1 Institute of Reproduction and Development, Monash University, Victoria, Australia and 2 Department of Obstetrics and Gynecology, PO Box 7039, Clinical Centre, Ultuna, S-750 07, Uppsala, Sweden
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Abstract |
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Key words: albumin/macromolecule/mouse embryo/PVA/viability
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Introduction |
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In attempts to standardize culture conditions, synthetic macromolecules such as polyvinylalcohol (PVA) have been suggested as alternatives to replace the albumin in embryo culture medium (Bavister, 1981; Bavister, 1995
). While PVA has been used extensively for successful culture of the hamster embryo in culture (Bavister, 1981
; Bavister, 1995
), substitution of serum albumin with PVA in culture systems for other species such as the cow has been questioned (Thompson et al., 1998
). In contrast, glycosaminoglycans (GAGS) are found at high concentrations in the fluid of the female reproductive tract of several mammalian species (Lee and Ax, 1984
; Yanagishita, 1994
; Laurent et al., 1995
; Zorn et al., 1995
; Legge et al., 1996
). One of these GAGS hyaluronan, a linear polysaccharide of alternating D-glucuronic acid and N-acetyl-D-glucosamine residues, increases in concentration at the time of implantation in the mouse embryo (Carson et al., 1987
). Furthermore, both human (Campbell et al., 1995
) and bovine (Valcarcel et al., 1999
) embryos have the surface receptor for hyaluronan, CD44, throughout development from the oocyte to the blastocyst stage. Importantly, Wheatley and colleagues observed a strict temporospatial regulation of CD44 expression in post-implantation mouse embryos at the sites of hyaluronan-mediated morphogenesis, indicating a role for receptorligand interaction (Wheatley et al., 1993
). In addition, hyaluronan also has an important role in the maintenance of human sperm motility (Sbracia et al., 1997
). It is therefore most plausible that hyaluronan has a role in early embryo development.
The aim of this study was to examine the role of hyaluronan in sequential culture media and to compare this with the effects of other macromolecules. Part of this study was presented in Abstract form at the annual ESHRE meeting in Edinburgh, 1997.
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Materials and methods |
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Embryo culture
Embryos were cultured in groups of 10 in 20 µl drops of medium under mineral oil (Lane and Gardner, 1992). All embryos were cultured at 37°C for 48 h in DM2 to the 8-cell stage, followed by culture for a further 48 h in DM3 to the blastocyst stage in 5% CO2, 7% O2, and 88% N2.
Determination of inner cell mass and trophectoderm cell number
Embryo cell number and allocation to the inner cell mass (ICM) and trophectoderm (TE) were determined using differential nuclear staining with the dyes propidium iodide and bisbenzimide (Hardy et al., 1989). Blastocysts were incubated in 0.5% pronase in H-DM1 to dissolve the zona pellucida. Embryos were then washed in H-DM1 and incubated in picrylsulphonic acid (Sigma) for 10 min at 4°C before a further wash and 10 min incubation in 0.1 mg/ml of Anti-DNP BSA (ICN Technologies, Costa Mesa, CA, USA) at 37°C. Following incubation with the antibody, embryos were again washed in H-DM1 and incubated in a 1:5 dilution of guinea-pig serum (ICN Technologies) containing 25 µg/ml of propidium iodide (Sigma) in H-DM1 for 7 min. Embryos were subsequently placed in 25 µg/ml bisbenzimide (Hoechst 33258, Sigma) in ethanol overnight at 4°C. The following morning, differential staining of nuclei was determined under UV light using a fluorescence microscope.
Embryo transfer
Blastocysts were transferred to pseudopregnant F1 (C57BL/6 x CBA/Ca) female mice (1 day asynchronous). Treatments were allocated to each uterine horn using random numbers. Five blastocysts were transferred to each uterine horn. Embryos were transferred to the uterus in the H-DM3 supplemented with the same macromolecule as the culture medium in which they were developed. On day 15 of pregnancy, implantation rates, fetal development, and normalcy were assessed. Fetal age was determined using a scoring system (Lane and Gardner, 1994).
