Human Reproduction, Vol. 15, No. 1, 157-164,
January 2000
© 2000 European Society of Human Reproduction and Embryology
Culture of one-cell hamster embryos with water soluble vitamins: pantothenate stimulates blastocyst production
Department of Animal Health and Biomedical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA
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Abstract |
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The effects of water-soluble vitamins, singly or in combinations, on development of hamster 1-cell embryos were examined in a protein-free, chemically defined culture medium, HECM-6. Pantothenate significantly stimulated blastocyst development compared to the vitamin-free control and to every other single vitamin, except thiamine. Ascorbic acid, biotin, choline, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine had no detectable stimulation or inhibition on cleavage stage development or morula/blastocyst formation. When combinations of vitamins were tested, embryo development was either unchanged or significantly greater than in the control, but never significantly greater than development with pantothenate alone. A dose response to pantothenate indicated that 3 µmol/l was the optimum concentration. After embryo transfer, the percentage of live fetuses recovered per 100 1-cell embryos cultured in HECM-6 plus pantothenate (now designated HECM-9) was 24%, significantly higher than the 11% recovered from 100 1-cell embryos cultured in HECM-6 alone. This is the first report to show a stimulatory effect of a single vitamin on in-vitro development of preimplantation embryos in any mammalian species.
Key words: development/embryo/pantothenate/vitamins
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Introduction |
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As numerous studies with a wide variety of species have shown, determination of the nutritional requirements for preimplantation stage (1-cell to blastocyst) embryos is a prerequisite to achieving normal development consistently in vitro (discussed in Bavister, 1995). The hamster 1-cell embryo has become a well-established model for investigating in-vitro requirements for development. Many constituents of conventional cell culture media have been re-examined using the hamster to devise media that address the specific needs of the developing embryo. For example, removing inorganic phosphate from the culture medium eliminated the characteristic `2-cell block' to hamster embryo development in vitro (Schini and Bavister, 1988
). Removal and/or adjustments of traditional energy substrates (glucose, pyruvate and lactate) supplied to the embryo in vitro also significantly improved embryo development in vitro (Seshagiri and Bavister, 1989; McKiernan et al., 1991), and certain amino acids were found to inhibit development while others stimulated it (McKiernan et al., 1995).
In contrast to the extensive studies with substrates and amino acids, the role of vitamins for normal embryo development in vitro has not been thoroughly examined, although as with somatic cells, these important nutrients may be expected to play a key role in the embryo's metabolic balance and general nutrition. Vitamins are organic nutrients required in small amounts by mammalian cells; however, most mammals have lost the ability to manufacture them and so vitamins must, therefore, be supplied as nutrients (Baum, 1978; Stryer, 1981). Many water-soluble vitamins are precursors of co-enzymes that are necessary for numerous biochemical reactions in the cell. Vitamins involved in metabolic reactions include riboflavin and niacin as precursors of FAD and NAD respectively, pantothenate as a component of co-enzyme A and thiamine which is required for the conversion of pyruvate to acetyl CoA. The co-enzyme pyridoxal phosphate is an essential catalyst for the transamination and deamination of amino acids. Biotin is a co-enzyme in carboxylation reactions for the biosynthesis of purines and fatty acids. Ascorbic acid is an antioxidant, while choline and inositol are components of phosphoglycerides found in most membranes of higher organisms. Since these processes are so essential for normal cell functions and maintenance, it would appear that vitamins are indispensable nutrients for cultured cells including embryos.
