Amino acids promote human blastocyst development in vitro

Human Reproduction Vol.16, No.4 pp. 749–756, 2001

Amino acids promote human blastocyst development in vitro F.Devreker1,2,5, K.Hardy4, M.Van den Bergh2,3, A.S.Vannin2,3, S.Emiliani2,3 and Y.Englert2,3 1Scientific

Collaborator at the Belgian National Funds for Scientific Research, 2Clinic of Fertility, Department of Obstetrics and Gynaecology, Hospital Erasme, Route de Lennik 808, 1070 Brussels, 3Laboratory for Biology and Psychology of Human Fertility, the Faculty of Medicine, Free University of Brussels, Belgium and 4Department of Reproductive Science and Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, UK 5To

whom correspondence should be addressed at: Clinic of Fertility, Department of Obstetrics and Gynaecology, Hospital Erasme, Route de Lennik 808, 1070 Brussels, Belgium.

Supplementation of culture media with amino acids has been shown to benefit preimplantation embryo development in several species. This randomized study analysed the in-vitro development of human embryos obtained after IVF in the presence or absence of a combination of amino acids from the 2- to 4-cell stage to the blastocyst stage. A total of 129 human embryos was randomly distributed between three serum-free chemically defined sequential media: (i) glucose-free Earle’s balanced salt solution (EBSS) with glutamine (Gln) prior to morula stage, supplemented with glucose for blastocyst formation; (ii) glucose-free EBSS with glutamine and non-essential amino acids (AA) for cleavage stage development, and supplemented with all 20 AA for blastocyst formation (Earle’s⍣AA); and (iii) a sequential commercial medium containing amino acids (K-SCIM). Embryos were individually cultured for successive periods of 24 h. On day 6 of development, blastocysts were differentially labelled and the numbers of trophectoderm and inner cell mass cells, mitoses and dead cells were examined. Blastocyst development was similar for the three sequential media. The mixture of AA significantly increased total blastocyst cell numbers from 61.8 ⍨ 4.2 with Earle’s⍣Gln to 99.3 ⍨ 8.4 with Earle’s⍣AA and 100.2 ⍨ 9.4 with K-SCIM (P ⍧ 0.005). This increase was present in both the trophectoderm and inner cell mass lineages (P < 0.02). Furthermore, the dead cell index was significantly lower with Earle’s⍣AA (P ⍧ 0.047). Key words: amino acids/cell death/cell numbers/human blastocyst

Introduction Suboptimal culture conditions are a possible cause of developmental arrest in vitro, and for low embryo viability post-transfer. Evidence for reduced embryo viability include a delayed cell doubling time (Bowman and McLaren, 1970), lower blastocyst cell numbers (Gardner and Lane, 1993), a decrease of the dry mass of the embryos (Turner et al., 1990), acceleration of protein degradation (Jung, 1989), lower incorporation of labelled amino acids (Jung et al., 1987), and a reduction in oxidative metabolism with the production of large amounts of lactate (Gardner and Leese, 1990; Gott et al., 1990; Leese et al., 1993). Furthermore, embryos grown in vitro have a reduced viability post-transfer (for a review, see Bavister, 1995). 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 © European Society of Human Reproduction and Embryology

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) 749

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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 nonessential 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. Materials and methods Source of embryos Women underwent ovulation induction with human menopausal gonadotrophin (Humegon®; Organon, Oss, The Netherlands) in combination with LH-releasing hormone agonist (Buserelin®; Hoechst, Hounslow, Middlesex, UK). Ovulation was induced by injection of 10 000 IU of human chorionic gonadotrophin (HCG; Profasi®, Serono; Pregnyl®, Organon), and oocyte retrieval was carried out 34–36 h later. 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 16 h after insemination by the presence of two pronuclei (day 1). On the

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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 艋1/3 or 艌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, 100⫻; Sigma) and essential amino acids (MEM-ESS, 50⫻; 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). Embryo culture Embryos were allocated evenly by block randomization to three sequential media: (i) control medium lacking glucose and supplemented with glutamine for cleavage stage development with the addition of glucose for blastocyst development at a final concentration of 1 mmol/l (Earle’s⫹Gln; n ⫽ 42); (ii) Earle’s with Gln and Eagle’s non-essential amino acids for cleavage stage development, and Earle’s with all 20 Eagle’s amino acids for blastocyst development (Earle’s⫹AA; n ⫽ 44); and (iii) a commercially available sequential medium based on the composition of the human tubal fluid medium (Quinn et al., 1985) and supplemented with amino acids (K-SCIM; n ⫽ 43). Embryos were cultured in the cleavage medium until day 4. On the morning of day 4, embryos were washed three times in the next culture medium and subsequently transferred to a fresh drop of their respective second step medium. Embryos were individually incubated for sequential 24 h incubation periods between days 2 and 6 post-insemination in 5 µl drops of medium overlaid with paraffin oil (Devreker et al., 1998). At the end of the culture period, each embryo was moved to a fresh incubation drop for the next 24 h. 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

