1 Department GYN/OB Stanford University Medical Center, Stanford, CA 94305 and 2 Fertility Center of San Antonio, San Antonio, USA
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Abstract |
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Key words: blastocyst/development/embryo culture/implantation/sequential culture
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Introduction |
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Our approach to blastocyst production follows the sequential strategy, but employs a protein-supplemented, glucose/phosphate-free variant of human tubal fluid medium (`P1 medium'; Pool et al., 1998), containing the single amino acid taurine for the first culture interval. A modified Ham's F-10 (`blastocyst medium'; Behr, 1997
) was used for the second culture period. Both media are commercially available, are supplemented with synthetic serum substitute (SSS) and blastocysts are transferred without zona removal. In this report, we show that viable human blastocysts can be produced efficiently with P1/blastocyst medium used sequentially, suggesting that neither glucose nor a complex array of amino acids is required by early cleavage-stage preimplantation embryos during the first culture period.
The production of viable blastocysts using a simple, commercially-available sequential culture system represents an important goal in the advancement of clinical IVF. The utility of the system described in this report is still not fully known since the rate of blastocyst production was scored only on day 5 and largely by culturing supernumerary embryos after embryo transfer of the best grade embryos on day 3. However, there appears to be no glucose or inorganic phosphate requirement in the first interval. Further, the inclusion of taurine as the only amino acid in the first interval is consistent with the production of viable blastocysts as demonstrated by implantation following embryo transfer.
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Materials and methods |
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Results |
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Discussion |
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Since the first medium used in the culture sequence was completely devoid of both glucose and inorganic phosphate, neither appear to be required in the first culture interval. On the contrary, a complete lack of glucose/phosphate in culture medium for the first 3 days has been shown to improve pregnancy rates significantly when compared to a similar medium containing both (Quinn, 1995; Rawlins et al., 1997
; Barrett et al., 1997
; Pool et al., 1998
). Furthermore, culturing embryos for 3 days in a glucose/phosphate-free environment gave significantly increased pregnancy rates over 2 days of culture prior to transfer (Carrillo et al., 1998
). Conaghan et al. (1993) showed a slight enhancement of development of human embryos to the eight-cell stage by the omission of glucose compared to the inclusion of 1 mM glucose. Conaghan also demonstrated that blastocysts had more cells if glucose was omitted at the early cleavage stages. An important consideration regarding metabolic roles for glucose, other than as an energy substrate, has been raised by Gardner and Lane (1997), namely that glucose is a key entity in several crucial anabolic pathways that lead ultimately to the generation of triacylglycerols, phospholipids, mucopolysaccharides, glycoproteins and purines both for nucleic acids and the reduced oxidation-reduction co-enzyme, nicotine adenine dinucleotide phosphate.
Although the possibility exists that the low level (<0.5 mM) of glucose seen in the Fallopian tube at midcycle (Gardner et al., 1996) is required to support these anabolic needs, there are data demonstrating the existence of intracellular sources of glucose within the oocyte that could also function in these pathways. For example, when glucose and pyruvate consumption and the production of lactate were measured in single human embryos through non-invasive technology, more lactate was produced than could be accounted for by glucose uptake (Gott et al., 1990
), suggesting that the remaining lactate was derived from endogenous sources, such as glycogen. Additionally, the uptake of glucose by human embryos in medium containing 1 mM glucose is less than 10 pmol/embryo/h prior to the 16-cell stage (Hardy, 1993
). To date, no studies have demonstrated the advantage of including glucose at any concentration in the first culture interval (3 days) whereas significant improvements, both to embryogenesis and pregnancy upon transfer of cleavage-stage embryos, have been reported when glucose is omitted completely. However, the inclusion of glucose in the second culture interval (days 35) is an absolute requirement for the efficient production of viable blastocysts (Gardner and Lane, 1997
) and is consistent with the results from glucose uptake studies of human blastocysts in vitro (Hardy, 1993
).
A potential role for glucose in fertilization has been suggested by several investigators. Quinn (1995) noted that more spermatozoa were required in glucose-free medium to achieve a fertilization rate equivalent to that of glucose-containing medium. Gardner and Lane (1997) likewise have recommended that either sperm number or glucose concentration be increased for fertilization in G1 medium to overcome the reduction in sperm velocity and fertilization reported by Quinn. Mahadevan et al. (1997) also indicated that fertilization and sperm movement is decreased in Ham's F10 medium devoid of glucose compared to that medium containing glucose. No reduction in the fertilization rate was seen in P1 medium when compared to human tubal fluid (HTF) medium (Graham and Pool, 1998) or Ham's F10 (unpublished observations). The decline in fertilization reported by others in glucose-free medium is therefore not attributable solely to the absence of glucose, but must involve other medium components, making the observed phenomenon medium-dependent. Alternatively, the taurine included in P1 medium may exert a positive influence on the fertilization process, thus masking any detrimental effects that might be mediated by the absence of glucose.
