Hum. Reprod. Advance Access originally published online on October 12, 2007
Human Reproduction 2007 22(12):3059-3068; doi:10.1093/humrep/dem318
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Impact of hyperglycemia on early embryo development and embryopathy: in vitro experiments using a mouse model
Academic Unit of Reproductive and Developmental Medicine, University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK
1 Correspondence address. Tel: +44-114-2268537; Fax: +44-114-2261074; E-mail: r.b.fraser{at}sheffield.ac.uk
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Abstract |
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BACKGROUND: The aim of this study is to model the processes of early embryopathy seen in human pregnancy complicated by maternal hyperglycemia secondary to maternal diabetes using a mouse embryo culture system.
METHODS: Female mice were superovulated and mated in pairs. Two-cell embryos were harvested from the oviducts and cultured in vitro in KSOM medium (synthetic oviductal medium enriched with potassium) supplemented with 0.2, 5.56, 15.56 or 25.56 mM D-glucose. Cell proliferation, differentiation and apoptosis were assessed. Experiments were performed in constant, embryos exposed to a particular concentration of glucose (0.2, 5.56, 15.56 or 25.56 mM) from harvest to either Day 5 post fertilization (pf) or Day 8 pf, and fluctuating, embryos exposed to alternate high 25.56 mM and normal 5.56 mM concentrations of glucose between harvest and Day 5 pf, glycemic culture.
RESULTS: Expected levels of blastocyst formation and hatching were seen at 0.2 and 5.56 mM concentrations of glucose but both were impaired at higher concentrations (
2, P < 0.005; P < 0.001). Total cell numbers (P < 0.002) and cell allocation to the inner cell mass (P < 0.01) were reduced, but with no evidence of enhanced apoptosis in the hyperglycemic cultures. Variation in hyperglycemic exposure of the embryos on Days 2, 3 and 4 showed no adverse effects of hyperglycemia up to 24 h, but 48 and 72 h exposures were equally embryopathic (P < 0.01).
CONCLUSIONS: Hyperglycemic exposure for >24 h is toxic to early embryo development. These findings may explain the lower than expected implantation rates and higher than expected rates of congenital abnormality and early pregnancy loss seen in patients with diabetes, particularly those with poor diabetic control.
Key words: embryopathy/hyperglycemia/in vitro/mouse embryo/diabetes
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Introduction |
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The St Vincent Declaration of the European Association for the Study of Diabetes and the World Health Organization set a target for women with diabetes of achieving pregnancy outcomes similar to those of non-diabetic women. One area in which this target is not being achieved is the prevention of congenital malformation in the offspring. Collected series confirm that embryopathy associated with maternal diabetes remains an important clinical problem. In women with pre-existing diabetes, the rate of major malformations is 4–5 times the rate in non-diabetics. This excess has been associated with relative maternal hyperglycemia since the original observations of Miller et al. (1981)
which reported that malformations were a feature of poor periconceptional plasma glucose control manifested by high levels of glycated hemoglobin (HbAlc) measured in the first trimester of pregnancy.
A large body of clinical data including case series and quasi-randomized experiments has demonstrated that improved periconceptional control is associated with a reduction in fetal malformation rates in women offered preconceptional counselling and enhanced diabetic control during pregnancy. The meta-analysis of Ray et al. (2001) reported a mean fall from 7.7 to 2.4% for major malformations, a 68% reduction. Of particular interest is the Diabetes in Early Pregnancy Study (Mills et al., 1988) that reported a significant reduction in fetal malformations in women who enrolled before pregnancy and were subjected to an enhanced preconceptional diabetes control program. It was observed, however, that the malformation rate in these women was still more than twice that of non-diabetic matched controls, and surprisingly that malformations were still seen amongst the better controlled women as judged by lower individual HbAlc levels. High post-prandial peak excursions of plasma glucose can occur in apparently well-controlled subjects. The difference between pre- and post-prandial concentrations of glucose in apparently well-controlled women with diabetes can be 10-times greater compared to non-diabetics (Kyne-Grzebalski et al., 1999). Since the concentration of glucose in tubal and uterine fluid is directly related to the levels in plasma (Leese, 1981), we have hypothesized that in well-controlled diabetic women the embryo may still be exposed to intermittently fluctuating high levels of glucose during development inside the female reproductive tract. We also hypothesized that exposure to fluctuating levels of glucose, as well as absolute hyperglycemia, may induce further stress on the preimplantation embryo, compromising development and contributing to the increased incidence of fetal malformations seen in apparently well-controlled diabetes. Since the publication of the studies analysed by Ray and colleagues, therapeutic advances in diabetes care suggest that safer, better-targeted periconceptional control programs may be devised. The first of these is the introduction of basal-bolus type self-management programs such as DAFNE (DAFNE Study Group, 2002) which might allow a safe enhancement of diabetic control at the woman’s own initiative. Secondly, and perhaps complementary to this, is the availability for use in pregnancy of new insulin analogues. These can be administered to modulate the high post-prandial peak excursions of plasma glucose seen in apparently well-controlled subjects with diabetes.
