1 Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115 and 2 Beth Israel Deaconess Hospital, Boston, MA 02115, USA
3 To whom correspondence should be addressed. E-mail: john_biggers{at}hms.harvard.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are several variants of two-step protocols for the culture of preimplantation embryos. Gardner (1994) first advocated a two-step protocol to remove ammonium and pyrrolidone-5-carboxylic acid, the breakdown products of glutamine (Gln), which arise spontaneously in all media containing Gln (review: Biggers et al., 2004a
). Renewing the medium, however, has potentially favourable and unfavourable effects. It may re-supply beneficial compounds that have been significantly depleted during the initial culture period. Conversely, it may remove any beneficial compounds secreted by the cells during the initial period, such as growth factors. Two-step culture protocols can also involve media in which the second medium differs in composition from the first medium. This can involve the mere addition of a substance, e.g. the addition of glucose to medium CZB, which contains no glucose (Chatot et al., 1989
). Alternatively it can involve the omission of a single substance from the first medium, as was done by Lane and Gardner (1995) in which the second medium (DM1) is the first medium (DM2) less EDTA (Table I). The EDTA was considered toxic in the later stages of preimplantation development. More complex two-step procedures can also involve several additions and subtractions of compounds from the first medium to give the second medium, e.g. as is done using medium G1.1 and medium G1.2 (Gardner et al., 1998
) (Table I). In these protocols the embryos may be subjected to stress by being transferred to a new chemical environment. The results summarized in this paper describe the effects of renewing potassium-enriched simplex optimized medium supplemented with glucose and amino acids (KSOMgAA), the effects of removing EDTA from KSOMgAA and the effects of replacing medium G1.2 with medium G2.2 and DM2 with DM1.
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Zygotes
Donor females (CF1 mice, 68 weeks old; Harlan Sprague Dawley, Indianapolis, IN, USA) were injected i.p. with 5 IU of pregnant mares serum gonadotrophin (P.G.600; Intervet, Inc., Millsboro, DE, USA) followed 48 h later with 5 IU HCG. C57BL/6J ¥ DBA/2J (BDF) male mice, 211 months old were also obtained from Harlan Sprague Dawley or The Jackson Laboratory, Bar Harbor, ME, USA. Animals were maintained in accordance with the guidelines of the Institutional Animal Care and Use Committee of Harvard Medical School.
Culture
Embryos were cultured in sets of 12 per micro-drop in a tri-gas atmosphere of 5% O2, 6% CO2 and 89% N2, as reported previously (Biggers and McGinnis, 2001). Embryos cultured using a one-step protocol for 4 days (144 h post-HCG) were cultured in 50 µl drops under oil (Sigma embryo-tested light mineral oil). Embryos cultured using a two-step protocol, where the medium was renewed or changed at 48 h (72 h post-HCG), were incubated in 20 µl drops under oil, following the recommendation of Gardner and Lane (1996).
Embryo evaluation
In one experiment embryos were graded at 48 h post-HCG at x40 magnification on a warmed microscope stage at 35°C (Wild dissecting microscope), for stage of development including 1-, 2-, 3 4-, 8-cell and morula. In all experiments embryos were observed 96, 120 and 144 h post-HCG and graded for the stage of development including compaction, blastocoel formation and hatching. These times correspond approximately to 72, 96 and 120 h in culture.
Differential ICM and TE cell counts
After 144 h of culture, blastocysts were stained with polynucleotide-specific fluorochromes to differentially stain inner cell mass (ICM) and trophectoderm (TE) cells using a modification of a method originally described by Handyside and Hunter (1984, 1986
) and Papaioannou and Ebert (1988)
. The details of the modified method are described by Biggers et al. (2000)
.
Embryo transfer
Pseudopregnant CD1 female mice (Taconic, Albany, NY, USA) were produced by mating CD1 females to vasectomized CD1 males. Cultured blastocysts and compact morulae (4th day of culture) were transferred into day 3 pseudopregnant females. Embryos produced in KSOMgAA were injected into one uterine horn at random and embryos produced in G1.2/G2.2 into the other uterine horn. Fetuses were collected from the pregnant mice on the 12th day after transfer for gross examination and body weight measurements.
