Articles Growth of human preimplantation embryos in vitro

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1 RBMOnline - Vol 2. No Reproductive BioMedicine Online webpaper 2000/008 on web 7 Mar 2001 Articles Growth of human preimplantation embryos in vitro Rajvi Mehta obtained her Master s degree in Microbiology and Doctorate in Applied Biology from the University of Bombay, India. Her doctoral thesis was on The Biology of the Human Oocyte in Women Undergoing IVF-ET. She is currently the Science Director of Hope Infertility Clinic, Bangalore and the Executive Director of Reproductive Biotechnologies Pvt. Ltd. Her research interests include ovarian folliculogenesis, early embryonic development and environmental effects on male fertility. Dr Rajvi Mehta Rajvi H Mehta Hope Infertility Clinic, 12 Aga Abbas Road, Bangalore , India Tel ; Fax ; mehtat@vsnl.com Abstract The human oocyte fertilizes and develops into embryos in the Fallopian tube and reaches the uterus only after compaction. However, for several years embryos that were developed following in-vitro fertilization (IVF) were transferred into the uterus on day 2 or 3 at the 4 8 cell stage in contrast to the in-vivo situation where they would be present in the Fallopian tube. Earlier attempts to grow embryos in vitro for 5 to 6 days were not always successful. Attempts were therefore made to understand the in-vivo environment of the Fallopian tube where the early embryonic development occurs. This article reviews the studies carried out to understand the composition of fluids in the Fallopian tube specifically with reference to the energy metabolites lactate, pyruvate and glucose; it also covers how the formulation of culture media for human IVF and embryonic development were modified over the years based on some classical work done on embryo culture in laboratory animals. Keywords: blastocysts, culture media, embryos, IVF, sequential media Introduction For nearly 20 years, it had been a common practice to transfer 4- to 8 cell human embryos, which develop following in-vitro fertilization (IVF), into the uterus. This was in contrast to the in-vivo situation where the 4- to 8 cell embryos are present in the Fallopian tube and enter the uterus only after compaction. It was thought that the pregnancy and implantation rates could be improved if one could transfer embryos at the blastocyst stage. Such a hypothesis was further strengthened when Bustillo et al. (1984) achieved 60% implantation rates in women receiving naturally formed blastocysts obtained by flushing from the uterine cavity of embryo donors. Thus, the need for growing human embryos beyond the 8-cell stage in vitro was recognized but was not easily achievable. This may possibly be due to the metabolic block exhibited by human embryos at the 4 8 cell stage and/or the inability of the available formulations of culture media to support further embryonic development. There were a few reports of human embryos grown up to the blastocyst stage but these were more of an exception rather than a rule (Steptoe and Edwards, 1978; Bolton and Braude, 1987; FitzGerald and DiMattina, 1992). IVF culture media: the early days The culture media commonly used for human IVF were either the simple salt solutions such as Earle s balanced salt solution (EBSS), or the complex media such as Ham s F-10 which were either supplemented with maternal or fetal cord serum. These media can be considered to be at opposite ends of the spectrum. EBSS being a simple salt solution is composed of just glucose and electrolytes while Ham s F-10 contains all the essential and non-essential amino acids, vitamins, growth factors, co-enzymes in addition to glucose and electrolytes. Human eggs fertilized in both these types of media, and developed into embryos which on transfer resulted in pregnancies and therefore it was difficult to comprehend whether all the additional components present in Ham s F-10 were in any way beneficial. Both these types of media had been formulated long before human IVF was carried out and were meant for culturing somatic cells. They did not take into consideration that the needs of the gametes and embryos may be different from those of somatic cells.

