Recent advances in the production of viable human embryos in vitro

RBMOnline - Vol 4. No 3. 294–302 Reproductive BioMedicine Online; www.rbmonline.com/Article/338 on web 11 February 2002

Reviews Recent advances in the production of viable human embryos in vitro*

Dr Thomas Pool

Thomas ‘Rusty’ Pool was the featured editor in a previous issue of Reproductive BioMedicine Online (Volume 4, number 2). Briefly, his academic career has been wideranging, including a BSc in agriculture and an MA from Sam Houston State University in his native Texas. His thesis research focused upon gametogenesis in invertebrates and he was awarded his PhD in developmental biology from the University of Virginia in Charlottesville, Virginia in 1976. Dr Pool began collaborative studies on in-vitro fertilization in non-human primates in 1982. This work culminated in the live birth of the first cynomolgous monkey produced by IVF and the first live birth of a non-human primate resulting from the transfer of a frozen–thawed embryo produced by IVF. In 1990, he helped found the Fertility Center of San Antonio, a private, office-based programme and currently serves as the Scientific Director. He is active in the ASRM, the American Association of Bioanalysts (AAB) and the Pacific Coast Fertility Society. His research interests include improving embryo culture media and the in-vitro growth environment. He now serves as a member of the editorial board of Reproductive BioMedicine Online.

Thomas B Pool Fertility Center of San Antonio, San Antonio, Texas 78229, USA Correspondence: Tel: 210-614-3232; Fax: 210-692-1210; e-mail: [email protected]

Abstract The introduction of stress to embryonic blastomeres through inappropriate culture conditions results ultimately in the loss of viability. Retention of normal metabolic function in human preimplantation embryos, as well as those of other mammalian species, has been improved by the use of stage-appropriate culture media wherein energy substrates and amino acids are provided in a temporally evolving sequence. While the time dependence of nutrient exposure to embryos has received wide attention, spatial considerations in the embryonic microenvironment have received none. The manner in which media are presented to embryos, the rate at which media are changed, the rate at which cell products are removed and the macromolecular influences upon embryonic microenvironments have received far less attention in the experimental literature. Recent advances in micro-scale engineering allows for the rapid production of matrices containing culture channels slightly larger than the dimensions of preimplantation embryos. Microfluidic systems hold great promise for providing physical configurations yielding significantly reduced volumes but simultaneously providing control over the dynamics of media change and waste removal via fluid flow with time. Additionally, macromolecules may be presented from fixed sites in a minimum volume of solvent thus allowing us to test the importance of space and geometry in facilitating the physical and chemical effectiveness of the embryonic milieu. Keywords: embryo culture, sequential media, macromolecules, microfluidics, plasticity

Introduction

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The success rates from in-vitro fertilization and embryo transfer in humans have risen dramatically since the birth of Louise Brown in 1978, because of improvements in ovulation induction medications and regimens and improvements in culture technology, including culture media. The improvements realized through the use of better culture media, however, are relatively recent given the approximate 23-year history of clinical IVF. Although embryo-specific culture media, developed in a host of animal models, have been widely available to fertility clinics over this time period, most began operation by employing a single medium that was borrowed from somatic cell culture technology, either in the form of a simple salts, such as Earle’s Balanced Salt solution (EBSS) or a complex medium such as Ham’s Nutrient Medium F10. Through the work of Quinn et al. (1985) and Menezo et *Paper based on contribution presented at the Alpha meeting in New York, USA, September 2001.

al. (1984), media designed explicitly for human embryo culture became available in the mid 1980s, a boon for the then current practice of transferring cleavage-stage embryos to the uterus, but not for blastocyst production. Co-culture was introduced into the clinical setting as a way to satisfy the demands for higher pregnancy rates and remained in vogue for nearly 10 years as the method of choice for producing viable blastocysts for fresh transfer or for cryopreservation. There are now, however, commercially available media systems, formulated on the basis of the temporally evolving nutritional requirements of the human preimplantation embryo, which routinely allow for the growth of viable blastocysts without the need for supplementation with whole serum or the use of somatic cell support (Gardner, 1994; Gardner and Lane, 1997; Pool et al. 1998; Gardner et al. 2000; Pool and Martin, 2001) for comprehensive reviews).

Reviews - Production of viable human embryos in vitro - TB Pool

The goal of this summary is to identify some of the recent advances in culture media and technology that are facilitating the routine production in vitro of late-stage, viable human embryos, to describe the unifying principles of sequential culture technology that are emerging from an analysis of clinical outcomes and to suggest future improvements to current culture technology.