Experimental design and statistical analysis
The results of three randomized block experiments are described in this paper, where each block is a separate replicate of the treatments. Experiment 1 compared four protocols: KSOMgAA, KSOMgAA/KSOMgAA, G1.2 and G1.2/G2.2, cultured until 144 h post-HCG. Experiment 2 was an analogous experiment using four treatments that compared the effects of culturing zygotes for 144 h post-HCG in KSOMgAA, KSOMgAA/EDTA-free KSOMgAA, DM2 and DM2/DM1. Experiment 3 compared the effects of culturing in KSOMgAA and G1.2/G2.2, followed by the transfer of embryos into the uterus of surrogate mothers. In all experiments the experimental unit consisted of 12 zygotes.
Two sets of observations were made. Set 1 concerns the morphological development of the zygotes into 2-cell stages, and blastocysts by 96, 120 and 144 h post-HCG, including the numbers of zona-enclosed blastocysts, hatching and completely hatched blastocysts. These are categorical observations. Before applying exploratory graphical and statistical analyses, the data were re-expressed as the numbers of embryos that at least develop into blastocysts, which at least start to hatch and at least completely hatch. The data on the numbers of blastocysts that developed were examined using the parametric generalized linear model (McCullagh and Nelder, 1989), assuming that the errors follow a binomial distribution. An analysis of deviance partitioned the variation into between the replicates, the treatments and the replicate x treatments interaction, respectively. If the interaction was not significant at the P = 0.05 level the data were pooled over replicates. The blastocysts that developed by 144 h post-HCG were then classified into the numbers of blastocysts that remained enclosed in the zona pellucida, those that had partially hatched and those that had completely hatched. The asymptotic or exact KruskalWallis test of significance with one singly ordered variable was used to test whether these distributions differed significantly (Mehta and Patel, 2001
). Set 2 concerns the numbers of ICM and TE cells in each blastocyst that had developed 144 h post-HCG in each culture condition. The distributions are displayed using notched box plots. The boxplots show the 10th, 25th, 50th (median), 75th, 90th percentiles; observations outside of the 10th and 90th percentiles are considered outliers. The notches on the box plots are the 95% median confidence limits. Two medians are significantly different if their confidence limits do not overlap. The errors associated with the cell counts are assumed to be formed by a multiplicative process. Thus the data have been submitted to an analysis of deviance assuming lognormally distributed errors (McCullagh and Nelder, 1989
), partitioning the variation into between the replicates, the treatments and the replicate x treatments interaction. The analysis is summarized as a two-way analysis of variance (ANOVA) table. If the interaction was not significant at the P = 0.05 level the data were pooled over replicates.
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 computed. The effects of the two media were considered significantly different if the confidence limits did not include 1.
All statistical analyses were done, with one exception, using the S-Plus 5 package (Insightful, Seattle, WA, USA). The exact KruskalWallis test for the analysis of a one-way ordered 2 x n contingency table, and the exact analysis of stratified 2 x 2 contingency tables was the exception analysed using StatXact 6 (Cytel Software Corporation, Cambridge, MA, USA). A difference in all analyses is considered statistically significant if P < 0.05.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Development during the first phase
The distributions of the numbers of embryos that remained as 1-cell, developed to 24-cell embryos, 58-cell embryos and compacted morulae by 72 h post-HCG, when cultured in KSOMgAA and G1.2, are summarized in a 2x4 contingency table (Table II). There was no significant difference between the rates of development in KSOMgAA and G1.2 during this initial period of culture.