2 Over the last two decades, a two-pronged approach has been used to redefine culture media for human embryonic development. These are based on (i) an understanding of the metabolic events in early human embryos and (ii) determining the role of specific components of the complex media (such as vitamins, growth factors, certain amino acids, antibiotics etc.) on embryonic development by addition of the same to simple salt solutions or deleting the same from complex media (Bastias et al., 1993; Gardner and Lane, 1998; Devreker et al., 1998) Both these approaches have led to the generation of newer formulations which have subsequently resulted in the sequential culture of human embryos in different media. These approaches have led to an improvement in embryonic development as reflected by the number of embryos which now reach the blastocyst stage in vitro. Transfer of blastocysts in turn has led to an improvement in pregnancy and implantation rates to as high as 89% and 60% respectively (Khorram et al., 2000). Milki et al. (2000) evaluated 100 consecutive patients undergoing IVF-embryo transfer and reported that pregnancy rates following blastocyst transfer were 68% against 46% when the embryos were transferred on day 3. The implantation rates were also 48% as compared with 20% with 4 8 cell embryos transferred on day 3. Earlier, Gardner et al. (1998) also reported that the pregnancy and implantation rates were doubled when blastocysts were transferred as compared with the transfer of 8- cell embryos on day 3. This article reviews how a better understanding of human embryo metabolism has resulted in an improvement in the formulation of culture media. Understanding the metabolism of human preimplantation embryos Metabolism and development of human preimplantation embryos has been studied by indirect means, by characterizing the composition of the in-vivo environment where the early preimplantation embryo develops, viz. the Fallopian tube secretions (Leese, 1995) The tubal environment: composition of the tubal fluids It was thought that embryos should be cultured in an environment as close as possible to the endogenous environment in which the embryos normally develop. Attempts to mimic the endogenous environment were thus first made by Tervit et al. (1972) in the sheep. Later Quinn et al. (1985) formulated a culture medium similar to the then known composition of the human tubal fluid. With an improvement in the techniques of sampling and analysing micro-quantities of fluids, it was possible to know more about the composition of tubal fluid. Dickens et al. (1995) collected fluids from vascularly perfused preparation of the Fallopian tube and analysed them using highly sensitive ultramicrofluorometric assays. Using this technique, Dickens et al. (1995) estimated the concentration of glucose, lactate and pyruvate in excized Fallopian tubes from women undergoing hysterectomy. The mean concentrations of glucose, lactate (L+ isomer) and pyruvate respectively were: 0.53, 8.58 and 0.17 mm. These findings, however, need to be interpreted with caution since Fallopian tubes were obtained from women having an abnormal reproductive status who were undergoing hysterectomy for their problem. Gardner et al. (1996) also determined the values of the energy metabolites in the human tubal fluid using a suction pipette to collect very small volumes of luminal fluids from naturally cycling patients who were being investigated for infertility. It was found that the pyruvate concentrations in the oviduct remained constant throughout the cycle at 0.24 mm. Lactate (L+) increased from 0.49 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. Cumulus cells collected with oocytes retrieved for IVF readily consumed glucose in vitro, producing mainly lactate. The authors concluded that the early human embryos are exposed to an in-vivo environment 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 utilization in such events as secretory activity, muscular and ciliary movement (Gardner et al., 1996). Thus, these studies did play an important role in understanding the metabolic requirements of human oocytes and embryos and led to the formulation of culture media which could effectively be used for growing human embryos up to the blastocyst stage in vitro (Quinn et al., 1995; Gardner, 1998; Jones et al., 1998; Gardner and Lane, 1998). However, all these studies were preceded by pioneering work on animal models. It is on the basis of this work that culture media for human preimplantation embryos could be formulated. These studies have been reviewed in details (Bavister, 1995; Biggers, 1998). Determining the effects of specific components of media on in-vitro embryo development Embryos, like any other type of cells, must be grown in media which basically should contain: a source of energy primarily lactate, pyruvate or glucose; proteins, as a source of amino acids or directly amino acids; additional nutrients in the form of growth factors and vitamins; antibiotics and a ph indicator. Amino acids Amino acids in embryo culture media play a dual role. They serve as a substrate during protein synthesis as well as an energy source. However, it is not yet clear as to which of the 20 amino acids are absolutely essential for preimplantation human embryo development or whether excess amino acids can have any detrimental effect. The issue on which amino acids are required, useless or harmful has been extensively debated in one of the World

