Human Reproduction, Vol. 17, No. 2, 457-462,
February 2002
© 2002 European Society of Human Reproduction and Embryology
Preimplantation exposure to high insulin-like growth factor I concentrations results in increased resorption rates in vivo
1 Department of Obstetrics and Gynecology and 2 Department of Cell Biology and Physiology, 4911 Barnes–Jewish Hospital Plaza, Washington University School of Medicine, St Louis, MO 63110, USA
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Abstract |
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BACKGROUND: Women with polycystic ovarian syndrome suffer increased rates of miscarriage. Elevated insulin and insulin-like growth factor I (IGF-I) concentrations have been implicated. Here, we hypothesize that the high concentrations of IGF-I result in miscarriage, represented by decreased normal pregnancy rates and increased resorption rates in a mouse model. METHODS: In-vitro studies: 2-cell embryos were cultured in either 1.3 or 130 nmol/l IGF-I; or 500 nmol/l IGF-I receptor (IGF-IR) sense and antisense oligoprobes for 72 h. Embryos were then transferred into pseudo-pregnant ICR females. In-vivo studies: IGF-I-containing slow-release pellets or mock pellets were implanted within the uterine horn in ICR female mice. For both studies, the recipient females were killed on day 14.5 and the numbers of normal implantation sites versus resorption sites were recorded. RESULTS: In-vitro studies: blastocysts cultured in low IGF-I exhibited significantly higher normal implantation rates than blastocysts cultured in high IGF-I concentrations (P < 0.01). Blastocysts cultured in IGF-IR sense oligoprobes exhibited a significantly higher normal implantation rate than blastocysts cultured in antisense oligoprobes. In-vivo studies: mice implanted with IGF-I-containing pellets exhibited significantly lower normal implantation rates as compared with mock-pellet controls (P < 0.01). CONCLUSIONS: High preimplantation IGF-I concentrations in vitro or in vivo lead to increased resorption rates in the mouse.
Key words: apoptosis/IGF-I/IGF-IR/mouse model/polycystic ovarian syndrome
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Introduction |
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Models of excess insulin-like growth factor I (IGF-I) either in vivo or in vitro demonstrate deleterious effects on the murine preimplantation embryo (Moon et al., 1990
; Katagiri et al., 1996). Raising IGF-I concentrations either by gonadotrophins during ovulation induction or by implanting slow release pumps containing IGF-I adversely affects developmental progression to the blastocyst stage in mice with a larger percentage of degenerated embryos. The blastocysts that do form have smaller numbers of cells. This effect is reversed with anti-IGF-I antibody infusions or somatostatin analogues (Katagiri et al., 1997). Women with polycystic ovarian syndrome (PCOS) exhibit hyperinsulinaemia, leading to a decrease in insulin-like growth factor binding protein (IGFBP)-1 and -3, thus resulting in elevated bioactive IGF-I concentrations. These women also experience significantly higher rates of pregnancy loss (Sagle et al., 1988; Balen et al., 1993; Tulppala et al., 1993). It is not clear whether this effect is due to high androgen concentrations, elevated gonadotrophin concentrations or high insulin and IGF-I concentrations in serum and locally in tubal or uterine fluid.
Previously, we have shown that exposure of murine blastocysts to elevated IGF-I or insulin concentrations in vitro induces apoptosis primarily in the inner cell mass (Chi et al., 2000). This increase in apoptosis is the result of a decrease in IGF-I receptor expression and is related to a decrease in insulin-stimulated glucose uptake possibly via the expression of the only insulin-regulated glucose transporter in the blastocyst, namely GLUT8 (Carayannopoulos et al., 2000). We have shown previously that maternal hyperglycaemia induces a decrease in basal glucose uptake (Moley et al., 1998a) in contrast to a decrease in insulin-stimulated glucose uptake as seen here. This decrease in basal glucose uptake triggers an increase in BAX-dependent apoptosis (Moley et al., 1998b; Chi et al., 2000). We have also shown that this glucose-induced apoptosis manifests later in pregnancy as an increase in resorptions and congenital malformations (Chi et al., 2000). In this work we postulate that the increase in apoptosis induced by IGF-I results in an increase in miscarriages, represented by a decrease in normal pregnancies and an increase in resorption rates in the mouse model. To test this hypothesis, we took both an in-vitro approach involving embryo transfer of embryos exposed to elevated concentrations of IGF-I and an in-vivo approach in which we raised uterine horn IGF-I concentrations with implanted pellets. Pregnancy outcomes were examined under both conditions.