Extended embryo culture: promise and problems The benefits to programmes of assisted reproduction of being able to routinely produce and transfer viable blastocysts are numerous and have been discussed in depth in the literature (Bavister and Boatman, 1997; Behr, 1997; Gardner and Lane, 1997; Desai, 1998; Gardner and Schoolcraft, 1998; Menezo et al. 1998; Pool et al. 1998; Quinn, 1998; Tsirigotis, 1998). The restoration of spatial and temporal synchrony of uterus and embryo, the opportunity for better embryo selection, the potential for sophisticated diagnostic testing, such as preimplantation genetic diagnosis (PGD) or metabolic screening of embryos, and the realization of improved implantation rates are some of the more compelling reasons for blastocyst transfer. The most important benefit, however, is the retention of high clinical pregnancy rates with a simultaneous reduction in the incidence of multiple gestation through the transfer of fewer late-stage embryos. The immediate need to reduce multiple pregnancy in human assisted reproductive technology has been illustrated succinctly by Schnorr and Jones (2001) in a recent review. In less than 20 years in the United States, the incidence of twin deliveries has risen by 52% whereas single births rose by only 6%. The incidence of triplet and higher order gestations, however, increased by 404% over the same time period. Most of these multiple gestations arose through fertility therapy. Although the goals of blastocyst production are well defined, the classic approach of using a single medium throughout the entire culture period has proven ineffective, as it fails to recognize that the physiology of preimplantation embryos is dynamic. The earliest efforts to produce viable human blastocysts for transfer demonstrated this concept clearly. For example, Bolton et al. (1991) used EBSS supplemented with heat-inactivated patient serum to grow embryos of patients who were under 35 years of age, with tubal occlusion as the sole source of infertility, for either 2 or 5 days before transfer of a maximum of three embryos. The transfer of cleavagestage embryos on day 2 resulted in a pregnancy rate of 24% per embryo transfer, a live-birth rate of 16% per transfer and an implantation rate of 9%. After 5 days of culture, 70% of the embryos reached the morula stage and 40% became blastocysts but the pregnancy and live-birth rates per transfer were only 10% and 10%, respectively, with an implantation rate of 7%. In a later study, Huisman et al. (1994) examined the production of late-stage embryos using a mixture of EBSS and Ham’s Nutrient Mixture F-10 in a ratio of 17:3. This investigation involved the culture of nearly 6000 embryos and analysed the outcomes of over 2000 transfers. When pregnancy and implantation rates were compared overall, no differences were seen statistically between 2, 3 and 4 days of culture. When implantation rates were stratified by the

normality of growth rates on days 2, 3 and 4 of transfer, a different story emerged. The percentage of embryos that maintained either a normal or advanced developmental rate on days 2 and 3 was similar (60.5% and 54.2%, respectively) but the percentage of embryos showing normal or advanced development by day 4 fell precipitously to 18.4%. These embryos implanted at a rate of 41% compared with a significantly reduced rate of 11% for growth-retarded embryos transferred on day 4. Implantation rates for normally developing embryos transferred on days 2 and 3 were significantly lower at 18.2% and 19.2%, respectively. From both studies, it is clear that the efficiency of embryonic production upon extended culture in a single medium drops, but that viability falls even more precipitously. Of equal clarity though is the finding that four out of five embryos fail to demonstrate a normal developmental rate in vitro using the culture conditions or single medium approach.

The physiological basis of sequential culture Data from a wide variety of mammalian embryonic systems clearly support the notion that the preimplantation period is divisible into discrete stages with specific characteristics, and humans are no exception. Leese (1995) indicates that preimplantation development may conveniently be divided into two phases, early and late, where the early embryo is undifferentiated, non-vascularized, shows no net growth, is controlled by maternal RNA and is largely insensitive to exogenous hormones and growth factors. The late preimplantation embryo is under genomic regulation and shows net growth, differentiates trophoblast and inner cell mass and responds to extracellular modulators of growth, such as hormones and growth factors. Metabolic control of the embryo, in this consideration, can be categorized into intrinsic and extrinsic levels. Pool and Martin (2001), have shown that human preimplantation development falls into at least three, discrete stages when as few as four variables are considered (predominate energy source, level of genetic regulation, mode of cell cycle regulation, amino acid requirement). The recent development of sequential culture media, such as media G1 and G2 developed by David Gardner (Barnes et al., 1995), is based on the notion that maintaining viability of late-stage human embryos is fostered, at least in part, by exposing embryos to nutrients in culture that are encountered by embryos in vivo in a temporally similar manner. Other versions of contemporary sequential media, such as P-1 medium formulated by this author (Pool et al., 1998) although ultimately conforming to this model, were devised independently by modifying an existing medium, human tubal fluid (HTF), based solely upon prior results obtained in animal models. Blastocyst Medium, as developed by Behr et al. (1999), likewise is a modification of Ham’s F10 that was shown to yield viable blastocysts when used as a second medium following initial culture in P-1 medium. There are now a number of commercially available sequential media combinations available to IVF laboratories, and G1/G2 and P-1/Blastocyst Medium are offered simply as examples of diverse formulae that work by addressing blastomere homoeostasis. As Leese et al. (2001) explain, the metabolic

