ART Laboratories. First Generation of Media for ART. Historical Perspective of Culture Media Development PATRICK QUINN

Transcription

1 Journal of Andrology, Vol. 21, No. 5, September/October 2000 Copyright American Society of Andrology Review of Media Used in ART Laboratories Andrology Lab Corner PATRICK QUINN From SAGE BioPharma, San Clemente, California. As the development of culture media for assisted reproductive technology (ART) evolves, paralleling other advancements in the field, we find ourselves at a critical point at which increasing specialization and phase-specific design is the clear goal to help improve outcomes in human in vitro fertilization (IVF). This leads us to the question, Is there an ideal culture medium? Clearly, the goal is to emulate the in vivo environment as closely as possible. Because this milieu is dynamic compared with the virtually static in vitro environment, a single ideal ART medium seems elusive. The constantly changing in vivo environment embodies a specific chemical composition at every stage of embryonic development, together with factors such as embryonic density and a maximal O 2 /CO 2 environment, to name just a couple of critical parameters. Today s most advanced culture media are being developed to address these and other physiological issues, and to provide the most appropriate components at various stages of the IVF process. An interesting evolution has occurred in the development of these media, which provides perspective for the future of this clinical art. Historical Perspective of Culture Media Development Culture of cells derived from normal tissue was first practiced by Arnold (1887; see Waymouth, 1972). The title of father of tissue culture technique has been bestowed on Harrison ( , 1908) on the basis of his work on living nerve fibers; in those studies he used frog lymph as a supporting and nutrient medium. In an extensive review on the topic of the construction of tissue culture media, Waymouth (1972) reminds us of the work Ringer, Locke, and Tyrode, published in the late 19th and early 20th centuries. The names of these scientists are synonymous with physiological solutions, but their work may Correspondence to: Patrick Quinn, PhD, HCLD, SAGE BioPharma, 944 Calle Amanecer, Suite L, San Clemente, CA ( patrick. quinn@sagebiopharma.com) Received for publication May 3, 2000; accepted for publication May 3, not be familiar to some readers. Waymouth s review (1972) solves that problem; she points out that the work of Ringer, Locke, and Tyrode invoked the idea of a physiological salt solution as the basis for the development of defined media designed on the imitative principle. Such an approach has continued with the development of ART media for human and other mammalian species. First Generation of Media for ART The development of culture media for human IVF has paralleled other aspects of ART, such as controlled ovarian stimulation, micromanipulative procedures, and genetic diagnosis. An extensive review on this topic covering various aspects of media composition and use is found in a treatise by Edwards and Brody (1995). Culture media initially used for human IVF were those used for the incubation of somatic cells (eg, Ham s F10, Earle s) or those that were also first developed for somatic cell and tissue culture and then adapted for IVF and embryo culture in laboratory animals (eg, Tyrode s T6, WM 1 ). One way, then, to classify IVF culture media is on the basis of whether media were initially developed for use with somatic cells and tissues and subsequently used for IVF, or whether they were developed specifically for IVF and embryo culture. Another manner by which to categorize IVF culture media is whether they are complex media containing a mixture of inorganic salts, energy sources, amino acids, vitamins, and other cofactors (eg, Ham s F10, Menezo s B2 and B3 media, Eagle s modified essential medium [MEM], Behr s Blastocyst Medium, Gardner s G2) or whether they are simple balanced salt solutions containing energy sources (eg, Earle s, Tyrode s T6, WM 1, Pool s P1, Quinn s HTF, Gardner s G1). In general, complex media, in addition to containing most components that may be required for fertilization and embryo development in vitro, also contain components that have a negative effect on in vitro development. Some examples of this phenomenon are the ill effects of hypoxanthine in Ham s F10 medium that are involved in generating excessive amounts of reactive oxygen species (Loutradis et al, 1987) and a negative effect of nicotinamide on mouse embryo development (Tsai and Gardner, 1994) and vitamin B 12 on rabbit blastocyst expansion (Kane, 1988). Conversely, simple culture media may lack some essential