Amino acids increase human blastocyst cell numbers

Table I. Culture media compositiona Component

NaCl KCl MgSO4.7H2O NaH2PO4.2H2O CaCl2.2H2O Penicillin (IU/ml) NaHCO3 Na pyruvate Glucose Lactate Glutamine NESS ESS EDTA Taurine Glycine Osmolarity

Earle’s⫹Gln

Earle’s⫹AA

K-SICM

Step I

Step II

Step I

Step II

Step I

Step II

118.16 5.37 0.81 1.01 1.80 97.5 25.01 0.47 – – 1 – – – – – 284

118.16 5.37 0.81 1.01 1.80 97.5 25.01 0.47 1 – 1 – – – – – 284

112.9 4.98 0.77 0.96 1.7 97.5 25.01 0.47 – 14.04 1 0.1 – – – – 284

112.9 4.98 0.77 0.96 1.7 97.5 25.01 0.47 1 14.04 1 0.1 ⫹ – – – 284

⫹ ⫹ ? low ? ? 25.01 ? – ? ⫹ ⫹ – ⫹ ⫹ ⫹ 285

⫹ ⫹ ? low ? ? 25.01 ? ⫹ ? ⫹ ⫹ ⫹ – ⫹ ⫹ 290

aSubstrate

concentrations are expressed in mmol/l. Step I: culture medium used from day 2 until day 4. Step II: culture medium used for blastocyst formation, from the morning of day 4 until day 6. AA ⫽ amino acids; ESS ⫽ Eagle’s essential amino acids; NESS ⫽ Eagle’s non-essential amino acids.

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-dinitrophenolbovine 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 15–20 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: Mitotic cell index ⫽ (no. of metaphases/total no. of cells)⫻100 Dead cell index ⫽ (no. of dead cells/total no. of cells ⫹ no. of dead cells)⫻100 Statistical analysis The number of embryos that reached the blastocyst stage was compared using χ2 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 (Mann–Whitney) 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).

Results A total of 129 embryos at the 2- to 4-cell stage and donated by 33 patients was randomly and evenly distributed between the three media. The distribution of embryo quality and cleavage stage was not different between the three groups (Figure 1). The mean (⫾ SEM) score was 3.3 ⫾ 0.2 for the embryo cohort and 4.3 ⫾ 0.2 for the transferred embryos. The implantation rate for the 73 sibling transferred embryos was 751

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Figure 1. Distribution of embryo score (left panel) and cell number (right panel) for the spare embryos included in the experiment.

Table II. Total cell number for day 6 blastocysts cultured in the presence or absence of a mixture of amino acids Media

n

Earle’s⫹Gln

17

Earle’s⫹AA

24

K-SICM

24

Total cell number 61.8 ⫾ 4.2a (35–98) 99.3 ⫾ 8.4 (32–175) 100.2 ⫾ 9.4 (29–208)

Mitotic index

Dead cell index

0.9 ⫾ 0.4

8.8 ⫾ 1.5b

0.6 ⫾ 0.2

4.6 ⫾ 0.6

0.7 ⫾ 0.2

7.0 ⫾ 1.0

Values are mean ⫾ SEM; values in parentheses are ranges. aCell number is significantly lower compared with cell number with Earle’s⫹AA or K-SCIM (P ⫽ 0.005). bDead cell index is significantly higher compared with dead cell index with Earle’s⫹AA (P ⫽ 0.041).

Figure 2. Human blastocyst development in the presence of Earle’s⫹Gln (j; n ⫽ 42), Earle’s⫹AA (u n ⫽ 43) or K-SCIM (z n ⫽ 44). M ⫽ morula; B ⫽ blastocyst.

24.6%. Only nine of the 33 couples had embryos suitable for freezing. Overall, 68 (52.7%) embryos developed to the blastocyst stage. A similar proportion of embryos reached the morula stage: 50% with Earle’s⫹Gln, 63.6% with Earle’s⫹AA, and 62.8% with K-SCIM (Figure 2). The percentage of embryos reaching the blastocyst stage was not different for the three media: 40.5% with Earle’s⫹Gln, 58% with Earle’s⫹AA, and 59% with K-SCIM (Figure 2). Overall, of the embryos that reached the blastocyst stage, 71% with Earle’s⫹Gln, 72% with Earle’s⫹AA and 77% with K-SCIM did so by day 5. The proportion of embryos that reached the 8-cell stage by 68 h post-insemination was similar between the three treatment groups: 72% with Earle’s⫹Gln, 69.5% with Earle’s⫹AA, and 52% with K-SCIM. No difference was observed between the three media for the proportion of blastocysts that were expanded, or that began to escape from the zona pellucida. Some 29% of blastocysts began to escape from the zona with Earle’s⫹Gln, 52% with Earle’s⫹AA, and 35% with K-SCIM. 752

