1 Scientific Collaborator at the Belgian National Funds for Scientific Research, 2 Clinic of Fertility, Department of Obstetrics and Gynaecology, Hospital Erasme, Route de Lennik 808, 1070 Brussels, 3 Laboratory for Biology and Psychology of Human Fertility, the Faculty of Medicine, Free University of Brussels, Belgium and 4 Department of Reproductive Science and Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, UK
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Abstract |
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Key words: amino acids/cell death/cell numbers/human blastocyst
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Introduction |
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The development of simple chemically defined media has allowed the analysis of specific requirements of the preimplantation embryo as it develops from the 1-cell to the blastocyst stage. The first evidence that amino acids could play an important role in embryo development was obtained from studies on mouse (Brinster, 1965), hamster (Gwatkin and Haidri, 1973
; Carney and Bavister, 1987
), rat (Zhang and Armstrong, 1990
; Kishi et al., 1991
) and rabbit (Kane and Foote, 1970
) embryos. It was shown that four amino acids including glutamine, phenylalanine, methionine and isoleucine supported the first cell division of hamster embryos (Gwatkin and Haidri, 1973
). Rabbit embryos could develop in the absence of exogenous energy substrates up to the morula stage, but required amino acids for blastocyst formation and hatching (Kane and Foote, 1970
).
Subsequent experiments have analysed the role of amino acids in preimplantation development of mouse (Chatot et al., 1989; Mehta and Kiessling, 1990
; Gardner and Lane, 1993
; Lane and Gardner, 1997a
,b
), bovine (Liu and Foote, 1995
; Pinyopummintr and Bavister, 1996
; Steeves and Gardner, 1999
), hamster (Carney and Bavister, 1987
; Bavister and McKiernan, 1993
), sheep (Gardner et al., 1994
) and human (Gardner and Lane, 1997
) embryos. These studies have determined that some amino acids can stimulate while others can inhibit the embryo development (Bavister and McKiernan, 1993
). Furthermore, temporal and differential effects of amino acids have been shown for mouse (Lane and Gardner, 1997b
) and bovine (Steeves and Gardner, 1999
) embryos. It was shown that Eagle's non-essential amino acids and glutamine decreased the time of the first three cell divisions of mouse embryos (Lane and Gardner, 1997a
) and stimulated blastocyst formation in vitro (Lane and Gardner, 1997b
).
In contrast, Eagle's essential amino acids were inhibitory when present before the 8-cell stage, but promoted blastocyst development and cell number when present after the 8-cell stage (Lane and Gardner, 1997b). A combination of non-essential amino acids and glutamine before the 8- to 16-cell stages and all amino acids after the 8- to 16-cell stages was found to be the best combination to improve bovine embryo viability in vitro (Pinyopummintr and Bavister, 1996
; Steeves and Gardner, 1999
). A similar combination of amino acids has been shown to improve human embryo viability in vitro and to increase embryo viability post-transfer (Gardner et al., 1998a
,b
; Jones et al., 1998
).
It has been shown, however, that events occurring during the early stages of development can affect later fetal growth (Leese et al., 1998). For example, spontaneous amino acid breakdown generates significant amounts of ammonium ions that can induce cerebral anomalies in mouse embryos (Lane and Gardner, 1994
). Furthermore, in domestic species components present in the culture medium can affect the cell number ratio between the trophectoderm and inner cell mass, inducing larger fetuses and newborns (reviewed by Leese et al., 1998).
Little is known about the effect of amino acids on human embryo differentiation in vitro. Recently, it has been shown that glutamine was beneficial for human embryo development in vitro while taurine, when present after the 4-cell stage, did not further enhance embryo development when compared with glutamine (Devreker et al., 1998, 1999
). In the current randomized study, the development of sibling human preimplantation embryos was compared in the presence or absence of pooled amino acids in sequential media. Subsequently, the effect of amino acids on cleavage rate, development to the blastocyst stage, numbers of cell allocated to the trophectoderm and inner cell mass, total cell numbers, cell division and cell death was analysed.
