Hum. Reprod. Advance Access originally published online on November 25, 2005
Human Reproduction 2006 21(3):766-773; doi:10.1093/humrep/dei385
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Fatty acid metabolism in human preimplantation embryos
1 Embryonic Programming Group, Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB and 2 Department of Obstetrics & Gynaecology, Aberdeen University, Aberdeen AB9 2ZD, UK
3 To whom correspondence should be addressed. E-mail: p.haggarty{at}abdn.ac.uk
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Abstract |
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BACKGROUND: Little is known of fatty acid metabolism in human embryos. This information would be useful in developing metabolic tests of embryo quality and improving embryo culture media. METHODS: The fatty acid composition of human embryos and their ability to accumulate 13C labelled fatty acids was assessed in relation to the stage of development using gas-chromatography and combustion-isotope-ratio-mass spectrometry. RESULTS: Compared with embryos which did not develop beyond the 4-cell stage, those that did had significantly higher concentrations of the unsaturates, linoleic (12% versus 3%; P = 0.02) and oleic (14% versus 7%; P = 0.02), and a lower concentration of total saturates (62% versus 77%; P = 0.04). There was uptake of both 13C linoleic and palmitic, but the developmental pattern was different for each fatty acid. The net accumulation in pmol/embryo/24h for palmitic was 1 at the 2-cell to <8-cell stage, 4 at the 8-cell–morula stage and negligible at the blastocyst stage. For linoleic, there was little net accumulation at the 2-cell to <8-cell stage, 8 (8-cell–morula stage) and 17 pmol/embryo/24h (blastocyst stage). CONCLUSION: Preimplantation human embryos actively take up individual fatty acids at different rates at different stages of development. The high unsaturated concentration at the later stages of development may be explained by preferential uptake of linoleic acid.
Key words: embryo/fatty acids/IVF/linoleic acid/stem cells
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Introduction |
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Despite recent advances in IVF technology, live births per treatment cycle remain relatively low at 21% (Human Fertilisation and Embryology Authority, 2002
). In an effort to optimize pregnancy rates, most IVF units transfer more than one embryo, thereby increasing the number of multiple pregnancies with a consequent influence on perinatal mortality and morbidity. The ability to choose the ‘best’ embryo for transfer should ultimately allow implantation of a single embryo of superior quality within the uterus thereby improving pregnancy rates whilst reducing the chances of a multiple pregnancy. Leese and colleagues have studied the possible use of amino acid and carbohydrate metabolism as predictors of embryo quality (Leese, 1987; Leese et al., 1993; Houghton et al., 2002) and have recently reported the value of assessments of amino acid metabolism in the prospective selection of the most viable embryo(s) for replacement in an IVF cycle (Brison et al., 2004). Evidence from animal studies suggests that individual fatty acids may affect normal oocyte maturation, fertility and embryo development (Waterman and Wall, 1988; Nonogaki et al., 1994; Khandoker and Tsujii, 1999a; Kim et al., 2001). Therefore, there may be merit in developing a similar predictive approach for the fatty acids. Knowledge of the pattern of fatty acid metabolism in relation to development is important for other reasons. The desire to move away from plasma-based media to synthetic mixtures of known origin provides an opportunity to influence the fatty acid mixture in ways which may better promote early stage embryo development. Knowledge of the metabolic requirements of the embryo at later stages of preimplantation development following transfer may also contribute to the success of IVF by identifying factors which are important in the maternal milieu. Information on late stage metabolism is also relevant to the creation of viable blastocysts for the production of human stem cell lines.
The aim of this study was to investigate the relationship between embryo fatty acid metabolism and the stage of development achieved. The fatty acid composition of human embryos and their ability to accumulate 13C labelled fatty acids were assessed in relation to the stage of development. Embryo selectivity for individual fatty acids was evaluated by determining the differential accumulation of unsaturated [13C]-linoleic acid and a representative saturated fatty acid, [13C]-palmitic acid.
