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Endocrinology Vol. 147, No. 7 3344-3355
Copyright © 2006 by The Endocrine Society

Maternal Insulin-Like Growth Factors-I and -II Act via Different Pathways to Promote Fetal Growth

Amanda N. Sferruzzi-Perri, Julie A. Owens, Kirsty G. Pringle, Jeffrey S. Robinson and Claire T. Roberts

Research Center for Reproductive Health, Discipline of Obstetrics and Gynecology, University of Adelaide, Adelaide, South Australia 5005, Australia

Address all correspondence and requests for reprints to: Claire T. Roberts, Research Center for Reproductive Health, Discipline of Obstetrics and Gynecology, University of Adelaide, Adelaide, South Australia, Australia 5005. E-mail: claire.roberts{at}adelaide.edu.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The placenta transports substrates and wastes between the maternal and fetal circulations. In mice, placental IGF-II is essential for normal placental development and function but, in other mammalian species, maternal circulating IGF-II is substantial and may contribute. Maternal circulating IGFs increase in early pregnancy, and early treatment of guinea pigs with either IGF-I or IGF-II increases placental and fetal weights by mid-gestation. We now show that these effects persist to enhance placental development and fetal growth and survival near term. Pregnant guinea pigs were infused with IGF-I, IGF-II (both 1 mg/kg·d), or vehicle sc from d 20–38 of pregnancy and killed on d 62 (term = 69 d). IGF-II, but not IGF-I, increased the mid-sagittal area and volume of placenta devoted to exchange by approximately 30%, the total volume of trophoblast and maternal blood spaces within the placental exchange region (+29% and +46%, respectively), and the total surface area of placenta for exchange by 39%. Both IGFs reduced resorptions, and IGF-II increased the number of viable fetuses by 26%. Both IGFs increased fetal weight by 11–17% and fetal circulating amino acid concentrations. IGF-I, but not IGF-II, reduced maternal adipose depot weights by approximately 30%. In conclusion, increased maternal IGF-II abundance in early pregnancy promotes fetal growth and viability near term by increasing placental structural and functional capacity, whereas IGF-I appears to divert nutrients from the mother to the conceptus. This suggests major and complementary roles in placental and fetal growth for increased circulating IGFs in early to mid-pregnancy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE PLACENTA IS a multifunctional organ that forms the interface between the fetal and maternal circulations. It is essential for fetal growth as it supplies the developing fetus with oxygen and nutrients, transporting them from the mother into the umbilical circulation. Abnormalities in placental structural development can impair placental function, reducing substrate supply to the fetus, and may result in intrauterine growth restriction (1). It is estimated that placental dysfunction accounts for 70–80% of growth-restricted newborns (2), currently affecting 6% of pregnancies in developed countries (3) and up to 40% in developing countries (4). Intrauterine growth restriction is associated with perinatal morbidity and mortality (5, 6) and increases the risk of poor health in childhood and adult life (7). In addition, impaired placental trophoblast invasion of the maternal uterine vasculature and/or poor placental function are implicated in other major pregnancy complications, such as miscarriage (8), preeclampsia (1), placental abruption (9), and preterm labor (10, 11). Therefore, it is imperative that we understand the factors essential for regulating placental functional development to identify causes of such diseases and as a basis for the development of therapeutics.

The IGF-I and -II have been implicated in placental structural and functional development. Igf2 overexpression in mice causes placental and fetal overgrowth (12), whereas Igf2 gene deletion reduces placental weight by 17% on d 13.5 and 25% on d 16.5 of gestation, with a fetal weight reduction of 40% from d 16.5 (term = 19 d) (13, 14). In addition, placental amino acid transporter expression is altered by Igf2 deficiency in mice (15). Ablation of the placental-specific Igf2 promoter (P0) in mice reduces placental weight and adversely affects placental structural differentiation and transport capacity, with reduced fetal weight evident 2 d later (16, 17). The latter reduction in fetal weight was comparable to that induced by global Igf2 gene ablation, suggesting that the effects of Igf2 deficiency on fetal growth are mediated by actions on the placenta in mice.

In contrast, Igf1 gene ablation in mice does not alter placental weight but reduces fetal weight, indicating that IGF-I is important in the fetus (14, 18). IGF-I may modulate placental nutrient capacity because IGF-I administration to pregnant rats, or increased endogenous expression in pregnant mice, increases the weight of the fetus but not that of the placenta (19). IGF-I stimulates glucose and amino acid uptake in cultured human placental trophoblasts (20, 21, 22) and promotes placental nutrient uptake and metabolism when infused into fetal sheep (23, 24, 25). Moreover, exposure to IGF-I inhibits release of vasoconstrictors, such as thromboxane B2 and prostaglandin F2 {alpha}, in human term placental explants (26), which may increase placental blood flow and delivery of nutrients for the growth of the fetus.

