Conceived and designed the experiments: JLMRL RGS VVH HJL. Performed the experiments: VVH RGS PB-A JLMRL. Analyzed the data: JLMRL VVH RGS PB-A. Wrote the paper: VVH RGS PB-A DR AG-A HJL PEJB JLMRL.
The authors have declared that no competing interests exist.
Elevated concentrations of serum non-esterified fatty acids (NEFA), associated with maternal disorders such as obesity and type II diabetes, alter the ovarian follicular micro-environment and have been associated with subfertility arising from reduced oocyte developmental competence. We have asked whether elevated NEFA concentrations during oocyte maturation affect the development and physiology of zygotes formed from such oocytes, using the cow as a model. The zygotes were grown to blastocysts, which were evaluated for their quality in terms of cell number, apoptosis, expression of key genes, amino acid turnover and oxidative metabolism. Oocyte maturation under elevated NEFA concentrations resulted in blastocysts with significantly lower cell number, increased apoptotic cell ratio and altered mRNA abundance of
Up-regulated lipolysis, a feature of metabolic disorders such as obesity and type II diabetes, results in increased plasma non-esterified fatty acid (NEFA) concentrations
Using a bovine model we have demonstrated that elevated serum NEFA concentrations are reflected in the follicular fluid of the pre-ovulatory ovarian follicle
Epidemiological studies have shown that lipolysis-linked maternal metabolic disorders, such as obesity and type II diabetes
It is widely acknowledged that appropriate oocyte development governs several sequential and critical steps in meiosis, fertilization and early cleavage
In the present study, we hypothesized that elevated NEFA concentrations are a key metabolic factor in the relationship between maternal metabolic disorders and subfertility, through a negative effect on the oocyte. To address this, we have used a bovine oocyte in vitro culture model to investigate whether elevated NEFA concentrations during bovine oocyte maturation influence subsequent embryo phenotype.
We report that elevated NEFA concentrations, specifically Oleic Acid (OA), Palmitic Acid (PA) and Stearic Acid (SA), during oocyte maturation have negative consequences for the resulting preimplantation embryo, measured 8 days later at the blastocyst stage. These include altered blastocyst gene expression and reduced embryo quality, determined in terms of energy and amino acid metabolism; known markers of embryo viability. Our data provide evidence of a mechanism for metabolic deregulation appearing in the preimplantation embryo as a consequence of elevated NEFA concentrations during oocyte maturation and help to explain the higher rate of early pregnancy loss and miscarriage
Supplementing maturation medium with different combinations of NEFA had no significant effect on cleavage rates but based on the odds ratios, maturing oocytes in medium with elevated Stearic Acid (HIGH SA) or in medium supplemented with a combination of elevated Stearic Acid (SA), Palmitic Acid (PA) and Oleic Acid (OA) concentrations (HIGH COMBI) resulted in a significant reduction in the number of oocytes reaching the blastocyst stage at day 7 post insemination (p.i.) (
n (%) | CONTROL | HIGH SA | HIGH PA | HIGH OA |
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133 (55.4) | 126 (52.1) | 155 (63.8) | 194 (67.8) |
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60 (25.0)a | 45 (18.6)b | 42 (17.3) b | 64 (22.4) a |
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60 (45.1)a | 45 (35.7)a | 42 (27.1) b | 64 (33.0)b |
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125.8±29.4 a | 105.4±24.7b | 118.5±34.5 a | 122.7±23.9 a |
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0.09±0.05a | 0.18±0.08b | 0.20±0.12b | 0.16±0.08 b |
Oocytes (n = 1024; three replicates) were matured in maturation medium supplemented with 1) physiological NEFA = control (150 µM of total NEFA, i.e. OA, SA and PA); 2) elevated Stearic Acid = HIGH SA (75 µM SA); 3) elevated Palmitic Acid = HIGH PA (150 µM PA) and 4) elevated Oleic Acid = HIGH OA (200 µM OA). Data marked with different superscripts per row are significantly different between treatments (
Total blastocyst cell number was significantly lower in the HIGH COMBI (104.7±26.1) and the HIGH SA (105.4±24.7) group compared with their control counterparts (125.8±29.4) (
We next investigated the expression of key genes representative of cellular processes linked to apoptosis, development, quality and metabolism in the blastocyst. Surprisingly, the expression pattern of many of these genes did not differ between embryos arising from oocytes maturated with HIGH SA or the controls (
Blastocysts were derived from oocytes matured under control, HIGH SA and HIGH COMBI conditions (n = 192; five replicates). Bars with different superscripts are significantly different between treatments with P<0.05.
