Is the mouse a good model for the human with respect to the development of the preimplantation embryo in vitro?

Is the mouse a good model for the human with respect to the development of the preimplantation embryo in vitro? Patrick Quinn 1 ' 2 and Frederick C.Horstman 1 ' 3 Advanced Reproductive Technologies, Inc., San Clemente and 2 IVF Laboratory, Alvarado Hospital Medical Center, San Diego, California, USA 3 To whom correspondence should be addressed at: 944 Calle Amanecer, Suite L, San Clemente, CA 92673, USA A comparison has been made of various aspects of preimplantation development of mouse and human embryos in vitro. Changes in substrate utilization follow similar patterns in both species. This similarity in metabolic parameters between the two species has facilitated the use of the mouse as a model to study the formulation of culture media to be used at different stages over the preimplantation period from fertilization to the fully expanded blastocyst stage. It has also prescribed the mouse embryo as a practical tool for quality control testing of the laboratory system in human in-vitro fertilization. Aspects of the physiology of both species that require further study are the physiological levels of endogenous inorganic phosphate in the female reproductive tract, the requirement for inorganic phosphate in culture medium, the specificity of the amino acid requirements for optimal development before and after compaction and the importance of including EDTA in culture medium. Key words: culture/embryo transfer/ivf/mouse/preimplantation embryo 'The initial stimulus to the introduction of human IVF arose from studies in mice' (Edwards and Brody, 1995) Introduction Some of the studies alluded to in the quotation given above include those on the superovulation mice (Fowler and Edwards, 1957; Edwards and Gates, 1959) and the subsequent transfer of embryos (Sirlin and Edwards, 1959). The pioneering work on human oocyte maturation, fertilization and embryo development in vitro undertaken by Edwards, Steptoe and Purdy (reviewed by Edwards and Brody, 1995) and Trounson and Wood (reviewed in Trounson and Gardner, 1993) owed a lot to studies that had been undertaken on mice from the time when preimplantation embryo development in vitro had been initiated by Whitten in 1956 and improved by the extensive undertakings of Brinster in the 1960s Human Reproduction Volume 13 Supplement 4 1998 European Society for Human Reproduction and Embryology 173

P.Quinn and F.C.Horstman (reviewed by Brinster, 1972). Further work by Bavister, Wales, Whittingham, Hoppe, Biggers and many others contributed to the initial success of the culture of human embryos in vitro. The work of Bavister with hamsters, and of others with non-human primates and domestic mammalian species has also contributed to our ability to improve human embryo culture but, in our opinion, our present day ability to culture human embryos in vitro would not be as advanced as it is without quality control (QC) and research studies utilizing mouse embryos. In this review, we will discuss some of the physiological aspects of preimplantation development in mice and humans, how mouse embryos can be used in QC assessment of the culture system in an assisted reproductive technology programme, the positives, negatives and alternatives when comparing mouse embryo development in vitro to that in humans and, finally, a brief consideration of whether it is effective to extend human embryo culture in vitro to the end of the preimplantation phase of development. Physiology Timing and size Of the readily available mammalian preimplantation embryos, most investigators would have to agree that the temporal sequence of events of cleavage, compaction, blastocoele formation and blastocyst differentiation, as they occur in the mouse, provide a good model for these events in the human preimplantation embryo. Data related to cleavage rates in vitro for the human (Steptoe et al, 1971; Edwards et al, 1981; Trounson et al, 1982) and mouse (Harlow and Quinn, 1982) are available and show an average cell cycle time of 13-16 and 10 h, respectively, over the first three or four cleavage divisions. Of course, culture conditions, and especially medium composition, can have a profound effect on cell cycle time, as has been reported for the hamster (see Bavister, 1997), mouse (Quinn, 1995) and is also most likely to occur with human embryos. Unfortunately, no data are currently available for this in humans. In general, mouse zygotes reach the fully expanded blastocyst stage after 84-96 h of culture and human embryos take another 24-30 h. With the improved media formulations now available, the in-vitro development time sequences in both the mouse and human are probably almost equivalent to the development rates in vivo. Similarly, although the differences are obvious and affect numerous physiological parameters, the size of mouse embryos makes them useful as practice models for various research studies, including micromanipulation, before applying these techniques to human models (e.g. micromanipulative techniques reviewed in Cohen et al, 1992). The average diameter of mouse embryos at 70 Lim is about half that of human embryos, thus making the volume of the human embryo ~8-fold larger than the mouse embryo. Metabolism Perhaps more so than any other physiological parameters such as developmental timing and size discussed above, the metabolism of energy substrates and 174