Previous work from this laboratory showed that although particular vitamins, especially inositol, choline and pantothenate, stimulated zona escape or `hatching' by hamster blastocysts, they had no demonstrable effects on embryo development per se starting from the 8-cell or morula stages (Kane et al., 1986; Kane and Bavister, 1988a,b). In these studies, no information was obtained on possible vitamin effects on development of earlier stages. The apparent lack of effect of vitamins on advanced stages of embryo development may have been due in part to the use of inferior culture media, as shown by the relatively low incidence of blastocyst development in these studies. Only ~50–70% of (in-vivo produced) 8-cell embryos reached the blastocyst stage in vitro using medium TLP-PVA (Kane and Bavister, 1988a,b), doubtless due to the presence of glucose and phosphate; when these compounds were eliminated from the medium, 80–90% of 8-cell embryos reached the blastocyst stage (Seshagiri and Bavister, 1989). In addition, because the earliest stages of embryo development (zygote and early cleavage stages) have markedly different nutritional needs to those of late cleavage and differentiating (morula/blastocyst) stages (Brinster and Troike, 1979; Gardner and Lane, 1996; Pinyopummintr and Bavister, 1996), a need to re-examine the role of vitamins on development from the 1-cell stage is indicated. The present work was undertaken with a more refined culture medium, HECM-6 (McKiernan et al., 1995), to examine the effect of 10 water-soluble vitamins either singly or in combination on development of hamster 1-cell embryos to the blastocyst stage and on their subsequent fetal development after embryo transfer.
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Materials and methods |
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Embryo collection and culture
Mature, cycling, female hamsters were super-stimulated with a weight-dependent dosage (10 IU if <90 g, 15 IU if 91–115 g, 20 IU if 116–140 g, 25 IU if >141 g) of pregnant mare's serum gonadotrophin (PMSG) on the day of post-oestrus discharge (day 1) and mated to fertile males 3 days later (oestrus, day 4). One-cell embryos were collected 10 h post-egg activation, i.e. at 1400 on the following day (Bavister et al., 1983
). Embryos were distributed to 50 µl treatment drops of culture medium under a mineral oil overlay (Sigma Chemical Co., St Louis, MO, USA; no. M-8410) in a manner defined as `controlled pooling', whereby embryos from one female were distributed in equal number to all treatments, then an equal number of embryos from another female were distributed to the same treatment drops (McKiernan et al., 1991). Embryos were incubated for 72 h at 37.5°C in a humidified atmosphere of 10% CO2:5% O2:85% N2.
Embryos were cultured in medium HECM-6 containing no vitamins (control) and in HECM-6 containing one or more of the following vitamins (Sigma: 10 µmol/l L-ascorbic acid (A-4034), 5µmol/l D-biotin (B-4639), 5µmol/l choline chloride (C-1879), 3µmol/l folic acid (F-7876), 3µmol/l myo-inositol (I-5125), 5µmol/l niacinamide (N-3376), 3µmol/l D-pantothenate (P-5155), 1 µmol/l pyridoxal HCl (P-9130), 1 µmol/l riboflavin (R-4500), and/or 3 µmol/l thiamine (T-4625) (Table I). The concentrations of vitamins used in these experiments were based on those in Ham's F10 medium (Ham, 1963; Kane and Bavister, 1988b).
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Experimental design
A previously used design (Kane and Bavister, 1988b
) was used for the first experiment to test 3 µmol/l inositol, 3 µmol/l pantothenate and/or 5 µmol/l choline, singly or in combination, on hamster 1-cell embryo development in vitro. This generated several more experiments to determine which of 10 vitamins (either singly or in groups) were stimulatory or inhibitory. These experiments included: an examination of the four vitamins required as co-factors for the pyruvate dehydrogenase complex; a survey of all 10 vitamins; a dose–response (1, 3, 10, 30 and 1000µmol/l) to pantothenate; an experiment where pantothenate was paired with choline, folic acid, riboflavin and thiamine; and an experiment in which pantothenate was present in combination with choline + thiamine, riboflavin + thiamine, choline + riboflavin, choline + riboflavin + thiamine and a final treatment containing the six vitamins that gave >40% blastocyst development in the survey experiment (choline, folic acid, inositol, pantothenate, riboflavin and thiamine). Experiments were replicated a minimum of four times with at least four females per replicate.
