The benefits of extended culture

G.M. Jones1 and A.O. Trounson

Centre for Early Human Development, Institute of Reproduction and Development, Monash University, Level 5, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria, Australia, 3168

It is common practice in most human assisted reproduction programmes around the world to carry out embryo transfers on day 2 of culture and, very occasionally, on day 3. However, the need for change has been raised (Tsirigotis, 1998Go). Embryos are almost always transferred to the patient's uterus at the early cleavage stage even though, in vivo, the embryo does not normally enter the uterus until the morula stage (Croxatto et al., 1972Go), which is closer to day 4 of culture. In rodents and domestic ruminants, premature exposure of early cleavage stage embryos to the uterine environment results in the embryos failing to develop further and a poor pregnancy outcome (Moore and Shelton, 1964Go; Holst and Braden, 1972Go). However, in both ruminants (Hasler et al., 1987Go) and mice (Bronson and McLaren, 1970Go), deferring uterine transfer to the blastocyst stage results in very high pregnancy rates.

In the human, premature exposure of early cleavage stage embryos to the uterine environment does not appear to severely compromise embryo development, as pregnancy rates of 20–40% are now routinely reported. However, these pregnancy rates are usually attained following transfer of multiple embryos and the resulting implantation rates are low (10–20%). This indicates that 80% of the early cleavage stage embryos selected for transfer following assisted reproductive techniques (ART), either have no further developmental competence, or at least no developmental competence in the uterine environment. Alternatively, the uterine endometrium may remain unreceptive to transferred embryos, limiting both pregnancy and implantation rates (Rogers et al., 1986Go).

Early cleavage stage embryos are usually selected for transfer on the basis of morphological criteria, e.g. number of blastomeres, symmetry of blastomeres, granularity and degree of extracellular fragmentation. In several studies, these features have been shown to be weakly correlated with pregnancy rates (Puissant et al., 1987Go; Steer et al., 1992Go; Roseboom et al., 1995Go). However, by day 2 in culture, embryo development is governed primarily by presynthesized maternal transcripts and stored messages rather than the true embryonic genome (Braude et al., 1988Go).

Deferring embryo transfer to day 3 (or beyond) of culture in vitro, allows selection of embryos that have at least demonstrated the potential of continued development under embryonic genomic control. As early as 1989 it was reported that deferring embryo transfer to day 3, or later, of culture in vitro, does not compromise embryo developmental competence (Van Os et al., 1989Go). It has been further demonstrated that deferring embryo transfer to days 3, 4 or 5, does not significantly reduce pregnancy rates (Huisman et al., 1994Go).

So, why do ART clinics persist in performing embryo transfer on day 2 of culture, when there is abundant evidence to suggest that there is very little chance of identifying and selecting a developmentally-competent embryo among the cohort available for transfer? Historically, this arose from the fear that prolonged in-vitro culture of embryos in suboptimal conditions would compromise their viability. Early attempts to extend culture to the blastocyst stage of development tended to confirm these fears, as only 10% of patients achieved a pregnancy following transfer of blastocyst stage embryos on day 5 of culture with an implantation rate of only 7% (Bolton et al., 1991Go). However, in 1992, Ménézo and co-workers reported the first pregnancies following transfer of blastocysts developed using a Vero cell co-culture system (Ménézo et al., 1992Go). Although the pregnancy rate was not increased, they reported a higher implantation rate following blastocyst transfer, in comparison with the transfer of early cleavage stage embryos on day 2 (52.8 and 40.4% for pregnant patients respectively). Perhaps the most encouraging feature of this report was the high rate of development of zygotes to the blastocyst stage in a group of difficult in-vitro fertilization (IVF) cases (>50%), with only 3% of patients failing to develop a single blastocyst and, therefore, failing to have a transfer. Since this report, others have confirmed the development of viable human blastocysts using various co-culture techniques (Plachot et al., 1993Go; Frydman et al., 1993Go; Schillaci et al., 1994Go; Olivennes et al., 1994Go; Janny and Ménézo, 1994Go; Fong et al., 1997Go).

