1112 days. Thus, the fetuses that developed were 1415 days post-hCG. At this time the females were killed and the numbers of implantation sites and fetuses counted in each horn. All the fetuses were assessed for gross abnormalities and weighed.
Differential ICM and TE cell counts
The remaining cultured blastocysts obtained 144 h post-hCG were fixed and differentially stained to determine the cell count of the inner cell mass (ICM) and trophectoderm (TE). The method is based on modifications of a method originally described using polynucleotide-specific fluorochromes (Handyside and Hunter, 1984, 1986
; Papaioannou and Ebert, 1988
). The complete method of differential staining has been described elsewhere (Biggers et al., 2000
). The numbers of non-mitotic, mitotic, pyknotic and fragmented nuclei were also counted.
Experimental design and biometrical considerations
Zygotes were produced by IVF in either Gln-mKSOMAA or GlyGln-mKSOMAA, and then transferred in groups of 12 to Gln-KSOMgAA and GlyGln-KSOMgAA for the rest of the culture period, respectively. Groups of 12 zygotes, produced by fertilization in vivo (controls), were also cultured simultaneously in Gln-KSOMgAA and GlyGln-KSOMgAA. The experimental unit was thus a droplet containing 12 zygotes. The four treatments were compared in a randomized block experiment with unequal numbers of experimental units in each cell. Four replicates were done. The number of intact 1-cell embryos that were exposed to sperm 24 h post-hCG were assumed to be fertilized. The 2-cell embryos were cultured for a further 72 h (96 h post-hCG) at which time the numbers of blastocysts that had developed were counted. The drops containing embryos were then divided at random into two groups. In the first group the morulae and blastocysts that had developed were transferred into the uteri of surrogate mothers. The second group was cultured for a further 48 h (144 h post-hCG) and at 120 and 144 h post-hCG the numbers of zona-enclosed blastocysts, partially hatched and completely hatched blastocysts, were counted. The blastocysts that developed by 144 h post-hCG were then stained for counting the numbers of ICM and TE cells and pyknotic and fragmented nuclei.
Two sets of observations were made. Set 1 were the categorical scores that recorded the morphological development of the zygotes into 2-cell stages, blastocysts by 96, 120 and 144 h post-hCG, including the numbers of zona-enclosed blastocysts, hatching and completely hatched blastocysts. Before applying exploratory graphical and statistical analyses, the data were re-expressed as the numbers of embryos that at least developed into blastocysts, at least started to hatch and at least completely hatched. The data was then analyzed using the parametric generalized linear model (McCullagh and Nelder, 1989), assuming that the errors follow a binomial distribution. The second set of observations were the counts of ICM and TE cells in each blastocyst that had developed 144 h post-hCG in each culture condition. The marginal distributions are displayed using either barplots or notched box plots. The boxplots show the 10th, 25th, 50th (median), 75th and 90th percentiles; observations outside of the 10th and 90th percentiles are considered outliers. The notches on the boxplots are the 95% median confidence limits. Two medians are significantly different if their confidence limits do not overlap. In the cell counts the errors are assumed to be formed by a multiplicative process. Thus the data have been submitted to an analysis of variance (ANOVA) assuming lognormally distributed errors. The numbers of dead cells are assumed to follow a Poisson distribution and have also been analyzed by ANOVA after transformation to the logarithmic scale.
The embryo transfer data (embryos that at least implanted and the numbers of fetuses) were treated as two sets of stratified 2 x 2 contingency tables (Mehta and Patel, 2001), each stratum being one mother. The exact probability that the sets were homogeneous was calculated. If P>0.05, the pooled exact estimate of the common odds ratio and its confidence limits (P=0.95) was then computed. The effects of the two media were considered significantly different if the confidence limits did not include 1.
All but one of the statistical analyses were done using the S-Plus 5 package (Insightful, Seattle, WA). The exact analysis of stratified 2x2 contingency tables was computed using StatXact 5 (Cytel Software Corporation, Cambridge, MA). A difference in all analyses is considered statistically significant if P<0.05.