Embryo transfer experiments were conducted to determine post-implantation viability of blastocysts that had developed in the control medium (HECM-6) and in HECM-6 plus pantothenate. Recipient females were mated to vasectomized males the day 1-cell embryos were collected from donor females. The female hamster has two uteri, each with its own cervix opening separately into the vagina (Magalhaes, 1968), therefore, no embryos will migrate from one uterine horn to the other. Taking advantage of these separate uteri, 8–10 blastocysts cultured in control medium were transferred to one uterine horn and an equal number of morphologically similar blastocysts cultured in HECM-6 plus pantothenate were transferred to the other uterine horn as previously described (McKiernan and Bavister, 1994). Embryos were transferred into 18 females. Recipient females were examined for fetuses 11 days post-transfer (day 14 of gestation, 2 days before term).
Data collection and analysis
Embryos were examined at 48 and 72 h post-egg activation for morphological development to 8-cell stage and to morula and blastocyst stages respectively. Percentages of 8-cell embryos, morulae/blastocysts and blastocysts were calculated from the total number of embryos cultured. After culture, embryos were either fixed and Hoechst-stained for determining cell numbers (Boatman et al., 1988) or embryos were transferred to pseudopregnant recipient females to determine post-implantation viability.
In all statistical analyses, proportions were transformed using an arc sine square root transformation and mean nuclear number data were transformed using a square root transformation. These transformations were used to control for possible heterogeneity of variance (Snedecor and Cochran, 1980). Treatment differences were determined using a two-way analysis of variance (ANOVA) using the GLM procedure of SAS (SAS Institute, 1989). Treatments were compared using Fisher's least significant difference test (LSD); significance was set at P < 0.05. Embryo transfer data were analysed using a paired t-test.
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Results |
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Hamster 1-cell embryos cultured in the presence of inositol, choline and/or pantothenate had similar developmental responses up to the 8-cell stage at 48 h. At 72 h, while development to morulae/blastocysts was similar in all treatments, a significant increase in blastocyst formation was observed in embryos cultured with 3 µmol/l pantothenate (73%) compared with the control (41%, Table II
). Blastocyst development in pantothenate alone was significantly higher compared to control, choline, inositol, choline plus inositol, inositol plus pantothenate and all three vitamins together. Inositol produced the fewest blastocysts and, in the presence of pantothenate, inositol significantly inhibited the beneficial effect of pantothenate. Choline plus pantothenate was not different from pantothenate alone, nor from the control. There was no significant difference in mean cell number between all treatments.
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Four vitamins, pantothenate, niacinamide, riboflavin and thiamine are co-enzymes required for the pyruvate dehydrogenase complex. The effect of these four vitamins, singly and in combination, was examined in the second experiment (Table III
). No vitamin treatment had a significant effect on 8-cell embryo development at 48 h or on morula/blastocyst development at 72 h of culture. However, pantothenate significantly stimulated blastocyst development (68%) compared with control medium (37%), or medium with niacinamide (31%), riboflavin (38%), thiamine (39%), and all four vitamins in combination (57%). Compared to control embryos, the mean cell number of embryos cultured in the four vitamins, either singly or as a group, was not significantly different. Pantothenate and niacinamide produced embryos with a mean cell number of 22, which was significantly greater than the mean cell number for embryos cultured in riboflavin and thiamine (18).
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A survey of 10 water-soluble vitamins revealed significant differences between treatments at all levels of development analysed (Table IV
). Medium containing pantothenate significantly stimulated development to the 8-cell stage at 48 h of culture compared with control and all other vitamins tested except for thiamine. After 72 h of culture, development to the morula/blastocyst stage was significantly stimulated by pantothenate compared with control. Morula/blastocyst development of embryos cultured in choline, thiamine and all 10 vitamins was not significantly different from pantothenate, nor from the control. Blastocyst development was significantly higher in the treatment containing pantothenate (67%) compared with control medium (37%) and all other treatments except thiamine (53%) and all 10 vitamins (58%). No vitamin treatment, however, significantly inhibited development. Embryos cultured in ascorbic acid, biotin, niacinamide and pyridoxal produced low numbers of blastocysts but these values were not significantly different from the control. There was no significant difference in mean cell number between treatments tested.