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This study was approved by Grampian Research Ethics Committee and licensed by the UK Human Fertilisation and Embryology Authority (RO118). The 233 embryos used in this study were donated with consent by couples attending the Assisted Reproduction Unit in Aberdeen for IVF or ICSI treatment.
Two studies were carried out using different grades of embryos. In both studies, the embryos used were destined to be destroyed but for different reasons. In the fatty acid composition study, the embryos used were fresh but deemed unsuitable for clinical use on the basis of quality or the presence of three pronuclei on the day after insemination. In the fatty acid labelling experiment, where fatty acid uptake was measured isotopically, the previously frozen embryos were deemed suitable for clinical treatment but were no longer required by patients and were approaching the end of their allowed storage life and destined to be destroyed.
Stimulation
Women underwent ovarian down-regulation and stimulation of follicular growth and were monitored by ovarian ultrasonography and serum estradiol (E2) measurement according to a local protocol (Bhattacharya et al., 2001). The dose of FSH was adjusted according to the individual response. HCG (Profasi: 10,000 IU; Serono Bedford Cross, Middlesex, UK) was administered when at least three follicles of diameter 
17 mm were visible. Oocytes were recovered in an outpatient procedure under sedation 
36 h after injection of HCG.
Fertilization and initial embryo culture
Standard laboratory protocols for IVF and ICSI were followed. All oocyte and embryo incubations were carried out in Universal IVF medium (Medicult, Redhill, Surrey, UK) overlaid with mineral oil (Sigma, Gillingham, Kent, Poole, Dorset, UK) at 37°C in a humidified atmosphere of 5% CO2 in air. The fatty acid composition of the culture media used in these studies is not made available by the manufacturers.
After follicular aspiration, cumulus–oocyte complexes were washed free from blood in Universal IVF medium and incubated in organ culture dishes (Falcon, UK; Franklin Lakes, NJ, USA) or four-well plates (Nunc PO Box 280, Denmark) in 750 µl aliquots of the same medium (1–5 oocytes per group) until insemination (IVF) or ICSI. For IVF, the oocytes were inseminated 40 h after HCG administration with sperm prepared to a density of 1 million/ml. Oocytes for ICSI were denuded and only metaphase II oocytes were injected with prepared sperm by microinjection techniques. All oocytes were checked 18–20 h after insemination/injection and normally fertilized zygotes were transferred to fresh culture drops. After selection of embryos for embryo transfer and cryopreservation on day 2/3, the remaining embryos considered unsuitable for treatment were used in the fatty acid composition study.
Freezing and thawing of embryos
Embryos donated for this research had been frozen at the pronucleate to 8-cell stages using methods described previously (Lassalle et al., 1985). Briefly, embryos in 1.5M 1,2-propanediol (PrOH) and 0.1M sucrose were cooled slowly (0.3°C per min) in mini-straws to –30°C, before rapid cooling to –196°C in liquid nitrogen for storage. Straws were warmed in air at room temperature (20–22°C) for 40 s, then immersed in water at 25°C until the ice melted (5 s). The contents of each straw were immediately expelled into the centre drop of a four-well plate and the morphology of the embryo(s) recorded. The embryos were pipetted through decreasing concentrations of PrOH and sucrose (0.2M) at 5 min intervals. Finally, they were returned to phosphate-buffered saline (PBS) + human serum albumin (HSA) for up to 15 min before assessment of morphology. Embryos with 50% of cells surviving were assigned to the fatty acid metabolism study.
Fatty acid composition study
Embryos were assigned to research on day 2 or 3 post insemination. They were cultured in 750 µl drops of Universal IVF medium under oil for up to 5 further days. Embryo morphology was scored daily under a dissecting microscope. The criteria assessed were: number of blastomeres; regularity of blastomere size; and degree of cytoplasmic fragmentation. Culture was terminated when: development arrested (no change in appearance for 24 h); there was an increase in fragmentation; or signs of cells beginning to degenerate; or when an embryo reached the blastocyst stage.