Mouse embryos as models for the human intermediates in mouse and human embryos share many similarities, as well as several differences. Thus, early preimplantation embryos of both species rely on endogenous energy sources such as ATP and glycogen for the first several mitotic cell cycles (Quinn and Wales, 1973; mouse data reviewed by Wales, 1975; Van Blerkom et al., 1995), utilizing pyruvate, lactate and, if necessary, amino acids to maintain intermediary substrate and cofactor levels. Then at around compaction (about the 8-cell stage in both species) and shortly thereafter when the blastocoel cavity begins to form, a switch to what has been termed aerobic glycolysis (Barnett and Bavister, 1996; Lane and Gardner, 1996) ensures the supply of the necessary increased energy demand. These changes have been studied extensively by culture experiments in which embryo development has been assessed when energy substrates in the medium have been varied, radioactive substrates have been used to measure uptake and metabolism, ultramicroassays have been used to measure the utilization and production of substrates, intermediates and endproducts and, finally, the endogenous changes in the concentration of energy substrates and other metabolic components in the reproductive tract fluids have been measured (reviewed by Barnett and Bavister, 1996; Quinn, 1997). To summarize, it appears that in both species, the early embryo during the first half of preimplantation development relies on pyruvate, to a lesser extent lactate and to some extent glutamine and other non-essential amino acids (Quinn, 1995; Gardner and Lane, 1996). The presence of inorganic phosphate (Pi) and glucose and Pi together appears to be inhibitory, although glucose by itself does not have a very big negative effect (reviewed by Leese, 1995; Barnett and Bavister, 1996). During the later half of preimplantation development, the utilization of exogenous glucose increases in both species and its presence in culture medium at this stage, together with glutamine and possibly other amino acids, is beneficial for development in vitro and subsequently in vivo (Conaghan et al., 1993; Gardner and Lane, 1996). The formulation of the current generation of new culture media (e.g. Basal XI and D3+ HTF, Quinn/Advanced Reproductive Technologies, Inc; G1-S1/G2-S2, Gardner/Scandinavian IVF; PI, Pool/Irvine Scientific) generally reflects these physiological parameters. There are several anomalies and differences that require discussion and further study. Physiological endogenous levels of Pi in reproductive tract fluid Although total elemental phosphorus has been measured by X-ray probe analysis (Borland et al., 1980), we do not know the Pi concentrations, which could be quite low. Leese (1997) recently asked some major questions concerning the role of Pi in preimplantation embryo development. To paraphrase his questions, he pointed out we do not know how Pi enters embryos, what is the intracellular concentration of Pi and how is this changed in the absence of extracellular Pi; similarly, what is happening with mitochondrial Pi, and what influence does Pi have on enzymatic activity of the likes of pyruvate kinase, hexokinase, phosphofructokinase, etc. It has also been shown that Pi influences cell cycle 175

P.Quinn and F.C.Horstman 100-90 - 80-70 - % 60 - FEBS 50 40-30 - 20-10 - 0 - L 1-1 11 1 _j 1, _ _ i 1 1 L 1 111 i p 0 2.78 5.56 Glucose cone mm HO mm Pi 0.37 mm Pi D1.85 mm Pi i i Figure 1. Interaction between glucose and phosphate (Pi) in a 3X3 factorial experiment for the development of zygotes from CF1 female mice to fully expanded blastocysts (FEBs). The results are the mean of five replicates, and each treatment contained a total of between 85 and 107 zygotes. No EDTA or glutamine was present in any of the media. Glucose and Pi were inhibitory, both individually and together. proteins (Haraguchi et al., 1996) and we know that ATP levels decline with preimplantation development, presumably releasing intercellular Pi. All these topics are in need of more thorough investigation. The negative effect of the combination of Pi and glucose This subject has also been recently reviewed by Biggers et al. (1997). Here again, opinions differ. Biggers et al. (1997) and Gardner et