Protein-Free Medium for Embryo Transfer

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Although considerable progress has been made in our ability to culture the human embryo, there has been relatively little research on the role and composition of the embryo transfer medium. The media used for embryo culture and transfer are almost pure aqueous solutions supplemented with protein, usually at 5 to 10%. This protein is usually in the form of serum albumin or whole serum. However, such media even when supplemented with protein bear little similarity to the fluids of the female reproductive tract, which are more viscous in nature. In an attempt to increase the viscosity of the transfer medium Menezo et al., (1989) used 1% placental collagen. However, there was no increase in implantation rates as a result of its use. Although albumin is the most abundant macromolecule in the female reproductive tract, glycosaminoglycans (GAGS) are found at high concentrations in the fluid of the female reproductive tract of several mammalian species (Zorn et al., 1995). 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, human embryos have the surface receptor for hyaluronan, CD44, throughout development from the oocyte to the blastocyst stage (Campbell et al., 1995). In a study on mouse embryo development and transfer Gardner et al., (1999) determined that hyaluronan could substitute for serum albumin in culture media. However, it was found that at the time of transfer the presence of hyaluronan in the medium resulted in a significant increase in implantation rates. This benefit did not depend on the presence of any macromolecule in the culture medium. Embryos cultured to the blastocyst stage in the absence of any protein/macromolecule benefited from the presence of hyaluronan in the transfer medium. There are several possible means by which hyaluronan could facilitate implantation; hyaluronan has been shown to increase cell-cell adhesion and cell-matrix adhesion 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 and by interaction with EGF. The latter point is of particular interest given the stimulatory effect of EGF on implantation in the mouse. 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) with 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 or serum, to the uterine lumen will result in the slow dispersal of the medium and embryo with the luminal contents. In conclusion, the polysaccharide hyaluronan may prove to be effective in facilitating improvements in embryo transfer. As hyaluronan can be obtained from bioengineered bacteria in an endotoxin-free form, transfer media can be standardized and biological variation eliminated.

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References

CAMPBELL S, SWANN HR, APLIN JD, ET AL. CD44 is expressed throughout pre-implantation human embryo development. Hum. Reprod. 10, 425-430, 1995.

CARSON DD, DUTT A. AND TANG J-P. Glycoconjugate synthesis during early pregnancy: hyaluronate synthesis and function. Dev. Biol. 120, 228-235, 1987.

GARDNER DK, LANE M AND RODRIGUEZ-MARTINEZ H. Fetal development after transfer is increased by replacing protein with the glycosaminoglycan hyaluronate for embryo culture and transfer in the mouse. Hum. Reprod. 14, 1999.

MENEZO Y, ARNAL F, HUMEAU C, ET AL. Increased viscosity in transfer medium does not improve the pregnancy rates after embryo transfer. Fertil. Steril. 52, 680-682, 1989.

ZORN TMT, PINHAL MAS, NADER HB, ET AL. Biosynthesis of glycosaminoglycans in the endometrium during the initial stages of pregnancy of the mouse. Cell. Mol. Biol. 41, 97-106, 1995.