At the end of culture, embryos were washed through at least 3 changes of PBS (100 µl to 1 ml per wash) to remove any traces of medium containing fatty acid and pipetted in a small volume of PBS into a chromatography v-vial (Agilent, Cheadle, Cheshire, UK) and stored at –80°C prior to analysis. The same volume of PBS, without embryos, was retained from the final wash to measure background levels of fatty acids. Embryos were washed in PBS without any macromolecular supplement as both polyvinyl alcohol (PVA) (Sigma) and ‘bovine fatty acid free’ serum albumin (Sigma) were found to contain trace amounts of fatty acids sufficient to distort the results.
Both embryos and the corresponding ‘background’ samples for each embryo were pooled prior to fatty acid analysis. Embryo samples in which the ‘background’ concentration of a fatty acid was found to exceed the embryo value by >1% was rejected as being unreliable. Two groups of embryos (21 embryos in total) were excluded from the analysis on this basis.
Fatty acid labelling study
After thawing, embryos were pipetted singly into drops (15 µl) of Blastassist media (Medicult Ltd, Redhill, Surrey, UK) with or without the addition of either [U-13C]-linoleic acid or [1-13C]-palmitic acid overlaid with oil and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Embryo-free control drops were incubated alongside the drops containing embryos to measure any non-specific fatty acid degradation/appearance.
Labelled media were prepared by adding [U-13C]-linoleic acid (Cambridge Isotope Labs, Andover, MA, USA) or [1-13C]-palmitic acid (Isotec Inc, Miamisburg, OH, USA)—provided at 99 Atom %—to Blastassist media B1 and B2. The [U-13C]-linoleic acid, supplied as the methyl ester, was hydrolysed first and taken up in ethanol. The [1-13C]-palmitic acid was also taken up in ethanol. On the day before incubation, the labelled fatty acid in ethanol was taken to dryness under a stream of nitrogen in previously autoclaved vials, Blastassist media B1 and B2 were added, and the mixture allowed to equilibrate overnight at 4°C. Control media were prepared in the same way using ethanol but without any additional fatty acid. The addition of labelled fatty acid increased the relative concentrations of palmitic acid in the Blastassist media from 19 to 30 mol % and linoleic acid from 18 to 29 mol %.
Embryos from each patient were allocated at random to culture in the three treatment groups: (i) Blastassist medium containing [U-13C]- linoleic acid; (ii) [1-13C]- palmitic acid; or (iii) no added stable isotopes (control). After thawing the embryos were washed in Blastassist medium B1 (1–3 cell stages) or B2 (4-cells) with or without added isotope before culture in drops of the same medium.
After 48 h culture, all developing embryos were transferred into B2 media containing the same fatty acid supplement. Embryos were scored every 24 h by counting and recording the number of cells, size of cells and the percentage of fragmentation. The medium was changed every 48 h for embryos which continued to develop. Culture was terminated when either an embryo degenerated, development arrested (no increase in cell number for 24 h), the embryo reached the blastocyst stage or on day 7 post insemination. In separate tests, the mineral oil used in the incubations had no measurable effect on the fatty acid composition or the stable isotope concentration of the labelled fatty acids in the media.
At the end of culture, the embryos were washed through at least four changes (50 µl drops per wash) of PBS + 0.1% fatty acid free BSA to remove all traces of 13C labelled fatty acid and oil. Each embryo was pipetted in a small volume of PBS + BSA into a glass gas chromatography vial and stored at –80°C prior to analysis. The same volume of PBS, without an embryo, was also taken from the final wash and pipetted into a gas chromatography phvial for measurement of background levels of 13C fatty acids. Unlike the fatty acid composition studies, the presence of trace amounts of unlabelled fatty acid during analysis does not affect the detection of isotope. In this experiment, the priority was to remove the 13C labelled fatty acid and, for ease of pipetting, fatty acid free albumin was added to the PBS at a concentration of 0.1%.