The placenta is exposed to IGFs from multiple sources, including those produced locally and those circulating within the fetus and mother. Maternally derived IGFs may have a major influence on placental development, particularly in women and in guinea pigs where circulating IGFs are substantial (27, 28). Indeed, the IGF axis in guinea pigs is very similar to that of humans (29), whereas rats and mice do not have circulating IGF-II postnatally. The placenta in guinea pigs is more similar to the human placenta than that of other nonprimate species being hemomonochorial, although it is labyrinthine rather than villous in structure. The guinea pig placenta is comprised of a labyrinth, which contains both fetal capillaries and maternal blood sinuses and provides the means for exchange between the two circulations and an interlobium that is comprised of syncytiotrophoblast and maternal blood sinuses, and is the site where much of the metabolic activity of the placenta is thought to occur (30). In the human placenta, exchange and endocrine functions are performed in the placental villi (31). In addition, placental trophoblast cells in guinea pigs are highly invasive and, like those in humans, engage in interstitial and endovascular invasion of the decidua. They remodel the uterine spiral arterioles to permit the large increase in blood flow to the placenta (32, 33) that is essential for placental growth and subsequent function and therefore fetal growth.

In the guinea pig, major structural determinants of placental function are strongly predicted by maternal IGF-II concentration in mid-pregnancy and by maternal IGF-I in late pregnancy (34, 35). Furthermore, in this species, food restriction reduces maternal plasma IGF concentrations (36) that correlate with delayed structural and functional maturation of the placenta and with reduced fetal growth (34, 35, 37). The structural defects in the placenta of food-restricted guinea pigs are similar to those seen in placentas from women with preeclampsia (34). In addition, reduced maternal plasma IGF-I in pregnant women is associated with placental dysfunction and small-for-gestational-age (38, 39) or growth-restricted infants (40).

Consistent with these adaptive changes in maternal IGFs regulating placental development, maternal supplementation with IGF-I or IGF-II in early to mid-pregnancy in the guinea pig increases placental and fetal weights by mid gestation (41). Therefore, we suggest that the increased maternal production of both IGFs in early pregnancy is an important adaptation to pregnancy, which promotes placental functional development and consequently fetal growth. Whether anabolic effects of an increased abundance of maternal IGFs in early pregnancy on the placenta would persist into late gestation and affect the fetus is currently unknown. Therefore, the aim of this study was to determine the effects of maternal IGF-I and -II supplementation in early to mid-pregnancy on placental development and fetal growth and viability near term.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
This study was approved by the University of Adelaide, Animal Ethics Committee. Virgin guinea pigs (IMVS colored strain, approximately 500 g, 3–4 months old) were housed individually in the University of Adelaide Medical School Animal House. Guinea pigs were provided with food and water ad libitum. Females were examined daily for estrus indicated by a ruptured vaginal membrane (complete estrous cycle lasts approximately 15 d) and mated naturally with a male. The day a copulatory plug was observed was designated as d 1 of pregnancy. From 2 wk before mating, body weight was monitored three times weekly. Females were assigned to three groups of similar mean weight at mating.

On d 20 of pregnancy (term 69–70 d), females were anesthetized with atropine sulfate (0.05 mg/kg, sc; Apex Laboratories, Sydney, Australia), xylazine hydrochloride (4 mg/kg, im; Troy Laboratories, Sydney, Australia), ketamine hydrochloride (25 mg/kg, ip; Troy Laboratories) and administered local analgesia with lignocaine hydrochloride (Troy Laboratories). A 200-µl mini osmotic pump (Alzet 2002; Alzet, San Francisco, CA) was surgically inserted sc. Minipumps had previously been prepared to deliver vehicle (0.1 M acetic acid) (n = 7) or 1 mg/kg·d IGF-II (n = 7) or IGF-I (n = 9) (human recombinant protein; GroPep Pty. Ltd., Adelaide, Australia) for 18 d at a flow rate of 0.51 µl/h.

On d 62 of pregnancy, guinea pigs were killed by overdose of sodium pentobarbitone (Lethobarb; Virbac, Sydney, Australia). Viable and resorbing implantation sites were counted and the uterus and its contents, viable fetuses, and placentae were weighed. Fetal biparietal diameter, abdominal circumference, and crown-to-rump length were measured. A 3-mm mid-sagittal placental slice was fixed in 4% paraformaldehyde for structural analysis. Analyses of body composition were performed on the mothers and all fetuses to determine the absolute and relative weights of adrenals, kidneys, pancreas, liver, spleen, heart, brain, lungs, gastrointestinal tract, reproductive tract, biceps, triceps, gastrocnemius and soleus muscles and retroperitoneal, perirenal, and interscapular adipose tissues. Skin and carcass weights of the dams and carcass weight of the fetuses were also recorded.