al. (1996) recommend glucose be included in culture medium, whereas Leese suggests that Pi should be provided but that glucose can be omitted, at least initially (Conaghan et al. 1993; Leese, 1995, 1997). We are inclined to believe that the presence of Pi and the combination of Pi and glucose in culture medium for both human and mouse embryos is detrimental (Quinn, 1995, 1997) and ponder why some media formulations include Pi (e.g. S1/S2, KSOM). To us, it seems that the absence of Pi, at least for the early stages of culture, would be more appropriate, and this is the rationale we have used in the formulation of our Basal XI HTF medium. An example of the negative effects of glucose and phosphate on the development in vitro of CF1 mouse zygotes is shown in Figure 1. The most striking observation in this experiment was that the absence of both glucose and Pi from the medium gave the best result. Specificity of amino acids Although the inclusion and requirement of amino acids in culture media for mouse embryos has been extensively studied by Gardner (reviewed by Lane and Gardner, 1997a; Gardner and Lane, 1997), similar studies with human embryos do not exist. Unfortunately, the studies of Lane and Gardner (1997a) were undertaken with media containing Pi and the inhibitory effect of this compound 176

Mouse embryos as models for the human may have necessitated the presence of a greater variety of amino acids in the media than is actually necessary. Presence of EDTA in culture media It appears likely that EDTA is beneficial for the mouse (reviewed by Gardner and Lane, 1996; Quinn, 1997) and human preimplantation embryo (Quinn, 1995; Quinn et al., 1995), as well as the embryos of some other mammalian species. As well as chelating trace amounts of toxic divalent cations, including ferrous ions that can contribute to reactive oxygen species, a recent study by Lane and Gardner (1997b) also showed that EDTA inhibits the enzyme 3-phosphoglycerate kinase, thus reducing glycolytic activity in the early embryo. Increased glycolysis in the early embryo appears to be associated with poor development (Gardner and Lane, 1996). Specific glucose requirements by mouse and human embryos in vitro Although not specifically associated with early embryo development in vitro, the requirement for glucose for mouse and human fertilization in vitro appears directly contradictory. Thus glucose is required by the mouse (Hoppe, 1976) but is not essential for the human (Quinn, 1995), although sperm kinematics and subsequent fertilization rates are compromised in the human in the absence of glucose if sperm concentration is suboptimal. As stated previously, the first part of cleavage seems to do best in both species in the absence of glucose or with much lower concentrations than were originally used in HTF, T6 and Ham's F10 media (e.g. 0-0.5 mm versus 2.8-6.1 mm). There are contradictory reports as to whether there is an absolute requirement for glucose in the latter part of preimplantation development in the mouse. Thus Chatot et al. (1989), Martin and Leese (1995) and Gardner and Lane (1996) all contend that glucose is required for blastocyst formation in vitro and that the viability of murine blastocysts is maintained in the presence of glucose. On the other hand, Lawitts and Biggers (1991), Quinn (1995) and Scott and Whittingham (1996) have all reported that glucose is not essential for the development of mouse embryos to the blastocyst stage in vitro. We (P.Quinn and F.C.Horstman, unpublished data) have found that the source of protein added to the culture medium can influence the transition of the embryos from the morula to the blastocyst stage. It is probable that the amount of glucose and other substrates bound to the protein may influence the outcome. It may also be likely that the presence of EDTA and amino acids in the medium (Quinn, 1995; Gardner and Lane, 1996) and the level of oxygen used in the atmosphere could affect the requirement for glucose to obtain the blastocyst stage in the mouse. Whether such phenomena also occur with the human embryo is unknown but it has been observed that the complete preimplantation development of human zygotes to fully expanded blastocysts can occur in vitro in the absence of exogenous glucose in Basal XI HTF medium, using glucose-free exogenous protein, and that these blastocysts have given rise to live births following cryopreservation, thawing and subsequent transfer (Quinn, 1995 and unpublished data). 177