The mechanisms of diabetes-induced embryopathy have been the subject of many studies usually based on experimental diabetes in rodent models. These have shown that abnormal maternal fuel metabolism and hyperglycemia associated with diabetes serve to impair embryogenesis as early as the preimplantation stages of development (Moley, 1999). In vivo studies have demonstrated morphological, cellular and biochemical changes in early embryos flushed from the reproductive tract of spontaneous and chemically induced diabetic mice and rats. Blastocysts (Day 4 of pregnancy) have a higher degree of nuclear fragmentation, a reduced number of inner cell mass cells (ICM) and an increased incidence of apoptosis in the ICM compared to those from non-diabetic animals (Pampfer et al., 1990, 1997; Vercheval et al., 1990; De Hertogh et al., 1991a; Moley et al., 1991, 1998a; Lea et al., 1996). Trophoblast outgrowth in vitro is also impaired (Pampfer et al., 1994a; Leunda-Casi et al., 2001). In addition, De Hertogh et al. (1991a) have also shown that the total number of embryos formed is reduced in diabetic mice. Interestingly, the administration of insulin to these animals appears to prevent diabetes-induced embryopathy although the total number of embryos is still significantly lower compared to non-diabetic mice (Moley et al., 1991; De Hertogh et al., 1991b).
In vitro experiments using rodent models have also attempted to identify the teratogenic agents and associated underlying mechanism(s) that underpin diabetic embryopathy during the preimplantation period. Critical factors include hyperglycemia, but also high levels of ketones, reactive oxygen species and tumor necrosis factor alpha (Zusman et al., 1987; Pampfer et al., 1994b, 1994c, 1995, 1997; Moley et al., 1994, 1996; Ornoy et al., 1996; Wuu et al., 1998). Embryo culture experiments in the presence of high concentrations of exogenous glucose (20–52 mM) have been shown to inhibit blastocyst development, cell proliferation and cell differentiation, as well as increase apoptosis and affect intra-embryonic metabolite levels (Zusman et al., 1985; Diamond et al., 1989; Pampfer et al., 1990; De Hertogh et al., 1991a; Moley et al., 1996; Moley, 2001). Moreover, a hyperglycemic environment in vitro is associated, paradoxically, with down regulation of Glucose Transporter 1, 2 and 3 in the blastocyst, reduced glycolytic activity, and a relative drop in intra-embryonic free glucose (Moley et al., 1998b; Leunda-Casi et al., 2001; Moley, 2001). In turn, this has been shown to increase levels of reactive oxidative species and stimulate expression of pro-apoptotic genes (p53 and Bax) (Moley et al., 1998b; Keim et al., 2001; Leunda-Casi et al., 2001) and intracellular apoptotic effectors (caspase-3 and caspase-activated deoxyribonuclease) (Hinck et al., 2001), resulting in a reduction in ICM numbers.
The minimum exposure time during the preimplantation period required to induce irreversible effects during the peri- and post-implantation stage of development is yet to be determined. Moreover, there has been little attempt to define the periods of preimplantation development that are susceptible to hyperglycemia, in particular whether this sensitivity occurs before, as well as after, activation of the embryonic genome. Prior to activation of the embryo’s own genome, which occurs at the 2–4 cell stage in the mouse, the embryo is reliant on stores of mRNA and proteins inherited from the oocyte (Braude et al., 1979; Flach et al., 1982). An understanding of the synchronization of such factors contributing to teratogenesis is important for establishing a mouse model to further investigate the underlying mechanisms associated with diabetes induced embryopathy.
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Materials and Methods |
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Animals
Outbred mice (CD1 strain; Harlan, Loughborough, UK) were housed with a 12 h light:12 h dark photoperiod, with light commencing at 07:00 h. Food and water were available ad libitum. Females (2–3 weeks old) were superovulated by an i.p. injection of 5 IU Pregnant Mare Serum Gonadotrophin (PMSG, Intervet, UK) per mouse followed by a second i.p. injection 46 h later of 5 IU HCG (Intervet, UK). After injection with HCG the mice were mated, in pairs, with 6–8-week-old male mice (CD1 strain; Harlan, Loughborough, UK). The following morning, the female mice were separated, and this was deemed to be Day 1 post fertilization (pf). The use of animals in these procedures was approved by the South Sheffield Research Ethics Committee, and carried out in accordance with the Home Office Animals (Scientific Procedures) Act 1986.