The exact role of inorganic phosphate (Pi) in preimplantation embryo development is not clear. The presence of Pi in mammalian embryo culture medium, however, has been associated with embryo arrest in vitro (Bavister, 1995). Matsumoto and Sugawara (1998) recently showed that short term exposure of Pi to rat embryos during the early cleavage stage (first to second cleavage) retarded embryo development and resulted in a reduced blastocyst development rate. Long-term exposure resulted in embryo arrest. Their experiments revealed that this inhibition of development was not due to inhibition of histone H1 kinase activity or maturation-promoting factor activity. Until further studies reveal the requirement or benefit of the presence of Pi in the early culture interval, we suggest its omission from the medium in the first culture phase (up to day 3).
Another problem solved by sequential culture systems is meeting the changing amino acid requirements of preimplantation embryos in culture. These requirements have been studied exhaustively in a mouse model system (Gardner and Lane, 1997). The most pregnancies following embryo transfer were obtained when medium for the first culture interval contained Eagle's non-essential amino acids and the second medium contained all 20 Eagle's amino acids, essential and non-essential (Lane and Gardner, 1997
). Sequential culture of human embryos using this same approach has been successful (Gardner et al., 1997), although the exact amino acid requirement for human embryos has not been determined. In contrast to G1 medium, which contains Eagle's non-essential amino acids, P1 medium contains only the single amino acid, taurine. Taurine has been shown, however, to promote embryogenesis to advanced stages in the hamster, even when it is included as the only amino acid in the medium (Bavister and McKiernon, 1993
). We now show that medium containing only taurine as the sole amino acid can suffice as the first medium in a sequential system that includes a complex medium (blastocyst medium) as the second component.
As mentioned above, the exact requirements of amino acids and the roles they play in the early culture interval are not known for human embryos. However, there has been speculation that amino acids may function other than as nitrogen sources in the first interval, playing a more physical role, perhaps as osmolytes (Gardner and Lane, 1997). This is of particular interest since taurine has been shown to serve as an effective osmolyte in both human and mouse embryos. Dumoulin et al. (1997) preloaded oocytes and embryos with [3H]-taurine and showed that taurine is released by embryos that have to change cell volumes either by extracellularly-induced or intracellularly-occurring osmotic imbalances. As the authors indicate, its chemical inertness, lipophobic properties and ionic neutrality at physiological pH make taurine an ideal molecule to be retained within the cell at high concentrations, all of which underlie its suitability as an osmolyte. Taurine has also been described as having other protective functions, such as serving as an antioxidant. Li et al. (1993) have shown that the inclusion of taurine can increase the proportion of rabbit zygotes reaching the blastocyst stage and increase blastocyst cell number in protein-free medium to an extent equal to the inclusion of an optimal concentration of superoxide dismutase (600 IU/ml).
There are possibly other amino acids besides taurine that alone can promote embryogenesis to advanced stages. Devreker et al. (1998) measured an increase in the percentage of human embryos reaching the blastocyst stage when glutamine was included in a glucose-free variant of Earle's Balanced Salt Solution, although the total cell number was not increased. The viability of these blastocysts upon transfer, unlike those produced with taurine in the first culture interval in the present study, has yet to be demonstrated.
What has become clear in studies both from animal models (Pollard et al., 1995; Lane and Gardner, 1997
) and in humans (Gardner et al., 1998
) is that a complex array of amino acids is required in the second culture interval to produce blastocysts efficiently. Pollard et al. (1995) compared the growth of pig zygotes in combinations of simple and complex media. Not only was a sequential approach of simple followed by complex medium superior to either medium used alone, but simple medium supplemented with whole serum in the second interval failed to produce as many hatching blastocysts as minimum essential medium. The blastocyst medium used in the present study not only contains a complex array of amino acids, but also vitamins and nucleic acid precursors. The necessity for these components has yet to be examined in our hands, although their inclusion does not appear to compromise viability. Desai et al. (1997) also used a modified minimum essential medium (
MEM) to grow spare human embryos to blastocyst. Their medium was supplemented with 10% SSS as in this report. They reported a 37% blastocyst rate compared to the 54% we have using our sequential approach.
This report demonstrates that a simple system of sequential culture generated acceptable blastocyst development (54%) with `leftover' embryos, without the use of feeder cells or conditioned medium. Recognizing the deferential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex glucose containing medium (blastocyst medium) for subsequent blastocyst development. In a small series of thawed and fresh blastocysts, we have demonstrated that the blastocysts derived from this culture system are viable and able to generate clinical pregnancies. A larger trial with fresh blastocyst transfer is currently underway.
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Notes |
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References |
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Submitted on April 24, 1998; accepted on October 22, 1998.