Measurement of maternal circulating IGF-I, IGF-II, and IGF binding proteins (IGFBPs)
In an additional cohort of guinea pigs (vehicle, n = 5; IGF-I, n = 5; IGF-II, n = 3), mothers were killed on d 35 of pregnancy, while the minipumps were still active by overdose of sodium pentobarbitone. Maternal blood was collected by cardiac puncture and centrifuged at 2500 rpm for 15 min at 4 C, then plasma was recovered and stored at –20 C.

Plasma IGF-I and IGF-II proteins were dissociated from their binding proteins (IGFBPs) by size exclusion high pressure liquid chromatography performed at pH 2.5, as previously described (42, 43). From each acidified plasma sample, four fractions were eluted from the column, and fraction 1, which contained only IGFBPs, and fraction 3, which contained only the IGFs, were collected for later analysis. The IGF fraction 3 was analyzed by specific RIAs for IGF-I and IGF-II concentrations as previously described (42, 44).

Recombinant human IGF-I and IGF-II (GroPep Pty. Ltd.) were used as standards and for preparation of radiolabeled ligands. IGF-I was measured by RIA using rabbit antihuman IGF-I (MAC Ab 89/1; GroPep Pty. Ltd.) at a final dilution of 1/60,000 and a monoclonal mouse antirat IGF-II antibody (kind gift from Dr. K. Nishikawa, Kanaza Medical University, Ishakawa, Japan) was used at a final concentration of 1/500 to measure IGF-II by RIA. Cross-reactivity of IGF-II in the IGF-I RIA was less than 1% (44) and that of IGF-I in the IGF-II RIA was less than 2.5% (45). Both IGF-I and IGF-II amino acid sequences are remarkably conserved across species. Guinea pig IGF-I and IGF-II have previously been shown to have 100% amino acid sequence identity to those of human (46, 47), whereas guinea pig IGF-II has only one amino acid different to that of the rat (48). We have previously reported that the recoveries of IGF-I and IGF-II are more than 95% for these assays (28). The minimal detectable concentrations of IGF-I and IGF-II were 6.64 and 9.48 ng/ml, respectively. The samples were analyzed in a single RIA, where the mean intraassay coefficients of variation were 3.7 and 5.6% for IGF-I and IGF-II RIAs, respectively.

The total IGFBP binding capacity in the maternal circulation was indirectly measured as the interference of the IGFBPs in fraction 1 in the IGF-I RIA, as previously described (42). The ratio of IGFs to IGFBPs provided an index of IGF bioavailability in the maternal circulation.

Placental histology
Mid-sagittal slices of placentae that had been fixed in 4% paraformaldehyde overnight were washed in 1% PBS, dehydrated, and embedded in paraffin wax, then 5-µm sections were stained with Masson’s Trichrome (49). From each dam, one to three placentae were randomly selected for histological assessment. The cross-sectional areas of the placental interlobium (germinative region) and labyrinth (exchange region) were measured in complete mid-sagittal sections using an Olympus BH-2 microscope with x2 objective and x3.3 ocular lenses and video image analysis software (Video Pro; Leading Edge, Adelaide, Australia). The proportion (percentage) of each region in the placenta was then estimated by dividing the cross-sectional area of that region by the total mid-sagittal cross-sectional area of the placenta. An estimate of the volume of these regions was then calculated by multiplying their proportion by total placental weight.

Structure of the placental exchange region (labyrinth)
To distinguish cell types within the placental labyrinth, mid-sagittal sections of placenta were double-labeled with mouse antibodies to human vimentin (3B4; Dako, Glostrup, Denmark) and human pan cytokeratin (C2562; Sigma, Sydney, Australia) to identify fetal capillaries and trophoblast, respectively, and then stained with eosin to aid the identification of maternal blood spaces. This employed a triple layer technique for each antibody, performed sequentially. Sections were deparaffinized and brought to water. For antigen retrieval, sections were incubated at 37 C for 15 min in 0.03% protease (Sigma). Endogenous peroxidase activity was quenched by incubating with 3% hydrogen peroxide in water for 30 min. Sections were then washed in three changes of PBS for 5 min each and blocked for nonspecific binding with serum-free protein block (Dako) for 10 min without washing. 3B4 antibody diluted 1:50 with 10% normal guinea pig serum and 1% BSA was applied first and incubated overnight in a humidified chamber at room temperature. Sections were washed as above, and biotinylated goat antimouse IgG secondary antibody (Dako) was applied for 30 min, followed by washing. Streptavidin conjugated to horseradish-peroxidase (Rockland Immunochemicals, Pottstown, PA) was applied for 40 min, then sections were washed as above. 3B4 binding was visualized by incubating with diaminobenzidine with 2% ammonium nickel (II) sulfate (Sigma) to form a black precipitate. The process was then repeated for the second primary antibody (C2562) diluted 1:50 with PBS, 10% normal guinea pig serum, and 1% BSA, but nickel was omitted from the chromogen, leaving a brown precipitate. Negative controls used irrelevant mouse IgG in place of the primary antibodies or the primary antibody diluent on its own.