Preimplantation embryo retrieval and culture
Female mice were killed on Day 2 pf or Day 4/5 pf to obtain 2-cell embryos or blastocysts, respectively. Two-cell embryos were retrieved by flushing the oviducts, whilst blastocyst stage embryos were retrieved by flushing the uterine horns with either warm (37°C) synthetic oviductal medium enriched with potassium (mKSOM) or warm (37°C) synthetic oviductal medium enriched with potassium and 50% reduced amino acids (KSOM) using a 32 gauge stainless steel hypodermic needle (Popper and Sons, NY, USA). Different culture media were used initially to determine which was the most suitable for culturing embryos to the blastocyst stage. Two-cell embryos were cultured in groups of 10–15 in 50 µl droplets of KSOM medium supplemented with 0.2, 5.56, 15.56 or 25.56 mM D-glucose, and overlaid with mineral oil. The range of concentrations chosen taken into account the concentration of glucose measured in mouse oviduct fluid (5.10 ± 0.20 mM) (Gardner and Leese, 1990) and in rodent diabetic serum (23.3 ± 0.5 mM) (Pampfer et al., 1997). It was postulated that embryos are exposed to glucose levels in this range within the female reproductive tract. Any increase in osmolarity caused by the addition of higher concentrations of glucose to culture medium was compensated for by adjusting the concentration of sodium chloride accordingly. Embryos were cultured in a humidified incubator gassed with 5% CO2 in air at 37°C (Martin and Leese, 1995, 1999). To prevent the build-up of toxic ammonium ions, each embryo was transferred to a fresh pre-equilibrated drop of culture medium once each day until Day 5 pf.
Peri-implantation embryo culture
Hatched blastocysts (Day 5pf) were cultured in groups of 10–15 on a defined extra cellular matrix of fibronectin at 37°C in 5% CO2 in air until Day 8 pf. Briefly, a four-well plate was prepared by incubating round, glass, sterile cover slips with a 10 µg/ml fibronectin (Sigma) solution in phosphate-buffered saline overnight at 4°C. Wells were washed thoroughly before transferring zona-free blastocysts to warm, pre-equilibrated KSOM medium.
Assessment of preimplantation embryo development (Days 2–5)
Four parameters were used to assess preimplantation embryo development in vitro on Day 5 pf: (i) morphological development; (ii) cell proliferation; (iii) cell death and (iv) cell differentiation.
Morphological development
Embryos were assessed daily for cellular morphology, including cytoplasmic granularity, fragmentation, blastomere size and shape. Embryos were also assessed daily for their developmental stage, i.e. the proportion of embryos reaching the blastocyst stage and the percentage of these hatching. A low proportion of embryos reaching blastocyst stage is indicative of compromised development.
Cell proliferation
On Day 5 pf, the total cell number of blastocysts was measured using the fluorochrome nuclear stain Bisbenzimide Hoechst No 33258 (Sigma) (Martin et al., 1998). Briefly, embryos were incubated in acid tyrodes solution (Sigma) to remove the zona pellucida before being washed in ethanol and incubated in 50 µg/ml bisbenzimide at 4°C overnight. Embryos were washed thoroughly in ethanol before being mounted in glycerol and examined using an Olympus BH2-RFCA fluorescence microscope (Fig. 1A). A low cell number at the blastocyst stage can be associated with reduced embryo viability (Desai et al., 2000).
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Cell death (apoptosis)
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to determine the proportion of cells undergoing apoptosis employing the Fluoroscein-FragEL DNA Fragmentation Detection Kit (Oncogene) (Fig. 1B). Embryos incubated in DNAse I, to generate double strand breaks, were used as a positive control. Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to determine total cell number. An increase in the dead cell index (number of apoptotic cells/total cell number) is associated with a decrease in embryo quality (Brison and Schult, 1997
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Cell differentiation
The allocation of cells to the two cell populations of the blastocyst, the ICM and trophectoderm (TE), was measured using antibody mediated complement-lysis employing the fluorochromes propidium iodide and bisbenzimide (Hewitson et al., 1996) (Fig. 1C). A threshold number of ICM cells are required for normal fetal development. Too few ICM cells are incompatible with normal fetal development (Van Soom et al., 1997).
Assessment of peri-implantation embryo development (Days 6–8)
Morphological development
On Day 8 pf morphological development of peri-implantation embryos was assessed as the percentage of blastocysts remaining attached to the cover slip after gentle agitation, and the proportion of blastocysts with signs of trophoblast outgrowth. The degree of trophoblast outgrowth (indicated by a flattened area of the attached embryo) was initially assessed using a semi-quantitative method. Embryos were graded as having none; slight; moderate or significant outgrowth (Fig. 2). The degree of outgrowth was also quantified by measuring the total area of spreading using image analysis equipment (Scion) (Martin et al., 1998; Martin and Leese, 1999). Trophoblast development in vitro is considered to be a marker of implantation potential (Pampfer et al., 1994a; Leundu-Casi et al., 2001).
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Cell proliferation
Cell number in attached blastocysts was determined by removing and inverting cover slips onto a droplet of Vectashield with DAPI. Blastocysts were examined under fluorescent light. Cell number was approximated as cell density was high.
Cell death (apoptosis)
TUNEL was used to determine the proportion of cells undergoing apoptosis, as described earlier.