|
Morphological development reached by 144 h post-HCG in KSOMgAA, KSOMgAA/KSOMgAA, G1.2 and G1.2/G2.2
The data from the three replicates have been pooled and summarized in Table IIIa. The rates of blastocyst formation were very similar in media KSOMgAA and KSOMgAA/KSOMgAA, reaching a rate of formation of 89% by 144 h post-HCG. There was also no significant difference between the rates of blastocyst formation in media G1.2 and G1.2/G2.2, both reaching a rate of
82%. Development of blastocysts was slightly slower using the G1.2 and G1.2/G2.2 protocols compared with the KSOMgAA and KSOMgAA/KSOMgAA protocols. In summary, high yields of blastocysts were obtained by 144 h post-HCG using all four protocols. The distributions of the zona-enclosed blastocysts, hatching and hatched blastocysts at 144 h post-HCG observed using the four protocols are shown in Table IIIb. Statistically, the four distributions are significantly different (P = 0.008). The significance is due to variation in the pattern of hatching using the G1.2, G1.2/G2.2 protocols and the KSOMgAA, KSOMgAA/KSOMgAA protocols. The G1.2 and G1.2/G2.2 protocols supported a majority of blastocysts that started to hatch and completed hatching by 144 h post-HCG. In contrast, a greater percentage of blastocysts cultured using the KSOMgAA, KSOMgAA/KSOMgAA protocols started to hatch but fewer had completed hatching by 144 h post-HCG.
|
|
Numbers of ICM and TE cells in blastocysts that developed by 144 h post-HCG in KSOMgAA, KSOMgAA/KSOMgAA, G1.2 and G1.2/G2.2
The ICM data have been pooled over replicates to generate the box plots shown in Figure 1a. The numbers of ICM cells vary over a wide range (073). The notches of the distributions overlap for blastocysts developed in KSOMgAA and KSOMgAA/KSOMgAA indicating no significant differences between the medians of these two distributions. A t-test based on the ANOVA shows that there is no significant difference between the means of the two distributions (P = 0.597). In contrast, the notches of the distributions for blastocysts that developed throughout in G1.2 and in G1.2/G2.2 respectively just fail to overlap, showing that the number of ICM cells which develop is slightly less when cultivated in G1.2 compared with G1.2/G2.2. A t-test shows that the means of the distributions are significantly different (P = 0.008). Only one trophoblastic vesicle was seen in this experiment in an embryo cultivated in G1.2.
|
The distributions of the ratios between the numbers of ICM and TE cells are shown in Figure 1c. Statistical analyses demonstrate no significant differences between these ratios in blastocysts cultured in KSOMgAA, KSOMgAA/KSOMgAA and G1.2/G2.2. The ratio is significantly lower in blastocysts cultured in G1.2, demonstrating that relatively fewer ICM cells developed compared to TE cells.
Development in KSOMgAA, KSOMgAA/EDTA-free KSOMgAA, DM2 and DM2/DM1 (experiment 2)
The development of mouse zygotes by 144 h post-HCG using four culture protocols was compared. The protocols were culture of embryos in: (i) KSOMgAA until 144 h post-HCG with no renewal of the medium; (ii) KSOMgAA for 48 h followed by culture to 144 h post-HCG in EDTA-free KSOMgAA; (iii) DM2 until 144 h post-HCG with no renewal of the medium; (iv) DM2 for 48 h post-HCG followed by culture to 144 h post-HCG in DM1. Three replicates were done. Eighteen experimental units were allotted to each treatment in replicates 1 and 2, and 15 experimental units in replicate 3. The experimental design was similar to that described in experiment 1, the results being obtained in two phases.
Development during the first phase
The distributions of the numbers of embryos that remained as 1-cell, developed to 2-cell embryos, 34-cell embryos, and 8-cell embryos plus compacted morulae after 48 h of culture in KSOMgAA and DM2 are summarized in a 2x4 contingency table (Table IV). There was a highly significant difference between the rates of development in KSOMgAA and DM2 during this initial period of culture, the embryos cultured in KSOMgAA developing at a faster rate.
|
Morphological development reached by 144 h post-HCG in KSOMgAA, KSOMgAA/EDTA-free KSOMgAA, DM2 and DM2/DM1
The data from the three replicates have been pooled and summarized in Table Va. The rates of blastocyst formation were very similar in media KSOMgAA, KSOMgAA/EDTA-free KSOMgAA and DM2/DM1 throughout the 144 h period of culture. The rate of blastocyst formation in medium DM2 was similar to the other media for the first 120 h post-HCG but fell off slightly in the next 24 h, causing a significant difference between the yields of blastocysts that developed by 144 h post-HCG (P = 0.02). In summary, high yields of blastocysts were obtained by 144 h post-HCG using all four protocols, the yield of blastocysts in medium DM2 (
73%) being only
8% less than development in the other three protocols (
80%).