As shown in
A. Amino acid ‘turnover’ of day 7 blastocysts as calculated by summing all amino acids produced and consumed on a per-embryo basis. Comparison was done between blastocysts (n = 135; three replicates) derived from oocytes matured under control, HIGH SA and HIGH COMBI conditions. B. Overall mean profiles of individual amino acids by blastocyst in the three treatments. Bars with different superscripts are significantly different between treatments (P<0.05).
The profiles of individual amino acids showed that significantly higher levels of serine (
Given the observations that exposing oocytes to elevated NEFA impacted on blastocyst quality, gene expression patterns and amino acid metabolism, we next investigated the oxygen consumption by these embryos. Using a non-invasive real time assay of oxygen consumption, we found that blastocysts arising from oocytes matured under HIGH COMBI conditions consumed significantly less oxygen than control embryos (
A. Mean oxygen consumption of day 7 blastocysts. Blastocysts (n = 66; three replicates) were derived from control-, HIGH SA- and HIGH COMBI-exposed oocytes. Different superscripts indicate significant differences between treatments (P<0.05). B. Mean pyruvate, glucose and lactate consumption of blastocysts. Blastocysts (n = 84; five replicates) originated from oocytes matured in control, HIGH SA and HIGH COMBI maturation medium. Different superscripts indicate significant differences between treatments (P<0.05).
Elevated serum NEFA concentrations, arising from up-regulated lipolysis, have been implicated as a key factor in the association between metabolic imbalances, cellular dysfunction and related pathologies
Support for the bovine as model of early mammalian reproduction, especially of the human, has grown steadily over the last decade. The human and bovine are single ovulators and there are close similarities between human and bovine ovarian function and oocyte characteristics, in contrast to major differences in ovarian physiology and reproductive function between rodents and humans
Our data show that elevated NEFA exposure during oocyte maturation does not impair the ability of zygotes to reach the 2-cell stage. However, there is a reduction in the number of the HIGH NEFA-exposed oocytes capable of forming blastocysts. In bovine and human embryos, the developmental processes of the first 3-to-4 cleavage divisions occur under the control of maternally-derived mRNAs and proteins stored in the oocyte, until major embryonic genome activation (EGA) occurs at the 8–16 cell stage. The reduction in blastocysts formed indicates that NEFA-exposure during oocyte development has a significant negative impact on post-genome activated development as well as on the pattern of gene transcription after EGA. Data from recent studies on an obese female mouse model
The total cell number in blastocysts from HIGH NEFA-exposed oocytes was significantly reduced and the corresponding apoptotic cell ratio increased, compared with control counterparts. It is generally recognized that saturated NEFA, such as PA (C16:0) and SA (C18:0), can directly exert negative effects on cell viability
NEFA exposure during oocyte maturation also affected the gene expression pattern of the resulting blastocysts. A significant increase in relative mRNA abundance of the HIGH COMBI embryos was found for the
In 2002, the ‘quiet embryo hypothesis’ was presented
It is increasingly recognized that oocyte and embryo metabolism are closely linked with subsequent developmental capacity
Surprisingly, HIGH COMBI embryos consumed significantly more lactate compared with control embryos. One explanation for this might be that NEFA exposure results in an imbalance of the intracellular oxidation-reduction (REDOX) potential, as previously reported
Such an alteration in REDOX status may affect the activity of REDOX sensitive transcription factors; for example, the amount of
In conclusion, our data show that maternal metabolic conditions, associated with elevated NEFA during oocyte maturation, may compromise fertility through a reduction in oocyte developmental competence and the viability of the subsequent embryo.