Measurement of media and embryo fatty acid composition
The absolute and relative concentrations of non-esterified fatty acids (NEFA) in the culture media were determined by gas chromatography following the addition of an internal standard (C22:1; Sigma, Poole, Dorset, UK). For the embryos, only the relative concentration was measured as absolute quantitation would have required the use of valuable embryos to simply test the appropriate concentration of internal standard to use.
Lipid was extracted from the unlabelled media, pooled embryos and pooled background media samples using chloroform: methanol (2:1 v/v) containing butylated hydroxytoluene essentially as described previously (Bligh and Dyer, 1959).
Physical exclusion of mineral oil was difficult to achieve when removing embryos from media and the presence of mineral oil in the lipid extracts reduced the quality of the gas chromatography traces. Therefore, an additional step was developed to remove the last traces of mineral oil by applying the lipid to a high performance thin layer chromatography (HPTLC) plate (Waters, Elstree, Hertfordshire, UK) and developing this in 1% ether in hexane.
Following trans-esterification and methylation, the fatty acid methyl esters from the embryos were separated and analysed on a DB23 (30 m, 0.25 mm i.d., 0.25 µm film) column (J&W Scientific, Folsom, CA, USA) via cool on-column injection at 40°C on a Hewlett-Packard 56890 series II gas chromatograph (Hewlett-Packard Ltd, Cheshire, UK) fitted with a flame ionization detector (temperature programme; 40°C for 5 min, 25°C/min to 160°C, 1°C/min to 210°C, 10°C/min to 220 for 20 min.). Peaks were integrated with Hewlett-Packard Chemstation® software and identified by reference to known standards.
Measurement of 13C uptake
Following incubation in labelled media, the fatty acids present in the embryos were extracted and methylated essentially as in the composition studies. The 13C fatty acid composition was determined by gas-chromatography-combustion-gas-isotope-ratio-mass-spectrometry (GC-C-IRMS) on a Thermo Finnigan Delta Plus XL (Thermo Finnigan, Bremen, Germany) after separation on a CP-Sil88 50 m x 0.25 mm i.d. column.
Isotope ratio mass spectrometry is extremely precise in the measurement of isotopic abundance (<1 ppm), but it is less sensitive to absolute levels of fatty acid than the flame ionization detection used in the composition studies. It was therefore necessary to add carrier fatty acid (10 µg each of unlabelled linoleic and palmitic acids) prior to extraction. In addition, a third labelled fatty acid (10 µg 13C C21:0 at 1.09 Atom %) was added as an internal standard to monitor the reproducibility of the mass spectrometry results.
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Results |
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The primary carrier for non-esterified fatty acid (NEFA) in blood is human serum albumin (HSA) and this is normally present in human plasma at 3.5–5% (w/v). HSA was present in the incubation medium at a concentration of 0.2% (w/v). No other source of fatty acid is listed as a component of the culture medium; therefore, it must be concluded that the fatty acids in the medium simply reflected those already bound to the added albumin. The concentration of NEFA in the Blastassist medium was measured at 24 µM. We have previously measured a mean NEFA concentration of 530 µM in fasting plasma taken at the time of oocyte recovery during IVF treatment (unpublished data), suggesting that both albumin and the fatty acids are present in the culture medium at a concentration
1/20th of that in human plasma. The relative concentrations (mol %) of the individual fatty acids to total NEFA in the Blastassist medium used in the fatty acid uptake studies were as shown in Table I. There was no evidence for a difference in the fatty acid concentration or relative composition of the Blastassist B1 and B2 media. The relative concentrations (mol %) of the individual fatty acids to total NEFA in the Universal medium used in the composition studies was also similar to the Blastassist composition (Table I).
