Culture of viable human blastocysts in defined sequential ... · mass appears to require the essential group of amino acids (i.e. those required by somatic cells in culture), whilst

Culture of viable human blastocysts in definedsequential serum-free media

David K.Gardner1'3 and Michelle Lane2

'Colorado Center for Reproductive Medicine, 799 East Hampden Ave.,Suite 300, Englewood, CO 80110, and department of Animal Health andBiomedical Sciences, University of Wisconsin, Madison, WI 53706, USA

3To whom correspondence should be addressed

In human in-vitro fertilization (IVF), embryos are routinely transferred tothe uterus on either day 2 or day 3 of development, resulting in a 10-15%implantation rate. However, in other mammalian species, the transfer ofcleavage stage embryos, which normally reside in the oviduct, to the uterusresults in a significantly lower implantation rate compared with blastocysts.It is therefore proposed that, in order to increase implantation rates inhuman IVF, one has to move to extended culture and transfer at theblastocyst stage. The transfer of blastocysts will not only help synchronizethe embryo with the female tract but will facilitate the identification of thoseembryos with little or no developmental potential. In order to culture viableblastocysts it is important to use more than one culture medium to cater forthe changing requirements of the preimplantation embryo as it develops anddifferentiates. If sequential culture media are not used, one can obtainblastocysts but their resultant viability is low. The use of sequential serum-free media in human IVF has resulted in >50% of embryos becomingblastocysts with an implantation rate of -50%. Further advances in humanembryo culture should come from the replacement of protein with theglycosaminoglycan hyaluronate, which is more suitable than albumin insupporting implantation in the mouse, and which will eliminate biologicalvariation and possible contamination from blood products. With the routineculture of human blastocysts will come the introduction of non-invasive testsof embryo viability, capable of identifying those blastocysts most likely todevelop from a given cohort. As the implantation rate of blastocysts is higherthan that of the cleavage stage embryo, fewer embryos will be required fortransfer in order to establish a successful pregnancy, thereby reducing thenumber of multiple gestations and increasing the overall efficiency ofhuman IVF.Key words: blastocyst transfer/human metabolism/viability

Introduction

There are considerable advantages of being able to culture the human embryo to theblastocyst stage before transfer in human in-vitro fertilization (IVF). Advantages

148 © European Society for Human Reproduction and Embryology Human Reproduction Volume 13 Supplement 3 1998

Culture of human blastocysts in serum-free media

include: (i) an increased implantation rate due to the identification of thoseembryos with little if any developmental potential and the synchronization ofthe embryo with the female tract; (ii) a decrease in the number of embryosrequired at the time of transfer in order to achieve a pregnancy; (iii) a decreasein the number of multiple gestations; (iv) an ability to undertake cleavagestage embryo biopsy without the need for cryopreservation when the biopsiedblastomere has to be sent to a different locale for analysis; (v) facilitating theintroduction of trophectoderm biopsy, which means the removal of non-embryonictissue for analysis, which in itself has both ethical and religious implications;and (vi) allowing the introduction of quantitative methods of embryo viabilityassessment, as at the blastocyst stage embryo physiology is being analysed andnot that of the oocyte (which is the case when the cleavage stage embryos areexamined after their brief growth in vitro).

So why is the human embryo not routinely transferred at the blastocyst stagein human IVF? The main reason is that culture media have tended to be toosimplistic, with conventional embryo culture media being based on a balancedsalt solution with added carbohydrates as energy sources. At the other end ofthe spectrum several laboratories use tissue culture medium such as Ham's F-10or minimal essential medium (MEM), which were designed to support somaticcell lines in vitro, and do not reflect the actual requirements of the mammalianpreimplantation embryo and may be considered too complex. The problem ofwhich medium composition is the optimum has been further complicated by theuse of a single medium in an attempt to support all stages of development. Asthe zygote and blastocyst have differing physiology and metabolism theysubsequently have different requirements in culture. It is therefore proposed thatin order to optimize embryo development in culture one has to formulate morethan one culture medium to support embryo development in vitro.