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pathways supporting early embryonic development are not strict, obligatory ones. Instead, embryos have ‘preferred’ pathways, retaining the capacity to use alternative nutrients and pathways if the preferred ones are unavailable. This gives the early embryo the ability to remain viable in the face of suboptimal nutrient availability, a quality known as embryonic plasticity, and explains why several different sequential culture systems, although differing in composition, have proved to be equally successful in supporting the growth of viable blastocysts in vitro (Gardner et al., 2000). Although the rationale behind sequential media is that a single medium cannot meet the dynamic physiological needs of the embryo, what evidence suggests that the nutritional environment of the preimplantation human embryo in vivo changes in significant way? Hardy (1993) measured glucose and pyruvate uptake in human embryos and showed that glucose uptake was constant from the 2-cell to 16-cell stage, then more than doubled to 24 pmol per embryo per hour at the blastocyst stage. Conversely, pyruvate uptake was seen to increase from the 2-cell to 4-cell stage, then peak at the morula stage. Conaghan et al. (1993) subsequently showed that excluding pyruvate in the presence of 1 mmol glucose allowed only 16% of spare 2- to 4-cell embryos to progress to blastocysts, whereas restoring pyruvate to 0.47 mmol while lowering glucose to 0.5 mmol, or eliminating it completely, resulted in 48% of embryos to reach the blastocyst stage. Measures of viability, however, were not made in this study. The most important study providing direct evidence that human embryos experience profoundly different nutritional environments during preimplantation embryogenesis in vivo was conducted by Gardner et al. (1996). Metabolite concentrations, including glucose, pyruvate and lactate, were measured in the reproductive tract in human females at different stages of the menstrual cycle. From these measurements, it became clear that cleavage-stage embryos, while in the Fallopian tube around mid-cycle, are exposed to a low glucose (0.5 mmol) environment but transcend into a high glucose environment (3.15 mmol) as late-stage embryos in the uterine cavity. Additionally, the levels of pyruvate and lactate are higher in the Fallopian tube at mid-cycle than in the uterus, with pyruvate concentrations dropping by one-third (0.32 mmol to 0.1 mmol) and lactate falling by about a half (10.5 mmol to 5.87 mmol). These data provided the basis for metabolite concentrations in Gardner’s G1 and G2 media. As indicated above, the concentrations of these same metabolites used in P-1 medium were derived from animal embryo data. Pyruvate and lactate levels were included in HTF, the progenitor medium of P-1, by Quinn et al. (1985) based upon experiments with mouse embryos. The omission of glucose and phosphate in P-1 was based upon earlier work in the hamster embryo by Schini and Bavister (1988) and Barnett and Bavister (1996).

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Despite there being two unrelated routes to formulating G1 and P-1 media, the ultimate concentrations of pyruvate and lactate are very similar and both feature reduced glucose levels (0.5 mmol in G1 or total elimination in P-1) relative to earlier formulations used for human embryo culture. A more detailed review of the evolution of energy substrates used in human embryo culture media is given by Graham and Pool (1997).