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The relative concentrations of the different fatty acids in the media were generally similar to that found in plasma, although the relative concentration of linoleic acid (C18:2n-6) was lower and that of oleic acid (C18:1) considerably higher than we measure in the NEFA of fasting samples taken from women undergoing IVF treatment in Aberdeen. The origin of the human albumin used in the medium was not specified, but this difference could possibly be due to the use of albumin from non-fasted subjects or from individuals consuming different diets.
Embryo studies
Relatively few viable embryos were available for study and those that were represent a valuable and finite resource. As no information was available on the magnitude of human embryo fatty acid metabolism, we opted to perform the various measurements in groups of pooled rather than single embryos. This greatly restricted the statistical analysis which could be applied to the results but ensured that we would be able to detect any biologically significant changes in composition and metabolism with stage of development.
Embryo fatty acid composition
A total of 136 embryos unsuitable for clinical use, including 14 embryos derived from abnormally fertilized oocytes, were donated for this experiment. Eight embryos were lost during washing after culture due to the difficulty of pipetting in PBS with no macromolecular supplement and 21 were excluded from the analysis on the basis of the fatty acid background relative to embryo concentration exclusion criteria. Sixty-three embryos did not develop past the 6-cell stage and were pooled into four batches prior to lipid extraction. A total of 44 embryos developed beyond the 6-cell stage, of which three formed morulae and 10 reached the blastocyst stage. These embryos were pooled into three batches prior to lipid extraction (a single 2-cell stage embryo was included in one of the batches in error).
The fatty acid composition of embryos is shown in Table II. The values for C10:0 and C12:0 are only approximate as these had to be integrated manually due to their proximity to the solvent front. The embryos were relatively high in saturated fats (62–77%) with around half of this consisting of palmitic acid (C16:0). The next most common group was the monounsaturates with oleic acid (C18:1) making up the greatest fraction. The next most common was linoleic acid (C18:2n-6), followed by arachidonic acid (C20:4n-6) and docosahexaenoic acid (C22:6n-3).
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The most striking change with stage of development was in the relative concentration of the saturated fatty acids and linoleic acid (Fig. 1). The fatty acid composition of embryos which failed to develop past the 4-cell stage was generally similar to that previously reported in oocytes which failed to fertilize (Matorras et al., 1998). Embryos which developed beyond the 4-cell stage had significantly higher concentrations of the unsaturates, linoleic acid (P = 0.019) and oleic acid (P = 0.022), and a lower concentration of total saturates (P = 0.036). The long chain polyunsaturates, arachidonic acid and docosahexaenoic acid, were also higher in the later stages of development but these differences were not significant. The low proportion of embryos reaching the blastocyst stage reflects the relatively poor quality of the embryos available for this analysis.
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•) and total saturated fatty acid (SFA 
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Embryo 13C fatty acid uptake
To understand more about the processes underpinning the developmental changes in composition, embryos were incubated with stable isotope (13C) labelled fatty acids. Given the marked and opposing changes in the composition of saturated fatty acids and linoleic acid, the fatty acids chosen for study were 13C linoleic acid and 13C palmitic acid as the major saturated fatty acid.
A total of 134 frozen embryos no longer required for treatment and donated for research were used in six replicate experiments. Twenty-eight embryos did not survive thawing leaving 116 embryos to be cultured; 39 embryos in 13C linoleic augmented media, 39 in 13C palmitic augmented media, and 38 in medium without added stable isotopes.
For the subsequent analysis, embryos at a similar stage of development were pooled into four groups for each treatment: (i) those that did not develop in culture (8–11 embryos per treatment); (ii) those that arrested at <8-cell stage (11–17 embryos per treatment); (iii) 8-cell –morula (7–9 embryos per treatment); and (iv) blastocysts on day 6/7 post insemination (7–9 embryos per treatment).
The addition of labelled fatty acid increased the relative concentrations of palmitic acid from 19 to 30 mol % and linoleic acid from 18 to 29 mol % in the incubation media. It was therefore of interest to compare the stage of development achieved by embryos under these conditions. The proportion of embryos developing to blastocyst was not significantly different between the groups: 23% (9 out of 39) for the linoleic acid augmented media; 18% (7 out of 39) for the palmitic acid augmented media; and 18% (7 out of 38) for the control group. The key finding here is that the addition of stable isotope labelled fatty acid was not detrimental to development.