The two key nutrients utilized by the developing embryo are carbohydratesand amino acids. The significance and role of these will be discussed in turn.

Carbohydrates

The human embryo undergoes a change in its nutrient preference as it develops,pyruvate being the preferred nutrient of the cleavage stage embryo, with glucosebecoming increasingly utilized as development to the blastocyst proceeds (Hardyet al., 1989). Interestingly, such changes in carbohydrate uptake mirror theconcentration of available nutrients within human oviduct and uterine fluids, inwhich pyruvate and lactate are at their highest concentration in the oviduct atthe time when the embryo is present, while glucose concentration is reduced(0.5 mM). In contrast, the concentration of pyruvate and lactate are lower in theuterus, but glucose concentration is at its highest (3.15 mM) (Gardner et al.,1996a). In conventional embryo culture media, such as human tubal fluid (HTF)(Quinn et al., 1985), and Earle's medium (Edwards, 1981), glucose can impairhuman embryo development in culture (Conaghan et al., 1993; Quinn, 1995).

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An explanation for this observation has been derived from work on the embryosfrom other mammalian species. Glucose is not the preferred energy substrate ofthe cleavage stage embryo and yet it appears that in conventional embryo culturemedia, glucose is actually utilized by the cleavage stage embryo at the expenseof oxidation (Menke and McLaren, 1970; Gardner, 1998a; Gardner and Leese,1990; Seshagiri and Bavister, 1991; Gardner and Lane, 1993a; Gardner andSakkas, 1993). This phenomenon is similar to that reported for certain types oftumour cell metabolism, and is known as the Crabtree effect (Koobs, 1972).

Importantly, however, this impairment of metabolic function is not evidentwhen specific amino acids and the chelator EDTA are present in the culturemedium. Both amino acids and EDTA act through independent mechanisms tosuppress glycolytic activity in the early embryo (Gardner and Lane, 1993a; Laneand Gardner 1997a; Gardner 1998a). Furthermore, they act in synergy to minimizeany adverse affect of glucose on the cleavage stage embryo. At the blastocyststage the embryo can readily utilize glucose as both an oxidative and glycolyticenergy source. The mechanisms underlying this metabolic switch have beendocumented elsewhere (Biggers et al., 1989, Leese, 1991; Rieger, 1992; Gardner1998a,b).

However, two important considerations regarding the use of glucose by theembryo post compaction are worth discussing. Firstly glucose will have animportant role in biosynthesis, as it is involved in the generation of ribosemoieties for nucleic acid synthesis and the NADPH required for the biosynthesisof lipids and other complex macromolecules. Secondly, the work of Hewitsonand Leese (1993) indicates that the inner cell mass generates its energypredominantly from glycolysis. Therefore a suppression of glycolysis by eithersubstrate (glucose) limitation or the inhibition of an enzyme in the pathway (forexample by EDTA), may well interfere with the development of the inner cellmass, thereby affecting subsequent fetal development. Indeed, this does appearto be the case. When mouse zygotes were cultured to the blastocyst stage in theabsence of glucose, although the resultant blastocysts implanted in the uterusafter transfer, significantly more fetuses were lost in comparison with the controlblastocysts which had developed in the presence of glucose (Gardner and Lane,1996). Similarly, if mouse embryos were cultured to the blastocyst stage in thecontinual presence of EDTA, which suppresses glycolysis, then resultant fetaldevelopment was significantly lower than from those blastocysts which had beenexposed to EDTA for the first 48 h of culture from the zygote (Gardner andLane, 1996). Gardner et al. (1997a) went on to demonstrate that the presence ofEDTA in the culture medium for the first 72 h of culture significantly improveddevelopment of cattle embryos to the 8-cell stage. However, if the resultant 8-cell embryos were left in the presence of EDTA, then the developing blastocystshad a significantly smaller inner cell mass, and the number of cells in thetrophectoderm was unaffected. Such data highlight the differing metabolism ofthe cleavage stage and post compaction embryo and supports the hypothesis thatdifferent culture conditions are required at different stages of development

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in order to satisfy the changing requirements of the embryo (Gardner andLane, 1993b).