The vast majority of these reports investigated the possibility of producing viable blastocysts from patients who had experienced repeated implantation failures following transfer of early cleavage stage embryos. Pregnancy rates of 37–50% and implantation rates of 18–20% were consistently reported for this group of patients. Why, then, has blastocyst development using co-culture technology not been adopted for the treatment of the `difficult IVF patient' defined as those experiencing repeated implantation failure following transfer of morphologically `good quality' embryos on day 2? Perhaps it is due to the inherent difficulties, workload and expense of maintaining the feeder or support cells in culture. There is also the potential risk of transmitting infective agents to the embryos from feeder cell lines, particularly those derived from human oviduct cells, bovine cells and primate-derived cell lines. More recently, new culture systems have been devised to develop viable human blastocysts using specially-designed complex media or new generation sequential media which cater to the embryo's changing metabolic requirements as it develops from the zygote to the blastocyst stage.

All reports of extended culture using more complex media have reported high rates of development to the blastocyst stage, regardless of whether all the embryos or only those surplus to the needs of transfer, were cultured to the blastocyst stage (36–66%) (Muggleton-Harris et al., 1995Go; Scholtes and Zeilmaker, 1996Go; Desai et al., 1997Go; Jones et al., 1998aGo,bGo; Gardner et al., 1998Go; Rijnders and Jansen, 1998Go). The ability of the zygote to develop to the blastocyst stage does not necessarily reflect the viability of the embryo. Bolton and co-workers successfully cultured 40% of all zygotes to the blastocyst stage in simple culture medium (Bolton et al., 1991Go), but these blastocysts had very little viability (an implantation rate of only 7%). Similarly, Winston and co-workers reported that 39% of embryos cultured in simple culture medium developed to the blastocyst stage, but that 61% of the blastocysts analysed had very low cell numbers which they believed to be inconsistent with viability (Winston et al., 1991Go). The importance of recent reports is that not only do zygotes develop to the blastocyst stage in high numbers in the new sequential culture medium without the need for co-cultured somatic cells, but that the blastocysts which develop, result in high implantation and pregnancy rates following transfer (Gardner et al., 1998Go; Jones et al., 1998aGo,bGo).

To date, the only randomized trial which has been published, comparing the success rates of transfer of early cleavage stage embryos on day 3 with the transfer of morula–blastocyst stage embryos on day 5 using a single complex culture medium for the entire culture period, showed that implantation rates were not significantly different, indicating that prolonged culture does not have a deleterious effect on embryo quality (Scholtes and Zeilmaker, 1996Go). However, transferring cavitating early blastocysts through to expanding blastocysts on day 5, produced a significantly higher implantation rate, in comparison with transferring good quality 8-cell embryos on day 3. This indicates that the selection advantage offered by extended culture will help to reduce the number of embryos required for transfer, while maintaining high pregnancy rates. As a consequence, one highly desirable outcome will be the reduction in the number of multiple IVF pregnancies.

It appears, therefore, that one of the major barriers to extending embryo culture beyond 2–3 days has been overcome. With some confidence, it is now possible to grow large numbers of human blastocysts without compromising their full developmental competence.

By increasing the optimization of culture conditions in vitro, the concern that embryos should be returned to the patient's uterus as soon as possible to prevent loss of viability in vivo, becomes less important than the opportunity to select embryos for increased developmental competence. Indeed, it is probable that the environment in utero is more hostile to the early embryo than the optimum conditions in vitro. Uterine secretions, immune cells and molecules and other adverse conditions may result in greater embryonic wastage in vivo than in vitro. At present, ~80–90% of the embryos selected for transfer on days 2 or 3, fail to survive to implantation. It is also known that embryos deemed to be unsuitable for transfer or cryopreservation on days 2 to 3 of culture are capable of continued development to the blastocyst stage in vitro (Bolton et al., 1989Go; Dokras et al., 1993Go; Rijnders et al., 1998) and will implant after transfer to the uterus (Harper et al., 1997Go). Conversely, many of the embryos deemed to be of good quality on days 2–3 of culture, either fail to develop to the blastocyst stage or develop to blastocysts but are not selected for transfer at this later stage as morphologically superior blastocysts have developed from their (apparently) less superior cohort embryos (Harper et al., 1997Go). It is also apparent from group embryo cultures that only some of the embryos within the group, exposed to identical culture conditions, are capable of continuing development to blastocysts. It has been postulated that the capacity of an embryo to develop into a blastocyst is primarily dependent upon the activation of a competent embryonic genome (Scholtes and Zeilmaker, 1998Go).

