Culture and selection of viable blastocysts: a feasible proposition for human IVF?

 Human Reproduction Update 1997, Vol. 3, No. 4 pp. 367–382 European Society for Human Reproduction and Embryology Culture and selection of viable blastocysts: a feasible proposition for human IVF? David K.Gardner1,3 and Michelle Lane1,2 1Institute of Reproduction and Development, Monash University, Monash Medical Centre, Clayton, Victoria 3168, Australia and 2Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin 53706, USA TABLE OF CONTENTS Introduction Why transfer embryos at the blastocyst stage? Embryo physiology Influence of culture volume and embryo grouping Types of embryo culture media Carbohydrates Amino acids Amino acids and ammonium: a catch 22? Chelators Serum: friend or foe? Formulation of physiological culture media Requirement for quality control Assessment of embryo viability Conclusions and future scenarios Acknowledgements References 367 367 369 370 370 371 371 375 376 376 377 377 377 379 379 379 In human in-vitro fertilization (IVF) embryos are routinely transferred to the uterus on day 2 or day 3 of development. Resultant implantation and pregnancy rates are disappointingly low, with only ~10% of embryos transferred leading to a live birth. The ability to culture embryos to the blastocyst stage should help to resolve this problem by synchronizing the embryo with the female reproductive tract, and by identifying those embryos with little developmental potential. Co-culture has offered a possible means of producing blastocysts capable of high implantation rates. However, recent developments in the field of embryo physiology and metabolism have led to the formulation of new sequential serum-free culture media capable of supporting the development of viable blastocysts in several mammalian species, including the human. It is therefore proposed that blastocyst transfer should be considered for routine use in human IVF. The high viability of blastocysts cultured in the appropriate sequential media means that fewer embryos are required for transfer to achieve a pregnancy, culminating in fewer multiple births. Furthermore, the development of suitable non-invasive tests of embryo viability should further increase the overall success of human IVF by the ability to select before transfer those blastocysts most able to establish a pregnancy. Key words: blastocyst transfer/implantation rate/ metabolism/viability Introduction This paper aims to highlight the recent developments in embryo culture systems which have resulted in an ability to produce highly viable blastocysts from the zygotes of several mammalian species, including the human. Furthermore, a non-invasive method to quantify blastocyst viability prior to transfer is proposed. It is envisaged that blastocyst transfer in human in-vitro fertilization (IVF) will result in an increase in implantation and pregnancy rates and decrease the number of embryos required for transfer in order to achieve a pregnancy. It is beyond the scope of this paper to review the history of embryo physiology and culture. Several in-depth accounts of this have been published over the past decade (Biggers, 1987; Biggers et al., 1989; Leese, 1991; Rieger, 1992; Gardner and Lane, 1993a; Bavister 1995). Why transfer embryos at the blastocyst stage? It is an accepted global practice in human IVF to transfer embryos on day 2 (around the 4-cell stage) or on day 3 (around the 8-cell stage) of development. However, it is 3To whom correspondence should be addressed at: Colorado Center for Reproductive Medicine, 799 East Hampden Avenue, Suite 300, Englewood, Colorado 80110, USA. Tel: (303) 788–8300; Fax: (303) 788–8310 368 D.K.Gardner and M.Lane important to note that, in vivo, such cleavage stage embryos reside in the Fallopian tube and not in the uterus. The significance of this observation is that in other mammalian species the transfer of cleavage stage embryos to the uterus does not result in high pregnancy rates when compared with embryos transferred post-compaction or at the blastocyst stage (Bavister, 1995). Indeed, the premature replacement of the human embryo to the uterus may account in part for the low implantation rates associated with human IVF. Implantation rates of 10–15% are routinely reported in the literature, with only ~10% of embryos transferred proceeding to term. Data to date on the replacement of human cavitating morulae and/or blastocysts on day 4 or 5 of development indicate that such embryos have a higher implantation rate (Huisman et al., 1994; Olivennes et al., 1994; Ménézo and Ben Khalifa, 1995). In the case of the blastocyst, implantation rates twice those of cleavage stage embryos have been reported (Scholtes and Zeilmaker, 1996). Such data therefore support the hypothesis that the transfer of later stage embryos will increase implantation and pregnancy rates per embryo transferred. In support of this hypothesis is the study by Buster et al. (1985), in which human blastocysts developed in vivo and flushed from the uterus were transferred singly to recipient patients. In this case an implantation and pregnancy rate of 60% per blastocyst transferred was reported. Potential advantages of blastocyst culture and transfer in human IVF therefore include: (i) synchronization of the embryo with the female tract leading to increased implantation rates, thereby reducing the need for multiple embryo transfers; (ii) assessment of viability of an embryo before transfer. This can be achieved by both the identification of those embryos with little developmental potential, as manifest by slow development or degeneration in culture (Dawson et al., 1995; Ménézo and Ben Khalifa, 1995), and by the introduction of non-invasive tests of developmental potential to select the most viable embryos from within a cohort for transfer (Gardner and Leese, 1987, 1993; Lane and Gardner, 1996). Furthermore, culture of the human embryo beyond the 4–8-cell stage, the time at which the genome is activated (Braude et al., 1988), will facilitate the quantification of true embryonic markers as opposed to those inherited from the oocyte, i.e. after the 8-cell stage one is assessing embryo physiology, while prior to this the physiology of the cleavage stage embryo reflects that of the oocyte; (iii) an increase in the time available between cleavage stage embryo biopsy and embryo transfer. This is of particular importance where the biopsied material has to be sent to a separate locale for analysis; and (iv) facilitation of the introduction of trophectoderm biopsy for the screening of genetic diseases. Trophectoderm biopsy represents the earliest form of genetic diagnosis of non-embryonic material. The question is, therefore, why are embryos not routinely transferred at the cavitating morula or blastocyst stages? The answer stems in part from an inability to maintain the mammalian embryo in culture for more than a couple of days without compromising its viability. It is important here to different (...truncated)