Amino acids

Until relatively recently, amino acids have not been considered to be importantregulators of human embryo development. However, a resurgence of interest intheir role in mammalian embryo development leads one now to conclude thatamino acids are amongst the most important of regulators for the early embryo.The physiological basis for the inclusion of amino acids in embryo culturemedium stems from the fact that amino acids are abundant within the fluid ofthe female reproductive tract (Miller and Schultz, 1987; Gardner and Leese,1990; Moses et al, 1997), and that the oocyte and embryo maintain an endogenouspool of amino acids through specific carriers on the plasma membrane (Schultzet al, 1981; Miller and Schultz, 1987; Van Winkle, 1988). Interestingly, thoseamino acids which are most abundant in the oviduct are those with the greatestbeneficial effect on the cleavage stage embryo (Bavister and McKiernan, 1993;Gardner and Lane, 1993c). These amino acids include; alanine, glutamate,glutamine, glycine, proline and serine. Together, these amino acids have beenshown to significantly decrease the time that the mouse embryo takes to completethe first three cleavage divisions and undergo compaction (Lane and Gardner,1997b), whilst decreasing the time it takes to form a blastocyst (Gardner andLane, 1993c). This group of amino acids also shares a striking homology withthose amino acids present in Eagle's non-essential amino acids (Eagle, 1959),i.e. those amino acids not required by somatic cells in culture.

Importantly, those amino acids not present at high levels in the oviduct, whichare classified as essential by Eagle (1959), confer no beneficial effect to thecleavage stage embryo (Gardner and Lane, 1993c; Lane and Gardner 1997c).Indeed, exposure of the mouse embryo to the essential amino acids, at theconcentrations in Eagle's medium, prior to the 8-cell stage results in a loss ofviability (Lane and Gardner, 1994). Interestingly, however, like the switch theembryo undergoes regarding its utilization of carbohydrates, there is also a switchin the requirements for amino acids. This switch seems to stem specifically fromthe different requirements of the two cell types in the blastocyst. The inner cellmass appears to require the essential group of amino acids (i.e. those requiredby somatic cells in culture), whilst the trophectoderm utilizes the non-essentialamino acids and glutamine (Lane and Gardner, 1997c). The reason for thisobserved switch in amino acids has been attributed to the roles that specificamino acids have in embryo physiology (Gardner and Lane, 1997; Gardner1998a; 1998b). In essence the non-essential amino acids serve as regulators ofenergy metabolism, osmolytes and intracellular pH buffers prior to compaction.Such data like that obtained for the utilization of carbohydrates support thehypothesis that more than one culture medium is required for the optimaldevelopment of the preimplantation embryo in culture.

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Development of sequential culture media

In light of the dynamics of embryo physiology and the resultant changes inrequirements for both carbohydrates and amino acids, it is proposed that optimaldevelopment of the mammalian embryo in culture will require more than a singleculture medium. Rather, two or more will be required to meet the changingrequirements of the embryo. To this end, two culture media, designated growth1 and growth 2 (Gl and G2) were formulated to support the growth of thehuman pronuclear embryo to the blastocyst stage (Gardner, 1994; Barnes et al,1995; Gardner and Lane, 1997). The Gl medium is based on the levels ofcarbohydrates present in the human Fallopian tube at the time when the cleavagestage embryo is present. This medium also contains those amino acids whichhave been shown to stimulate development of the cleavage stage embryo (i.e.the non-essential amino acids and glutamine). The chelator EDTA is also present,not only to sequester any toxic divalent cations present in the system, but alsoto help minimize glycolytic activity of the embryo, thereby minimizing metabolicperturbations.