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Materials and methods |
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Preincubation and insemination or intracytoplasmic sperm injection of oocytes, and embryo culture before transfer, were carried out in custom-made Earle's balanced salt solution (EBSS) containing 5.56 mmol/l glucose and supplemented with 25 mmol/l sodium bicarbonate (Sigma, Bornem, Belgium), 0.33 mmol/l pyruvic acid (Sigma) and 0.5% human serum albumin (Red Cross, Brussels, Belgium), under a gas phase of 5% CO2, 5% O2 and 90% N2 (Van den Bergh et al., 1995). Normal fertilization was confirmed 16h after insemination by the presence of two pronuclei (day 1). On the morning of embryo transfer (day 2), embryos were examined and the number of cells determined. Each embryo was given a numerical score on the basis of embryo morphology and cleavage rate (Puissant et al., 1987
). An embryo with regular blastomeres and no cytoplasmic fragments was awarded 4 points, an embryo with uneven blastomeres and one or two cytoplasmic fragments was awarded 3 points, and an embryo with uneven blastomeres and cytoplasmic fragments of the embryonic surface
1/3 or
1/3 was awarded 2 points or 1 point respectively. A further 2 points were added if the embryo had reached the 4-cell stage. A maximum of three embryos with the best morphology and at the most advanced stage of development were selected for transfer and the remaining embryos of score 5 or 6 were selected for cryopreservation. After the patient's informed consent had been obtained, embryos unsuitable for transfer or freezing were allocated to the study.
The study was approved by the research ethics committee of the Hospital Erasme, Free University of Brussels.
Preparation of culture media
The basic modified EBSS was prepared as described previously (Devreker et al., 1998) without glucose. Media were prepared weekly from individual stock solutions using MilliQ water and stored at 4°C (Table I
). Separate 100-strength concentrated solutions of pyruvate, glutamine and NaHCO3 were prepared freshly before the media were made. The osmolarity of the media was checked before the addition of human serum albumin (HSA). All media were supplemented with 1 mmol/l glutamine (Sigma) and 0.5% HSA. Non-essential amino acids (minimal essential medium; MEM-NESS, 100x; Sigma) and essential amino acids (MEM-ESS, 50x; Sigma) solutions were added to the medium to create a final dilution of 1:100 and to avoid too high a pH variation and too high an ammonium ion formation. Media were filtered with a 0.22 µm Millipore filter (Sterivex-GV, Bedford, MA, USA). Dishes containing 5 µl drops overlaid with paraffin oil (Sigma) were set up each day and equilibrated for at least 2 h in an atmosphere of 5% CO2, 5% O2 and 90% N2 at 37°C before each experiment. Preparation of culture media, culture and scoring of embryos were performed by the same operator throughout the study. The commercial medium (K-SCIM, Sydney IVF, Queensland, Australia; complete composition unknown) was purchased from Cook IVF (Queensland, Australia).
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A blastocyst is formed by a vesicle of trophectoderm (TE) surrounding a fluid-filled cavity and a small group of inner cell mass (ICM) cells. Blastocyst morphology depends on the degree of expansion of the blastocele and the appearance of the TE and ICM. Early blastocysts had a blastocele of less than half of the volume of the embryo. Blastocysts had a blastocele of more than half of the volume of the embryo. Expanded blastocysts had a cavity that completely fills the embryo and stretched the zona pellucida. Hatching blastocysts were blastocysts in which the TE had begun to herniate through the zona. Good blastocysts had a well-defined ICM and a TE formed of many `sickle-shaped' cells
Differential labelling of ICM and TE nuclei
The numbers of cells in the TE and ICM of expanded blastocysts were counted on the morning of day 6 as described previously (Hardy et al., 1989a). TE nuclei were specifically labelled with the fluorochrome propidium iodide (Sigma) during antibody-mediated complement lysis (ICN).