Another crucial facet of culture medium design involves the inclusion of an array of amino acids that are appropriate for a given embryonic stage. Gardner and colleagues have long noted the striking similarity between the amino acids found in the oviduct of the mouse and those described experimentally and categorized as ‘non-essential’ by Eagle. In a host of meticulous investigations with mouse embryos, best summarized by Lane and Gardner (1997), the nature of an evolving amino acid requirement during preimplantation development is described. By testing the effects of including amino acids in groups, using the nomenclature of Eagle, a preferential sequence of exposure to amino acids was defined, which maximized not only embryogenesis but also viability, upon subsequent embryo transfer. The addition of nonessential amino acids plus glutamine in G1 medium, followed by the inclusion of all 20 amino acids in G2, yielded not only the production of viable blastocysts in the mouse model, but in humans as well (Barnes et al. 1995). The importance of including amino acids in both culture intervals of the sequential approach has been recently confirmed directly in humans by Devreker et al. (2001). In this study, 129 human embryos were randomized into three experimental arms that compared different sequential systems. In the first arm, EBSS without glucose was supplemented with only glutamine for the first culture interval. The second culture interval (days 4–6) was done in the same medium, but in the presence of 1 mmol glucose. In the second group, glucose-free EBSS was supplemented with non-essential amino acids plus glutamine for the first interval and all 20 amino acids plus glucose and glutamine for the second interval. The third arm used a commercially available media system (K-SCIM) based upon the formula of HTF, supplemented with amino acids in a manner similar to the second arm. The percentage of embryos reaching the blastocyst stage was not statistically different between each combination, but total cell number in day-6 blastocysts was significantly lower when glutamine was the sole nitrogen source. Additionally, an analysis of apoptotic nuclei, characterized by the presence of discrete clusters of nuclear fragments labelled with fluorescent dye, allowed for the calculation of a dead cell index from embryos of each arm. This index was significantly lower in EBSS with the complete array of amino acids. In earlier work, Bavister and McKiernan (1993) examined the ability of single amino acids to support embryogenesis of hamster embryos and compared the results obtained with those seen when glutamine is included as the sole nitrogen source. It is interesting that those amino acids yielding significantly improved results, although tested as single additives, were similar to the non-essential group of Eagle. The most significant improvement, however, was observed when the amino acid taurine was used as the sole nitrogen source. For this reason, taurine was included in the formula of P-1 medium. Blastocyst Medium, which is most often used as the second medium in sequence with P-1, contains a complete array of all 20 amino acids. In all instances, the characteristics of sequential media used for human IVF have been improved by the inclusion of a macromolecule, although the exact roles of macromolecules have been woefully underestimated.

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Macromolecules and plasticity of the embryonic microenvironment It is evident that the design and application of stageappropriate culture media in a temporally appropriate manner, coupled with strict attention to maintaining constant physical conditions in the embryo culture system, reduce the chance for introducing an imbalance in blastomere homoeostasis. Embryonic plasticity provides metabolic flexibility but it has limits, and thus it is the breadth of intrinsic plasticity that determines viability under suboptimal conditions in vivo and in vitro. One emerging concept, however, deserves further consideration and it is that the ability to respond to adversity, introduced during the culture of early cleavage-stage embryos, is not the same upon continued culture in vitro as it is in vivo following embryo transfer. There are examples where viable blastocysts are generated in vivo, as evidenced from pregnancy, from cleavage-stage embryos of low quality by uterine transfer on day 3 whereas they are not by continued culture to day 5 or day 6 in vitro (Racowsky et al., 2000). It seems, therefore, that plasticity exists in the embryonic microenvironment and that this extra-embryonic plasticity is a quality of the uterine environment, not duplicated in vitro by some contemporary culture methods. The authors have postulated that native macromolecules of the reproductive tract, such as mucins and other glycosaminoglycans, may not only yield physical-chemical conditions that support cellular homoeostatic mechanisms (Pool and Martin, 1994), but also provide chemical responsiveness to imbalances in embryonic physiology, rendering an element of responsiveness or plasticity to the extracellular microenvironment (Pool et al., 1998; Pool, 2001). This idea is not novel and Hunter (1994) has offered a similar and even extended view of macromolecular influences on preimplantation embryogenesis. That our knowledge of embryo physiology and our ability to generate viable late-stage human embryos in vitro have increased profoundly as a result of using defined components in culture media is clear. However, the use of a single macromolecule, such as albumin, as a supplement to embryo culture media, although consistent with the strategy of definition, may well simultaneously eliminate potential microenvironmental plasticity. In fact, the addition of any array of macromolecules in solution, where the behaviour of bulk water predominates, may fail to mimic macromolecular–solute interactions that exist in vivo. The preimplantation embryo in vivo encounters macromolecules from fixed sites as well as those in solution. One of the more ubiquitous classes of macromolecules in the reproductive tract is mucins, a polydisperse collection of glycoproteins encoded by at least 10 genes that are characterized by non-ideality and exist either in secreted forms or in those anchored into epithelial cells by a cytoplasmic tail. Given the dense packing of O-linked sugars emanating from the single peptide backbone of mucins via serine and threonine residues, the most frequent embryo–macromolecular interaction with molecules from this family of glycoproteins is an embryo–carbohydrate interaction, not an embryo–protein interaction.