In contrast, G2 medium is based on the levels of carbohydrates present in thehuman uterus and contains both non-essential and essential amino acids tofacilitate both blastocyst development and differentiation. EDTA is not presentin medium G2 as it appears to selectively impair inner cell mass developmentand function, culminating in a loss of viability (Gardner and Lane, 1996; Gardneret al, 1997a). Both Gl and G2 media are supplemented with serum albumin.Serum is not required nor desired in embryo culture systems, especially thosedesigned to support blastocyst growth (Gardner and Lane, 1993b; Gardner, 1994).The beneficial effects of using sequential media to support mouse embryodevelopment in culture are shown in Figure 1. One of the most important thingsto come from this work is the observation that it is possible to generate healthylooking blastocysts in culture which unfortunately have little if any furtherdevelopmental potential. This stems from the fact that different components ofthe culture system affect different aspects of embryo development. When mouseembryos are cultured in Gl medium for the entire preimplantation period tothe blastocyst, although the embryos form healthy looking blastocysts, mostimplantations are lost, i.e. they did not have a sufficient inner cell mass to forma viable fetus (Gardner et al, 1997b; Lane and Gardner, 1997c). The lack ofadequate inner cell mass development stems from both the lack of sufficientglucose and the presence of EDTA (both affecting glycolysis) and the omissionof essential amino acids. In contrast, those embryos that were switched to G2medium after 48 h of culture formed blastocysts at the same rate and of equivalentmorphologies, but due to the development of a significant inner cell mass, veryfew implantations were lost thereby maintaining a very high pregnancy rate.

The observation that a well-formed blastocyst does not necessarily equate toa viable blastocyst is an important one, and one which can perhaps explain whyprevious attempts to culture the human embryo to the blastocyst stage haveresulted in low implantation rates after transfer. For example, Bolton et al. (1991)

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Figure 1. Effect of sequential culture media on the development of F] (C57BL/6XCBA/Ca) mouse zygotesin vitro. Zygotes were collected at 20 h post-administration of human chorionic gonadotrophin (HCG). Allmedia were supplemented with bovine serum albumin (BSA, 2 mg/ml). All embryos were transferred tofresh medium after 48 h of culture, with the exception of embryos in medium Gl, where the embryos weretransferred to either medium Gl or G2. To compensate for this, twice the number of embryos were originallycultured in medium Gl, although only a designated 50% of these embryos were used in the statisticalanalysis of the 44-52 h data set. (a) Embryo cell number after 44, 48 and 52 h of culture. Values are means± SEM. n = 200 embryos/medium. Media: Gl (solid bar); human tubal fluid (HTF; open bar); Ham's F-10(hatched bar). **Significanfly different from other media, P <0.01. (b) Embryo development after 72 h ofculture, n = 150 embryos/medium. G1/G2; embryos cultured for 48 h in medium Gl and then transferred tomedium G2. Blastocyst (solid bar), hatching blastocysts (as a percentage of total blastocysts; open bar). Likepairs are significantly different; a c d P <0.05; bP <0.01. (c) Embryo development after 92 h of culture, n =150 embryos/medium. G1/G2; embryos cultured for 48 h in medium Gl and then transferred to medium G2.Blastocyst (solid bar), hatching blastocysts (as a percentage of total blastocysts; open bar). Like pairs aresignificantly different; ab-cP <0.05. Significantly different from medium Gl and G1/G2; **P <0.01. (d) Cellallocation in the blastocyst after 92 h of culture, n = 150 embryos/medium. G1/G2; embryos cultured for 48h in medium Gl and then transferred to medium G2. Trophectoderm (solid bars), inner cell mass (openbars). Significantly different from other media; *P <0.05; **P <0.01. (e) Viability of cultured blastocysts;n = at least 60 blastocysts transferred per treatment. G1/G2; embryos cultured for 48 h in medium Gl andthen transferred to medium G2. Implantation (solid bar), fetal development per implantation (open bar). Likepairs are significantly different; a 4 P <0.05; b-cP <0.01

obtained a very respectable 40% rate of blastocyst development from humanpronuclear embryos. However, the resultant implantation and pregnancy rate wasonly 7%. The medium used in this study was Earle's supplemented with pyruvateand 10% maternal serum. Clearly then, such media are not suitable for extendedhuman embryo culture.