|
|
Numbers of ICM and TE cells in blastocysts that developed by 144 h post-HCG in KSOMgAA, DM2, KSOMgAA/EDTA-free KSOMgAA and DM2/DM1
The analysis of deviance of the ICM data shows significant main effects of replicates (P = 0.001) and protocols (P < 0.0001) and also a very significant replicate x protocol interaction (P = 0.001). The interaction is due to variation between the magnitudes of the differences between the effects of the four protocols. Nevertheless the patterns of the differences are similar so that the data have been pooled over replicates to generate the box plots shown in Figure 2a. The data show that the numbers of ICM cells vary over a wide range (058). Further, the notches of the distributions, which indicate the approximate confidence limits for the medians, overlap for blastocysts developed in KSOMgAA, KSOMgAA/EDTA-free KSOMgAA and DM2/DM1 indicating no significant differences between the effects of these three protocols. The notch of the distribution for blastocysts that developed throughout in DM2 is well below the others indicating that the number of ICM cells that develop under these conditions is much less. A small part of the variation in the ICM count data is due to failure of the ICM to develop, giving rise to trophoblastic vesicles. The numbers of these vesicles observed in embryos that developed in KSOMgAA, KSOMgAA/EDTA-free KSOMgAA , DM2/DM1 and DM2 were one (1/83: 1.2%), two (2/99: 2.0%), two (2/72: 2.8%) and seven (7/69: 10.1%) respectively.
|
The distributions of the ratios between the numbers of ICM and TE cells are shown in Figure 2c. Statistical analyses (not shown) demonstrate no significant differences between these ratios in blastocysts cultured in KSOMgAA, KSOMgAA/EDTA-free KSOMgAA and DM2/DM1. The ratio is significantly lower in blastocysts cultured in DM2 demonstrating that relatively fewer ICM cells developed compared to TE cells.
Implantation and fetal development rates of transferred embryos cultured in either KSOMgAA or G1.2/G2.2 (experiment 3)
Blastocysts and compacted morulae for embryo transfer were obtained from zygotes cultured for 96 h post-HCG using either the one-step protocol KSOMgAA or the two-step protocol G1.2/G2.2. The remainder of the embryos were cultured until 144 h post-HCG. Those embryos grown to 144 h post-HCG in KSOMgAA yielded 89% blastocysts containing median numbers of 31 and 126 ICM and TE cells respectively, while for those grown in media G1.2/G2.2, 81% of zygotes had developed into blastocysts containing median numbers of 29 and 88 ICM and TE cells respectively. The hatching rates of the blastocysts were similar using the two media. Compact morulae and blastocysts were transferred into 30 recipient females of which 21 became pregnant. Forty-eight and 28 fetuses developed using the KSOMgAA and G1.2/G2.2 protocols respectively. No gross abnormalities, such as exencephaly, were observed in either group of fetuses.