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Materials and methods |
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Embryo recovery and culture conditions
Animals were killed by cervical dislocation at 48 h after HCG administration and mating. Two-cell and 4-cell embryos were obtained by flushing dissected uterine horns and ostia as described previously (Hogan et al., 1991
; Moley et al., 1998a). The embryos were then immediately placed in human tubal fluid (HTF; Irvine Scientific, CA, USA) containing 0.25% bovine serum albumin (BSA, fraction V; Sigma Chemical Co., MO, USA) and cultured in groups of 20–30 at 37°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 24, 48, 62 and 72 h. The culture conditions included: (i) control HTF; (ii) HTF with added 1.3 nmol/l IGF-I (Sigma); or (iii) 130 nmol/l IGF-I (Sigma).
Expression of IGF-I receptor (IGF-IR) protein by immunoprecipitation and Western immunoblot analysis
The pooled embryos were then solubilized for 30 min at 4°C in a HEPES buffer (50 mmol/l HEPES, 1 mmol/l EDTA, 150 mmol/l NaCl, 1 mmol/l Vanadate, 1% BSA, 1% Triton, pH 7.4) containing protease inhibitors. The supernatants were then immunoprecipitated overnight with a rabbit polyclonal anti-mouse IGF-I receptor (IGF-IR) antibody against the 
-subunit and not cross-reactive with the insulin receptor (1:1000; Upstate Biotechnology, NY, USA). Immune complexes were then bound to Protein-A Sepharose beads (Upstate Biotechnology) and washed extensively. The samples were then added to 2xsample buffer, subjected to 7.5% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to nitrocellulose. IGF-IR was then detected using an antibody against the ß-subunit of IGF-IR (1:1000; Santa Cruz). 125I-Labelled goat anti-rabbit IgG was used as the secondary antibody. A PhosphorImager SI (Molecular Dynamics) and NIH Image (version 1.60) analysers were used to quantify radioactive bands. All experiments were performed in triplicate.
Blastocyst transfer following exposure in vitro to high and low IGF-I
Embryos were obtained and cultured to blastocyst stage as described above. A total of eight blastocysts were then transferred into recipient pseudo-pregnant ICR female mice as previously described (Hogan et al., 1991). The mice were killed on day 14.5 and the numbers of normal implantation versus resorption sites were recorded. Implantation rate represents normal gestational sacs divided by total number of sacs including resorptions.
IGF-I pellet transfer. Uterine luminal and blood IGF-I concentration determination
Recombinant human IGF-I was obtained from R&D Systems (MN, USA). IGF-I pellets containing 65 µg were obtained from Innovative Research of America (FL, USA). These pellets were designed to release ~5 µg/day in order to mimic our in-vitro conditions. The pellets were placed in the uterine lumen by making a small incision cephalad to the bifurcation of the uterine horn. Using fine forceps, a single IGF-I pellet was placed within the uterine lumen. The abdomen was then closed and the mice were recovered on a heating pad. A similar experiment was repeated using inert pellets (mock) to be used as controls.
Seven days following transfer of the pellets the animals were killed and proper placement of the pellet was confirmed at this time. The uterine horns with the attached oviducts were then dissected and placed in a 50 µl droplet of HTF under oil. The uterine luminal fluid was then flushed using 200 µl of HTF within 5 min after killing. The uterine luminal fluid IGF-I concentrations were assayed using radioimmunoassay. A dilution factor of 4 µl tubal fluid in 200 µl was used to calculate the final concentration (Wales and Edirisinghe, 1989). The minimum detectable concentration of the IGF-I radioimmunoassay kit is 
17 ng/ml with an intra-assay coefficient of variation of 8.3%. Similar experiments were done in the control mice. Blood was obtained by cardiac puncture in all killed animals and corresponding serum IGF-I concentrations detected by radioimmunoassay were obtained.
In-vivo pregnancies following exposure to IGF-I and mock pellets
IGF-I and control (mock) pellets were transferred as described above. Three days following the surgery the pellet-containing mice were allowed to mate with proven fertile male ICR mice. These mice were assessed for evidence of estrous and spontaneous plugging each day. If no plugging was evident by day 7 following surgery, the female mice ovaries were stimulated with pregnant mare's serum gonadotrophin and ovulation was induced using HCG as described above. The mice were killed on day 14.5 and the numbers of normal implantation sites versus resorption sites were recorded.
Blastocyst transfer following in-vitro exposure to IGF-IR sense and antisense oligonucleotides.