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This approach of using sequential culture media has now been applied in pilotclinical studies at two IVF programmes, one in Colorado, USA (Colorado Centerfor Reproductive Medicine) and one in Melbourne, Australia (Monash IVF). Inboth programmes the use of sequential embryo culture media has resulted inblastocyst development of ^50% with a significant increase in subsequentimplantation rate (Gardner et al, 1997b, 1998; Jones et al, 1998). Data to dateat the Colorado Center for Reproductive Medicine after 50 blastocyst transfersshow that an implantation rate of ~50% can be achieved, resulting in an ongoingpregnancy rate of 70% with a mean of 2.6 blastocyst transferred. Importantly,all the babies born from this culture system are normal. Clearly, now that culturesystems have been tested and the viability of the resultant blastocysts established,it is important to reduce the number of embryos transferred down to two, theultimate goal being the transfer of a single blastocyst. Large-scale randomizedtrials are now required to validate the findings of these pilot studies.

There remain two important issues regarding human embryo culture andtransfer in human IVF; the use of blood products in assisted conception and theneed to identify the most viable embryos for transfer.

Alternatives to protein in embryo culture medium

There is a possible risk of infection arising from the use of blood products inIVF. Not only does the use of pooled blood to generate serum albumin alwaysrepresent a cause for concern, but batches of serum albumin vary significantlyin their embryotrophic ability, thus making batch to batch comparisons verydifficult and highlighting the problem of biological variation. This very problemhas led to the search for alternative macromolecules to serum albumin. Thesynthetic polymers polyvinylalcohol and polyvinylpyrollidone have both beenused in assisted reproductive technologies (Bavister, 1995). However, neither canbe considered as a physiological alternative to protein, and the teratologicalnature of these compounds has not been fully evaluated. Furthermore, the truerole of serum albumin in embryo culture has yet to be fully elucidated, althoughit is plausible that albumin helps to bind and stabilize growth factors.

The presence of macromolecules in an embryo culture system serves tofacilitate manipulation of gametes and embryos, which would otherwise adhereto the plastic culture vessel. A possible alternative to albumin is the glycosamino-glycan, hyaluronate. Not only do the levels of hyaluronate increase in the uterusaround the time of implantation (Zorn et al, 1995), but the human embryoexpresses the receptor for hyaluronate throughout preimplantation development(Campbell et al, 1995). Although hyaluronate is a glycosaminoglycan, unlikeother glycosaminoglycans such as heparin, it has no protein moieties and cantherefore be considered as a polysaccharide. This, therefore, removes both theproblem of variation and contamination, as hyaluronate can be synthesized in apure form. In preliminary trials of hyaluronate in mouse embryo culture andtransfer, it was found that not only could hyaluronate (0.5 mg/ml) readily replace

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serum albumin for in-vitro cultures, but more importantly it significantly increasedthe implantation rate of resultant blastocysts (Gardner et al., 1997c). Hyaluronatesupported a significantly higher implantation rate than albumin. Furthermore,this increase in implantation rate could be attributed to the transfer medium alone.

When embryos were cultured in appropriate sequential media similar to Gland G2, there was no evident benefit in vitro from the presence of anymacromolecule/protein. Embryos formed blastocysts at the same rate in mediumdevoid of any macromolecule, and the blastocysts had inner cell mass andtrophectoderm cells equivalent to those embryos cultured with either albuminor hyaluronate. However, when embryos were pre-equilibrated in mediumsupplemented with 0.5 mg/ml hyaluronate for 5 min prior to transfer in mediumcontaining hyaluronate, the resultant implantation rate was equivalent to thatobtained for embryos cultured for the entire preimplantation period in thepresence of hyaluronate (Gardner et al., 1997c).

The beneficial effect of hyaluronate on the embryo and implantation may beattributed to one or more roles of hyaluronate; these include the ability to forman anti-viral and anti-immunogenic layer around the embryo, an ability to increaseangiogenesis and perhaps most importantly to facilitate the rapid diffusion of thecontents of the transfer medium (the embryo) into the fluid of the uterus. Asuterine 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 theslow mixing of the medium and embryo with the lumenal contents. In contrast,the transfer of an embryo in a hyaluronate solution will facilitate diffusion ofthe embryo into the lumenal environment. Furthermore, hyaluronate may beinvolved in the initial phases of attachment of the blastocyst to the endometrium.