Cell differentiation
The proportion of trophoblast cells exhibiting a giant nuclear phenotype was used as a marker of differentiation (Pampfer et al., 1994a). The distribution of phagocytosed fluorescent beads was used to assess whether there was an ICM present in the embryos on Day 8 pf (Nishioka et al., 1994; Albieri et al., 1996; Grandjean et al., 1997, Rassoulzadegan et al., 2000). Briefly, embryos were cultured in the presence of fluorescent beads for 2 h and then washed in culture media to remove any beads, which had not been phagocytosed. The embryos were then fixed and mounted in DAPI and the number and distribution of beads was analysed. The presence of an ICM was indicated by an area of the attached embryo being devoid of beads. ICM, in contrast to TE/Trophoblast (TB), do not phagocytose the fluorescent beads (Fig. 3). The density of beads was used to indicate what proportion of the two discrete groups of TE cells where present, with a higher bead density indicating more cells of the mural TE type and a lower bead density indicating more cells of the polar TE type. The mural TE generates primary trophoblast giant cells and the polar TE generates the ectoplacental cone and the extra-embryonic ectoderm and later much of the fetal part of the placenta (Rassoulzadegan et al., 2000).
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Determination of the teratogenic susceptible periods during preimplantation development
To determine the stage at which exposure to teratogenic glucose concentrations (as determined from the preceding experiment) is most detrimental to preimplantation development, embryos were exposed to teratogenic levels of glucose for either 24 or 48 h at differing times throughout preimplantation development. Embryos were cultured in a physiological level of glucose (5.56 mM) at all other times. Embryos exposed to 5.56 mM glucose from Day 2 to 5 pf served as controls. Embryo development was assessed on Day 5 pf using parameters as before.
All these experiments were repeated at least five times. Positive and negative controls were included each time. Data were then pooled before analysis.
Statistical analysis
Statistical differences between control and diabetic distribution patterns (i.e. percentages) were analysed using chi-squared test. Mean values (e.g. total cell numbers) between the control and treatment groups were compared using unpaired Student’s t-test or one-way analysis of variance where appropriate.
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Results |
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Choice of preimplantation culture system
Preliminary experiments were carried out to determine the optimal culture system to examine the effects of exposure of pre and peri-implantation embryos to teratogenic concentrations of glucose. Embryos were cultured from the 1- to 2-cell stage, in two different culture media, mKSOM or KSOM (containing 50% reduced concentration of amino acids).
No effect of increasing glucose concentration was observed on morphological development of 1-cell embryos to the blastocyst stage, in either mKSOM or KSOM. This was attributed to the low rate of blastocyst development. A phenomenon known as the 2-cell block which is well documented in embryos cultured from the 1-cell stage (Robi et al., 1988; Schini et al., 1988; Minami et al., 1992; Goddard et al., 1983; Liu et al., 1995) is probably responsible for the low rates of blastocyst development observed here. However, increasing concentrations of glucose (0.2–25 mM) did result in significant difference in blastocyst development when embryos were cultured from the 2-cell stage in both mKSOM and KSOM. As the rates of blastocyst development on Day 5 pf were significantly greater in KSOM [61.2% (66.3% including expanded blastocysts)] compared to mKSOM (45.6%). KSOM was selected as the medium for further experiments.
In order to establish effective in vivo and in vitro controls, Day 2 pf embryos were cultured in KSOM with 5.56 mM glucose, which is equivalent to the rodent physiological glucose concentration, until Day 5 pf and compared to in vivo embryos harvested on Day 4/5 pf. Initially, blastocysts were retrieved in the morning of Day 5 and assessed without culturing. It was found, however, that by Day 5 the majority (>90%) of blastocysts had already attached and very few embryos could be flushed from the uterine horns. In an attempt to increase the yield, blastocysts were retrieved on Day 4.5 and again assessed without culturing. Again, however, it was found that between 30 and 50% of blastocysts had already attached and only a few embryos were able to be flushed from the uterine horns. Finally, to further increase the yield, blastocysts were retrieved on Day 4 and assessed without culturing. There were no problems encountered with retrieval at this stage. All the blastocysts (Day 4, 4.5, 5 in vivo) were assessed and compared to each other and to Day 5 in vitro blastocysts. Owing to the reduced number of animals involved, Day 4 in vivo blastocysts were used as the in vivo control. Moreover, all parameters assessed on Day 5 pf embryos cultured in vitro were comparable to their in vivo counterparts on Day 4 pf (data not shown). As there was no statistical difference between Day 4 in vivo and Day 5 in vitro embryos, it was decided to use Day 5 in vitro embryos as the only control for further experiments.
Determination of the concentrations of exogenous glucose that cause embryopathy during the pre- and peri-implantation periods
Effect on preimplantation development
Exposure of preimplantation embryos to increase the concentrations of glucose resulted in significantly fewer blastocysts, and fewer blastocysts hatching, by Day 5 pf compared to control embryos (cultured in 5.56 mM glucose) (Table I).
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A significant effect on cell allocation on Day 5 pf was observed. There was a significant reduction in both the number of ICM cells and the number of TE cells with the increasing concentrations of glucose on Day 5 pf. The ICM:tE ratio was also significantly different to the control culture (5.56 mM) with increased or decreased concentrations of glucose on Day 5 pf (Table II).