Statistical analysis
Data for embryo morphology and viability were assessed using linear-logistic regression in which the error distribution was assumed to be binomial. Replicate of experiment was fitted as a factor. The null hypothesis was tested using the log-likelihood ratio statistic. Data for cell number, ICM and TE cell numbers were assessed using analysis of variance. Between-treatment differences were assessed using the Bonferroni procedure for multiple comparisons. A P value of 0.05 was considered significant.
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Results |
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Effect of different macromolecules on embryo development in culture
There was no difference in morula/blastocyst and blastocyst development between embryos cultured with no macromolecule, hyaluronan, or BSA. However, significantly fewer embryos cultured with dextran or PVA formed morula/blastocyst or blastocysts (Table III). Blastocyst hatching was significantly increased by culture with both 0.5 mg/ml hyaluronan and BSA compared with all other treatments (Table III
).
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Effect of macromolecules on blastocyst viability
Culture with hyaluronan (P < 0.01), hyaluronan together with BSA (P < 0.01), or PVA or dextran (P < 0.05), significantly increased implantation rates compared to culture with BSA or no macromolecule. However, fetal development was only significantly increased by culture with hyaluronan or hyaluronan together with BSA (Table IV). Fetal weight was not affected by the presence of macromolecules. It was notable that almost a quarter of fetuses derived from embryos cultured in the presence of dextran were retarded by up to 24h. Furthermore, 12% of the fetuses derived from embryos cultured with PVA were grossly abnormal, exhibiting skeletal and tissue degeneration.
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Discussion |
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Hyaluronan at low concentrations (0.5 mg/ml) has been demonstrated to increase the development of porcine and bovine embryos in culture (Miyano et al., 1994; Stojkovic et al., 1999
). While hyaluronan could be used as the sole macromolecule, an interaction between hyaluronan and albumin in the culture medium on embryo development in culture was evident. Specifically, trophectoderm cell number and hatching were highest when both macromolecules were present in the culture medium. As both hyaluronan and albumin are present at high concentrations in the female reproductive tract (Carson et al., 1987
; Leese, 1988
) this observation highlights the importance of emulating the physiological environment when designing culture media for the mammalian embryo. It is evident that albumin has a role to play in embryo development in culture, even though it conferred no apparent benefit post-transfer. Therefore a source of albumin that is not isolated from mammalian material, but rather produced in a recombinant form is highly desirable. Preliminary results from our laboratory indicate that recombinant albumin is a promising source for albumin supplementation for mammalian embryo culture media (Gardner and Lane, unpublished observations).
Addition of hyaluronan to the culture medium in the presence of BSA not only resulted in the highest rates of blastocyst development and blastocyst cell number, subsequent implantation rates and fetal development were increased. However, interestingly, addition of hyaluronan to the transfer medium only (blastocysts were cultured in the absence of any macromolecule) significantly increased the rate of implantation and fetal development compared to transfer medium without macromolecule. In the mouse uterus, there is a fivefold increase in the concentration of hyaluronan on the day of implantation. This increase was not observed in mated but non-pregnant mice (Carson et al., 1987; Zorn et al., 1995
). Furthermore, there was no corresponding increase in the concentration of other glycoproteins indicating that the increase was specific to hyaluronan (Carson et al., 1987
). The observed increase in implantation and fetal development when hyaluronan was added to the transfer medium are consistent with a role in embryo and endometrium interaction. There are several possible means by which hyaluronan could facilitate implantation: hyaluronan has been shown to increase cellcell adhesion and cellmatrix adhesion (Turley and Moore, 1984
), and so may function during the initial stages of apposition and attachment of the blastocyst and endometrium. However, even though the human embryo may specifically bind hyaluronan, it is plausible that the beneficial effects of hyaluronan may be manifest through other indirect routes. For example, hyaluronan can promote angiogenesis by both its degradation products (West et al., 1985
) and by interaction with epidermal growth factor (EGF) (Sato et al., 1991
). The latter point is of particular interest given the stimulatory effect of EGF on implantation in the mouse (Paria et al. 1991
). Hyaluronan may also confer some degree of viral protection and anti-immunogenic properties (Laurent and Fraser, 1992
). Alternatively, the beneficial effect of hyaluronan in the transfer medium may be a physical phenomenon, by facilitating rapid diffusion of the contents of the transfer medium (the embryo) into the fluid of the uterus. As uterine fluid is a viscous solution, the transfer of a relatively aqueous solution, such as culture medium with albumin, to the uterine lumen will result in the slow dispersal of the medium and embryo with the luminal contents. In contrast, the transfer of an embryo in a hyaluronan solution will facilitate dispersal of the embryo into the luminal environment.