Four of the pregnant females had implantations and/or fetuses in only one horn and were excluded from the statistical analysis. These results are summarized in Table VI. The analyses show that the data within each group of mothers formed homogeneous groups and can be pooled. The common odds ratio showed that the numbers of implantations in each group were not significantly different. In contrast significantly fewer fetuses developed using the G1.2/G2.2 protocol compared with the KSOMgAA protocol. The distributions of the fetal body weights are shown in Figure 3. There is no significant difference in body weight of the fetuses that developed using the KSOMgAA and G1.2/G2.2 protocols.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of removing EDTA from medium DM2
Early attempts to culture newly fertilized mouse ova, particularly from outbred and inbred strains, were frustrated by the 2-cell block (review Biggers, 1993). Abramczuk et al. (1977)
overcame this block by adding EDTA to Whittens medium (Whitten, 1971
). Although the 2-cell block was not absolute in the strains of mice they used, the addition of EDTA spanning a range of concentrations increased the numbers of blastocysts that developed. There have been several independent studies published that confirm the ability of EDTA to overcome the 2-cell block (Hoshi and Toyoda, 1985
; Loutradis et al., 1987
; Fissore et al., 1989
; Nasr-Esfahani et al., 1990
). Gardner and Lane (1996)
, however, reported that the body weights of fetuses that developed from blastocysts produced in vitro in the presence of EDTA and transferred to surrogate mothers were significantly lower than those that developed from blastocysts in DM2/DM1. The practice of not including EDTA in the second phase of culture in two-step protocols stems from this observation. Our results confirm that the presence of EDTA in DM2 during the second phase of culture has deleterious effects, shown by a significant reduction in the rate of hatching and significant reductions of the numbers of ICM and TE cells. This reduction in the numbers of cells in the blastocysts may presage the reduced fetal weights reported by Gardner and Lane (1996)
. The cause of the inhibitory effects on the development of the mouse blastocyst is unknown. The development of the bovine blastocyst is also inhibited by EDTA, possibly by the inhibition of glycolysis when there is a rapid increase in the utilization of glucose, presumably as an energy source (Gardner et al., 2000
). A similar increase in the uptake of glucose begins in the mouse at the morula stage (review: Biggers et al., 1989
) whose utilization could be inhibited by a high concentration of EDTA.
The reason that EDTA is not deleterious in KSOMgAA but deleterious in DM2 on the development of the blastocyst may well be a concentration effect. The majority of the concentrationresponse curves, determined independently by Abramczuk et al. (1977), Loutradis et al. (1987)
and Fissore et al. (1989)
, suggested that most of the embryos overcame the 2-cell block when 510 µmol/l EDTA is added to media. Higher concentrations were also effective and toxicity did not occur until concentrations >200 µmol/l were used. Chatot et al. (1989)
used 100 µmol/l EDTA in a new medium they produced for the culture of mouse preimplantation embryos called CZB, and Gardner and Lane (1996)
used 100 µmol/l EDTA in medium DM2. Lawitts and Biggers (1991)
exploited the experimental strategy called sequential simplex optimization to determine simultaneously the optimum concentrations of all components in the medium in order to take account of all the possible interactions between the effects of the several components. The result was medium SOM (Lawitts and Biggers, 1992
) (the forerunner of KSOM) in which the optimum concentration of EDTA was one order of magnitude less (10 µmol/l) than that used by other investigators. Possibly this lower concentration of EDTA does not lead to the adverse effects that have been observed when 100 µmol/l EDTA is included in the media.
Development in G1.2 and G1.2/G2.2
The proportions of blastocysts that formed in G1.2 and G1.2/G2.2 were not significantly different, as well as those which hatched (P = 0.352). Thus the components and their concentrations in G1.2 are able to support the morphological structure of the blastocyst throughout the culture period without replacement. The changes in the composition of the environment produced by replacing G1.2 with G2.2 do not appear to affect the morphological development. In contrast, the numbers of ICM cells which formed in blastocysts that developed only in G1.2 is significantly less than those that develop when G1.2 is replaced with G2.2 48 h after the beginning of the culture period. Unfortunately the compositions G1.2 and G2.2 were trade secrets when our work was done. Only recently have the compositions of these media been revealed (Gardner and Lane, 2002). Several differences exist between the compositions of the two media, so it is impossible to pin-point the cause of the effects on ICM numbers. In our hands the numbers of ICM and TE cells produced in the blastocysts were larger using KSOMgAA than G1.2/G2.2 in contrast to the results reported by Lane and Gardner (2003)
. The importance of such differences should not be overemphasized since, in using commercially prepared media, changes could occur during the inevitable shipping and long storage after preparation.