Development of blastocyst viability assays

Conventional embryo culture media induce considerable cellular trauma in thedeveloping embryo. One of the key manifestations of this is an abnormal patternof energy metabolism. As the developing embryo requires sufficient levels ofcellular energy for DNA replication, biosynthesis and mitosis, any aberrations inthe embryo's metabolism has dire consequences for subsequent development andviability. Studies on both cattle and mice have determined that there is arelationship between both the rate and normality of nutrient utilization anddevelopmental potential. Renard et al. (1980) observed that day 10 cattleblastocysts with a glucose uptake >5 jig/h developed better in culture and gaverise to more pregnancies than blastocysts with a glucose uptake <5 Jig/h.Subsequently, Gardner and Leese (1987) used the non-invasive technique ofultramicrofluorescence to measure glucose uptake by individual day 4 mouseblastocysts prior to their transfer to recipient females. Those blastocysts thatwent to term had a significantly higher glucose uptake in culture than thoseembryos that failed to develop after transfer. Unfortunately these studies were

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retrospective and as such could not conclusively demonstrate whether it waspossible to identify viable embryos prior to transfer using metabolism as a marker.

However, a study of the metabolism of day 7 cattle blastocysts before andafter cryopreservation showed that it was possible to identify those blastocystscapable of re-expansion in the hours immediately post-thaw. Those blastocystswhich survived the freeze-thaw procedure had a significantly higher glucoseuptake and lactate production than those embryos which did not re-expand andsubsequently died (Gardner et al, 1996b). Of greater significance however, wasthe observation that there was no overlap in the distribution of glucose uptakeby the viable and non-viable embryos, suggesting that it may therefore bepossible to use metabolic criteria for prospective selection of viable embryos(Gardner et al, 1996b). Following on from this study, Lane and Gardner (1996)performed a prospective trial in which day 5 mouse blastocysts were classifiedas either viable or non-viable according to their glycolytic activity. It was foundthat those blastocysts which exhibited a pattern of glycolytic utilization similarto that of embryos developed in vivo had a developmental potential of 80%,whilst those blastocysts which exhibited an excessive lactate production (i.e.aberrant glycolytic activity), had a developmental potential of only 6%. As such,this data supports the hypothesis proposed by Rieger (1984) that embryonicmetabolism can be used to successfully identify viable embryos in culture priorto transfer, thereby significantly increasing pregnancy rates. Now that the routineculture of the human embryo to the blastocyst stage is achievable, non-invasiveassessment of blastocyst metabolism may be used to identify those embryos withthe highest developmental potential prior to transfer.

Conclusions

Using sequential serum-free culture media it is possible to culture the humanpronuclear embryo to the blastocyst at acceptable frequencies. More importantly,using this approach the resultant blastocysts have a high implantation rate,culminating in a high pregnancy rate and significantly reducing the need for thetransfer of multiple embryos, thereby reducing the number of multiple gestations.

The next generation of embryo culture media are likely to be protein free,using such macromolecules as hyaluronate as a physiological alternative to serumalbumin. Such media will have inherently less biological variation and shouldsignificantly reduce the potential for infection in the culture system.

With the advent of blastocyst culture and transfer in human IVF, it should befeasible to further reduce the number of embryos transferred whilst maintaininghigh pregnancy rates, by the implementation of non-invasive methods of assessingembryo metabolism prior to transfer.

Acknowledgements

It is with great pleasure we thank our mentors; Henry Leese, John Biggers, Bob Edwards, BarryBavister and Don Rieger for their valuable insight and continuous support of our research over

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the years. We would also like to thank Alan Trounson for giving us laboratory space in his Centreand the intellectual freedom to develop an independent research program. Finally we are indebtedto William Schoolcraft, Colorado, for his vision and encouragement.

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Culture of viable human blastocysts in defined sequential ... · mass appears to require the essential group of amino acids (i.e. those required by somatic cells in culture), whilst