2 Quinn Media in ART Labs components that may enhance in vitro development of embryos. As discussed later, the addition of a single amino acid or a mixture of amino acids to simple media has led to dramatic improvement in embryo development and viability. The question arises as to which is the best approach in designing media for ART (ie, does one select a complex medium, ascertain what the deleterious components are and eliminate them, or does one start with a simple medium and then add components that are believed to be embryotrophic?). The general consensus is that the second approach has been more worthwhile. We are now at a point at which specific components are known to have certain effects at different stages of preimplantation embryonic development. The first specific medium for human IVF was human tubal fluid (HTF; Quinn et al, 1985), which was formulated on the then-known chemical composition of human tubal fluid (Lippes et al, 1972; Borland et al, 1980) with unknown factors based on what had worked with mouse embryos (eg, lactate and pyruvate levels). This strategy had been successfully used by Tervit et al (1972) to construct a medium, synthetic oviduct fluid (SOF), for the culture of cattle and sheep embryos. Subsequently, HTF medium became extensively used for human IVF in many laboratories around the world. It has since been recognized that the original HTF medium formulation is not the ultimate medium for human IVF. We have made improvements to the original HTF formulation, as have others over the last few years (Quinn, 1995; Gardner et al, 1996). Nevertheless, useful information was obtained from our initial studies (Quinn et al, 1985) with HTF medium. First, it appeared that the potassium ion level in T6 medium was too low, and when it was raised, as in HTF medium, both mouse and human embryos tended to show better development rates. Second, this study and its predecessor (Quinn et al, 1984) clearly established the mouse 1-cell assay as an effective quality control bioassay for monitoring IVF culture media and disposable items used for human IVF. Subsequent modifications of this assay using protein-free medium and zygotes from randombred mice (Quinn, 1995) have sensitized the assay even further. This assay has been used extensively for embryo proficiency testing for ART laboratory accreditation in the United States (Quinn et al, 1998a). Both HTF and SOF fall into the simple culture medium category, and improvements to both have involved the addition of amino acids, among other components. In addition, with HTF, we have removed glucose and phosphate. Second Generation: Energy Sources and Other Components More sensitive methods of directly sampling tubal fluid (Gardner et al, 1996), collection of fluid from vascularly 611 perfused preparations of fallopian tube (Dickens et al, 1995) and, in particular, highly sensitive ultramicroflourescent assays (Leese and Barton, 1984; Gardner and Leese, 1990) that have been used to assay metabolites in fluids and enzyme activities in embryos, have all contributed to a greatly expanded knowledge base with regard to the concentration of metabolites in human reproductive tract fluids. For example, Dickens et al (1995), using vascular perfusion of excised fallopian tubes from women undergoing hysterectomy, found that the mean rate of accumulation of tubal fluid was 48 L per hour. The mean concentrations of glucose, lactate (L isomer) and pyruvate were 0.53, 8.58, and 0.17 mm, respectively. There were no correlates between the concentration of these metabolites and the length of perfusion, cannulation time, patient age, or condition. Perhaps because of patients age (mean 43 1 year) and abnormal reproductive status (all were undergoing hysterectomy for various reasons), it could be suggested that the material and the results were not physiologically normal. A more recent study by Gardner et al (1996), however, confirms these values for the 3 energy metabolites in human tubal fluid and gives further data on changes over the menstrual cycle and levels in uterine fluid. In their study, Gardner and colleagues used a suction pipette to collect small volumes (0.5 L) of luminal fluids from naturally cycling patients who were being investigated for infertility. It was found that pyruvate levels in the oviduct remained constant throughout the cycle at 0.24 mm. Lactate (L ) increased from 4.9 mm in the follicular phase to 10.5 mm at ovulation, and then decreased to 6.2 mm in the luteal phase. Glucose decreased from 3.1 mm in the follicular phase to 0.5 mm in mid-cycle, and subsequently increased to 2.3 mm in the luteal phase. In uterine fluid, the concentrations of pyruvate, lactate, and glucose remained constant throughout the cycle at values of 0.1, 5.9, and 3.2 mm, respectively. Cumulus cells collected with oocytes retrieved for IVF readily consumed glucose in vitro, producing mainly lactate. It was evident to the authors that the early human embryo would be exposed to an in vivo environment that was high in lactate and low in glucose. The decrease in glucose in oviduct fluid around the time of ovulation is consistent with it being a major energy source of the oviduct, for consumption in such events as secretory activity, and muscular and ciliary movement (Gardner et al, 1996). Claims by Gardner et al (1996) that the levels of lactate and glucose in HTF medium have little homology with those present in human oviduct fluid are quite erroneous. The lactate concentration in HTF is 21.4 mm of a mixture of D and L isomers, which is equivalent to 10.7 mm L( ) isomer, nearly identical to the 10.5 mm L( ) level measured in mid-cycle oviduct fluid by Gardner et al (1996). It is notable that only the L isomer of lactate is biologically active; however, it can be conceded that perhaps