Expression of IGF-IR was blocked with the antisense oligoprobes in order to determine whether decreased expression of IGF-IR recreates the increased resorptions as seen with high IGF-I concentrations. Two-cell embryos were cultured for 72 h in 0.5 µmol/l IGF-IR antisense (5'-TCC TCC GGA GCC AGA CTT) or sense (5'-AAG TCT GGC TCC GGA GGA) oligodeoxynucleotides corresponding to codons 21–26 of the signal sequence of the -subunit IGF-IR preceding the proreceptor sequence. These oligodeoxynucleotides have been used previously in rodent models and are known to block expression successfully (Resnocoff et al., 1994). The blastocysts were then transferred as described as above and the mice were killed on day 14.5 and the numbers of normal implantation sites versus resorption sites were recorded.
Statistical methods
Implantation rate represents normal gestational sacs divided by total number of sacs including resorptions.
Differences between the groups of blastocyst exposed to: low IGF-I, high IGF-I, sense and antisense oligonucleotides and control were compared by unpaired Student's t-test. By power analysis, using Epi-Info (Version 6.04 B), it was calculated that a total of 12 fetuses were needed in the transfer studies based upon an -error of 0.5 and ß-error of 0.90. For all individual experiments there were at least five fetuses in the control animals. Results are expressed as means ± SEM of at least three separate experiments.
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Results |
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As seen in Figure 1
, a progressive decrease in IGF-IR protein expression over time was evident in embryos exposed to high (130 nmol/l) IGF-I. These effects were time dependent and mean (± SEM) decreases in expression compared with expression at 24 h were 89 ± 5% at 48 h, 76 ± 3% at 62 h and 67 ± 5% at 72 h. The decrease at 72 h is similar to what has been reported previously (Chi et al., 2000). No further decrease was seen after 72 h (data not shown). Exposure to 1.3 nmol/l IGF-I had minimal non-significant effects on IGF-IR expression. These findings suggest that at the time of onset of apoptosis at the blastocyst stage, the IGF-I-induced down-regulation of IGF-IR expression is occurring maximally.
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Preimplantation exposure to high IGF-I conditions in vitro adversely affects pregnancy outcome
Blastocysts exposed to low IGF-I concentrations, before being transferred to pseudo-pregnant recipients, exhibited significantly higher normal implantation rates (69.2 ± 12.4) than blastocysts exposed to high IGF-I (17.3 ± 4.4) (Figure 2
). Blastocysts exposed to high IGF-I exhibited significantly higher resorption rates (79.4 ± 9.7) than blastocysts exposed to low IGF-I (31.3 ± 14.4) (n = 5 experiments, P < 0.03).
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Measurement of IGF-I concentrations in the tubal luminal fluid and serum
In order to test the in-vivo effects of high IGF-I, pellets containing IGF-I and placebo were placed in the uterine horn. Both the luminal fluid and the serum showed distinct profiles following the implantation of the IGF-I-containing pellets and the placebo pellets in the uterus (Figure 3
). The serum IGF-I concentrations in the pellet mice averaged 427 ± 70 ng/ml versus 58 ± 11.3 ng/ml in the mock animals. These serum concentrations were checked on day 7 following surgery. The tubal fluid IGF-I concentrations averaged 939 ± 70 ng/ml in the IGF-I pellet mice and were below the level of detection (
17 ng/ml) in the mock animals. These concentrations mimic our in-vitro concentrations and are within the physiological range, since in a previous publication we determined that the IGF-I concentrations in control non-human primates range from 150–490 ng/ml (Chi et al., 2000).
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Preimplantation exposure to high IGF-I conditions in vivo adversely affects pregnancy outcome
Among mice containing pellets, two mock pellet mice and two IGF-I pellet mice plugged spontaneously. One mock and two IGF-I pellet mice did not go into estrous by 4 days, and underwent ovulation induction and were plugged. Results from these seven mice indicated a significant difference in implantation rates between mice containing mock versus IGF-I pellets with a 100% normal implantation rate among mocks compared with 37 ± 14% normal implantation rate among the IGF-I pellet mice (P = 0.004) (Figure 4
). The pregnancies, both resorptions and normal gestations, were noted in both horns of the uterus and were equally distributed, suggesting that elevated local concentrations of IGF-I did not affect implantation.
