Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA
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Abstract |
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Key words: embryo culture/glucose/mouse preimplantation development/phosphate
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Introduction |
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The data presented previously (Schini and Bavister, 1988) on the joint effects of phosphate and glucose in overcoming the 2-cell block in hamsters suggested that glucose and phosphate inhibit development additively, but independently. In contrast, it was shown later (Reed et al., 1992a
) that the development of 8-cell rat embryos was inhibited only when glucose and phosphate were present in the medium together. A similar observation was made subsequently (Scott and Whittingham, 1996
) using two outbred strains of mice (CF1 and CD1). The results of a factorial experiment on the joint effects of glucose and KH2PO4 on the development of CF1 zygotes in a modified human tubal fluid medium have been graphically sketched (Quinn and Horstman, 1998
). These authors' illustration suggests that glucose inhibits development in the absence of phosphate but is barely inhibitory in the presence of 1.85 mmol/l phosphate. The possibility of the existence of a glucosexphosphate interaction was not tested statistically. No studies have been reported on the effects of varying the phosphate concentration in KSOM. Therefore, the effects of different combinations of KH2PO4 and glucose concentrations (in a factorial experimental design) on the development of outbred CF1 mouse zygotes cultured in KSOM were examined. A detailed statistical analysis of results from this type of experiment was necessary to determine whether the effects of glucose on the development of mouse preimplantation embryos cultured in KSOM are influenced by the concentration of KH2PO4. A multivariate approach was used to assess the joint effects of glucose and KH2PO4 on preimplantation development, by measuring: (i) the percentages of blastocysts that develop from zygotes; (ii) the percentages of blastocysts that initiate hatching; and (iii) the total cell counts in the blastocysts that developed after 144 h after human chorionic gonadotrophin (HCG) administration.
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Materials and methods |
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Donors
Zygotes were obtained by mating CF-1 female mice, 68 weeks old (Charles River Laboratories, Willmington, MA, USA) and BDF1 males, 211 months old (Charles River Laboratories, Raleigh, NC, USA). Females were stimulated with 5 IU pregnant mare's serum gonadotrophin, and superovulated 48 h later with 5 IU HCG. The oviducts were flushed 2226 h after HCG administration with a glucose/KH2PO4-free HEPES-buffered modification of KSOM, denoted FHM (Lawitts and Biggers, 1993), and embryos with two pronuclei were selected for culture. The medium used for embryo collection and holding prior to culture was also FHM.
Culture media
All culture media were formulated from KSOM (Table I). The medium, omitting glucose and KH2PO4, was prepared as a 2x solution (complete KSOM without the CaCl2), frozen in 50 ml culture tubes at 70°C for up to 3 months. On the day before embryo collection, 50 ml of KSOM was thawed and supplemented with CaCl2 (also stored as a frozen stock), glucose, KH2PO4 and H2O. Eighteen different media were prepared, each containing a specific combination of the concentrations of glucose and KH2PO4 shown in Table II
.
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Embryo evaluation
Embryos were observed at 96, 120 and 144 h post-HCG administration, on a Wild dissecting microscope fitted with a warmed stage at ~35°C using 40x magnification. They were graded for stage of development including compaction, blastocoele formation and hatching.
Total blastocyst cell counts
Embryos were fixed 144 h post-HCG administration in 3% formaldehyde for 15 min at 37°C, after which the nuclei were stained with the fluorochrome, Hoechst 33258 (1 µg/ml) in Dulbecco's phosphate-buffered saline (PBS) for 15 min at room temperature (Ebert et al., 1985). Blastocysts were mounted on glass slides, in groups of one to four, and covered with a mounting medium (50% glycerol, 50% PBS, 5 mg/ml sodium azide and 1 µg/ml Hoechst 33258). A glass coverslip was placed over the embryos and sealed in place with clear nail polish. Nuclei were counted at a magnification of 40x using an inverted Zeiss epifluorescence microscope with a 365 nm band pass excitation filter and a 420 nm long pass barrier filter.