P.Quinn and RC.Horstman Base + Gin & EDTA 3 Base + EDTA Base + Gin I... ;i Base + Pi & Glu I. ~~~1 Base + Glu Media i, i Base + Pi i ; 1 Base I ' 1 + EDTA & Gin J +Gln + EDTA -\ 1 1 1 h 0 10 20 30 40 50 60 70 80 90 100 % FEBs Figure 2. Development of zygotes from CF1 female mice to fully expanded blastocysts (FEBs) in various combinations of glucose (Glu; 2.8 mm), phosphate (Pi; 0.37 mm), EDTA (0.1 mm) and glutamine (Gin; 1.0 mm). The 'Base' medium was devoid of glucose, phosphate, EDTA and glutamine. These are the combined results from four separate experiments, each one replicated three to five times with a total of 66 to 122 zygotes in each treatment. Some treatments were repeated in up to three separate experiments. We have found that the use of mouse zygotes derived from random-bred CF1 females mated with B6C3 Fl males has provided a useful model to study some of the interactions between glucose (Glu), Pi, EDTA, and glutamine (Gin). A summary of the overall results from a series of experiments conducted to study these interactions is shown in Figure 2. Embryo development was inhibited in the presence of Glu and Pi. Omission of these two compounds allowed >80% of the zygotes to reach the expanded blastocyst stage (medium 'Base' in Figure 2). Addition of EDTA and Gin to Glu/Pi-free medium gave the maximum response (>90% blastocysts; top bar in Figure 2: we now refer to this medium as 'Basal XI HTF'). The addition of EDTA, Gin or both compounds to HTF medium containing Glu and Pi stimulated development; Gin alone had a more pronounced beneficial effect than EDTA alone (62 versus 31% blastocysts; control 14%), but both compounds together produced a blastocyst rate of 84%. Finally, an interesting observation was that in-house-produced HTF medium with Glu and Pi (medium Base + Pi & Glu in Figure 2) was less inhibitory than commercially prepared HTF (bottom bar, Figure 2; 33 versus 19%, respectively). This further highlights the usefulness of this assay using zygotes from randombred mice that show a partial 2-cell block in traditional culture media. Quality control testing using mouse embryos The mouse embryo bioassay has been the dominant QC assay used for human IVF over the last decade. Over 90% of the 159 assisted reproduction laboratories participating in the embryo proficiency testing survey conducted by the American Association of Bioanalysis (AAB) in May, 1997 used the 1- or 2-cell, fresh or frozen mouse embryo bioassay; 28% used human sperm motility (some laboratories ran both the mouse embryo and human sperm motility bioassays) and 2% 178

Mouse embryos as models for the human used the hamster sperm motility assay. This distribution of use may indicate not only the effectiveness of the mouse embryo QC assay over the other bioassays but could also be related to accessibility of the raw material; commercial sources of cryopreserved mouse embryos are readily available. More than 53% of the participating assisted reproduction laboratories used frozen 2-cell mouse embryos, 34% used fresh 1- or 2-cell mouse embryos and the remaining 13% used frozen mouse zygotes. There are, of course, questions regarding the sensitivity and specificity of the bioassays. The question of specificity can be basically broken down into two aspects: (i) does the bioassay detect basic cell toxicity? and/or (ii) does the bioassay measure specific aspects of preimplantation embryogenesis such as cleavage, compaction and blastocoele cavity formation? One also has to ask whether a bioassay involving fertilization in vitro per se may be more appropriate or also necessary, in addition to a bioassay involving embryo development, cell division and/or cell movement. Some negative aspects of the sperm motility bioassays are that they are not geared to assess cell division or other aspects of embryogenesis and, in the case of the hamster sperm motility assay, require additional components (penicillamine, hypotaurine and epinephrine) to be added to the medium that are essential for hamster sperm motility (Bavister and Andrews, 1988) but not for mouse or human sperm motility. On the other hand, the sperm motility assays have a short turn around time compared with the embryo bioassays. It has been noted that despite the usefulness of the mouse zygote QC assay in detecting inadequate washing of consumable items (Quinn et al., 1984) and gassing of the culture environment (Quinn et al., 1990), there was no correlation between the results of the QC assay with various batches of culture medium and human IVF events such as fertilization, cleavage and pregnancy rates (Quinn et al., 1984; Weiss et al., 1992). This observation has recently been confirmed in a much larger group of patients and involved several independent assisted reproduction programmes using the same commercial lots of culture medium. Alternatives to the QC assays in which embryo development or sperm motility are measured have been proposed. Mortimer et al. (1995) suggested an internal QC based on adequate and consistent fertilization, cleavage and pregnancy rates. Such an approach can be termed reactive rather than proactive and cannot correct problems before they arise although it can be useful to monitor the overall 'health' of an IVF laboratory on an ongoing basis. Are there benefits of prolonged culture? One of the basic premises we have always held is that conditions in vitro can never fully emulate those in vivo and that therefore it is better to replace embryos into the reproductive tract as soon as possible so that the deleterious effects of an inadequate culture system do not impinge on embryo viability. This was the rationale behind studies in which pronuclear human embryos were selected for replacement on day 1 of development (Quinn et al., 1990) and this approach 179