Although compelling, the ability to test the hypothesis that environmental chemical plasticity is mediated by glycosylated macromolecules of the reproductive tract is thwarted as much by engineering considerations as it is by biological limitations. Even if purified mucins and other glycosylated protein species were available for potential investigation, the physical presentation of these molecules to preimplantation embryos is problematic. To mimic the physical–chemical conditions present in vivo, a potential culture system must able to present an array of macromolecules, an assemblage that changes qualitatively with time, from fixed sites in a minimum volume of water. Only in this way can the thermodynamic non-ideality of solutions produced in vivo be approached in vitro. This concept is considered further in the ‘future considerations’ section of this review.

Outcomes in human assisted reproduction treatments using sequential culture technology An increasing number of reports now substantiate that sequential culture systems are capable of producing viable human blastocysts that yield superior pregnancy and implantation rates when compared with cleavage stage embryos. Jones et al. (1998) compared several media in different combinations to produce an effective sequential system for the production of viable blastocysts. In the most successful protocol, a modified HTF medium supplemented with human serum albumin, was used for the first interval and Gardner’s G2 medium was used for the second. A continuing pregnancy rate of 38.2% was seen following the transfer of fresh blastocysts, when this culture system was used in combination with a strategy of regrouping embryos with similar growth patterns and completely removing the zonae of blastocysts with pronase digestion before transfer. In the same month, Gardner et al. (1998a) published preliminary results with 23 patients comparing the results of day 3 (cleavage stage) versus day 5 transfer (blastocyst). The conversion rate to blastocysts was significantly higher when blastocysts were produced using G1/G2 media (66%) compared with Ham’s F10 supplemented with fetal cord serum followed by G2 medium (38%). Pregnancy rates for day 5 were 63% compared with 47% for day 3 transfer and fewer embryos per patient were transferred on day 5 versus day 3 (2.7 and 3.8, respectively). The implantation rates were 21% per embryo for day 3 and 45.5% for day 5 transfers. These observations were then expanded in a prospective, randomized trial of blastocyst transfer (Gardner et al., 1998b) involving 92 patients who showed moderate to good response to ovulation induction. Pregnancy rates were not different from day 3 (66%) to day 5 (71%), but the average number of embryos transferred was less on day 5 (2.2) versus day 3 (3.7). Implantation rates were 30.1% for day 3 and 50.5% for day 5 transfer. It should be noted that these results are from a selected patient population, namely ones that respond appropriately to ovulation induction. Other programmes use different inclusion criteria for blastocyst transfer, such as requiring a minimum number of eight-cell embryos on day 3, usually three or more. Using the latter criteria, Milki et al. (1999) reported that an

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equivalent implantation rate (47%) and ongoing pregnancy rate (58–62%) could be achieved when two blastocysts were transferred compared with three blastocysts. These results were obtained using a P-1/Blastocyst Medium, a system previously shown to yield 54% blastocysts from day 3 to day 5 when starting with five-cell or greater embryos on day 3 (Behr et al. 1999). In a subsequent study of 100 patients, Milki et al. (2000) reported significant improvements in ongoing pregnancy (46% versus 68%) and implantation rates (20% versus 47%) again comparing day 3 transfer with day 5. However, these too are selected patients and data such as these beg the question of the utility of blastocyst transfer in clinical IVF, without rigorous patient selection. Racowsky et al. (2000) suggest that the decision for transfer day should be based on culture performance on day 3, as no pregnancies were obtained in their hands if embryos with less than eight cells were grown until day 5 then transferred compared with transferring them on day 3 where a 33% pregnancy rate was seen. Other groups have reported conflicting results and have taken all patients to blastocysts transfer. Schoolcraft et al. (1999) applied no selection criteria to 174 patients where all embryos were grown to blastocyst and had only 3 patients fail to have an embryo transfer on day 5. The ongoing pregnancy rate was 66.3%, the implantation rate was 48%. Marek et al. (1999), reviewed 790 non-selected patients where day 3 outcomes (n = 477) are compared with those for day 5 transfer (n = 313), and saw the ongoing pregnancy rate increase from 36% to 44% and the implantation rate increase from 23% to 33% with day 5 transfer. The cancellation rate, however, also increased from 2.9% to 6.7%. Clearly, both patient and embryonic characteristics influence the success of blastocyst transfer. Langley et al. (2001) reported a higher blastocyst conversion rate from pronuclear embryos in egg donation patients (67%) compared with IVF patients (52%) using their own oocytes. The implantation rate was also lower in IVF patients (30%) compared with those receiving embryos from donated eggs (54%). Nonetheless, it is a certainty that clinical outcomes are dramatically improved by our ability to now culture late-stage, viable human embryos using sequential systems with day 5 transfer compared with the transfer of cleavage-stage embryos.