The labelling study was carried out in a novel way using carrier fatty acids to allow the detection of very low rates of fatty acid uptake with high precision. The sensitivity and precision of mass spectrometer response was checked by measuring the linearity of the enrichment signal down to a level equivalent to 1 pmol of fatty acid. The pattern of accumulation of 13C linoleic and palmitic acids is shown in Fig. 2A. The most striking thing about this graph is the rapid increase in the uptake of linoleic acid with stage of development and the relatively low level of uptake of palmitic acid at the later stages in particular. It is also noticeable that, even in embryos which failed to develop, there was a higher accumulation of linoleic than palmitic acid—though this baseline uptake is largely a function of the physicochemical properties of these fatty acids.
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The rate of uptake reflects the rate of cellular utilization (Haggarty, 2002
), but there is always some passive partition of fatty acids into cells and the greater accumulation of linoleic acid compared with palmitic acid is to be expected on the basis of known fatty acid solubilities in the aqueous phase and the dissociation constants for linoleic acid, palmitic acid and human albumin (Richieri et al., 1993). The uptake values obtained in non-developing embryos therefore represent a useful baseline against which to compare the uptake in developing embryos. In order to estimate rates of uptake in relation to developmental competence, the additional net accumulation of 13C at each stage of development was calculated per 24 h of incubation (Fig. 2B). This shows that there was a net accumulation of palmitic acid of 1 pmol/embryo/24h at the 2-cell to <8-cell stage and further accumulation of 4 pmol/embryo/24h at the 8-cell to morula stage. However, it is striking that thereafter there was no further accumulation of palmitic acid and even a possible net loss at the blastocyst stage. The developmental pattern for linoleic acid accumulation was quite different with a negligible net accumulation at the 2-cell to <8-cell stage, followed by a net accumulation of 8 pmol/embryo/24h at the 8-cell to morula stage, and a further doubling of the rate of uptake to 17 pmol/embryo/24h at the blastocyst stage.
For the composition studies, the average age of women in the groups arresting before and after the 6-cell stage was 34 years in both cases. The method of fertilization (IVF%/ICSI%) was 67/33 in the former and 50/50 in the latter. In the fatty acid uptake studies, the average age was 34 years (failed to develop), 33 years (>8-cell), 35 years (8-cell to morula), 34 years (blastocyst). The method of fertilization (IVF%/ICSI%) was 39/61, 35/65, 39/61 and 43/58, respectively. There was no evidence for a difference in maternal age in relation to stage of development achieved in either the composition or metabolism studies. Some workers have reported that ICSI may be associated with reduced potential for development (Archer et al., 2003), but there was no evidence for such an effect in the embryos studied here.
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Discussion |
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This study has demonstrated that the fatty acid composition of human preimplantation embryos and their rate of uptake of individual fatty acids varies with stage of development. The fatty acid composition of embryos which failed to develop past the 4-cell stage was generally similar to that previously reported in oocytes which failed to fertilize (Matorras et al., 1998
). However, embryos which developed beyond the 4-cell stage had significantly higher concentrations of the unsaturates, particularly linoleic acid (C18:2n-6) and oleic acid (C18:1n-9), and a lower concentration of total saturates. The long chain polyunsaturates, arachidonic acid (C20:4n-6) and docosahexaenoic acid (C22:6n-3), were also present in higher concentrations in the later stages of development but these differences were not statistically significant. Compared with the reported fatty acid composition for oocytes from cattle, pigs and sheep (McEvoy et al., 2000), early stage human embryos had a similar proportion of the important unsaturated fatty acids, 
-linolenic (C18:3n-3), arachidonic and eicosapentaenoic (C20:5n-3) and the saturated palmitic (C16:0). However, the concentration of oleic acid in early stage human embryos was only a third, and the concentration of linoleic acid only half, that in animal oocytes. There are also large differences between species in the stores of total lipids with bovine embryos being particularly rich in lipid (Ferguson and Leese, 1999; McEvoy et al., 2000). These differences emphasize the need to study human embryos directly in relation to fat metabolism.