Biometrical considerations
Concentrationresponse surfaces as models for chemically defined media
It was proposed previously (Biggers et al., 1957) that the responses of cells and organs cultured in chemically defined media can be modelled by a multidimensional concentrationresponse surface. This model is a generalization of the doseresponse line familiar in hormone biological assays. Later, it was suggested that a similar model be used to represent the response of preimplantation embryos in vitro in chemically defined media (Biggers et al., 1972
). An 11-dimensional concentrationresponse surface was assumed when sequential simplex optimization was used determine the concentrations of 10 components that were adopted in SOM (Lawitts and Biggers, 1991b
). In the present paper, it is assumed that the response of the zygote to joint changes in the concentrations of glucose and KH2PO4 can be represented by a two-dimensional surface. An estimate of the shape of part of this surface was obtained by using a 4x4 factorial design in which four concentrations each of glucose and KH2PO4 were used in all 16 combinations to span the region of interest.
Multivariate characteristics of the responses
Each embryo was observed at three different times, so that the responses were repeat (longitudinal) measurements (Diggle et al., 1994). At each time, the numbers of embryos that developed into three developmental stages: zona-intact blastocysts, partially and completely hatched blastocysts were counted, so that the data were also ordinal categorical responses (Clogg and Shihadeh, 1994
). Thus, the multivariate observations were serially correlated both longitudinally and ordinally. The raw data were re-expressed as the numbers of zygotes that developed at least into blastocysts and at least begin to hatch by 120 and 144 h post-HCG in each experimental unit. The response in each drop was then expressed as the percentage of blastocysts that developed from zygotes and the percentage of these blastocysts that initiated hatching. The mean percentage of the percentages in a series of drops was estimated as a weighted mean, weighting each observation in proportion to the number of blastocysts that formed in each drop. Total cell counts were made on all the blastocysts that developed.
General linear regression (McCullagh and Nelder, 1989)
All categorical responses just described were analysed using general linear regression by fitting the model:
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Boxplots (McGill et al., 1978)
All distributions were summarized using notched boxplots. The boxplots show the 10th, 25th, 50th (median), 75th and 90th percentiles and outliers. The notches on the boxplots were the median confidence limits. Two medians were significantly different if their confidence limits did not overlap. All computations were performed using S-Plus 2000.
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Results |
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Effect of KSOM and glucose/KH2PO4-free KSOM on blastocyst development and initiation of hatching
The percentages of zygotes that developed in KSOM at least to the blastocyst stage and at least starting to hatch by 120 and 144 h post-HCG are summarized in Table III. These levels of response are comparable with independent results using KSOM described elsewhere (Erbach et al., 1995
; Biggers et al., 2000
). The percentages of zygotes that developed in glucose/KH2PO4-free KSOM at least to the blastocyst stage and at least starting to hatch by 120 and 144 h post-HCG administration are also summarized in Table III
. The omission of glucose and KH2PO4 from KSOM had no effect on the percentages of zygotes that developed at least to blastocysts, or at least began to hatch.
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The total cell counts in blastocysts that developed from zygotes in 144 h in two of the replicates were counted. The mean total cell counts observed in each medium are shown in Table VIIa. The analysis of deviance (Table VIIb
) showed that the only significant effect was between replicates. The effects of glucose, KH2PO4 and their interaction were not statistically significant. The mean number of cells over all groups was 66. The median cell count in blastocysts produced in standard KSOM was similar (Figure 1
). In contrast, the median cell counts in blastocysts produced in glucose/KH2PO4-free KSOM were lower (Figure 1
).