Other elements of the culture system Measurements of oxygen tension in the reproductive tracts of a number of mammalian species is reported to be significantly lower than ambient air, ranging from 1.5% to 8%, which may explain the enhancement in embryogenesis that has been observed using reduced oxygen tensions during culture in a number of studies on a variety of species (Gardner and Lane, 1999).

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This is particularly well illustrated in the recent studies of Lim et al. (1999) where a significant stimulation of embryogenesis at the two-cell, 16-cell, morula and blastocyst stages was seen along with a significant improvement in the 8-cell to blastocyst conversion rate by growing bovine embryos in 5% O2, 5% CO2, 90% N2 versus 5% CO2 in air in a glucose-free medium

containing minimum essential medium (MEM) amino acids and haemoglobin. Although many clinical laboratories have elected to culture human embryos under low oxygen conditions for years, quantitative measures on the effects of low oxygen tension with respect to the efficiency of production of late-stage embryos have appeared only recently. Behr et al. (1999) showed equivalent blastocyst conversion rates from day 3 human embryos grown in 5% and in 20% oxygen; however, blastocyst cell number, cell allocation to the inner cell mass and viability were not scored. More recently, Dumoulin et al. (1999) compared embryo growth, pregnancy and implantation as a function of oxygen tension during culture in 1380 IVF cycles. No differences were seen in fertilization rate, embryo development on day 2, embryo development on day 3, pregnancy and implantation. A significant increase in blastocyst formation, derived from examining supernumerary embryos cultured to day 5 or 6, was seen under reduced oxygen conditions. Particularly instructive was the observation that a large number of blastocysts produced in the presence of 20% oxygen had abnormally low cell numbers. Gardner et al. (1999) looked at growth in 5% or 20% oxygen using thawed pronuclear human embryos and reported a significant increase in cell number on day 3 and at the blastocyst stage, although the number of blastocysts produced were equivalent. Meintjes et al. (2000) recently showed that significantly more patients had blastocysts cryopreserved when cultured under reduced oxygen concentrations compared with 20%. At the present time, available data suggest that superior performance characteristics of contemporary culture systems are realized under reduced oxygen tensions and, although it is assumed that low oxygen conditions suppress damaging oxygen radical formation, the mechanism behind this phenomenon has yet to be demonstrated conclusively. Although it appears that lowered oxygen concentrations produce favourable outcomes for cultured human embryos, the exact concentration of CO2 for use in culture systems, and thus the proper external pH, is medium dependent and potentially stage dependent. Dale et al. (1998), using fluorescence technology, measured intracellular pH (pHi) in unfertilized human oocytes to be 7.4. Further, they found fertilization to be pH dependent, peaking at 7.5. In additional experiments, they found pHi to be strongly dependent upon external pH, measuring an ability of oocytes to recover from alkalosis, but unable to recover from intracellular acidification induced by exposure to low external pH. From their work, they conclude that there is a mechanism in human oocytes for recovery from alkalinization, likely through a HCO3–/Cl– exchanger. In contrast, however, was the finding that human oocytes and early embryos are unable to regulate acid shock until the blastocyst stage of development, indicating that embryos are highly permeable to H+ and do not possess an antiporter system to remove them when in excess. Phillips et al. (2000) used alternative methods to measure pHi in human oocytes and cleavage-stage embryos (2-cell to 8cell) and found it to be stable, averaging 7.12. In contrast to the

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findings of Dale et al. (1998), the average pHi of metaphase II oocytes was 6.98. A HCO3–/Cl– exchanger was identified that prevents alkalosis with a set-point of pH 7.2–7.3. Also in the presence of HCO3–/CO2, they discovered what appears to be two mechanisms that prevent acidosis. One, a Na+/H+ antiporter, activates below pH 6.8 whereas the second, a bicarbonate-dependent mechanism, activates below 7.0.

(0.33 mmol) should be included. The amino acid taurine should be included at a minimum. Additional non-essential amino acids may be included but essential amino acids should be omitted from the first interval. First-interval media should include a protein source that is either albumin alone or in combination with α- and β-globulins.