Zona-free human blastocysts were incubated in 10 mmol/l trinitribenzenzsulphonic acid (Sigma) in ungassed M2 (Whittingham, 1971) supplemented with 4 mg/ml polyvinylpyrrolidone (Calbiochem 5295; Nottingham, UK) on ice for 45 min. Embryos were washed in three changes of M2+BSA and incubated in 0.1 mg/ml anti-dinitrophenol-bovine serum albumin (Anti-DNP-BSA; ICN Biomedical, Doornveld, Belgium) in M2+BSA at 37°C for 15 min. After further washing in M2+BSA, embryos were transferred to a 1:10 dilution of guinea pig complement serum (Sigma) in M2+BSA containing 0.01 mg/ml propidium iodide at 37°C for 1520 min. This fluorochrome can penetrate only lysed cells, and is thus excluded from viable ICM cells. The degree and evenness of lysis of outer TE cells were assessed by examination under a stereo microscope, and the blastocysts were finally fixed in a solution of absolute ethanol containing 0.05 mmol/l bisbenzimide (Hoechst 33258; Sigma) overnight at 4°C. TE nuclei would be labelled with propidium iodide and bisbenzimide, and ICM cells with bisbenzimide only. Since the emission spectra of the two fluorochromes differ, the labelled nuclei could be distinguished by the colour of their fluorescence and the numbers of TE and ICM cells counted. Labelled blastocysts were washed in absolute ethanol for 1 h before examination.
Differentially labelled embryos were mounted in glycerol, partially disaggregated and counted under fluorescence microscopy. An initial examination was carried out in whole mounts to check for even labelling of TE cells. Application of gentle sustained pressure to the coverslip disaggregated the nuclei so that they could be counted. Normal nuclei had a distinct nuclear outline, brightly staining nucleoli, and even shape. Cells in mitosis, with visible chromosomes, were clearly discernible and counted as single cells. Estimates of the number of dead cells were based on the presence of apoptotic nuclei characterized by discrete clusters of labelled nuclear fragments (Hardy et al., 1989a). These dead cells were not included in the overall total of cell numbers. Mitotic and dead cell indices were calculated as follows:
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Statistical analysis
The number of embryos that reached the blastocyst stage was compared using 2 analysis with Yates' correction. Differences in the distribution of total cell number and the numbers of TE and ICM cells in blastocysts, and distribution of the grade of embryos between the three groups were compared with the use of Wilcoxon rank-sum (MannWhitney) test. Analysis was performed with the use of the Statistical Package for the Social Sciences 7.0 for Windows 95 (Microsoft, Inc., Redmond, WA, USA).
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Results |
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Discussion |
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During development from the zygote to the blastocyst stage, the embryo undergoes several important developmental steps including activation of the embryonic genome, formation of tight junctions between cells (Hardy and Handyside, 1996), differentiation of the TE and the ICM, and formation of the blastocele cavity. These different developmental stages have different metabolic requirements (for reviews, see Leese, 1991; Bavister, 1995; Gardner and Lane, 1997). Before activation of the embryonic genome, embryo metabolism is low and relies mainly on maternal transcripts. At this stage, pyruvate is the major source of energy, and the embryo has little capacity to metabolize glucose (Hardy et al., 1989b
; Leese et al., 1993
). After activation of the embryonic genome, and as development proceeds, DNA replication and protein synthesis drastically increases. By that time, the embryo is able to metabolize glucose, and glucose and pyruvate uptake increases to satisfy the extra demands for energy (Hardy et al., 1989b
; Leese et al., 1993
). The late preimplantation embryo also requires a greater variety of nutrients, including amino acids, vitamins or fatty acids to support the different biosynthetic pathways (Barnett and Bavister, 1996
; Gardner and Lane, 1997
; Lane and Gardner 1997b
).
The culture of cell lines in vitro has shown that amino acids are a key constituent of cellular metabolism. Amino acids, present in high concentrations in female reproductive tract fluids, have been shown to improve preimplantation mammalian embryo development in vitro (for a review, see Bavister, 1995; Gardner and Lane, 1997). Furthermore, the amino acid content of mouse embryos has been shown to decrease when embryos were cultured in media lacking amino acids, resulting in a reduction of embryo viability (Van Winkle and Dickinson, 1995). The addition of amino acids to a synthetic industrial medium enriched with potassium (KSOM, a chemically defined medium; Lawitts and Biggers, 1991), enhanced the development of mouse embryos in vitro and increased gene expression to a similar level as embryos grown in vivo (Ho et al., 1995
). Non-essential amino acids and glutamine have been reported to reduce the time required for the first three cell divisions of mouse embryos (Lane and Gardner, 1997a
). Moreover, a medium supplemented with non-essential amino acids and glutamine to support early cleavage and with all amino acids to support post-compaction mouse embryo development has been shown to be the best combination to increase blastocyst formation, cell numbers in TE and ICM and blastocyst viability post-transfer (Lane and Gardner, 1994
, 1997a
). The presence of non-essential amino acids and glutamine was also beneficial for bovine embryo development (Pinyopummintr and Bavister, 1996
; Steeves and Gardner, 1999
).