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Increasing concentrations of glucose, however, had no significant effect on the percentage of apoptotic cells when examined using TUNEL on Day 5 pf (Table III). Positive control embryos consistently had a majority (>90%) of apoptotic cells.
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Effect on peri-implantation development
The effect of exposure of mouse peri-implantation embryos to increasing concentrations of glucose (0.2, 5.56, 15.56 and 25.56 mM) from Day 2–8 pf on morphological development was initially assessed using a semi-quantitative method. The number of attached embryos with none, slight, moderate or significant degrees of trophoblast outgrowth was determined. Increasing concentrations of glucose in embryo culture medium significantly increased (
2, P < 0.01) the degree of trophoblast outgrowth by Day 8 pf (data not shown). The effect of exposure to increasing concentrations of glucose on cell proliferation was investigated by estimating the total cell number of attached blastocysts. Exact total cell numbers were not possible to obtain due to the morphology of the attached embryos. Cells that could be seen were counted, whilst cells that were out of sight were estimated. As a result, cells were grouped into ‘bands’ of cell numbers. Cell numbers were then represented as the median value for that band (e.g. 1–25 = 13, 26–50 = 38). Increasing glucose concentrations resulted in significantly fewer cells in attached blastocysts by Day 8 (Table IV). There was no significant effect on the total surface area of Day 8 blastocysts, as quantified using Scion software (Table IV). There was a significant effect on cell density however with density decreasing as glucose concentration increases (Table IV).
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Increasing concentrations of glucose resulted in significantly fewer embryos containing an ICM (
2, P < 0.01) as indicated by an area of the embryo being devoid of fluorescent beads. In 0.2 and 5.56 mM glucose cultures, 80% of embryos contained an ICM whereas in 15.56 and 25.56 mM glucose cultures only 60.5 and 31.3%, respectively, had an ICM. The total uptake of fluorescent beads was also used to quantify the proportion of ICM to TE cells, with fewer beads per median cell number indicating less cells of the TE/TB type. Increasing concentrations of glucose were associated with an increase in the uptake of beads and an increase in bead density (Table V).
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Determination of the teratogenic susceptible periods during preimplantation development
The experiments outlined above suggest that embryopathic effects are maximal at 25.56 mM D-glucose. Mouse embryos were therefore exposed to 25.56 mM D-glucose for 24 and 48 h periods between Days 2 and 5 pf to examine whether relatively short and/or fluctuating exposure to supraphysiological levels of glucose can induce embryopathy.
The development of embryos cultured in the presence of 25.56 mM glucose for 24 h on Days 2, 3 or 4 (early 24 h, mid 24 h or late 24 h) was not significantly compromised when assessed on the basis of morphology, cell proliferation, differentiation and apoptosis on Day 5 pf (Tables VI and VII). However, development to the blastocyst stage was reduced following exposure to 25.56 mM glucose for a total of 48 h, this became significant when it involved exposure to fluctuating levels (Split 48; Fig. 4).
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Cell proliferation was significantly reduced in embryos exposed to 25.56 mM glucose for 48 h in the ‘late’ and ‘split’ experiments, but not in the ‘early’ exposure experiments (Table VI).
In comparison, no significant effect on cell apoptosis was observed when embryos were exposed to high glucose for a total of 48 h split between Days 2 and 4 (data not shown). However, significant effects on both cell allocation and total cell number were observed (Table VI). No effect on cell allocation was observed when embryos were exposed to 25 mM glucose for 48 h in the ‘early’ and ‘late’ exposure experiments.
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Discussion |
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We used an in vitro mouse model to investigate the effects of embryo exposure to continuous or fluctuating raised concentrations of glucose to mimic the pre- and post-prandial level changes observed in apparently well-controlled diabetes. The results of this study provide insight into the benefits from optimizing glycemic control before conception in diabetic women. The pathogenesis of hyperglycemia in diabetic teratogenesis has not previously been adequately explored in the mouse preimplantation in vitro model. Previous studies have not attempted to determine which concentrations of glucose induce embryopathy during the preimplantation period and we can find only one study of the effects of glucose concentration within this context, which was restricted to measurement of intra-embryonic metabolite levels (Moley et al., 1996
). In studying our culture system, we found expected levels of expanded blastocyst formation and hatching on Day 5 both at the 0.2 mM glucose concentration and the more physiological 5.56 mM concentration. Higher levels of glycemia were associated with a progressive fall in success of both processes. Total cell numbers and allocation to an ICM were both significantly reduced in the hyperglycemic culture.
Previously reported experiments with blastocysts from mice and rats with induced diabetes suggested increased rates of apoptosis—particularly affecting the ICM—leading to increased rates of malformation and absorption (Moley et al., 1998a). In contrast, induction of the anti-apoptotic factor Bcl-2 has been reported in response to hyperglycemia (28 mM versus 6 mM glucose) and a mechanism similar to this may explain the lack of increased apoptosis seen in our particular experiment (Pampfer et al., 2001).