The polyunsaturated fatty acids in general, and linoleic acid in particular, support a number of key developmental processes in the embryo. They act as the precursors of the eicosanoids and regulate the processes of endocytosis/exocytosis, ion-channel modulation, DNA polymerase inhibition and gene expression. Linoleic acid stimulates protein kinase C (Murakami et al., 1986), which is critical in cell growth and differentiation (Nishizuka, 1988), and it is probably the fatty acid which has been most implicated in animal fertility studies (Homa and Brown, 1992). The polyunsaturated fatty acids are also essential components of membrane lipids which increase rapidly with each cell division; even 2-cell divisions associated with the transition from the one to the four cell stage results in a 74% increase in membrane surface area (Pratt and George, 1989). This increasing requirement for membrane lipids with cell number is consistent with the observation that the concentration of linoleic acid increases with stage of development.
It is important to know the mechanism by which the change in composition occurs, but this is difficult to infer from the compositional measurements alone. Bovine embryos classified as category 1 (highest grade) on the basis of their cytoplasmic quality have greater concentrations of linoleic acid and lower concentrations of saturated fatty acids compared with grade 2/3 embryos (Kim et al., 2001). It may therefore be that the embryos studied here already had a more unsaturated fatty acid composition at the 
4-cell stage. If this is the case, it is likely that the effect originates in the oocyte since the same high saturate and low unsaturated composition found in poorly developing embryos was observed in oocytes which failed to fertilize (Matorras et al., 1998). Alternatively, the compositional changes could be a normal function of development rather than a marker of embryo quality, with the higher degree of unsaturation measured at the ultimate stage of development resulting from differential uptake and metabolism in the later stages of preimplantation development.
More information on possible mechanisms can be obtained from dynamic studies of fatty acid uptake by embryos. This was done here by measuring the uptake of two isotopically labelled fatty acids, 13C linoleic acid and the main saturated fatty acid—13C palmitic acid. The uptake of both fatty acids was very low prior to the 8-cell stage and increased with the stage of development for linoleic in particular. The overall trend indicated an early preference for uptake of the saturated fatty acid, but increasing uptake of linoleic acid and decreasing uptake of the saturated fatty acid with stage of development. This differential pattern of uptake during development could, in itself, explain the observed developmental changes in embryo fatty acid composition.
This general trend towards increasing uptake of linoleic acid in particular with stage of development is similar to that reported for other nutrients in a range of animal embryos. Also, the rates of uptake, varying from less than 1 pmol/embryo/24 h at the 2–4 cell stage to 17 pmol/embryo/24 h at the blastocyst stage, are of the same order of magnitude as those reported for other nutrients in animal embryos. For example, bovine and ovine embryos take up glucose and pyruvate at a rate of 20 and 500 pmol/embryo/24 h between the 2-cell and blastocyst stage (Gardner et al., 1993; Thompson et al., 1996). Given that the average fatty acid contains between three and nine times the amount of carbon as these low molecular weight nutrients, the absolute rates of carbon uptake are comparable.