3 Wide Conferences On Reproductive Biology (Elder and Elliott, 1999). There is a consensus that the early embryo (4-cell stage) can grow in the absence of any amino acids. However, there is no concurrence amongst the various research groups when it comes to the need of amino acids for the development of the 4 8-cell embryo into the blastocyst. This is because of lack of studies on the effects of specific amino acids (except glutamine) added to culture media on embryonic development. Devreker et al. (1998) were the first to demonstrate the beneficial effects of glutamine on human embryo development. Human embryos were cultured from the day 2 to the blastocyst stage in media supplemented with and without glutamine. In the presence of glutamine, 89% of the embryos reached the morula stage in contrast to the 68% in controls and 71% reached the blastocyst as compared with 54% in controls. This clearly indicated that glutamine was indeed beneficial. The beneficial effect of glutamine has been attributed to: (i) it being a source of energy by entering the tricarboxylic acid (TCA) cycle and generating several molecules of ATP in the process; (ii) serving as an organic osmolyte protecting embryos against the high salt concentrations; (iii) it being an important carrier of the amine group, which is necessary for the synthesis of ADP, RNA, DNA, amino acids and proteins; (iv) de-amination of glutamine resulting in the generation of glutamic acid, which is an important precursor for the generation of the other amino acids. The same group later showed that supplementation of medium with taurine has no additional benefit over glutamine. Taurine was specifically tried since it had shown beneficial effects when added to culture medium in which mouse embryos were grown (Dumoulin et al., 1992). Taurine supported development of 2 4-cell human embryos to the blastocyst stage, although it did not further augment the beneficial effects of glutamine (Devreker et al., 1999). Despite this advance, the role of the other essential and nonessential amino acids remains unclear. One of the approaches has been to add other amino acids in addition to taurine or glutamine to the culture medium on the rationale that the female genital tract contains more than one amino acid. Another approach has been to add only glutamine or taurine to culture media as their specific beneficial effect has been well documented. Blastocysts and high pregnancy rates have been achieved using both these approaches. Thus, until a comparative study on the relative merits of these media is performed, a conclusion cannot be reached about the advantages of one type of media over the other. The only possible conclusion is that the human embryo, like those from other species, has a degree of plasticity and can develop in a variety of culture media with differing composition of amino acids. A note of caution against the indiscriminate use of amino acids in culture medium would be the generation of ammonium ions. The source of NH 4 + is (i) the metabolism of amino acids and (ii) the breakdown of amino acids in the medium irrespective of the presence of any embryos (Gardner and Lane, 1993). The latter event would be the major source of ammonium ions. With the lack of data on the concentrations of ammonium ion which can be toxic to embryos, it is only prudent that the use of amino acids is restricted to those absolutely essential or those that are less labile. Thus, glutamine which is the most labile of amino acids, is often replaced by alanyl-glutamine which is less labile but is equally effective for embryo development. Energy substrates Unlike the ambiguity associated with the supplementation of medium with amino acids, the role of energy substrates is fairly well defined. Using the preimplantation mouse embryo as the animal model, it was demonstrated that pyruvate was indeed a preferred substrate in early stages of development (Biggers et al., 1967) and glucose, as a sole substrate, could not sustain development (Brinster, 1965; Brinster and Thomson, 1966). Therefore, pyruvate was incorporated into culture media for human embryos as early as 1981 (Edwards et al., 1981). There are several reports which clearly implicate the metabolic block at the 2-cell stage when mouse embryos are cultured in vitro. Deleting glucose from the culture medium during the first few cleavage divisions has indeed been found to be advantageous (Chatot et al., 1989) in mice as well as hamsters (Schini and Bavister, 1988). A similar situation in humans is the block at the 4 8-cell stage. Growing embryos in a glucose-free medium has resulted in improved implantation rates (Quinn et al., 1995). However, glucose is important in the fertilization medium (Mahadevan et al., 1997; Barak et al., 1998) as it possibly participates in sperm oocyte fusion (Urner and Sakas, 1996). Nutrients in culture medium are generally supplied at concentrations that are well above that present in the in-vivo environment. The effect of the provision of nutrients at concentrations much higher than those to which the embryos are exposed in vivo has often not been considered. The role of endogenous nutrients is also not taken into consideration. To understand the absolute necessity of energy metabolites, human embryos were exposed to various concentrations of glucose and pyruvate (Conaghan et al., 1993). It was clearly concluded that pyruvate was absolutely essential for early embryonic development. Absence of pyruvate, i.e. culture in the presence of only glucose resulted in developmental arrest in 84% of the embryos. Thus, this study clearly indicated that glucose alone could not support embryonic development. Pyruvate uptake is directly proportional to the concentration of pyruvate in the culture medium. Even the progress of embryos to blastocysts was higher when they were initially cultured in a medium devoid of glucose (82% versus 60%) and the number of cells within the blastocysts was also higher (99 versus 58) in medium devoid of glucose as compared with the medium supplemented with glucose. Lactate production did occur even in the absence of glucose and this clearly shows that the embryos were obtaining energy from an endogenous source which could vary between oocytes and embryos (Conaghan et al., 1993).