Our observation that increasing glucose concentration had no significant effect on the apoptotic cells numbers or proportion of apoptotic cells is in sharp contrast to previously published studies (Keim et al., 2001; Chi et al., 2002; Jimenez et al., 2003). This could be due to differences in experimental design, e.g. Jimenez et al. (2003) cultured embryos initially in normal glucose levels then increased the glucose levels for a period of time before assessing the level of cell death whereas we cultured the cells continuously in the various glucose concentrations before assessing the level of cell death. The stage at which the level of cell death is assessed also varies between papers.
In addition to the shift from ICM to TE cells associated with increasing glucose concentrations, the hyperglycemic environment stimulates trophoblastic overgrowth in vitro. Despite this there was a reduction in median cell number and cell density with increasing glucose concentrations. The relevance of these observations to viability and disordered embryogenesis will have to await re-implantation experiments, but the dramatic reductions in the numbers of embryos with an ICM, confirmed the profound adverse effects of hyperglycemia in culture.
Most importantly in the experiments where the glucose concentration was varied over 1 or 2 days, of Days 2, 3 or 4 of culture, we have demonstrated preservation of percentage blastocyst formation on embryos exposed to only 24 h of hyperglycemia, on any of the 3 days. There was a significant reduction in successful blastocyst formation in the split hyperglycemia experiment, Days 2 and 4 only. This reduction in blastocyst formation may be due to the significant reduction in total cell number and the significant change in the ratio ICM–TE cells we observed, as there was no evidence of a significant increase in cell death.
The type of fluctuation used in the split hyperglycemia experiment may most closely mimic the environment experienced by the human embryo in poorly controlled maternal diabetes. These findings may help explain the high rates of implantation failure and early pregnancy loss seen in women with diabetes. We plan to extend these experiments to study events following embryo transfer, using embryos exposed to glycemic fluctuation, to quantify absorption and malformation rates associated with early embryopathy in the non-diabetic mouse.
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Conclusion |
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This animal model offers great potential for studies of detailed time related aspects of disordered embryopathy in relation to maternal hyperglycemia in human pregnancy complicated by maternal diabetes.
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Funding |
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Sheffield Hospitals Charitable Trust (7814).
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References |
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Albieri A, Bevilacqua E. Indicuation of erythrophagocytic activity in cultured mouse trophoblast cells by phorbol myristate acetate and all-trans-retinal. Placenta (1996) 17:507–512.[CrossRef][Web of Science][Medline]
Braude P, Pelham H, Flach G, Lobatto R. Post-transcriptional control in the early mouse embryo. Nature (1979) 282:102–105.[CrossRef][Medline]
Brison DR, Schultz RM. Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor alpha. Biol Reprod (1997) 56:1088–1096.[Abstract]
Chi MMY, Hoehn A, Moley KH. Metabolic changes in the glucose induced apoptotic blastocyst suggest alterations in mitochondrial physiology. Am J Physiol Endocinrol Metab (2002) 283:E226–E232.
DAFNE Study Group. Training in flexible, intensive insulin management to enable dietary freedom in people with Type 1 diabetes: dose adjustment for normal eating (DAFNE) randomised controlled trial. BMJ (2002) 325:746–751.
De Hertogh R, Vanderheyden I, Pampfer S, Robin D, Dufrasne E, Delcourt J. Stimulatory and inhibitory effects of glucose and insulin on rat blastocyst development in vitro. Diabetes (1991) a40:641–647.[Abstract]
De Hertogh R, Vanderheyden I, Pampfer S, Robin D, Delcourt J. Maternal insulin treatment improves preimplantation embryo development in diabetic rats. Diabetologia (1991) b35:406–408.[CrossRef][Web of Science]
Desai N, Lawson J, Goldfarb J. Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage. Hum Reprod (2000) 15:410–418.
Diamond MP, Moley KH, Pellicer A, Vaughn WK, DeCherney AH. Effects of streptozotocin- and alloxan induced diabetes mellitus on mouse follicular and early embryo development. J Reprod Fertil (1989) 86:1–10.
Flach G, Johnson MH, Braude PR, Taylor RA, Bolton VN. The transition from maternal to embryonic control in the 2-cell mouse embryo. EMBO J (1982) 1:681–686.[Web of Science][Medline]
Gardner DK, Leese HJ. Concentrations of nutrients in mouse oviduct fluid and their effects on embryo development and metabolism in vitro. J Reprod Fertil (1990) 88:361–368.