Not all patients will have an embryo transfer if this is deferred to the morula to blastocyst stage of development. Failure to develop to the blastocyst stage is affected by the quality of the culture conditions used, but has also been reported to be influenced by the number of oocytes retrieved (Jones et al., 1998bGo; Scholtes and Zeilmaker, 1998Go) and a factor related to male infertility. Development to the blastocyst stage is compromised in cycles where male factor is the cause of infertility (Janny and Ménézo, 1994Go; Jones et al., 1998bGo). The negative association of maternal age and development to the blastocyst stage appears to be linked to the reduced number of oocytes retrieved (Jones et al., 1998bGo; Scholtes and Zeilmaker, 1998Go). A similar association has also been reported for transfer of early cleavage stage embryos (Dicker et al., 1991Go). Table IGo summarizes the main reports of the rate of failure to develop to the blastocyst stage with respect to patient selection criteria used.


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Table I. Summary of the reported rates of failure to develop to the blastocyst stage, with respect to the patient selection criteria
 
Patients undergoing ART procedures invest both financially and emotionally in the expected outcome of pregnancy. Repeated failure of implantation causes emotional distress, particularly when there is no explanation of the cause. It seems unethical and imprudent to continue ART treatment of patients without at least attempting some additional diagnostic assessment. Recently, it has been demonstrated that for patients with a poor pregnancy prognosis (>37 years of age, three or more previous IVF failures with embryo transfer or mosaic gonosomal karyotype), 57% of their embryos that appear to be of regular morphology and normal developmental rate, are chromosomally aneuploid (Gianaroli et al., 1997aGo,bGo; Magli et al., 1998Go). A further 16% of the embryos produced have abnormal cleavage rates and/or morphology. Elimination of the aneuploid embryos and transfer of selected normal embryos resulted in high pregnancy and implantation rates for this group of patients (Magli et al., 1998Go). This technology is presently expensive and not widely available. However, the option of extending culture to the blastocyst stage of development is available to all ART centres. Although development to the blastocyst stage is not a guarantee of chromosomal normality, the majority of the embryos that fail to continue to develop in extended culture show multiple aneuploidies for chromosomes X, Y, 16,18 and 21 (L.Gras, L.Gianaroli, G.Jones, C.Magli and A.Trounson, unpublished data). In conclusion, blastocyst transfer has earned its place in the armoury of technologies on offer to treat the infertile patient. Viable blastocysts can now be grown successfully in culture at high rates using well-defined culture systems. The pregnancy rate following blastocyst transfer will be higher than for transfer of the same number of early cleavage stage embryos. The resultant implantation rates are higher than for the transfer of early cleavage stage embryos and provide a realistic opportunity to reduce both the number of embryos transferred and, importantly, the number of multiple pregnancies following ART treatment. The improved success rates following blastocyst transfer are conferred by the advantage of selecting developmentally competent embryos. For those patients who produce only two or three embryos (which can be the case for patients of advanced maternal age), extending culture beyond day 2 or 3 offers no additional selection advantage. For those patients who have repeatedly failed to become pregnant following transfer of early cleavage stage embryos, extended culture offers the selection advantage of eliminating many severely aneuploid embryos. As more ART centres use the option of extended culture, the factors responsible for failure to develop to the blastocyst stage will be revealed. This, in turn, will lead to the development of new diagnostic procedures for patients whose entire cohort of embryos fails to develop to the blastocyst stage both in vitro and in vivo and provide a much-needed explanation of the failure of IVF to resolve their infertility.

Notes

1 To whom correspondence should be addressed Back

This debate was previously published on Webtrack 55, March 12, 1999

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