Clinical implications for human IVF
The results presented in this paper suggest that a two-step protocol may be sufficient for the culture of mouse zygotes to blastocysts but that it is not necessary. It is important to know whether this conclusion applies to the media used for the culture of the human preimplantation embryo to the blastocyst stage. Two studies suggest that this conclusion may be valid. Biggers and Racowsky (2002) showed that human zygotes can develop into blastocysts in 5 days with high efficiency when cultured using KSOM plus amino acids without renewal of the medium. Normal babies were born using this medium. Macklon et al. (2002)
cultured human zygotes in a 17:3 mixture of Earles balanced salt solution and Hams F10 medium supplemented with albumin which they called the Rotterdam medium. The zygotes developed with equal efficiency when they were cultured either in the Rotterdam medium using a one-step protocol or in G1.2/G2.2 using a two-step protocol. Further, renewing the Rotterdam medium after 48 h culture did not increase the efficiency. Unfortunately, the field is dominated by the strongly held opinion, originating in the writings of Gardner and Lane, that the two-step protocol is absolutely necessary to imitate the environment encountered by preimplantation embryos in situ (see several current reviews: Mehta, 2001
; Kolibianakis and Devroey, 2002
; Pool, 2002
; Quinn, 2004
). The popularity of this conviction is due to its intuitive appeal, but critical examination shows that it is not supported by direct experimental evidence (reviews: Biggers, 2003
; Summers and Biggers, 2003
).
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Biggers JD (1993) The culture of the mammalian preimplantation embryo. In Gianaroli L, Campana A and Trounson AO (eds) Implantation in Mammals. Raven Press, New York, pp 123136.
Biggers JD (2003) Fundamentals of the design of culture media that support human preimplantation development. In Van Blerkom J (ed) Essential IVF. Kluwer, Norwell, MA, pp 291332.
Biggers JD and McGinnis LK (2001) Evidence that glucose is not always an inhibitor of mouse preimplantation development in vitro. Hum Reprod 16,153163.
Biggers JD and Racowsky C (2002) The development of fertilized human ova to the blastocyst stage in medium KSOMAA: is a two-step protocol necessary? Reprod Biomed Online 5,133140.
Biggers JD, Gardner DK and Leese H (1989) Control of carbohydrate metabolism in preimplantation mammalian embryos. In Rosenblum IY and Heyner S (eds) Regulation of Growth in Development. CRC Press, Boca Raton, FL, pp 2032.
Biggers JD, Summers MC and McGinnis LK (1997) Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos. Hum Reprod Update 3,125135.
Biggers JD, McGinnis LK and Raffin M (2000) Amino acids and preimplantation development of the mouse in protein-free KSOM. Biol Reprod 63,281293.
Biggers JD, McGinnis LK and Lawitts JA (2004a) Enhanced effect of glycyl-l-glutamine on mouse preimplantation embryos in vitro. Reprod Biomed Online 9,5969.[ISI][Medline]
Biggers JD, McGinnis LK and Summers MC (2004b) Discrepancies between the effects of glutamine in cultures of preimplantation mouse embryos. Reprod Biomed Online 9,7073.[ISI][Medline]
Chatot CI, Ziomek CA, Bavister BD, Lewis JL and Torres I (1989) An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 86,679688.[ISI][Medline]
Fissore RA, Jackson KV and Kiessling AA (1989) Mouse zygote development in culture medium without protein in the presence of ethylenediaminetetraacetic acid. Biol Reprod 41,835841.
Gardner DK (1994) Mammalian embryo culture in the absence of serum or somatic cell support. Cell Biol Int 18,11631179.[CrossRef][ISI][Medline]
Gardner DK and Lane M (1996) Alleviation of the 2-cell block and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod 11,27032712.[Abstract]
Gardner DK and Lane M (2002) Development of viable mammalian embryos in vitro: evolution of sequential media. In Cibelli I, Lanza R, Campell K and West M (eds) Principles of Cloning. Academic Press, New York, pp 187213.
Gardner DK and Lane M (2003) Towards a single embryo transfer. Reprod Biomed Online 6,470481.[Medline]
Gardner DK, Schoolcraft WB, Wagley L, Schlenker T, Stevens J and Hesla J (1998) A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization. Hum Reprod 13,34343440.[Abstract]
Gardner DK, Lane MW and Lane M (2000) EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation. Mol Reprod Dev 57,256261.[CrossRef][ISI][Medline]
Handyside AH and Hunter S (1984) A rapid procedure for visualizing the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes. J Exp Med 231,429434.