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Results |
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Figure 1C shows the distributions of the ratio of the numbers of ICM and TE cells in blastocysts produced from control (in vivo) and in vitro-derived zygotes cultured in the presence of Gln-KSOMgAA and GlyGln-KSOMgAA. The presence of GlyGln has a greater relative stimulatory effect on the ICM cells than on the TE cells both in vitro and in vivo. For IVF-produced blastocysts the ratio of the two distributions of the ICM and TE is higher in blastocysts produced in GlyGln compared with Gln, but the notches overlap, showing that the effects of Gln and GlyGln are not significant. The ANOVAs (data not shown) of the logarithms of cell counts of the ratio of ICM/TE, which eliminates the variation between replicates, shows a statistically significant effect of replacing Gln with GlyGln in both treatment groups: fertilization in vitro (P = 0.002), and in vivo controls (P = 0.00009).
Blastocyst pyknotic and fragmented nuclei count
The distributions of the numbers of pyknotic and fragmented nuclei in blastocysts at 144 h post hCG cultured in KSOMgAA supplemented with either 2 mmol/l Gln or 2 mmol/l GlyGln are displayed in Figure 2. Figure 2A and C shows the distributions for blastocysts derived in vitro and cultured in Gln and GlyGln, respectively. Figure 2B and D shows the corresponding distributions for blastocysts derived in vivo. The number of pyknotic and fragmented nuclei is very variable, ranging from 0 to 19 cells. Visual comparison of the distributions of the proportions of pyknotic and fragmented nuclei in media containing either Gln or GlyGln suggests that those associated with GlyGln are more skewed to the left. This skewness is reflected in the medians of the distributions obtained using GlyGln which are less than those using Gln. The median number of pyknotic and fragmented nuclei was five for blastocysts derived from culture in GlyGln-KSOMgAA and approximately six or seven in Gln-KSOMgAA. An ANOVA (data not shown) demonstrated that the method of fertilization under our conditions had no effect on the numbers of pyknotic and fragmented nuclei. The only significant difference was due to effects of Gln and GlyGln.
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Since statistical analyses of the results using IVF showed that the data were homogeneous, they were pooled. The common odds ratios show that there is no significant difference in the incidence of implantations or fetuses that developed from zygotes produced in vitro using Gln or GlyGln (Table IV). Statistical analyses of the results using in vivo fertilization were also homogeneous and were pooled. The common odds ratios showed, in contrast, that there are significant differences in the numbers of implantations and fetuses when the zygotes were produced in vivo (Table IV). More implantations and fetuses were seen when embryos were cultured in Gln-KSOMgAA, compared with GlyGln-KSOMgAA.
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Discussion |
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Lane and Gardner (1994) cultured mouse zygotes in a medium containing 1 mmol/l Gln before transferring them to surrogate mothers. This concentration is commonly used in preimplantation culture media. Several cases of exencephaly were observed and were attributed to the accumulation of ammonium in the culture medium (Lane and Gardner, 1994
). During the culture of preimplantation embryos there are several potential sources of ammonium, including amino acid and protein metabolism/turnover by the embryo, spontaneous breakdown of the amino acids, most notably Gln, and perhaps from the breakdown/metabolism of albumin in the culture media. However, the relative contributions of all other sources are proportionately very small when compared to the chemical breakdown of Gln. Nakazawa et al. (1997)
showed that the addition of mouse embryos to their amino acid-supplemented culture media did little to increase the measured ammonium concentration. Further, Lane and Gardner (2003)
demonstrated that the rates of accumulation of ammonium in two embryo culture media that were supplemented with essential and non-essential amino acids and with AlaGln replacing Gln were negligible. The estimated ammonium production of preimplantation mouse embryos at the blastocyst stage is
1.0 pmol/embryo/h (Gardner and Lane, 1993
) and 4.3 pmol/embryo/h for bovine embryos. The latter higher value is attributable to the known higher levels of metabolic activity of bovine embryos (Orsi and Leese, 2004
). In our culture system, 12 mouse embryos are cultured in 50 µl droplets of culture media. Consequently, over 5 days of culture, the ammonium generated by the metabolism of the embryos would result in a final concentration of 0.0024 mmol/l, a value that is more than two orders of magnitude less than that derived from the breakdown of Gln in aqueous solution. This estimate is probably exaggerated, since amino acid utilization and turnover is lower in preimplantation embryos prior to compaction (Brinster, 1971
; Kaye et al., 1982
, Partridge and Leese, 1996
). We therefore estimate that <2% of the total ammonium is derived from all sources, other than the chemical breakdown of Gln. The rate of breakdown of Gln in aqueous solution follows first order kinetics, described by the equation:
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In our studies, the culture media are equilibrated overnight at 37 °C. Thus, at time zero, the start of the fertilization process in vitro, it is estimated that the medium contains 0.14 mmol/l ammonium. At the completion of fertilization, the ammonium concentration is expected to rise to 0.18 mmol/l. The zygotes were then transferred to fresh media containing 0.14 mmol/l ammonia. By 96 h post-hCG, when blastocysts were transferred to surrogate mothers, the ammonium will accumulate to 0.70 mmol/l, and by 144 h post-hCG, when blastocyst cell counts were done, it is expected to be 0.95 mmol/l. Our results show that these gradually increasing concentrations of ammonium do not adversely affect the development of the pre- and post-natally developing mouse embryo. These results agree with other results published elsewhere on the effects of Gln on the development in vitro of mouse zygotes recovered from the oviduct (Biggers et al., 2004a
). Hammon et al. (2000)
have also shown that bovine follicular fluid contains appreciable amounts of ammonium, and that exposure of bovine oocytes to ammonium (up to 0.35 mmol/l) did not affect their fertilization, subsequent cleavage or development to blastocysts or hatching blastocysts.