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Discussion |
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No evidence was obtained to show that the slight inhibitory effect of KH2PO4 on the percentages of zygotes that at least develop into blastocysts and that begin to hatch is dependent on the concentration of glucose in the medium. The effect of KH2PO4 is the same over all concentrations of glucose. A useful model of the distribution of the effects of an inhibitor on a population is a tolerance distribution (Finney, 1952). Specifically, let there be a threshold concentration of KH2PO4 for each zygote that uniquely inhibits blastocyst formation, and that these threshold concentrations are distributed say as a lognormal distribution. Then the slight inhibitory effect of KH2PO4 on blastocyst formation is due to the arrest of a small group of zygotes that are particularly sensitive to the compound. Presumably, concentrations of KH2PO4 >1.35 mmol/l would have blocked the development of more zygotes. Results from other studies (Haraguchi et al., 1996
, 1999
) have raised the possibility that there may be considerable differences between the sensitivities of different mouse strains to KH2PO4. The development of zygotes of the AKR/N strain were completely blocked at the morula stage by 1 mmol/l KH2PO4. The difference in sensitivity to KH2PO4 needs to be confirmed, however, by comparing the responses of the two strains using the same medium [Haraguchi et al. (1996) used a modified Whitten's medium, while KSOM was used in the current study]. Differences in sensitivity to KH2PO4 may be even greater between species. For example, the rat and hamster preimplantation embryo during the initial stages of cleavage seems to be particularly sensitive to phosphate (rat: Miyoshi et al., 1994; Miyoshi and Niwa, 1997; Yamada and Nishikimi, 1999; hamster: Lane et al., 1999).
Media dependency of the effects of glucose
It has been suggested (Biggers, 1993) that the effects of a compound in a medium may be differentially affected by the concentrations of other components. The combined results of several investigators on the effects of glucose and KH2PO4 in media for the culture of a single strain of mouse preimplantation embryos provide a good example of this possibility. Table VIII
shows the background composition of four media in which the effects of glucose and KH2PO4 on the development of outbred mouse CF1 zygotes have been studied. It has been shown in the present paper that the addition of glucose to one of these media (KSOM) has no inhibitory effect on development, and the addition of KH2PO4 has only a slight inhibitory effect. In contrast, the addition of both compounds in similar concentrations are inhibitory when added to the other three media [M16 (Lawitts and Biggers, 1991a; EMGP (Scott et al., 1993
); modified human tubal fluid (HTF; Quinn and Horstman, 1998)]. These observations on a single strain of mouse provide strong evidence that the effects of glucose and KH2PO4 may depend on the background composition of the media in which they are studied. The variable effects of glucose on preimplantation development of the sheep may well be due to the differences in the media used: Betterbed and Wright (1985) used Brinster's medium, Thompson et al. (1989) used medium SOF, McGinnis and Youngs (1992) used medium CZB, and Thompson et al. (1992) used medium SOF supplemented with amino acids.
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Media formulation
KSOM was formulated using the sequential simplex optimization technique to find a medium in which the 2-cell block did not occur (Lawitts and Biggers, 1991b, 1992
, 1993
). Optimun concentrations of glucose and KH2PO4 in which this block was minimal were found to be 0.20 mmol/l glucose and 0.35 mmol/l KH2PO4. These values, however, are likely to be ill-determined optima because the results described in this paper show that the concentrationresponse surfaces for glucose and KH2PO4 in the neighbourhood of these concentrations are, for all practical purposes, horizontal planes. When these conditions exist, the resolution of optimization strategies falls, since the sequential estimates of an optimum do not converge but tend to wander randomly about the surface (Walters et al., 1991
). What, in the light of the results reported in the present paper, should be the concentrations of glucose and KH2PO4 in a recommended version of KSOM?
It is suggested that in the case of glucose the `back to nature' principle enunciated earlier (Leese, 1998) be used, and a glucose concentration of 3.40 mmol/l be selected, which has been found in the oviduct of the mouse (Gardner and Leese, 1990
).