The second culture interval, days 3–5/6 In view of these findings, it is possible that Dale and colleagues did not use a low enough extracellular pH to detect the mechanisms that alleviate intracellular acidosis. From the work of Phillips et al. (2000), it appears that cleavage-stage human embryos possess mechanisms that enable intracellular pH to be maintained within the range of 7.0 to 7.3, consistent with their finding of an average pHi of 7.12. These data do not support the popular notion that the proper extracellular pH for culture medium is 7.4, and agrees with the empirical observation that embryogenesis is far more efficient at a reduced pH. Similarly, it is likely that metabolic stress is reduced when external pH falls within the range of internal pH without activation of the exchangers or antiporters. What remains to be determined is if there is a practical benefit of achieving fertilization at higher pH, as suggested by the data of Dale et al. (1998), followed by culture at reduced pH. It is clear, however, that excursions in pHi are detrimental to mammalian embryonic development, an important consideration in the design and application of contemporary culture technology. Experiments by Squirrell et al. (2001) in the hamster embryo have recently shown that acidification and alkalinization of pHi disrupt cytoplasmic organization and reduce developmental competence, thus establishing a critical link between cellular homoeostasis, cellular structural integrity and embryonic developmental potential.

Unifying principles for the extended culture of human embryos The development and application to clinical IVF of successful embryo culture systems has been extensively reviewed (Gardner, 1994; Gardner and Lane, 1997; Pool et al. 1998; Gardner et al. 2000; Pool and Martin, 2001). From these compilations, a few consistent principles for the culture of late-stage, viable human embryos have emerged (Pool and Martin, 2001). These are summarized below.

Sequential culture systems The physiology of the preimplantation embryo is dynamic. Components that are required at later stages of culture, such as elevated glucose levels or a complex array of amino acids, may produce suboptimal outcomes if included at earlier stages of culture. Therefore, the maximum number of viable, late-stage human embryos is produced through the use of a sequential culture system.

The first culture interval, days 1–3 In this first interval, glucose should be reduced to 0.5 mmol or omitted completely. In the presence of amino acids and EDTA, glucose at low concentration shows no adverse effect on embryo growth. Lactate (10.5 mmol L-form) and pyruvate

In the second interval, the glucose concentration should be raised to at least 2.5 mmol. A complex array of all 20 amino acids should be included, as well as pyruvate at a concentration of at least 0.1 mmol. The protein requirement is identical to that of the first culture interval.

Convergent evolution of embryo culture media Gardner et al. (2000) have recently suggested that culture media, such as G1 and P-1, although seemingly dissimilar in composition and derived through very different approaches, each work well because neither produce metabolic conditions exceeding the limits of developmental plasticity that underwrite embryonic viability. From this, it follows that other culture systems may well do the same.

Future considerations Perhaps the simpler goal in improving outcomes using cultured embryos, at least from the perspective of experimental design, is to attempt to identify the limits of developmental plasticity and those conditions that move embryos past those limits. To what extent clinical outcomes can be improved solely through enhanced culture systems is unclear, but appears to be somewhat limited. Although experimental investigations over the past 10 years to produce defined culture conditions have helped tremendously in establishing the nutritional requirements of preimplantation embryos through the use of defined media, they have simultaneously isolated the potential source of plasticity during development solely to the embryo, eliminating it from the microenvironment (Pool, 2001). The next several years will tell us if efforts aimed at improving clinical outcomes are best directed towards defining absolute nutritional requisites, restoring microenvironmental plasticity or selecting embryos for transfer based upon individual measures of viability. One major, recurring concern in the development of novel culture media and conditions for human embryos is the ultimate effect that culture components will have upon the health of embryos, fetuses and resulting offspring. That profound effects upon gene expression have been measured as a function of culture medium composition in the mouse (Khosla et al. 2001) and in cattle (Wrenzycki et al. 2001) serves as a dramatic ‘wake-up call’ to human embryologists. Indeed, the use of quantitative gene expression assays to measure embryonic response to medium components may well be not only the most effective means of improving efficacy, but also safety in the propagation of human embryos in vitro.