In highlighting the metabolic problems associated with obesity and their potential relationship with subfertility, our findings are consistent with public health recommendations which emphasise the importance of women being at healthy weight before starting a pregnancy.
For full details, see
The types and concentrations of free fatty acids used in the present study are based on bovine
Standard serum-free maturation systems are devoid of fatty acids, although the physiological environment, in which the oocyte matures
n (%) | standard serum-free maturation medium | physiologically relevant control medium |
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222 (76.2) a | 179 (64.8) a |
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88 (30.2) a | 89 (29.3) a |
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88 (39.6) a | 89 (49.7) a |
Cleavage rate at day 2 p.i., number of formed blastocysts at day 7 p.i. relative to the number of matured oocytes or to the number of cleaved zygotes. Oocytes (n = 595; three replicates) were matured in standard serum-free maturation medium devoid of all fatty acids
In preliminary experiments, we identified Stearic Acid (SA) as the most toxic NEFA for oocyte developmental competence (
For additional experiments we therefore focussed on the HIGH SA and the HIGH COMBI treatments. The following NEFA treatments were used in the present study:
All chemicals were purchased from Sigma®, unless otherwise stated. SA, PA and OA were dissolved in a stock solution of pure ethanol at concentrations of 25, 150 and 200 mM, respectively. These ethanol stock solutions were vortex-mixed for 4 min and diluted in working solutions to obtain the desired final concentration in maturation medium. The serum-free maturation medium consisted of TCM199 supplemented with 0.75% BSA free of fatty acids, 0.4 mM glutamine, 0.2 mM sodium pyruvate, 0.1 mM cysteamine, 50 µg/mL gentamycin and murine epidermal growth factor (mEGF, 20 ng/ml). All treatments were vigorously shaken for 45 min and filter-sterilised under aseptic conditions.
Cleavage (2 days post insemination (p.i.)), and blastocyst rates (7 days p.i.) were defined as the number of cleaved zygotes or formed blastocysts per oocyte matured, respectively. The number of blastocysts from cleaved zygotes was also recorded.
Cell number and apoptotic cell index (number of apoptotic cells over total cell count) were assessed by staining normal and expanded day 7 blastocysts with propidium iodide (PI) and with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), respectively, as described
Molecular biology procedures were carried out as previously described
The amino acid content of spent culture media containing day 7 blastocysts was determined by reverse-phase HPLC as previously described
Individual blastocysts day 7 p.i. were loaded into a PCR Glass micropipette (Drummond) and allowed to respire for 30 min to form an oxygen gradient. This oxygen gradient was measured in real time using a nanorespirometer (Unisense) and converted to oxygen consumption rate using SensorTrace Pro (Unisense) according to a previous study
Day 7 blastocysts were cultured individually for 3h in 4 µl droplets of modified SOF medium alongside empty control droplets. After the incubation period, the embryos were removed and spent culture droplets frozen at −80°C until analysis. Glucose, lactate and pyruvate utilization was determined by ultrafluorometric assays of spent medium as previously described
All statistical procedures were carried out with SPSS 15.0 (for Windows, Chicago, IL, USA), unless otherwise stated. Cleavage and blastocyst rates were compared between the three treatments using a binary logistic regression model. For the other parameters a mixed model ANOVA, taking treatment as fixed factor and replicate as random factor, was used to compare differences between the three groups. No data transformations were necessary for inequality of variance between groups or for achieving normality for any data with the exception of amino acid metabolism. Chi square tests were used to analyze the effect of blastocyst developmental stage on oxygen, glucose, pyruvate and amino acid profiles. Relative transcript abundance was analyzed using the SigmaStat (Jandel Scientific) software package using one-way ANOVA with multiple pair-wise comparisons using Student-Newman-Kleus method post-hoc.
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