3 612 Journal of Andrology September/October 2000 because of the high overall concentration of lactate, an abnormal ratio of pyruvate to lactate could prevail that might influence the redox potential in cells cultured in the medium. To circumvent this problem, we have decided to formulate our new series of sequential culture media with calcium L( ) lactate. So, for example, 2 mm calcium L( ) lactate contains the equivalent of 4 mm equivalents of L( ) lactate, which is equivalent to 8 mm equivalents of DL-lactate while maintaining the original calcium concentration as found in my current HTF media series (Quinn, 1995). Another advantage of using calcium lactate is that it is a dry salt, thus amenable to formulating lyophilized or powdered forms of medium, whereas sodium-dl-lactate is a syrup and cannot be lyophilized effectively. Glucose levels were reported to vary from 0.5 mm to 3.1 mm in oviduct fluid at different times of the menstrual cycle, and reached a constant level of 3.15 mm in uterine fluid (Gardner et al, 1996). These values are very similar to those in my original HTF formulation (Quinn et al, 1985) and, with changes instigated in my current versions of HTF (Quinn s HTF; Quinn s Basal XI HTF; and Quinn s D3 HTF for insemination, cleavage, and blastocyst development, respectively), offer a range of media with energy metabolites present at estimated physiological levels. It can also be said, and data presented by Gardner et al (1996) confirm, that a considerable amount of glucose in culture medium is metabolized to lactate. My use of a cumulus cell coculture system (1994) would tend to further decrease glucose levels in the medium. With these considerations, together with the inclusion of EDTA and glutamine and omission or lowering of phosphate ions in my new versions of HTF, very acceptable human embryo development rates in vitro and subsequent pregnancy rates have been generated (Quinn, 1995; Quinn et al, 1995). Finally, it must be remembered that in humans and many other mammalian species, pyruvate is an essential substrate for early embryo development and is included in all culture media at levels found to be present in tubal fluid (Conaghan et al, 1993). Evidence is now emerging concerning the influence of some of the ionic components in culture media on the overall function of embryonic cell activity. In particular, there are reports that an adequate ratio of calcium to magnesium is required to minimize the accumulation of internal calcium that may occur in the early embryo when exposed to the stress of in vitro culture (Lane et al, 1998). This can be achieved by raising the concentration of magnesium so that a Ca:Mg ratio of at least 1:1, rather than 4:1 is present. The increased levels of magnesium lower the increase in internal calcium which, if unimpeded, can have a negative effect on proper mitochondria and, hence, energy metabolism (Lane et al, 1998). Another problem involving the internal milieu of embryonic cells involves media components that alter the internal ph (phi) of embryos, and, in particular, the phi of embryos during freezing and thawing. Lane et al (1999) reported that in the presence of phosphate-buffered medium, the phi of embryos can be dramatically altered and because, prior to compaction, the embryo has no mechanism to remove this build-up of internal protons, the viability of such embryos is severely compromised. With HEPES-buffered media, however, no such negative phenomenon occurs (Lane and Gardner, 2000). This observation is therefore strong evidence for the use of HE- PES-buffered culture media for all procedures with gametes and embryos performed outside of an atmosphere lacking carbon dioxide (ie, in air). Such activities would include micromanipulation, cryopreservation, and embryo transfer. Such a recommendation has been made previously (Quinn et al, 1984, 1985) before the actual reason, as demonstrated by Lane and colleagues (1999), was evident. Antioxidants, Amino Acids, Glucose, and Phosphate Several new formulations of the original HTF medium have recently been published (Quinn, 1995). I now include EDTA and glutamine in all my culture media used for human and mouse embryos and gametes. The rationale for using these compounds was primarily based on reports showing their benefit for the culture of mouse embryos, particularly in those strains showing the so-called 2-cell block. EDTA probably acts in several ways, including 1) chelation of heavy metal divalent cations that may be present as trace contaminants in media components and plastic cultureware; 2) through the chelation of iron there would be a lowered generation of reactive oxygen species; 3) maintenance of enzyme activity such as adenyl cyclase, which generates camp; and 4) a recent study by Lane and Gardner (1997) also showed that EDTA will inhibit the enzyme 3-phosphoglycerate kinase, thus reducing glycolytic activity in an early embryo. Increased glycolysis in an early embryo appears to be associated with poor development (Gardner and Lane, 1996). Glutamine appears to be a readily available alternative energy source for embryos, which feeds into the tricarboxylic acid cycle; this would be beneficial if other avenues for entry of substrates into this cycle are restricted because of perturbations in glycolytic activity and mitochondrial metabolic pathways. A more stable source of glutamine (it can be autoclaved) in the form of the dipeptide alanyl-glutamine is being incorporated into our new series of sequential media. The use of mouse zygotes derived from random-bred CF1 females mated with B6C3 F1 males has provided a useful model to study some of the interactions between glucose, phosphate ions (Pi), EDTA, and glutamine. A summary of the overall results from a series of experi-