An additional advantage of hyaluronan is that although it is a glycosaminoglycan, unlike other glycosaminoglycans it is not covalently linked to a core protein, and can therefore be considered a polysaccharide (Figure 2). This therefore removes both the problems of variation and contamination, as hyaluronan can be synthesized and isolated in a pure form. Initial studies have shown that pure hyaluronan, obtained from a bacterial source, in the culture medium results in increases in mouse embryo development in culture and implantation rates post-transfer (D.K.Gardner and M.Lane, unpublished observations), indicating that pure hyaluronan is as effective as that refined from the rooster comb.
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Addition of dextran to the culture medium at 4 mg/ml also resulted in significantly reduced development to the blastocyst stage compared with no macromolecule. Dextran was used as it is a polyhydroxyl compound, which alters the solvent conditions of aqueous solutions to be more akin to the fluid of the reproductive tract, in a similar way to that in which - and ß-globulin have been proposed to act (Pool and Martin, 1994
). However, neither of these compounds is present in the female reproductive tract and cannot therefore be deemed as physiological. However, similar to PVA, blastocysts cultured with dextran had an increased viability compared to culture without macromolecule. Interestingly, however, 21% of the fetuses resulting after transfer were significantly delayed by around 24 h. Nevertheless it is possible that the single concentration used in this study was too high, and for optimum results it may be necessary to titrate the dextran concentration in the medium, and that at lower concentrations dextran may confer benefit to the embryos in culture.
Addition of PVA to the medium at the reported optimum concentration (Bavister, 1981; 1995
) as the sole macromolecule resulted in significantly reduced blastocyst formation and hatching compared to development without a macromolecule. Interestingly, implantation rates of resultant blastocysts in this study were increased by culture with PVA compared to culture without macromolecule, although fetal development rates were equivalent. It was found, however, that two of the resultant fetuses (12%) were abnormal. This observation necessitates further investigation.
In conclusion, in the appropriate sequential culture media containing amino acids, mouse blastocysts do not require any protein to develop into viable fetuses after transfer. The inclusion of hyaluronan (refined from rooster comb) in the transfer medium significantly increased implantation and fetal development, implicating a role for this glycosaminoglycan in the implantation process. It should be noted that this effect might not necessarily apply to human embryos. Although it is possible to culture the mammalian embryo to the viable blastocyst stage in the absence of any macromolecule, it would seem prudent to include macromolecules in the culture media for the following reasons: the presence of a macromolecule (i) greatly facilitates embryo manipulation, (ii) enables the binding and stabilizing of growth factors produced by the embryo, and (iii) enables the binding of potential toxins such as low levels of transition metals, thereby making culture conditions more robust. With the advent of recombinant albumin and fermented hyaluronan, such as that already available and used in ophthalmology, it is now possible to standardize culture media composition, while keeping the culture system physiological. Before the widespread introduction of such culture and transfer media in human assisted reproduction technology, prospective clinical trials are warranted.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed
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References |
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Submitted on April 14, 1999; accepted on July 12, 1999.