Handyside AH and Hunter S (1986) Cell division and death in the mouse blastocyst before implantation. Rouxs Arch Devel Biol 195,519526.[CrossRef]
Hoshi M and Toyoda Y (1985) Effect of EDTA on the preimplantation development of mouse embryos fertilized in vitro. Jpn J Zootech Sci 56,931937.
Kolibianakis ES and Devroey P (2002) Blastocyst culture: facts and fiction. Reprod Biomed Online 5,285293.[Medline]
Lane M and Gardner DK (1995) Removal of embryotoxic ammonium from the culture medium by in situ enzymatic conversion to glutamate. J Exp Zool 271,356363.[CrossRef][ISI][Medline]
Lane M and Gardner DK (2003) Ammonium induces aberrant blastocyst differentiation, metabolism, pH regulation, gene expression and subsequently alters fetal development in the mouse. Biol Reprod 69,11091117.
Lawitts JA and Biggers JD (1991) Optimization of mouse embryo culture media using simplex methods. J Reprod Fertil 91,543556.[ISI][Medline]
Lawitts JA and Biggers JD (1992) Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media. Mol Reprod Dev 31,189194.[CrossRef][ISI][Medline]
Lawitts JA and Biggers JD (1993) Culture of preimplantation embryos. In Wassarman PM and Depamphilis ML (eds), Guide to Techniques in Mouse Development. Methods in Enzymology, vol 225. Academic Press, San Diego, CA, pp 153164.
Loutradis D, John D and Keissling AA (1987) Hypoxanthine causes a 2-cell block in random-bred mouse embryos. Biol Reprod 37,111316.
Macklon NS, Pieters MHEC, Hassan MA, Jeucken PHM, Eijkemans MJC and Fauser BCJM (2002) A prospective randomized comparison of sequential versus monoculture systems for in-vitro human blastocyst development. Hum Reprod 17,27002705.
McCullagh P and Nelder JA (1989) Generalized Linear Models. 2nd edn, Chapman & Hall, London, pp 98148.
Mehta RH (2001) Growth of human preimplantation embryos in vitro. Reprod Biomed Online 2,113119.[Medline]
Mehta C and Patel N (2001) StatXact 5, Vol 2. Cytel Software Corporation, Cambridge, MA, pp 751771.
Nasr-Esfahani MH, Winston NJ and Johnson MH (1992) Effects of glucose, glutamine, ethylenediaminetetraacetic acid and oxygen tension on the concentration of reactive oxygen species and on development of the mouse preimplantation embryo in vitro. J Reprod Fertil 96,219231.[ISI][Medline]
Papaioannou VE and Ebert KM (1988) The preimplantation pig embryo cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro. Development 102,793803.
Pool TB (2002) Recent advances in the production of viable human embryos in vitro. Reprod Biomed Online 4,294302.[Medline]
Quinn P (2004) The development and impact of culture media for assisted reproductive technologies. Fertil Steril 81,2729.[ISI]
Sinawat S, Hsaio W-C, Flockart JH, Kaufman MH, Keith J and West JD (2003) Fetal abnormalities produced after preimplantation exposure of mouse embryos to ammonium chloride. Hum Reprod 18,21572165.
Summers MC and Biggers JD (2003) Chemically-defined media and the culture of mammalian preimplantation embryos: historical perspective and current issues. Hum Reprod Update 9,557582.
Summers MC, McGinnis LK, Lawitts JA, Raffin M and Biggers JD (2000) IVF of mouse ova in a simplex optimized medium supplemented with amino acids. Hum Reprod 15,17911801.
Summers MC, McGinnis LK, Lawitt, JA and Biggers JD (2005) Development of the mouse following IVF in media containing either l-glutamine or glycyl-l-glutamine. Hum Reprod, 20,13641371.
Whitten WK (1971) Nutrient requirements for the culture of preimplantation embryos in vitro. Adv Biosci 6,131141.
Whitten WK and Biggers JD (1968) Complete development in vitro of the preimplantation stages of the mouse in a simple chemically defined medium. J Reprod Fertil 17,399401.[ISI][Medline]
Submitted on April 1, 2005; resubmitted on June 11, 2005; accepted on June 23, 2005.
|