4 Quinn Media in ART Labs Development of zygotes from CF1 female mice to fully expanded blastocysts in various combinations of glucose (2.8 mm), phosphate (0.37 mm), EDTA (0.1 mm), and glutamine (1.0 mm). The Base medium was devoid of glucose, phosphate, EDTA, and glutamine. These are the combined results from 4 separate experiments, each one replicated 3 to 5 times with a total of 66 to 122 zygotes in each treatment. Some treatments were repeated in up to 3 separate experiments. From Quinn (1996). ments conducted to study these interactions is shown in the Figure. Embryo development was inhibited in the presence of glutamine and Pi. Omission of these 2 compounds allowed more than 80% of the zygotes to reach the expanded blastocyst stage (medium Base in the Figure). Addition of EDTA and glutamine to glucose/pifree medium gave the maximum response ( 90% blastocysts; top bar in the Figure). I now refer to this medium as Basal XI HTF ). The addition of EDTA, glutamine, or both compounds to HTF medium containing glucose and Pi stimulated development; glutamine 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 glucose and Pi (medium Base Pi and glucose in the Figure) was less inhibitory than commercially prepared HTF (Figure, bottom bar; 33% versus 19%, respectively). This further highlights the usefulness of this assay using zygotes from random-bred mice that show a partial 2-cell block in traditional culture media. Barnett and Bavister (1996) extensively considered the inhibitory effect of the glucose/pi combination in the early embryos of several mammalian species in a recent review of the topic. It should be remembered that it is actually the combination of glucose and Pi that is most inhibitory and not just, as is sometimes inferred, the presence of glucose. Also, my studies (1995) show that in mice, complete in vitro development from zygotes to completely hatched blastocysts can occur in the absence of glucose, but only if the medium contains an exogenous protein source. In the absence of protein, glucose is needed for blastocyst development. Exogenous protein used in 613 culture medium, especially serum albumin, is known to be a covert source of fatty acids, amino acids, and citrate (Barnett and Bavister, 1996). The situation in humans is unknown because in vitro embryo culture is rarely conducted in the complete absence of exogenous protein. We do know, however, that a high proportion (50%) of fully expanded blastocysts can develop in Basal XI HTF medium, which lacks both glucose and phosphate, especially when cumulus cells are used as a coculture support (Quinn and Margalit, 1996). Barnett and Bavister (1996) provide several hypotheses for suboptimal development of embryos in vitro that are centered on disturbed metabolism of embryos caused by abnormal culture environments. Although the exact mechanisms for the altered embryo metabolism are not known, increased glycolysis, altered substrate oxidation patterns, decreased respiration, and increased aerobic lactate production all occur in embryos produced in vitro (Barnett and Bavister, 1996). Barnett and Bavister (1996) present 2 hypotheses, which are not necessarily mutually exclusive. In the first, metabolic and culture deficiencies in vitro generate an increase in glycolytic activity that has detrimental consequences for embryo development. In a recent study of hexokinase activity in mouse embryos, Houghton et al (1996) suggest that the activity of this enzyme, the first enzyme of glycolysis, is diminished when Pi is omitted from culture medium. Omission of Pi would therefore be beneficial because this could lead to decreased glycolysis that is less detrimental to the embryo. In the second hypothesis, culture conditions directly affect mitochondria structure and function, leading to calcium ion mobilization, production of reactive oxygen species, or both. Effects of elevated Ca 2 i can be exacerbated by Pi and lead to damaged mitochondria membranes and uncoupling of oxidative phosphorylation (see Lane et al, 1998, 1999). However the mitochondrial damage occurs, responsibility for energy production is then displaced onto glycolysis that may be inadequate for continued embryo development. There is also recent direct evidence that Pi inhibits the cyclic dephosphorylation of the cyclic protein subunit, cdc2 (Haraguchi et al, 1996). If dephosphorylation of this subunit does not occur, then cell division is inhibited, and it is believed that this may be part of the reason for the 2- cell block in some strains of mice. In summary, the combination of glucose and phosphate is inhibitory to early cleavage-stage embryos of a number of mammalian species, including mouse, hamster, rat, and human. Initially, glucose was believed to be the major culprit, but it now appears that phosphate plays a significant role in the adverse effects of in vitro culture and that glucose is mainly a problem in the presence of high ( 0.35 mm) concentrations of phosphate (Quinn, 1996). The elemental concentration of phosphorus has been measured in human oviduct fluid using X-ray spectrometry