Goddard MJ, Praatt HP. Control of events during early cleavage of the mouse embryo: an anlysis of the ‘2-cell block. J Embryol Exp Morphol (1983) 73:111–133.[Web of Science][Medline]
Grandjean V, Sage J, Ranc F, Cuzin F, Rassoulzadegan M. Stage-specific signals in germ line differentiation: control of Sertoli cell phagocytic activity by spermatogenic cells. Dev Biol (1997) 184:165–174.[CrossRef][Web of Science][Medline]
Hewitson LC, Martin KL, Leese HJ. Effects of metabolic inhibitors on mouse preimplantation embryo development and the energy metabolism of isolated inner cell masses. Mol Reprod Dev (1996) 43:323–330.[CrossRef][Web of Science][Medline]
Hinck L, Van Der Smissen P, Heusterpreute M, Donnay I, De Hertogh R, Pampfer S. Identification of caspase-3 and caspase-activated deoxyribonuclease in rat blastocysts and their implication in the induction of chromatin degradation (but not nuclear fragmentation) by high glucose. Biol Reprod (2001) 64:555–562.
Jimenez A, Madrid-Bury N, Fernandez R, Perez-Garnelo S, Moreira P, Pintado B, De La Fuente J, Gutierrez-Adan A. Hyperglycaemia-Induced Apoptosis Affects Sex Ratio of Bovine and Murine Preimplantation Embryos. Mol Rep Dev (2003) 65:180–187.[CrossRef][Web of Science][Medline]
Keim AL, Chi MM, Moley KH. Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53. Mol Reprod Dev (2001) 60:214–224.[CrossRef][Web of Science][Medline]
Kyne-Grzebalski D, Wood L, Marshall SM, Taylor R. Episodic hyperglycemia in pregnant women with well-controlled Type 1 diabetes mellitus: a major potential factor underlying macrosomia. Diabet Med (1999) 16:702–706.[CrossRef][Web of Science][Medline]
Lea RG, McCraken JE, McIntyre SS, Smith W, Baird JD. Disturbed development of the preimplantation embryo in the insulin-dependent diabetic BB/E rat. Diabetes (1996) 45:1463–1470.[Abstract]
Leese H. The formation and function of oviduct fluid. J Reprod Fert (1981) 82:843–856.[CrossRef]
Leunda-Casi A, De Hertogh R, Pampfer S. Decreased expression of fibroblast growth factor-4 and associated dysregulation of trophoblast differentiation in mouse blastocysts exposed to high D-glucose in vitro. Diabetologia (2001) 44:1318–1325.[CrossRef][Web of Science][Medline]
Liu LPS, Chan STH, Ho PC, Yeung WSB. Human oviductal cells produce high molecular weight factor(s) that improves the development of mouse embryo. Mol Hum Reprod (1995) 10:2781–2786.
Martin KL, Barlow D, Sargent LL. Heparin-Binding Epidermal Growth Factor (HB-EGF) significantly improves human blastocyst development and hatching in serum-free medium. Hum Reprod (1998) 13:1645–1652.
Martin KL, Leese HJ. Role of glucose in mouse preimplantation embryo development. Mol Reprod Dev (1995) 40:436–443.[CrossRef][Web of Science][Medline]
Martin KL, Leese HJ. Role of developmental factors in the switch from pyruvate to glucose as the major exogenous energy substrate in the preimplantation mouse embryo. Mol Reprod Dev (1999) 11:425–433.
Miller E, Hare JW, Cloherty JP, Dunn PJ, Gleason RE, Soeldner JS, Kitzmiller J. Elevated maternal hemoglobin Alc in early pregnancy and major congenital anomalies in infants of diabetic mothers. N Engl J Med (1981) 304:1331–1334.[Web of Science][Medline]
Mills JL, Knopp RH, Simpson JL, Jovanovic-Peterson L, Metzger BE, Holmes LB, Aarons JH, Brown Z, Reed GF, Bieber FR, et al. Lack of relation of increased malformation rates in infants of diabetic mothers to glycemic control during organogenesis. N Engl. J Med (1988) 318:671–676.[Abstract]
Minami N, Utsumi K, Iritani A. Effects of low molecular weight oviductal factors on the development of mouse one-cell embryos in vitro. J Reprod Fertil (1992) 96:735–745.
Moley KH. Diabetes and preimplantation events of embryogenesis. Semin Reprod Endocrinol (1999) 17:13751.
Moley KH. Hyperglycemia and apoptosis: mechanisms for congenital malformations and pregnancy loss in diabetic women. Trends Endocrinol Metab (2001) l2:78–82.
Moley KH, Chi MM, Manchester JK, McDougal DB, Lowry OH. Alterations of intra-embryonic metabolites in preimplantation mouse embryos exposed to elevated concentrations of glucose: a metabolic explanation for the developmental retardation seen in preimplantation embryos from diabetic animals. Biol Reprod (1996) 54:1209–1216.[Abstract]
Moley KH, Chi MM, Mueckler MM. Maternal hyperglycemia alters glucose transport and utilization in mouse preimplantation embryos. Am J Physiol (1998) a275:E38–E47.[Web of Science][Medline]
Moley KH, Chi MM-Y, Kuudson CM, Karsmeyer SJ, Muekler MM. Hyperglycemia induces apoptosis in pre-implantation embryos through cell death affected pathways. Nat Med (1998) b4:1421–1424.[CrossRef][Web of Science][Medline]
Moley KH, Vaughn WK, DeCherney AH, Diamond MP. Effect of diabetes mellitus on mouse preimplantation embryo development. J Reprod Fertil (1991) 93:325–332.