In the light of these experiments, sequential media supplemented with amino acids have been evolved for human embryo culture in vitro. These media have been reported to support approximately between 46.5 and 60% blastocyst development of human embryos (Gardner et al., 1998a,b
; Jones et al., 1998
). The majority of the embryos had cavitated by day 5, and good implantation rates were obtained (45.550.5%; Gardner et al., 1998a,b). These studies assessed the beneficial effect of blastocyst transfers over cleavage stage embryos rather than the effect of amino acids on embryo viability as all media used contained a complex mixture of amino acids. The high implantation rates with blastocyst transfers could mostly result from the selection of viable embryos and synchronization between embryos and endometrium. Before undertaking blastocyst transfer clinically, evaluation of the effect of amino acids on blastocyst formation should be performed. The developmental potential of the early embryo may be disturbed without having any effect on its morphology. Culture conditions have been shown to affect the cleavage rate (Lane and Gardner, 1997b
), cell allocation to the TE and ICM (Devreker and Hardy, 1997
; Leese et al., 1998
), or embryonic genome expression (Ho et al., 1995
). Several components of culture media have been shown to induce anomalies in sheep (Thompson et al., 1995
), cow (Farin and Farin, 1995
) and mouse (Gardner and Lane, 1993
) fetuses.
The combination of amino acids used in the current study was chosen because it yielded mouse blastocysts with post-transfer viability similar to blastocysts developed in vivo (Lane and Gardner, 1997b) and supported human embryo development in vitro, resulting in highly viable blastocysts (Barnes et al., 1995
; Gardner et al., 1998a
,b
). Blastocyst development was similar for the three sequential culture media used in this experiment, and compares favourably with those previously reported for human embryos (Gardner and Lane 1997
; Devreker et al., 1998
, 1999
; Gardner et al., 1998a
,b
; Jones et al., 1998
), although in the present experiment only spare embryos were cultured. Moreover, 70% of the embryos that reached the blastocyst stage did so by day 5 compared with 20% (Jones et al., 1998
) or 92% (Gardner et al., 1998a
) reported previously. Blastocyst morphology was comparable for the three media. The proportions of poor, early, expanded and hatching blastocysts were not different, either with or without the mixture of amino acids. It is striking that sequential media which should correspond more closely to the changes in metabolic requirements of the embryo throughout the preimplantation period did not produce a higher percentage of blastocysts. This underlines that whatever the culture conditions, a proportion of embryos are abnormal and will not be able to develop beyond the morula stage because of chromosomal abnormalities (Plachot et al., 1987
; Kola et al., 1993
) or other metabolic defects (Edwards and Beard, 1999
). Optimization of the culture conditions will mainly reduce the proportion of embryos susceptible to the environment. This also confirms that blastocyst formation and morphology are poor criteria by which to measure the ability of a culture medium to produce viable embryos.
Cleavage rate is another parameter used to assess embryo viability (for a review, see Bavister, 1995). In the current experiment, the proportion of embryos that reached the 8-cell stage by day 3 (68 h post-insemination) or the morula stage by day 4 (92 h post-insemination) was, however, similar for the three treatment groups. This is in contrast to previous reports for hamster and mouse embryos. Hamster embryos cultured in vitro that reached the 8-cell stage more rapidly have been shown to produce a significantly higher proportion of blastocysts and to have a higher embryo viability post-transfer, even if the difference from the slow-cleaving embryos was only 3 h (McKiernan and Bavister, 1994). Amino acids have been shown to increase the cleavage rate of mouse embryos, producing blastocysts with higher viability (Lane and Gardner, 1997b
). As both the mitotic index and the cleavage rate were similar for the three media, the higher blastocyst cell numbers in the presence of a mixture of amino acids observed in the present study is probably due to the decrease in the proportion of cells undergoing cell death rather than to an acceleration of cleavage divisions.