4 Glucose is needed at the 8-cell stage, which corresponds to day 3 after insemination. The utilization of glucose is coupled with glycolysis as indicated by an upsurge in lactate production. This was one of the pioneering studies in understanding the energy requirements of preimplantation human embryos. Later, based on these observations, Gardner and Lane (1998) were the first to propose the use of sequential culture medium where embryos are grown in medium devoid of glucose until the 8-cell stage and later in a complex medium with glucose. Using such sequential media has led to a dramatic improvement in pregnancy and implantation rates. Inorganic compounds phosphates Inorganic salts form an intrinsic component of all types of tissue culture media. They are the major components contributing towards maintenance of its osmolality. Studies in laboratory animals have clearly shown that presence of phosphate along with glucose has an inhibitory effect on embryonic development (Seshagiri and Bavister, 1989; 1991). The exact mechanism responsible for this inhibitory effect is unclear. It is the combination of both glucose and phosphate that is most inhibitory. Nevertheless, this led to the development of low phosphate culture media for human embryos (Quinn et al.,1985) Chelating agents EDTA Ethylene diamine tetra-acetic acid (EDTA), a chelating agent, was never a component of the first generation of culture media used for human IVF and preimplantation embryonic development. Abramczuk et al. (1977) were the first to demonstrate that addition of EDTA to culture medium facilitated the development of zygotes in an outbred strain of mice. Subsequently, Mehta and Kiessling (1990) reported that the pregnancy rates following transfer of mouse embryos cultured in vitro in media supplemented with EDTA and amino acids gave equivalent pregnancy rates to those obtained in vivo. These findings were later corroborated by Gardner and Lane (1996) who reported on the beneficial effects of adding EDTA on mouse zygote development, especially in those strains on mice which show a 2-cell block in vitro. This observation was extrapolated to human IVF media and EDTA at the concentration of 0.1 mm is added to Quinn s (Quinn, 1995) and Gardner s (Barnes et al., 1995) media HTF and G1 respectively. The rationale behind the inclusion of EDTA in the culture media is as follows: (i) it acts as a chelator of heavy metal cations which may be present as trace contaminants in media components and plastic culture ware; (ii) it chelates iron and therefore reduces the generation of reactive oxygen species which in turn cause oxidative stress on the developing embryo; (iii) it chelates metallic ions which serve as co-factors for some of the enzymes involved in glycolysis. Inhibition of glycolysis is seen to be one of the reasons for some strains of mice passing through the 2-cell block. There is consensus on the beneficial effect of EDTA on the early stages of embryonic development. However, whether EDTA needs to be added to the blastocyst media remains controversial. Gardner (Barnes et al., 1995) and Menezo (Elder and Elliott, 1999) are of the opinion that EDTA needs to be totally removed from these media while Quinn s formulation (Quinn, 1995) contains EDTA but at lower concentration. This is an issue that needs to be resolved. Indicator phenol red Phenol red (phenolsulphonphthalein) is normally present in all tissue culture media for the purpose of detecting changes in ph of the media. The indicator changes from pink to colourless to yellow concomitant with the ph change from alkaline to acidic a visual indication of the culture environment especially while handling gametes and embryos. The necessity and advantages of phenol red in IVF culture medium is now being questioned. Some commercial manufacturers have completely deleted phenol red from their formulations while others have decreased its concentration despite lack of hard evidence. The issues raised against the use of phenol red are the presence of certain contaminants like heavy metals which are toxic to embryos and the reports on cytotoxic and oestrogenic activity of certain contaminants of phenol red on breast cancer-derived cell lines (Grady et al., 1991). The question that needs to be asked is: is it advisable to delete the ph indicator from the media because of the possibility of contaminants which have detrimental effect? Would it not be prudent to identify commercial sources which provide pure phenol red? Grady et al. (1991) have shown how the oestrogenic and cytotoxic activity of commercially available phenol red could be removed by