Moley KH, Vaughn WK, Diamond MP. Manifestations of diabetes mellitus on mouse preimplantation development: effect of elevated concentration of metabolic intermediates. Hum Reprod (1994) 9:113–121.
Nishioka K, Wagle JR, Rodriguez TJ, Maeta M, Kubo S, Dessens SE. Studies of human granulocyte phagocytosis stimulation by tuftsin. J Surg Res (1994) 56:94–101.[CrossRef][Web of Science][Medline]
Ornoy A, Kimyagarov D, Yaffee P, Abir R, Raz I, Kohen R. Role of reactive oxygen species in diabetes induced embryo toxicity: studies on pre-implantation mouse embryos cultured in serum from diabetic pregnant women. Isr J Med Sci (1996) 32:1066–1073.[Web of Science][Medline]
Pampfer S, de Hertogh R, Vanderheyden I, Michiels B, Vercheval M. Decreased inner cell mass proportion in blastocysts from diabetic rats. Diabetes (1990) 39:471–476.[Abstract]
Pampfer S, Wuu YD, Vanderheyden I, De Hertogh R. In vitro study of the carry-over effect associated with early diabetic embryopathy in the rat. Diabetologia (1994) a37:855–862.[Web of Science][Medline]
Pampfer S, Wuu YD, Vanderheyden I, DeHertogh R. Expression of tumor necrosis factor alpha (TNF alpha) receptors and selective effect of TNF alpha on the inner cell mass in mouse blastocysts. Endocrinology (1994) b134:206–212.
Pampfer S, Moulaert B, Vanderheyden I, Wuu YD, De Hertogh R. Effect of tumor necrosis factor alpha on rat blastocyst growth and glucose metabolism. J. Reprod Fert (1994) c101:199–206.
Pampfer S, Vanderheyden I, Wuu YD, Baufays L, Maillet O, DeHertogh R. Possible role for TNF-alpha in early embryopathy associated with maternal diabetes in the rat. Diabetes (1995) 44:531–536.[Abstract]
Pampfer S, Vanderheyden I, McCracken JE, Vesela J, De Hertogh R. Increased cell death in rat blastocysts exposed to maternal diabetes in utero and to high glucose or tumor necrosis factor-alpha in vitro. Development (1997) 124:4827–4836.[Abstract]
Pampfer S, Cordi S, Vanderheyden I, Van Der Smissen P, Courtoy PJ, Van Caurenberge A, Alexandre H, Donnay J, De Hertogh R. Expression and role of Bc1-2 in rat blastocysts exposed to high D-glucose. Diabetes (2001) 50:143–149.[Medline]
Rassoulzadegan M, Rosen BS, Gillot I, Cuzin F. Phagocytosis reveals a reversible differentiated state early in the development of the mouse embryo. EMBO (2000) 19:3295–3303.[CrossRef][Web of Science][Medline]
Ray JG, O’Brien TE, Chan WS. Preconception care and the risk of congenital anomalies in the offspring of women with diabetes mellitus: a meta-analysis. QJM (2001) 94:435–444.
Robl JM, Lohse-Heideman JK, First NL. Strain differences in early mouse embryo development in vitro: role of the nucleus. J Exp Zool (1988) 247:251–256.[CrossRef][Web of Science][Medline]
Schini SA, Bavister BD. Two-cell block to development of cultured hamster embryos is caused by phosphate and glucose. Biol Reprod (1988) 39:1183–1192.[Abstract]
Van Soom A, Boerjan ML, Bols PE, Vanroose G, Lein A, Coryn M, de Kruif A. Timing of compaction and inner cell allocation in bovine embryos produced in vivo after superovulation. Biol Reprod (1997) 57:1041–1049.[Abstract]
Vercheval M, De Hertogh R, Pampfer S, Vanderheyden I, Michiels B, De Bernardi P, De Meyer R. Experimental diabetes impairs rat embryo development during the preimplantation period. Diabetologia (1990) 33:187–191.[CrossRef][Web of Science][Medline]
Wuu YD, Pampfer S, Vanderheyden I, Lee KH, De Hertogh R. Impact of tumor necrosis factor alpha on mouse embryonic stem cells. Biol Reprod (1998) 58:1416–1424.
Zusman I, Ornoy A, Yaffe P, Shafrir E. Effects of glucose and serum from streptozotocin-diabetic and non-diabetic rats on the in vitro development of preimplantation mouse embryos. Isr J Med Sci (1985) 21:359–365.[Web of Science][Medline]
Zusman I, Yaffe P, Ornoy A. Effects of metabolic factors in the diabetic state of the in vitro development of preimplantation mouse embryos. Teratology (1987) 35:77–85.[CrossRef][Web of Science][Medline]
Submitted on July 12, 2007; resubmitted on August 28, 2007; accepted on September 12, 2007.
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