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A pantothenate dose–response experiment (0, 1, 3, 10, 30 µmol/l and 1 mmol/l) showed that 3 µmol/l pantothenate produced the highest percentage of blastocysts (70%) (Table V
). This value was significantly different from control (37%) but not significantly different from the other dosages examined. Development to blastocyst was not inhibited even with the highest, and most unphysiological dosage, of pantothenate (1 mmol/l).
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The following two experiments examined the effect of pantothenate alone and pantothenate in combination with vitamins that produced a blastocyst response greater than 45% in the survey experiment. The first of these experiments paired pantothenate with choline, folic acid, riboflavin and thiamine (Table VI
). There were no significant differences between treatments for development to the 8-cell stage at 48 h or morula/blastocyst stages at 72 h. For blastocyst development, pantothenate, pantothenate plus choline and pantothenate plus riboflavin were not significantly different from each other, but produced significantly greater development than control and pantothenate plus folic acid. The mean cell number for embryos cultured in pantothenate and pantothenate plus riboflavin was 22, not significantly different from control but significantly greater than the mean cell number for embryos cultured in pantothenate plus choline. The second of these experiments examined pantothenate alone or in combination with two or more vitamins (Table VII). The only significant effect observed was at the blastocyst stage where pantothenate alone produced significantly greater development than the control medium. All combinations of vitamins with pantothenate had very similar developmental responses and all were significantly greater than control medium but not significantly greater than with pantothenate alone. Mean nuclear number for these embryos was not significantly different between treatment groups.
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Post-implantation viability of embryos cultured in medium containing no vitamins and medium containing pantothenate was compared. One-cell embryos were cultured to the blastocyst stage in both treatments, then transferred to opposite uterine horns of pseudopregnant recipient females. Viable offspring were obtained from both treatments (Table VIII
). The number of fetuses produced from embryos cultured in HECM-6 + pantothenate was 41 (33%) compared to 29 (23%) produced from embryos cultured in HECM-6 alone. These percentages were not significantly different; however, due to the higher number of blastocysts produced in HECM-6 plus pantothenate, the number of live fetuses recovered per 100 1-cell embryos cultured in HECM-6 plus pantothenate was 24, more than double and significantly greater than the 11 fetuses recovered per 100 1-cell embryos cultured in HECM-6 alone.
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Discussion |
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Culture media that support normal development and viability of embryos subjected to in-vitro manipulations such as nuclear transfer will facilitate the success of mammalian embryo biotechnology. Culture media must support the needs of cultured cells. This was the driving philosophy used by Ham (1963) in the development of his medium F10 for the culture of a number of cell lines. Ham's F10 medium is a complex medium containing 46 ingredients (not including serum) and was an early favourite for human embryo culture in vitro. This medium, however, was not formulated for preimplantation embryos and was supportive of embryo development, usually after fortification with serum, in only a few species. Over the years, complex media have been slowly deconstructed, with careful analysis of the suitability of each component based on Ham's dictum (see Bavister, 1995). In this way the value of vitamins and amino acids for embryo development in vitro was established using rabbits (Kane and Foote, 1970
). In parallel to these efforts, embryo nutrient needs have been assessed using a constructive approach in which specific constituents are added to simple media that are little more than balanced salt solutions. These studies have produced novel media that have improved both the quality and quantity of embryos produced in vitro. A positive, cyclic relationship is unfolding between media and embryo development, in which culture media improvements provide new data on the requirements of embryos for development in vitro, thereby increasing the production of high quality, viable embryos, which in turn provide better material on which to determine factors affecting regulation of embryo development (Bavister, 1995).
The constructive method of media development has, in our laboratory, produced the chemically-defined, protein-free `hamster embryo culture medium' (HECM) series. Culturing embryos in a protein-free, completely defined medium provides an opportunity to examine effects of any compound on in-vitro development without confounding factors from serum components such as bovine serum albumin (BSA), which are notoriously variable in composition and properties (Kane, 1983; McKiernan and Bavister, 1992; Bavister, 1995). Incremental modifications of HECM during the past 10 years, incorporating changes in energy substrates and amino acids, have dramatically increased developmental responses, from only 26% of hamster 2-cell embryos developing through one cleavage division with HECM-1 (Schini and Bavister, 1988), to 27% of 1-cell embryos reaching blastocyst with HECM-3 (McKiernan et al., 1991), to 59% blastocysts from 1-cell embryos with HECM-6 (McKiernan et al., 1995). Each increment in development, with a concomitant reduction in the variability of responses, indicates an improvement in the medium, which forms the basis of the next series of experiments. The present study on the effects of vitamins on hamster 1-cell embryo development in vitro represents another logical step in this process.