In recent years, Gardner and Lane have modeled the alleged toxicity of Gln to mouse preimplantation development by studying the direct effects of added ammonium chloride to the culture medium (for review, see Lane and Gardner, 2003). In their experiments, ammonium chloride was added in high concentrations at the beginning of the culture period. Their protocols do not reproduce the conditions created by the slow accumulation of ammonium from the spontaneous breakdown of Gln, and is thus a poor model of the conditions that occur under commonly used culture conditions. For example, Lane and Gardner (2003)
have previously reported that the addition of exogenous ammonium to cultures of mouse preimplantation embryos resulted in a significant decrease in an embryo's ability to result in a viable pregnancy after embryo transfer, resulting in lower implantation rates, decreased fetal growth and an increased incidence of fetal anomalies. We observed no such findings either in the present study, or previous studies (Biggers et al., 2004a
), likely reflecting the well known ability of embryos to undergo post-transfer compensatory growth. We did observe a higher implantation rate and fetal viability for in vivo-derived embryos cultured in KSOMgAA containing 2 mmol/l Gln, when compared with GlyGln. This was not observed, however, in a much larger series of embryo transfer studies (Biggers et al., 2004a
), probably reflecting the relatively small group sizes in the present study. The problems involved with adding exogenous ammonium to embryo culture media and the reasons for the differences have been discussed in more detail elsewhere (Biggers et al., 2004b
).
The replacement of Gln with GlyGln in the fertilization and culture media, as expected, had no effect on the rate of development of the mouse embryos and also did not result in any gross abnormal development. Hagemann et al. (1998) observed no difference in fertilization rates of bovine oocytes or embryo development in media supplemented with either Gln or GlyGln.
Elsewhere, we have reported that replacing Gln with GlyGln in Gln-KSOMgAA increases the numbers of cells that develop in the blastocyst from zygotes produced in vivo, particularly the ICM cells (Biggers et al., 2004a). The results reported in the present paper using zygotes produced both in vitro and in vivo confirm this result. In addition, our results show that replacing Gln with GlyGln in Gln-KSOMgAA decreases the total numbers of pyknotic and fragmented nuclei observed in the blastocyst. This method does not distinguish between cells dying of oncosis or apoptosis. The median number of pyknotic and fragmented nuclei in blastocysts was not affected by the method of fertilization when cultured in GlyGln-KSOMgAA (median = 5). Statistically significant higher median pyknotic and fragmented nuclei counts were seen in blastocysts fertilized and cultured in Gln-KSOMgAA (in vitro: median = 7; in vivo: median = 6). Whether the higher numbers of pyknotic and fragmented nuclei in blastocysts produced in Gln-KSOMgAA is due to the breakdown of Gln is unknown. Kamjoo et al. (2002)
reported that the level of apoptosis in the ICM of mouse embryos was dependent on the culture media used and the choice of mouse strain.
Although there is no evidence that fertilization in vitro in the presence of Gln is detrimental to later development, there may be an advantage of replacing Gln with GlyGln because of the stimulation of the development of the ICM and the reduction in the number of dead cells. Elsewhere, we have presented evidence that AlaGln, now being added to several commercial media to overcome the accumulation of ammonium in preimplantation culture media, does not have the stimulatory effect on the ICM as GlyGln (Biggers, 2004a).
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Acknowledgements |
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References |
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Submitted on May 4, 2004; resubmitted on October 18, 2004; accepted on December 14, 2004.