5 614 Journal of Andrology September/October 2000 (Borland et al, 1980), but these values would have little relevance to the physiological amounts of inorganic phosphate ions in reproductive tract fluids. An estimate of phosphate ion concentration in fallopian tube and uterine fluids would help to clarify the role of this compound in early embryo development and assist in the construction of a more appropriate culture medium. The addition of antioxidants, such as EDTA, glutathione, and taurine, as well as specific amino acids such as glutamine, may help to alleviate some of the adverse culture environment effects. Other strategies such as coculture, maximizing embryonic density, and culture under an atmosphere of reduced oxygen may also be beneficial. Conditions of Use and Culture Strategies In addition to the actual composition of media used for ART, there are at least 2 further aspects that need to be qualified: at what stage of preimplantation development are the media being used, and under what conditions are they being used? There is no point in having a good medium if it is not used in the manner for which it was intended, or not used at a time in development for which it was intended, or both. Unfortunately, space does not allow elaboration on these aspects, but the following points should be kept in mind. The reader can follow up on each topic by consulting the indicated reference. Oil versus open culture (Quinn et al, 1985). Volume of medium and embryonic density (Lane and Gardner, 1992; Quinn, 1995). Time of gamete coincubation in IVF (Gianaroli et al, 1996; Quinn et al, 1998b). Oxygen and carbon dioxide concentrations (Quinn and Harlow, 1978; Gardner et al, 1999). Different media at different stages: sequential media (Gardner and Lane, 1997). Energy substrates, amino acids, glucose, phosphate, and EDTA (Quinn, 1995; Gardner and Lane, 1997). Growth factors and other embryonic stimulants (Lighten et al, 1998). Coculture (now a largely disbanded procedure, at least in North America with sequential media; Quinn and Horstman, 1997). Markers of embryonic viability (Gardner and Lane, 1997). Media for Sperm, Intrauterine Insemination, and IVF Insemination It took a number of decades of studies on mammalian sperm function from the 1940s to the 1960s before the beneficial effects of inclusion of macromolecules, and especially albumin in medium used for the suspension of spermatozoa, was realized. In the past 40 years, however, considerable progress has been made on designing media for handling spermatozoa. Specifically, for human spermatozoa and ART, we now have a very good idea of which are the best media and methods for preparing spermatozoa from semen, maintaining them for use in such procedures as intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI), and using them for the fertilization of oocytes in vitro. Space does not allow for an extensive review of this topic. Suffice it to say that density gradient separation, using polymer-coated colloidal silica (eg, Percoll, Pure- Ception, PureSperm, Isolate, etc) to minimize the generation of deleterious reactive oxygen species during sperm washing is now the norm. HEPES-buffered sperm suspension media are usually used for the final wash and resuspension of spermatozoa for IUI and ICSI, and bicarbonate buffered media, such as HTF, with supplements such as EDTA and taurine, have been found to give very good fertilization rates in IVF. Much of the historical development of media for these procedures up until several years ago can be found in Quinn (1993) and Mortimer (1990). Conclusion The development of culture media for human IVF remains on an evolutionary path. Waymouth (1972) stated that the design of media remains in part an empirical science and in part an art assisted by experience and intuition. She points out that in general, a medium designed for particular cells must come as close as possible to providing the nutritional and physical milieu that allows the unique genetic and epigenetic functional capacities of the cell to be expressed. This is certainly the case with preimplantation mammalian embryos, in which a small number of cells are combined in a group that will eventually grow and develop into a complete human being. This selfevident conclusion behooves us to continue the search for media and culture techniques that will allow the replacement of 1 human embryo at embryo transfer with the highest possible chance of continued development to birth as a normal baby. References Arnold J. Ueber Theilungsvorgänge an der Wanderzellen; ihre progressiven und regressiven Metamorphosen. Arch Mikrosk Anat. 1887;30: Barnett DK, Bavister BD. What is the relationship between the metabolism of preimplantation embryos and their developmental competence? Mol Reprod Dev. 1996;43: Borland RM, Biggers JD, Lechene CP, Taymor ML. Elemental composition of fluid in the human fallopian tube. J Reprod Fertil. 1980;58: Conaghan J, Handyside AH, Winston RML, Leese HJ. Effects of pyruvate and glucose on the development of human preimplantation embryos in vitro. J Reprod Fertil. 1993;99:87 95.