Pantothenate was the single most stimulatory vitamin affecting hamster 1-cell embryo development in vitro. Pantothenate significantly increased blastocyst development compared with the control and compared with every other single vitamin, except for thiamine. Development in medium containing combinations of vitamins (including pantothenate) was never significantly greater than when embryos were cultured in pantothenate alone. There appeared to be no synergistic interaction between pantothenate and any of the vitamins that were tested. It is not known at what stage pantothenate exerts its action on cultured embryos. Experiments carried out in our laboratory in which pantothenate was added at 0 h (1-cell stage), at 24 h (2-cell stage) or at 48 h (8-cell stage) of culture were inconclusive, and there was no significant difference between the three treatments (data not shown).
Embryo viability was significantly enhanced in the presence of pantothenate. The number of live fetuses produced per 100 1-cell embryos cultured in HECM-6 containing pantothenate was >2-fold greater than the number of live fetuses produced per 100 embryos cultured in HECM-6 alone.
The mechanism of the effect of pantothenate on blastocyst formation may be 2-fold. Pantothenate is an essential vitamin for the biosynthesis of co-enzyme A, which is a co-factor for a multitude of enzymatic reactions including the oxidation of fatty acids, carbohydrates, pyruvate, lactate and amino acids (Tahiliani and Beinlich, 1991). Satisfying the embryo's specific energy substrate needs is critical to successful development in vitro (discussed by Barnett and Bavister, 1996). In several species, glucose (at normal plasma concentrations) inhibits embryo development in vitro while pyruvate and/or lactate are the preferred energy substrates (Leese and Barton, 1984; Takahashi and First, 1992; Conaghan et al., 1993; Bavister, 1995). Increased glycolysis and decreased tricarboxylic acid (TCA) cycle activity are indicators of perturbed energy metabolism in cultured embryos (Seshagiri and Bavister, 1991; Gardner and Lane, 1993; Barnett and Bavister, 1996). Addition of vitamins and amino acids to the culture medium helps maintain in-vivo rates of glycolysis and oxidation, indicating that these compounds are key regulators of embryo metabolism (Lane and Gardner, 1998). Thus, the role of pantothenate in embryo development may be to encourage formation of acetyl-CoA, and hence to stimulate oxidative metabolism.
Enrichment of the embryo culture medium with pantothenate may also have a secondary role as a defence against free oxygen radicals. Free oxygen radicals can be generated in aerobic organisms, particularly in metabolically active mitochondria (Boveris et al., 1972). Cells preincubated with pantothenate contained less lipid peroxides and their plasma membranes were more resistant to permeabilization when exposed to UV radiation or hydroxyl radicals (Slyshenkov et al., 1996). Further work has shown that the protective effect of culture with pantothenate was not due to this vitamin scavenging oxygen free radicals but rather because it greatly increased the intracellular level of co-enzyme A. Co-enzyme A may facilitate removal of lipid peroxides by increasing metabolism of fatty acids and promote repair of the plasma membrane by activating phospholipid biosynthesis (Slyshenkov et al., 1996). Increased energy production coupled with structurally sound cell membranes should facilitate blastocele formation. In addition, by stimulating TCA cycle activity, pantothenate may indirectly increase the cellular supply of glutamate, a glutathione precursor (Slyshenkov et al., 1996). Supplementing culture medium with glutathione, which scavenges H2O2 and oxygen free radicals, relieved the 2-cell block in a mouse strain whose embryos were resistant to culture (Legge and Sellens, 1991), while synthesis of glutathione during oocyte maturation in vitro in the pig (Abeydeera et al., 1998) and cow (Luvoni et al., 1996; de Matos et al., 1996a,b) significantly improves subsequent blastocyst development.