The only equivalent information for fatty acids in animal embryos comes from radioisotopic studies where only relative changes in the metabolism of fatty acids to 14CO2 with stage of development is reported (Khandoker and Tsujii, 1998, 1999b). In the earliest of these studies, the oxidation of palmitic acid was shown to follow the same pattern of small but measurable rates of oxidation at the earliest stages, increasing with developmental stage. In this study, the highest rate of palmitic acid oxidation was observed at the blastocyst stage, when we observed a net loss of palmitic acid. This may indicate the selective channelling of previously accumulated palmitic acid towards oxidation for energy production at the blastocyst stage in human embryos. These authors also reported that the 14C fatty acid was incorporated into both triacylglycerols (the main storage lipid class) and phospholipids (the main membrane lipid class) 2 h after addition to 8-cell stage embryos, but they did not report the relative amounts in these two lipid classes (Khandoker and Tsujii, 1999b). Waterman and Wall (1988) reported uptake of 14C labelled oleic (C18:1n-9) and arachidonic (C20:4n-6) acids by rabbit zygotes and incorporation of label into both triacylglycerols and phospholipids—although, at this early stage of development, the preference was for incorporation into triacylglycerol.
Knowledge of the pattern of metabolism in relation to development is relevant to attempts to develop non-destructive tests of embryo quality. The study design described here required the destruction of embryos to allow the measurement of fatty acid uptake. However, the use of stable isotopes offers the possibility of non-destructive tests of metabolism by, for example, measuring the isotopic composition of the incubation media. Such metabolic studies have been carried out in animal embryos using radioisotopes such as 3H and 14C (Khandoker and Tsujii, 1998, 1999b), but the hazards associated with ionizing radiation would clearly preclude their use in human embryos destined for transfer. However, stable isotopes, such as the 13C used in this study, are always present in the environment and living systems. They are routinely used in human metabolism studies in vivo, including in pregnancy, and they present no known biological hazard to the embryo. There was no evidence here that they had any adverse effect on development and we know of no reason why they could not be safely used in IVF culture, although their use would have to be authorized by the relevant regulatory bodies. An alternative approach would be to infer embryo fatty acid metabolism by measuring its effect on the fatty acid profile of the incubation media as has been done successfully for the amino acids (Houghton et al., 2002; Brison et al., 2004). This approach would have the benefit of simultaneously evaluating the metabolism of all the fatty acids rather than a selected few, thereby providing better information on the metabolic status of the embryo.
Knowledge of the pattern of nutrient metabolism in relation to development is also relevant to the rational design of culture media nutrient composition. The fatty acid composition of the commercial media used here simply mimicked that of human plasma (as a result of the use of albumin as the sole source of fatty acids), yet the embryo is not in intimate contact with blood during the period of development corresponding to in vitro culture in IVF. Studies of rat embryos have demonstrated that development to blastocyst stage is promoted by the addition of the unsaturated fatty acids (oleic, linoleic and arachidonic), but not the saturated palmitic acid, to incubation media (Khandoker and Tsujii, 1999a). These results are consistent with the data presented here which indicate a preference for the unsaturated acids over the saturated palmitic acid in late preimplantation stage embryos and suggest that efforts to improve the composition of human embryo culture media could usefully focus on the ratio of linoleic to saturated acids.
The indication that the human embryo may have a high requirement for the unsaturated fatty acids, linoleic in particular, in the later stages of preimplantation development suggests that the availability of particular fatty acids in vivo, in the uterine horn and uterus prior to implantation, could potentially influence IVF success following transfer. It is known, for example, that body fat status is linked to fertility (Frisch, 1987; Zaadstra et al., 1993; Wass et al., 1997), an association which could be mediated via an effect on the availability of individual fatty acids.
The main drawback of this study was the need to pool samples prior to analysis to maximize the chances of gaining useful information from the finite and valuable resource of embryos surplus to clinical requirements. The data presented on the developmental changes in fatty acid metabolism in human embryos add useful basic information to the growing body of knowledge of early embryo metabolism. The data may also be useful practically in helping to develop non-destructive tests of embryo quality and improving culture media and the success of IVF treatment.
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Acknowledgements |
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The authors would like to express their gratitude to the couples who generously donated embryos for this research. The authors acknowledge financial support from the Wellcome Trust, Grampian Royal Hospitals Trust and SEERAD. Medicult UK donated some medium for part of the study.
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Submitted on February 2, 2005; resubmitted on October 9, 2005; accepted on October 21, 2005.
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