6 Quinn Media in ART Labs Dickens CJ, Maguiness SD, Comer MT, Palmer A, Rutherford AJ, Leese HJ. Human tubal fluid: formation and composition during vascular perfusion of the Fallopian tube. Hum Reprod. 1995;10: Edwards RG, Brody SA. Principles and Practice of Assisted Human Reproduction. Philadelphia: WB Saunders & Co; 1995: Gardner DK, Lane M. Alleviation of the 2-cell block and development to the blastocyst of CF1 mouse embryos: role of amino acids, EDTA and physical parameters. Hum Reprod. 1996;11: Gardner DK, Lane M. Culture and selection of viable human blastocysts: a feasible proposition for human IVF? Hum Reprod Update. 1997;3: Gardner DK, Lane M, Calderon I, Leeton J. Environment of the preimplantation human embryo in vivo: metabolite analysis of oviduct and uterine fluids and metabolism of cumulus cells. Fertil Steril. 1996; 65: Gardner DK, Lane M, Johnson J, Wagley L, Stevens J, Schoolcraft WB. Reduced oxygen tension increases blastocyst development, differentiation, and viability. In: Proceedings of the Conjoint Annual Meeting of the American Society for Reproductive Medicine and the Canadian Fertility and Andrology Society. Toronto, ON. Fertil Steril. 1999: 72(suppl 1):s30. Gardner DK, Leese HJ. Concentration of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J Reprod Fertil. 1990;88: Gianaroli L, Fiorentino A, Magli MC, Ferraretti AP, Montanaro N. Prolonged sperm-oocyte exposure and high sperm concentration affect human embryo viability and pregnancy rate. Hum Reprod. 1996;11: Haraguchi S, Naito K, Azuma S, Sato E, Nagahama Y, Yamashita M, Toyoda Y. Effects of phosphate on in vitro 2-cell block of AKR/N mouse embryos based on changes in cdc2 kinase activity and phosphorylation states. Biol Reprod. 1996;55: Harrison RG. Observations on the living developing nerve fiber. Proc Soc Exp Biol Med ;4: Harrison RG. Embryonic transplantation and development of the nervous system. Anat Rec. 1908;2: Houghton FD, Sheth B, Moran B, Leese HJ, Fleming TP. Expression and activity of hexokinase in the early mouse embryo. Mol Hum Reprod. 1996;2: Kane MT. The effects of water-soluble vitamins on the expansion of rabbit blastocysts in vitro. J Exp Zool. 1988;245: Lane M, Boatman DE, Albrecht RM, Bavister BD. Intracellular divalent cation homeostasis and developmental competence in the hamster preimplantation embryo. Mol Reprod Dev. 1998;50: Lane M, Gardner DK. Effect of incubation volume and embryo density on the development and viability of preimplantation mouse embryos in vitro. Hum Reprod. 1992;7: Lane M, Gardner DK. EDTA stimulates development of cleavage stage mouse embryos by inhibiting the glycolytic enzyme 3-phosphoglycerate kinase. Biol Reprod. 1997:56(suppl 1):131. Abstract 193. Lane M, Gardner DK. Blastomere homeostasis. In: ART and the Human Blastocyst. Final Program and Abstract Book. Dana Point, Calif: Serono Symposia USA Inc; 2000:11. Lane M, Ludwig TE, Bavister BD. Phosphate induced developmental arrest of hamster two-cell embryos is associated with disrupted ionic homeostasis. Mol Reprod Dev. 1999;54: Leese HJ, Barton AM. Pyruvate and glucose uptake by mouse ova and preimplantation embryos. J Reprod Fertil. 1984;72: Lighten AD, Moore GE, Winston RML, Hardy K. Routine addition of human insulin-like growth factor-i ligand could benefit clinical invitro fertilization culture. Hum Reprod. 