P.Quinn and F.C.Horstman was beneficial in salvaging pregnancy rates when culture conditions were inadequate because of poor gassing of the culture environment. However, it is well recognized that an adequate culture system can be used as a method to deselect those embryos that have a compromised potential for development in vivo because of genetic or other anomalies that only become apparent at later stages of preimplantation development (Quinn and Margalit, 1996). Extended culture also provides better chronological synchrony between the embryonic stage and the site of replacement at transfer, as well as giving more time and cells for preimplantation genetic diagnosis (Lui et al., 1993; Pickering and Muggleton-Harris, 1995). Strategies to optimize in-vitro conditions for prolonged preimplantation development involving culture media formulations have already been discussed. The strong positive role of pilot studies with mouse embryos in this strategy has been emphasized. Other tactics used in vitro in which preliminary studies with mouse embryos have indicated beneficial effects involve increasing the embryonic density to culture volume to maximize the positive effects of embryonic autocrine factors (Lane and Gardner, 1992; O'Neill, 1997). There is some indication that human preimplantation embryos also benefit from embryoproduced autocrine factors (Moessner and Dodson, 1994) and we routinely culture groups of human embryos together in small (30 jil) volumes of medium (Quinn, 1995; Quinn and Margalit, 1996). Nevertheless, the question has been asked 'If we are getting 60% pregnancy rates with embryo transfer on day 3 without 2 days of extra work (by extending culture to day 5), there needs to be something in it to add the trouble (of extended culture). In particular, culturing leftover (embryos) out to blastocysts on day 5 or day 6 always lands me on Saturday or Sunday to do the freezing' (Richard Rawlins, personal communication). Obviously, reducing multiple implantation and lowering spontaneous abortion rates by transferring embryos on day 5 must be demonstrated to support the use of this strategy on a routine basis. The transfer of human embryos on day 5 in combination with complete removal of the zona pellucida has been suggested as a means of increasing pregnancy rates in women who have repeatedly failed IVF when embryo transfer has been carried out at earlier stages (Trounson et al., 1997). One final aspect of human embryo culture that is assuming an increasing area of research and concern is the inclusion of polymers, traditionally serum albumin, in culture media for all aspects of assisted reproduction, from oocyte collection, through fertilization, cleavage, blastocyst culture, embryo transfer, cryopreservation and micromanipulation. Major problems have arisen however, because donors who have supplied the raw material used to produce the final albumin product have had increased risk of exposure to and, in one known case, even death because of lethal infective agents such as those causing Creutzfeldt- Jakob disease (CJD) and acquired immunodeficiency syndrome. Fortunately, a solution is at hand and, dare we say, the mouse has proved to be an excellent model in the development of alternative strategies to adding albumin to culture medium. Ann Kiessling and her group presented a paper at the 1997 ASRM meeting (Serta et al., 1997) in which they showed that protein is not necessary 180

Mouse embryos as models for the human in IVF culture medium containing EDTA and they conclude that the use of fully defined, protein-free culture medium will markedly improve the safety of embryos and mothers by avoiding exposure to potential pathogens in serum and albumin (such as CJD). We have confirmed some of Kiessling's work on a few patients and have also obtained fertilization and embryo cleavage in protein-free medium. In addition to a series of papers published from Kiessling's laboratory, we and others have also shown that mouse embryos and those of other species will cleave in protein-free medium, especially in the presence of EDTA (e.g. see Quinn, 1995). In another study with Ruth Margalit (unpublished), we have also shown that initial human sperm motility and velocity are not decreased in proteinfree medium and so this approach should be able to be used for intrauterine insemination