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The problem with the microdrop: spatial considerations in embryo culture As indicated earlier, finding improvements to embryo culture systems may be as much of an engineering problem as one involving chemistry. Attempts in our laboratory to generate conditions like those in vivo by modifying existing microdrop culture technology, where embryos are exposed to a twodimensional substrate below and a fluid phase that approaches ideality above have not proven fruitful, despite imposing chemical modifications of the culture substrate that are more akin to those encountered by embryos in the reproductive tract. The problem is two-fold but hinges on discovering and reproducing the correct macromolecular array in vitro. First, the presentation of high concentrations of a compound, such as macromolecular carbohydrates, is achieved in nature in a way that circumvents the problem of solubility serving as the limit to ultimate concentration. Oligosaccharides in the reproductive tract eminate from fixed sites, through O-linkage to the amino acids threonine and serine, so that the concentration of carbohydrates is determined genetically in the amino acid sequence of the gene encoding the glycoprotein. In current culture practice, even if an appropriate complex of macromolecules is tested in modified micro-drop culture as a substrate, the sheer volume of water comprising the smallest of drops will return the solute–solvent interactions to that seen in bulk water and thus, not duplicating those experienced in vivo. Despite significant advances over the past 10 years in understanding that the content of culture media is significant only in a temporal context, no attention has been paid to spatial considerations – the presentation of the microenvironment in three-dimensional space. There is no possibility of achieving this through microdrop culture technology.

The potential of microfluidic systems

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There is an emerging technology that provides a multitude of advantages over the physical design of contemporary embryo culture systems. This microfluidic system, designed by David J Beebe and colleagues from the Department of Biomedical Engineering, University of Wisconsin, is constructed on a microscale and offers the potential of embryo culture in exceedingly small volumes, but with flow of the culture medium. A system of channels, with dimensions in the micrometre range, are fabricated by liquid-phase photopolymerization, lithography and laminar flow and can be constructed to contain actuators, valves and sensors using the same construction platform (Beebe et al., 2000b). The fabrication platform, termed microfluidic tectonics (µFT), can fabricate complete systems in as little as 10 min. Recently, Zhao et al. (2001) have shown that fluid and gas flow can be controlled within the microchannels by patterning surface free energies in the channels using self-assembled monolayer chemistry in conjunction with multistream laminar flow or photolithography. As aqueous solutions are confined to channels with hydrophilic surfaces, large gas–liquid interfaces can be created, facilitating gas–liquid reactions in microfluidic systems. Finally, flow control is imparted within microfluidic channels by the fabrication of functional hydrogel structures during µFT. These hydrogels have the capacity to expand or contract in response to environmental stimuli and, thus, behave structurally as sensors and actuators (Beebe et al., 2000a). The

small scale of microfluidic systems greatly enhances the response times of hydrogels so that flow control is autonomous. The properties of microfluidic systems – small volumes, channel dimensions slightly larger than embryonic dimensions, flow of media, flow control of embryos within channels and environmentally stimulated activation of hydrogel sensors and actuators – collectively provide conditions that are decidedly more like those in vivo than conventional microdrop technology (Raty et al., 2000). In preliminary experiments, this group compared the development of two-cell ICR × B6SJL/F1 mouse embryos cultured in a microfluidic system fabricated in polydimethylsiloxane-Pyrex to those grown in conventional microdrop culture. The culture medium was M16 supplemented with 4 mg/ml of bovine serum albumin and the volume of medium in microchannels was 10 µl whereas that of microdrops was 30 µl. Culture in microchannels was associated with significant increases in the number of 16-cell embryos/morulae at 24 hours, blastocysts at 48 and 72 hours, hatched blastocysts at 72 and 96 hours and a reduction of degenerate embryos at 96 hours. These data are encouraging that a geometric configuration for embryo culture, characterized by simultaneous reduction of culture volume with medium flow, may be available for the culture of human preimplantation embryos. Glasgow et al. (2001) have recently described additional features of microfluidic systems that are useful for handling mammalian embryos. With channel dimensions of 160 to 200 µm deep, 250 to 400 µm wide at the top and 0–250 µm wide at the bottom, a flow rate of medium is established for mouse embryos that roll the embryos along the bottom of the channels at half the speed of medium flow. Channels are fabricated to connect to wells at channel ends and pressures can be manipulated in these wells, yielding control of flow direction and rate. Embryos therefore can be moved to various compartments for analysis, ‘parking’ and gradual exposure to new media or environmental compositions.

Patterning the molecular structure of surfaces Takayama et al. (1999) have used the laminar flow properties of microfluidic channels to pattern macromolecules to the surfaces of the channels. For example, an anchoragedependent cell line, BCE, was shown to selectively adhere to one side of a microfluidic channel that had previously been patterned via laminar flow with fibronection. This technology presents the experimental embryologist with tools to test systematically the effects of macromolecules in a spatial manner consistent with that seen in vivo and in the correct dimensions and ratio with extracellular water.

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