1998;13: Lippes J, Enders RG, Pragay DA, Bartholomew WR. The collection and analysis of human fallopian tubal fluid. Contraception. 1972;5: Loutradis D, John D, Kiessling AA. Hypoxanthine causes a 2-cell block in random-bred mouse embryos. Biol Reprod. 1987;37: Mortimer D. Semen analysis and sperm washing techniques. In: Gagnon C, ed. Controls of Sperm Motility: Biological and Clinical Aspects. Boca Raton, Fla: CRC Press; 1990: Quinn P. Sperm processing in assisted reproductive technology: male factor. Semin Reprod Endocrinol. 1993;11: Quinn P. Use of coculture with cumulus cells in insemination medium in human in vitro. J Assisted Reprod Genet. 1994;11: Quinn P. Enhanced results in mouse and human embryo culture using a modified human tubal fluid medium lacking glucose and phosphate. J Assisted Reprod Genet. 1995;12: Quinn P. Tubal fluid and its significance in human reproduction. In: Coutifaris C, Mastroianni L, eds. New Horizons in Reproductive Medicine: The Proceedings of the IX World Congress on Human Reproduction, Philadelphia, New York, NY: Parthenon Publishing Group; 1997: Quinn P, Harlow GM. The effect of oxygen on the development of preimplantation mouse embryos in vitro. J Exp Zool. 1978;206: Quinn P, Horstman FC. Co-culture or conditioned media can it be improved? In: Gomel V, Leung PCK, eds. Proceedings of the 10th World Congress on In Vitro Fertilization and Assisted Reproduction. Bologna, Italy: Monduzzi Editore; 1997: Quinn P, Keel BA, Serafy NJ Jn, Serafy NJ, Schmidt CF, Horstman FC. Results of the American Association of Bioanalysts (AAB) embryology proficiency testing (PT) program. In: Proceedings 54th Annual Meeting of the American Society for Reproductive Medicine. San Francisco, Calif: 1998a:O-267, S100. Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril. 1985;44: Quinn P, Lydic ML, Ho M, Bastuba M, Hendee F, Brody SA. Confirmation of the beneficial effects of brief coincubation of gametes in human in vitro fertilization. Fertil Steril. 1998b;69: Quinn P, Margalit R. Beneficial effects of coculture with cumulus cells on blastocyst formation in a prospective trial with supernumerary human embryos. J Assisted Reprod Genet. 1996;13:9 14. Quinn P, Moinipanah R, Steinberg JM, Weathersbee PS. Successful human IVF using a modified HTF medium lacking glucose and phosphate ions. Fertil Steril. 1995;63: Quinn P, Warnes GM, Kerin JF, Kirby C. Culture factors in relation to the success of human in vitro fertilization and embryo transfer. Fertil Steril. 1984;41: Tervit HR, Whittingham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil. 1972;30: Tsai FCH, Gardner DK. Nicotinamide, a component of complex culture media, inhibits mouse embryo development in vitro and reduces subsequent developmental potential after transfer. Fertil Steril. 1994;61: Waymouth C. Construction of tissue culture media. In: Rothblat GH, Cristofalo VJ, eds. Growth, Nutrition, and Metabolism of Cells in Culture. Vol 1. New York, NY: Academic Press; 1972: Andrology Lab Corner welcomes the submission of unsolicited manuscripts, requested reviews, and articles in a debate format. Manuscripts will be reviewed and edited by the Section Editor. All submissions should be sent to the Journal of Andrology Editorial Office. Letters to the editor in response to articles as well as suggested topics for future issues are encouraged.