extraction with diethyl ether. The ability to visually detect changes of ph in the media may not be essential but is indeed useful to the embryologist. ROS scavengers vitamin C It has been postulated that one of the reasons for the development block of embryos cultured in vitro may be the oxidative stress caused by the generation of reactive oxygen species (ROS). Therefore, Tarin et al. (1994) cultured human embryos in media supplemented with ascorbic acid, a known reactive oxygen species scavenger. There was no significant effect of ascorbate on fertilization, number of cells and embryo grade per embryo on days 2 and 3 after insemination of sibling oocytes cultured in control or media supplemented with ascorbate. Despite these results, a positive effect of ascorbate on fertilization and embryo development in vitro cannot be totally ruled out until the effects of other, non-physiological concentrations of ascorbate and longer-term embryo cultures (to the blastocyst stage) have been tested. Vitamins Although vitamins, especially of the B-Group, form a constituent of complex media, their role in preimplantation embryonic development remains equivocal. Not many studies have been done to determine the exact role or need of vitamins on human preimplantation embryo development. But data from other species may, to some extent, shed light on this topic. Vitamin E occurs naturally in cell membranes and protects the cells from oxidative stress. Attempts have been made to culture bovine embryos in the presence of vitamin E alone or along with vitamin C in the culture medium (Olson and Seidel, 2000). The number of zygotes that reached the expanded blastocyst

5 stage were higher in the presence of vitamin E. Interestingly, when these embryos were transferred and flushed 5 days later, they were 63% larger in surface area than the controls. Whether the larger embryos were normal or not remains questionable. While defining culture media, one needs to take into consideration the endogenous concentration of nutrients too. Reduced folate is an important substrate for the synthesis of thymidine which would be needed during the exponential increase in DNA during early cell division. O Neill (1998) has clearly demonstrated the absolute requirement of early embryos for reduced folate which is met entirely by endogenous sources and supplementation of media has no added benefits. McKiernan and Bavister (2000) were the first to report on the beneficial effects of an individual vitamin on hamster blastocyst development in vitro. Pantothenate significantly stimulated blastocyst development compared to the vitaminfree control and to every other single vitamin, except thiamine. Ascorbic acid, biotin, choline, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine had no detectable stimulation or inhibition on cleavage stage development or morula/blastocyst formation. However, one needs to be cautious before extrapolating these data towards defining culture media for human embryos. Hypoxanthine Hypoxanthine, a purine metabolite has been a constituent of Ham s F-10 - the first medium ever used for human IVF. Hypoxanthine is present in both ovarian follicular fluid as well as serum but surprisingly its concentration in Ham s F-10 is four times that in follicular fluid. Hypoxanthine had been implicated in the meiotic arrest of bovine, mouse and rhesus oocytes. However, its role in arresting the growth of embryos was first shown by Loutradis et al. (1987). In a classical study, they showed how % of mouse 2-cell embryos from in-bred mice progressed to the blastocyst stage when cultured in salt solutions, in contrast to only 15% of the embryos which were cultured in salt solution supplemented with Ham s F-10. By supplementing the salt solution with specific components of Ham s F-10, they could pin-point the component responsible: it was hypoxanthine. Later, Bastias et al. (1993) did a controlled trial with human embryos using Ham s F-10 with and without hypoxanthine. They reported that fertilization rates were higher (67% versus 53%) in medium without hypoxanthine and the rate of embryos cleaving without fragments was significantly higher in media without hpoxanthine (75% versus 35%). There have been other studies which have shown that use of media without hypoxanthine does result in improved clinical pregnancy and implantation rates in a human IVF programme as compared with media with hypoxanthine (Loutradis et al. 1993). However, these need to be interpreted with caution with particular reference to the role of hypoxanthine because, in addition to hypoxanthine, there were other components