Overall, 48 blastocysts were successfully double labelled. Due to incomplete dissolution of the zona pellucida, 17 remaining blastocysts were incompletely differentially labelled, with some of the TE nuclei being labelled only with bisbenzimide. However, in these embryos it was possible to determine the total cell count. The supplementation with a sequential mixture of amino acids increased the total cell number in day 6 blastocysts, for both Earle’s⫹AA (99.3 ⫾ 8.4; range: 32–175) and K-SCIM (100.2 ⫾ 9.4; range: 29–208) compared with Earle’s⫹Gln (61.8 ⫾ 4.2; range: 35–98) (P ⫽ 0.005; Table II). The distribution of the total cell number was similar for the two different media supplemented with sequential mixtures of amino acids, Earle’s⫹AA and K-SCIM (Figure 3). No difference was observed for the mitotic indices (Table II). The dead cell index was significantly higher for blastocysts cultured in the presence of only glutamine (8.8 ⫾ 1.5) compared with Earle’s⫹AA (4.6 ⫾ 0.6; P ⫽ 0.041). The proportion of cell death was not different between the two media supplemented with a mixture of amino acids (Table II). Both the TE (P ⫽ 0.012) and the ICM (P ⫽ 0.037) were significantly increased (by ~60%) for blastocysts cultured with Earle’s⫹AA or K-SCIM compared with glutamine alone

Amino acids increase human blastocyst cell numbers

Figure 3. Distribution of total cell numbers for day 6 human blastocysts cultured with Earle’s⫹Gln (upper panel), Earle’s⫹AA (centre panel) and K-SCIM (lower panel). The distributions are statistically different (Mann–Whitney test, P ⫽ 0.003).

(Table III). The percentage between the number of ICM cells to the total number of cells was similar for the three culture media: 30.38% for Earle’s⫹Gln, 30.08% for Earle’s⫹AA, and 31.90% for K-SCIM. These comparable ratios confirm that the rise in the cell numbers was present in both lineages. No differences were observed for the mitotic indices in both lineages between the three media (Table III). The proportion of TE cells undergoing cell death was lower for blastocysts cultured in the custom-made Earle’s⫹AA compared with the Earle’s⫹Gln, although the difference was not significant (Table II). Discussion This study reports the stimulatory effect of a complex mixture of amino acids on cell numbers in day 6 human blastocysts grown in vitro. The Earle’s⫹AA medium was based on a medium previously shown to improve human embryo development in vitro (Devreker et al., 1998). This basic medium was supplemented with Eagle’s non-essential amino acids and glutamine for cleavage stage development, and with all 20

Eagle’s amino acids for blastocyst development (Barnes et al., 1995). The control medium contained glutamine only for the cleavage stage, and glutamine and glucose for blastocyst development. The composition of the commercially available sequential medium was based on human tubal fluid medium (Quinn et al., 1985) and supplemented with Eagle’s nonessential amino acids, glutamine and taurine for the first step medium, and with all 20 Eagle’s amino acids and taurine for the second step medium (see Table I). Blastocyst development was similar for the three media. However, the two media supplemented with a sequential combination of amino acids increased total cell numbers in day 6 blastocysts by 60% compared with glutamine only. Half of the blastocysts had more than 100 cells for both custom-made and commercial media, the increase being present in both the TE and the ICM (Table II). The dead cell index was reduced in the presence of the mixture of amino acids; the decrease in cell death was most likely responsible for this dramatic increase in total cell numbers. 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 753

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Table III. Number of trophectoderm and inner cell mass cells in day 6 blastocysts cultured in the presence or absence of a mixture of amino acids Media

n

Earle’s⫹Gln

15

Earle’s⫹AA

18

K-SCIM

15

Trophectoderm

Inner cell mass

Cell number

Mitotic index

Dead cell index

Cell number

Mitotic index

Dead cell index

41.2 ⫾ 2.9a (26–50) 71.3 ⫾ 7.8 (37–138) 69.3 ⫾ 7.0 (28–111)

1.1 ⫾ 0.7

9.5 ⫾ 2.3

0.8 ⫾ 0.8

7.2 ⫾ 1.3

0.4 ⫾ 0.2

4.2 ⫾ 0.9

1.1 ⫾ 0.6

6.1 ⫾ 1.6

1.1 ⫾ 0.6

5.2 ⫾ 1.2

18.1 ⫾ 1.7b (9–35) 29.9 ⫾ 4.2 (20–79) 32.5 ⫾ 4.1 (10–49)

0.4 ⫾ 0.4

7.8 ⫾ 1.8

Values are mean ⫾ SEM; values in parentheses are ranges. aCell number is significantly lower compared with cell number with Earle’s⫹AA or K-SCIM (P ⫽ 0.012). bCell number is significantly lower compared with cell number with Earle’s⫹AA or K-SCIM (P ⫽ 0.037).

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 (45.5–50.5%; 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 posttransfer 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 754

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 posttransfer, 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

Amino acids increase human blastocyst cell numbers

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 invitro-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,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. Acknowledgements These studies were supported by the ‘Fondation Erasme’ and the Belgian National Fund for Scientific Research.

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