Lawitts and Biggers (1991b) described a medium denoted aKH2PO4 that contained no KH2PO4, and which supported a high rate of passage through the 2-cell block. This result was discussed by others (Bavister, 1995), and has been used to question the inclusion of 0.35 mmol/l KH2PO4 in SOM and its subsequent modification KSOM (Quinn, 1997
; Quinn and Horstman, 1998
). It has also been pointed out (Lawitts and Biggers, 1991b
) that this medium was obtained after only four cycles of the ongoing simplex optimization procedure. After a further 16 cycles, however, the medium converged to one referred to as SOM that contained 0.35 mmol/l KH2PO4 (Lawitts and Biggers, 1992
). The results reported in the present paper have demonstrated a statistically significant inhibitory effect of KH2PO4 in KSOM on development at least to the blastocyst stage and the initiation of hatching at 120 and 144 h post-HCG respectively (Tables IV and VI
; Figure 3
). Nevertheless, the effect of including KH2PO4 in concentrations ranging from 0.05 to 1.35 mmol/l still allows the development of
76% blastocysts by 144 h post-HCG administration (Table IV
, Figure 3
). The fitted concentrationresponse surface shows that raising the concentration of KH2PO4 from 0.05 to 0.35 mmol/l reduces the percentage of blastocysts formed from 87 to 83%. Similar findings were obtained when the observed response was the initiation of hatching. Thus, the effect of including 0.35 mmol/l KH2PO4 in KSOM is very small and may be of no biological significance. In many cells a high intracellular phosphate concentration is maintained by a balance between phosphate transporters in the cell membrane that import and export phosphate across the cell membrane. The nature of these transporters is unknown in the preimplantation embryo. At present, the concentration of phosphate in the mouse oviduct is not known, so the `back to nature' principle cannot be used as in the case of glucose. Nevertheless, it is reasonable to assume that phosphate is present in the mouse oviduct since its presence has been measured in the sheep (Restall and Wales, 1966
) and human (Lippes et al., 1972
). We believe it is important not to exacerbate the natural phosphate gradient across the cell membranes by eliminating KH2PO4 completely from the medium. It is recommended that the empirically determined concentration of 0.35 mmol/l KH2PO4 in KSOM, found by sequential simplex optimization, be retained until further information on the regulation of phosphate transport in preimplantation embryos becomes available.
The belief that glucose is physiologically inhibitory to early preimplantation embryos of all species, including man, has now attained dogmatic status. Recent publications recommend that low concentrations of glucose be used in media for the culture of human preimplantation embryos (Bavister, 1999; Coates et al., 1999
), and the putative beneficial effects of media containing low concentrations of these compounds are touted in advertisements of commercially available media for human IVF and embryo transfer (e.g. Scandinavian IVF Science AB, medium IVF-50TM; Irvine Scientific, Inc.). There is no doubt that glucose and KH2PO4 inhibit or adversely affect the early development of the human embryo in most of the media currently being used. The results of the present study suggest, however, that media could be found in which these compounds are not inhibitory in concentrations similar to those present in the natural in-vivo environment. In fact, an indication has been provided (Coates et al., 1999
) that such conditions could be found. A clinical study was carried out in which human preimplantation embryos were cultured in Earle's balanced salt solution with and without 5.5 mmol/l glucose. No significant effects of the glucose on pregnancy rates were observed. Adverse effects of the glucose on both the cleavage rate and grade of embryo were reported, which led the authors to suggest that `... a reduction of the glucose concentration of the medium used for embryo culture from the pronucleate stage to embryo transfer on day 2 or 3 is prudent'. Although the tests of significance show that the effects of glucose on cell number and embryo grade are both statistically significant, some caution should be exercised in suggesting that the effects are biologically significant, since the differences were only 5% based on the observation of two large group sizes (1283 exposed to glucose versus 1471 not exposed to glucose). A minimal difference of little physiological interest can be statistically significant if the sample size is sufficiently large (Lindsey, 1999
).
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Acknowledgments |
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Notes |
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References |
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Submitted on July 7, 2000; accepted on September 21, 2000.