which were added or deleted from the medium. Thus, there is a clear consensus that hypoxanthine should not form a constituent of embryo culture media and has been excluded from all the commercial formulations. Antibiotics Addition of antibiotics to culture media is not mandatory. But, they are usually added to minimize the risk of contamination either through handling, from the spermatozoa or the culture environment. The most commonly used antibiotics are penicillin, streptomycin and gentamicin and the concentrations at which they are used are based on toxicity studies carried out on cell culture. To date, there has been only one study which has evaluated the effects of antibiotics on embryonic development. Magli et al. (1996) cultured human embryos in three types of media. One contained penicillin and streptomycin at concentrations normally present in culture media (100 IU/ml penicillin and 50 µg/ml streptomycin); the second type of medium contained antibiotics at half the normal concentrations; and the third medium was devoid of any antibiotics. The embryos cultured in these media were evaluated 40, 64, 88 and 112 h postinsemination. It was observed that embryos cultured in antibiotic-free media had a higher rate of cell division, and the number of embryos which reached the blastocyst stage was also significantly higher as compared with those embryos cultured in antibiotic-containing media, irrespective of the concentration of the antibiotic used. It was concluded that supplementation of culture media with antibiotics had an adverse effect on the growth rate of preimplantation embryos, possibly because of interference with the cleavage events which delayed or blocked embryo development. Surprisingly, the authors concluded that antibiotics have an adverse effect but did not attempt to identify whether both penicillin and streptomycin individually have a detrimental effect on cell division. For example, penicillin specifically inhibits the synthesis of bacterial cell wall component, peptidoglycan, and therefore is unlikely to have an effect on early embryos; conversely, streptomycin belongs to the aminoglycoside group of antibiotics which affects protein synthesis and could be the one that is responsible for delayed cleavage events observed by Magli et al. (1996). If this is the case, then the same effect may be exhibited by gentamicin, as it too is an aminoglycoside antibiotic. However, no studies have been reported so far to study the specific effects of gentamicin and streptomycin on human embryo development. The effect of antibiotics on preimplantation embryo development remains unclear. Magli et al. s (1996) observation needs to be validated and formulations of media need to be varied so as to include the optimal non-toxic antibiotic. Recently, new formulations of the G series of media (Vitrolife Fertility Systems, Molndalsvagen 30, Gothenburg, Sweden) have been released, which are devoid of streptomycin and contain only penicillin. Protein Serum, either as maternal serum or fetal cord serum was added to culture media. Serum served as a source of proteins, growth factors and could also be considered as a sink which would remove some of the toxic metabolites from the embryonic environment. It was the most variable constituent of the media. With an improvement in understanding embryonic metabolism, most laboratories have switched over to the use of human serum albumin or synthetic serum substitute (Gardner, 1998). The use of serum has the inherent risk of transmitting viral diseases, which is reduced with the use of human serum albumin.

6 Conclusions The last few years have indeed seen great changes taking place in the formulations of IVF culture media. This has resulted in the ability of embryologists to grow blastocysts in vitro. Transfer of blastocysts has indeed improved pregnancy and implantation rates. Single embryo transfer to reduce the risk of multiple pregnancies is now seriously being considered. However, culture of human embryos is still not an exact science. Of course, as with any biological system, one can anticipate variations between patients and between embryos from the same individual. But at the moment, there are several questions unanswered, especially those concerning amino acids, antibiotics, ph indicators, vitamins and growth factors. A better understanding of the role of specific amino acids, vitamins and growth factors in embryo metabolism may lead to further changes in the formulation of culture media which may reflect in a further improvement in pregnancy and implantation rates. 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