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Down-regulation of the IGF-IR by sense and antisense oligonucleotides adversely affects pregnancy outcome
High IGF-I concentration leads to a down-regulation of the IGF-IR, thereby resulting in a decrease in receptor expression in the blastocyst (Chi et al., 2000
). We hypothesize that this decrease in receptor expression, which is responsible for apoptosis, manifests as an increased rate in resorptions as seen above. To confirm this hypothesis using a different approach, we exposed blastocysts in culture to sense versus antisense oligoprobes and pregnancy outcomes were measured. Blastocysts exposed to sense oligoprobe exhibited much higher normal implantation rates (72.33 ± 14.68) than blastocysts exposed to antisense oligoprobe (19.51 ± 11.91) (n = 4 experiments, P < 0.033). Blastocysts exposed to antisense oligoprobe exhibited much higher resorption rates (80.50 ± 11.91) than blastocysts exposed to sense oligoprobe (27.67 ± 14.68) (n = 4 experiments, P < 0.033) (Figure 5). The higher rate of pregnancy failure and resorptions in the antisense embryos when transferred back to foster mice indicates that IGF-IR expression is critical to embryo development and survival. These findings also support the hypothesis that the adverse effect of high IGF-I on the blastocysts resulting in pregnancy loss may be attributable to the effect on IGF-IR expression.
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Discussion |
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Elevated concentrations of IGF-I or insulin adversely affect the preimplantation embryo. Elevated concentrations of IGF-I result in a down-regulation of the IGF-IR in the preimplantation embryo exposed to high IGF-I concentrations, thus leading to significant apoptosis of the inner cell mass (ICM) or key progenitor cells of the embryo (Chi et al., 2000
) (Figure 6). It has been suggested that elevated concentrations of IGF-I increase early embryonic loss by impeding embryo survival (Katagiri et al., 1996). We have previously demonstrated that elevated IGF-I concentrations are associated with an increase in apoptosis via the BAX-dependent apoptotic cascade and that this event is secondary to a decrease in IGF-IR expression (Chi et al., 2000). This study extends our previous findings. First, these experiments demonstrate that the IGF-I-induced decrease in IGF-IR expression is time dependent, with the greatest down-regulation of IGF-IR occurring concurrently with the onset of apoptosis in the blastocyst. Second, these studies strongly suggest that this increase in apoptosis either in vivo or in vitro manifests as an increase in pregnancy resorption. These experiments in combination with our previous studies demonstrate that a decrease in embryo survival, due to IGF-I-triggered apoptosis and also to a decrease in IGF-I/insulin-stimulated glucose uptake, results in embryo resorption.
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Apoptosis in the preimplantation embryo is a normal event and occurs in several species including mouse, rat and human (Handyside and Hunter, 1986
; Pampfer et al., 1990; Sell et al., 1994). Programmed cell death at this stage of development is essential to help eliminate redundant cells in the ICM and are deficient in normal developmental capacity (Hardy et al., 1989). In human embryos apoptosis is evident in normal-appearing embryos but to a greater extent in arrested and fragmented embryos (Pierce et al., 1989; Sell et al., 1994). Studies have also shown increased cell death in murine preimplantation embryos undergoing retarded or suboptimal development. Our work (Moley et al., 1998b; Chi et al., 2000a,b) as well as others (Pamfer et al., 1990) suggests that uncontrolled apoptosis or programmed cell death occurring in the preimplantation embryo may lead to embryo demise. Therefore, signalling via this pathway may protect the early embryo by eliminating abnormal cells; however, a loss of balance in this tissue remodelling may lead to dysmorphogenesis or developmental arrest, resulting in early miscarriage as seen in this study.
The findings of this study have important clinical implications. Women with PCOS are known to be hyperinsulinaemic and hyperandrogenic and experience significantly higher rates of pregnancy loss (Sagle et al., 1988; Balen et al., 1993; Tulppala et al., 1993). Elevated concentrations of insulin lead to a decrease in the production of IGFBP-1 and IGFBP-3, thus resulting in increased concentrations of IGF-I (De Mellow and Baxter, 1988; Holy, 1990; Conover, 1992; Homburg et al., 1996; Morales, 1996). Our results suggest that elevated ambient concentrations of IGF-I may be responsible for the increased early embryonic loss seen in patients with PCOS. Altering the IGF-I and insulin concentrations in women with PCOS early in the preconception and preimplantation period may help to reduce the higher miscarriage rate associated with this condition.
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Acknowledgements |
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This work was supported in part by NIH through grants RO1 HD38061-01A1 (K.H.M.), and Washington University CNRU (NIH P30 DK56341) (K.H.M.).
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Notes |
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3 To whom correspondence should be addressed at: Departments of Ob/Gyn and Cell Biology and Physiology, Washington University School of Medicine, 4911 Barnes–Jewish Hospital Plaza, St Louis, MO 63110, USA. E-mail: moleyk{at}msnotes.wustl.edu
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accepted on September 25, 2001.
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