In contrast to the significant stimulatory effect of pantothenate, the remaining nine vitamins tested had no demonstrable effects on hamster 1-cell embryo development and responses were never significantly different from control values. Even though these vitamins showed no overt negative effects, simply supplying combinations of vitamins, such as the Ham's F-10 or minimum essential medium (MEM) vitamins requires caution. Mouse embryos showed reduced morula/blastocyst development and cell number in the presence of F-10 vitamins, and although MEM vitamins had a less drastic effect on morphological development, blastocyst cell number was also reduced (Tsai and Gardner, 1994). Specific vitamin requirements for preimplantation stage embryos, especially those grown in vitro, must be determined empirically. Few investigators have examined the effects of individual vitamins on preimplantation embryo development in vitro (Daniel, 1967; Kane, 1988; Kane and Bavister, 1988b; Shirley, 1989; Tsai and Gardner, 1994). In these studies, three species were used: rabbit, mouse and hamster. Common to all these studies, investigators found a majority of `neutral' vitamins, some that were required and some that inhibited development, while no vitamin had the same effect in all three species. Rabbit blastocysts required inositol, pyridoxal, riboflavin and niacinamide for expansion and growth (Daniel, 1967; Kane, 1988). Hamster 8-cell embryos required inositol, pantothenate and choline for blastocyst hatching (Kane and Bavister, 1988b). Mouse embryos do not appear to have any specific vitamin requirement; however, blastocyst cell numbers are reduced by physiological levels of niacinamide (Tsai and Gardner, 1994) and high levels of niacinamide, biotin, riboflavin, and ascorbic acid arrest development (Shirley, 1989). Perhaps general vitamin requirements of cultured embryos are met, to a large extent, by endogenous sources. A study by O'Neill (1998) indicated that mouse embryos had an absolute requirement for folate, shown by decreased development in the presence of methotrexate, an inhibitor of dihydrofolate reductase, although folic acid added to the medium had no beneficial effect on development.
When selected combinations of vitamins (each containing pantothenate) were tested in the present study, embryo development was unchanged or significantly greater than control (Tables VI and VII
). Indeed, the blastocyst-stimulating effect of pantothenate was the same regardless of the presence or absence of other vitamins. In general, any observed stimulation of embryo development with combinations of vitamins could be attributed entirely to the action of pantothenate because none of the responses were greater than with pantothenate alone. In keeping with our strategy for improving embryo culture media design, we now routinely incorporate pantothenate into the medium, and HECM-6 + pantothenate is designated HECM-9. In summary, different vitamins, individually or in combination, have neutral or stimulatory effects on blastocyst development of cultured 1-cell hamster embryos. The most striking finding in this study was that 3µmol/l pantothenate can account for virtually all of the embryo stimulatory effects observed. This is the first report of a single vitamin significantly stimulating blastocyst formation from cultured embryos in any mammalian species.
Pantothenate had a specific effect on blastocyst development, and therefore more transferable embryos were produced. Addition of this vitamin could have a significant effect on human embryo development in vitro, especially now that there is greater emphasis on blastocyst transfer. It remains to be seen if pantothenate can stimulate embryo development in species other than the hamster, especially in primates including human. However, a medium essentially the same as HECM-9 (Bavister's defined medium, BDM) has been used to support development of human IVF embryos from the zygote to 4-cell stage, with birth of healthy babies following embryo transfer (Rinehart et al., 1998 and unpublished data).
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Acknowledgments |
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We are grateful to Drs Jay Baltz, Billy Day, John Eppig, Michael Kane, Keith Latham, Randall Prather and Andrew Watson for their helpful comments on the manuscript and Dr Murray Clayton for help with statistical analyses. This work was done as part of the National Cooperative Program on Non-Human In-vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development, NIH, through cooperative agreement HD-22023.
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Notes |
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1 To whom correspondence should be addressed
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Submitted on May 4, 1999; accepted on September 13, 1999.
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