sperm preparations as long as the washed spermatozoa are inseminated reasonably soon after preparation (within ~60 min). We believe this will be an area of intensive clinical work over the next few years. Summary Some of the historical benefits of using mouse embryos as models for human preimplantation development in vitro have been discussed. In addition, personal bias plays some part in why we contend that the mouse is still a well-suited tool to monitor and improve the in-vitro conditions used for human embryos. The use of other species such as the hamster, non-human primates and domestic species is not to be overlooked, and obviously research on donated human embryos is essential. References Barnett, D.K. and Bavister, B.D. (1996) What is the relationship between the metabolism of preimplantation embryos and their developmental competence? Mol. Reprod. Dev., 43, 105-133. Bavister, B.D. (1997) Non-invasive evaluation of embryo quality and viability. In Gomel V. and Leung, P.C.K..(eds), In Vitro Fertilization and Assisted Reproduction. Monduzzi Editore, Bologna, pp. 163-169. Bavister, B.D. and Andrews, J.C. (1988) A rapid sperm motility bioassay procedure for qualitycontrol testing of water and culture media. J. In Vitro Fertil. Embryo Transfer, 5, 67-75. Biggers, J.D., Summers, M.C. and McGinnis, L.K. (1997) Polyvinyl alcohol and amino acids as substitutes for bovine serum albumin in culture media for mouse preimplantation embryos. Hum. Reprod. Update, 3, 125-135. Borland, R.M., Biggers, J.D., Lechene, C.P. and Taymor, M.L. (1980) Elemental composition of fluid in the human fallopian tube. J. Reprod. Fertil., 58, 479-482. Brinster, R.L. (1972) Cultivation of the mammalian embryo. In Rothblat, G.H. and Cristofalo, V.J. (eds), Growth, Nutrition, and Metabolism of Cells in Culture. Academic Press, New York, Vol. 2, pp. 251-286. Chatot, C.L., Ziomek, C.A., Bavister, B.D. et al. (1989) An improved culture medium supports the development of random-bred 1-cell mouse embryos in vitro. J. Reprod. Fertil., 86, 679-688. Cohen, J., Malter, H.E., Talansky, B.E. and Grifo, J. (1992) Micromanipulation of Human Gametes and Embryos. Raven Press, New York. Conaghan, J., Handyside, A.H., Winston, R.M.L. and Leese, H.J. (1993) Effects of pyruvate and 181

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Mouse embryos as models for the human Quinn, P. (1995) Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate. J. Assist. Reprod. Genet., 12, 97-105. Quinn, P. (1997) Tubal fluid and its significance in human reproduction. In Coutifaris, C. and Mastroianni, L. (eds), New Horizons in Reproductive Medicine. Parthenon Publishing, New York, pp. 255-260. Quinn, P. and Margalit, R. (1996) Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos. J. Assist. Reprod. Genet., 13, 9-14. Quinn, P. and Wales, R.G. (1973) The effect of culture in vitro on the levels of adenosine triphosphate in preimplantation mouse embryos. J. Reprod. Fertil., 32, 231-241. Quinn, P., Stone, B.A. and Marrs, R.P. (1990) Suboptimal laboratory conditions can affect pregnancy outcome after embryo transfer on day 1 or 2 after insemination. Fertil. Steril., 53, 168-170. Quinn, P., Warnes, G.M., Kerin, J.F. and Kirby, C. (1984) Culture factors in relation to the success of human in vitro fertilization and embryo transfer. J. Reprod. Fertil., 41, 202-209. Quinn, P., Moinipanah, R., Steinberg, J.M. and Weathersbee, P.M. (1995) Successful human in vitro fertilization using a modified human tubal fluid medium lacking glucose and phosphate ions. Fertil. Steril., 63, 922-924. Scott, L. and Whittingham, D.G. (1996) Influence of genetic background and media components on the development of mouse embryos in vitro. Mol. Reprod. Dev., 43, 336-346. Serta, R.S., Sakellariou, M. and Kiessling, A.A. (1997) Outcome of human embryos conceived and cleaved in protein-free culture conditions. Proceedings of the 53rd Annual Meeting of the American Society for Reproductive Medicine, Abstract P-225. Trounson, A.O. and Gardner, D.K. (1993) Handbook of In Vitro Fertilization. CRC Press, Boca Raton. Trounson, A., Jones, G., Kausche, A. et al. (1997) Blastocysts: their development in vitro and in vivo. J. Assist. Reprod. Genet., 14, 19S. Trounson, A.O., Mohr, L.R., Wood, C. and Leeton, J.F. (1982) Effect of delayed insemination on in-vitro fertilization, culture and transfer of human embryos. J. Reprod. Fertil., 64, 285-294. Van Blerkom, J., Davis, P. and Lee, J. (1995) ATP content of human oocytes and developmental potential and outcome after IVF-ET. Hum. Reprod., 10, 415-424. Wales, R.G. (1975) Maturation of the mammalian embryo: biochemical aspects. Biol. Reprod., 12, 66-81. Weiss, T.J., Warnes, G.M. and Gardner, D.K. (1992) Mouse embryos and quality control in human IVF. Reprod. Fertil. Dev., 4, 105-107. Whitten, W.K. (1956) Culture of tubal mouse ova. Nature, 111, 96. 183