The high blastocyst cell numbers in the presence of mixed amino acids compared favourably with those described previously for human embryos developed either in vitro or in vivo. The increase was present in both TE and ICM cells. A limited number of data are available for in-vivo-developed blastocysts. Blastocysts recovered after hysterectomy and estimated to be 5 days post-fertilization had 58 and 107 cells (Hertig et al., 1954), while others (Croxatto et al., 1972
) reported 186 cells in a human blastocyst obtained after uterus flushing. Several authors have reported lower mean total cell numbers for in-vitro-grown blastocysts under different conditions: 82 in the presence of glucose (Hardy et al., 1989a
), 99 in glucose-free medium (Conaghan et al., 1993
), 61 and 74 in the presence of glutamine or taurine respectively (Devreker et al., 1999
), 53 to 56 in human tubal fluid or with IVF50 medium (Scandinavian IVF Science AB, Gottenburg, Sweden) respectively (Dumoulin et al., 2000
), or 87 for blastocysts co-cultured with ampullary cells (Vlad et al., 1996
). Human blastocysts have also been obtained containing a mean of 79.6 cells after culture in Ham's F-10, a complex medium (Conaghan et al., 1998
). Others have reported either similar or higher total cell numbers; for example a mean of 96.8 cells for human blastocysts cultured in colony-stimulating factor medium (CSFM3) (Martin et al., 1998
), and means of 64 and 173 cells for expanded and hatching blastocysts respectively cultured in a complex sequential medium (S2; Scandinavian IVF Science AB, Gothenburg, Sweden) (Fong and Bongso, 1998). Blastocysts co-cultured with Vero cells and S2 medium had up to 246 cells (Fong and Bongso, 1998). The difference between the present results and those of the latter study could result either from the quality of the supernumerary embryos used, from the composition of S2 (which along with the 20 amino acids also contains vitamins, hormones and insulin), or for both reasons. Indeed, in the current study only embryos unsuitable for transfer or freezing were used, whereas others (Fong and Bongso, 1998) cultured a majority of good embryos. The relationship between cleavage embryo morphology and blastocyst cell numbers has been well illustrated (Hardy et al., 1989a
).
The beneficial effects of a mixture of amino acids on preimplantation embryo development in vitro parallel the observations of other species including bovine (Steeves and Gardner, 1999), mouse (Gardner and Lane, 1993
; Ho et al., 1995
; Lane and Gardner, 1997a
,b
), hamster (Carney and Bavister, 1987
; Bavister and McKiernan, 1993
), sheep (Gardner et al., 1994
; Walker et al., 1996
) and rat (Zhang and Armstrong, 1990
; Kishi et al., 1991
). Blastocyst cell numbers have been related to the embryo viability (Lane and Gardner, 1994
, 1997a
,Lane and Gardner, b
; Steeves and Gardner, 1999
). The higher cell numbers observed in the blastocysts cultured with a mixture of amino acids in the present study might reflect a higher embryo viability, although this needs to be confirmed by embryo transfers.
The advantage of custom-made culture media is the knowledge of the exact composition of the solution compared with commercial media. The effect of specific components on embryo development can therefore be analysed. In the case of Earle's+AA, the increase in cell number can be directly attributed to the supplementation of a sequential combination of amino acids. The composition of the commercial medium used in this study is based on the composition of the human tubal fluid (Quinn et al., 1985) and contained EDTA, glycine and taurine of unknown concentrations. The differences between K-SCIM and Earle's+AA also included concentrations of energy substrates. Although the beneficial effects of K-SCIM could not easily be related to the presence of a specific component, results obtained with Earle's+AA suggest that the increase in cell numbers with K-SCIM mainly resulted from the presence of the sequential mixture of amino acids.
In conclusion, supplementation of culture media with a mixture of amino acids significantly increased cell number in human blastocysts cultured in vitro. Future experiments comparing the transfer of blastocysts cultured in the presence or absence of a complex mixture of amino acids should confirm whether amino acids increase embryo viability post-transfer.
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Acknowledgements |
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Notes |
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References |
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Submitted on September 11, 2000; accepted on December 11, 2000.