The placental labyrinth was then morphometrically analyzed, as previously described (34). Briefly, the proportions (volume density) and volumes of the labyrinthine placental components were quantitated by point counting on 10 nonoverlapping fields with random systematic sampling using an Olympus BH-2 microscope with x20 objective and x3.3 ocular lenses. The weight of each component was estimated by multiplying the volume density by the weight of the placental labyrinth. The surface area per gram of placental labyrinth was quantitated using intercept counting and the total surface area of syncytiotrophoblast for exchange and arithmetic mean trophoblast thickness (the layer through which substrate exchange occurs) were calculated as previously described (34).

Protein localization of IGF receptors in the placenta on d 35 of pregnancy
To determine that the placenta expressed the type 1 and 2 IGF receptors at the time of treatment we localized them in placental sections from the cohort of guinea pigs that were killed on d 35 of pregnancy in which circulating IGFs had been quantified. Mid-sagittal slices of placentae were immuno-labeled with rabbit antibodies raised against human IGF1R (N-20, diluted 1:20; Santa Cruz Biotechnology, Santa Cruz, CA) and IGF2R (a kind gift from Dr. Carolyn Scott, Kolling Institute of Medical Research, Sydney, Australia; diluted 1:100). This employed a triple layer technique for each antibody performed on serial placental sections, as described above. Negative controls used irrelevant mouse IgG in place of the primary antibodies or the primary antibody diluent on its own.

Plasma metabolite and hormone concentrations
Maternal and fetal plasma glucose (glucose HK assay kit; Roche Diagnostics, Mannheim, Germany), free-fatty acids (WAKO Nefa C free fatty acid kit; NovoChem, Nieuwegein, The Netherlands), cholesterol (cholesterol CHOD-PAP assay kit; Roche Diagnostics), and triglycerides (triglycerides assay kit; Roche Diagnostics) were quantified with enzymatic assay kits using a COBAS Mira automated centrifugal analyzer (Roche Diagnostics). Maternal and fetal plasma {alpha}-amino nitrogen concentrations were determined using the ß-naphthoquinone sulfonate colorimetric assay as previously described (50). Maternal plasma estradiol (Ultra-Sensitive Estradiol; Diagnostic Systems Laboratories, Houston, TX) and progesterone concentrations (progesterone assay kit; Diagnostic Systems Laboratories) were quantified with RIA kits.

Statistics
To assess differences in fetal weight distribution between treatments, {chi}2 tests were performed using Microsoft Excel. All other data were analyzed using SPSS version 13 (SPSS, Chicago, IL). To assess differences in maternal weight gain, repeated measured ANOVA with Bonferroni post hoc tests were performed. To assess differences in maternal body composition, general linear model univariate ANOVA with Bonferroni post hoc tests were performed. To assess differences in fetal band placental parameters, linear mixed model repeated measures ANOVA with Bonferroni post hoc tests were performed with the mother as a subject and the fetus or placenta as the repeated measure. The number of viable pups per litter were used as a covariate when required. Data are expressed as mean ± SEM or estimated marginal mean ± SEM as required. Data were considered statistically significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exogenous maternal IGF treatment increases maternal plasma IGF-I and IGF-II
To determine the concentration of IGFs we achieved in the maternal circulation in response to this treatment, an additional cohort of guinea pigs was killed on d 35 of pregnancy, while the minipumps were still active. Exogenous IGF-I increased maternal plasma IGF-I by 340% (P = 0.001) and reduced that of IGF-II by 45% (P = 0.008; Fig. 1Go). Exogenous IGF-II did not alter plasma IGF-I concentrations but increased plasma IGF-II by 240% (P = 0.004; Fig. 1Go). In addition, the total apparent IGFBP activity in maternal plasma was not altered by exogenous IGF. Maternal IGF-I treatment increased the ratio of IGF-I to IGFBPs in plasma by 230% (P = 0.004), whereas IGF-II increased the ratio of IGF-II to IGFBPs in plasma by 125% (P = 0.04; Fig. 1Go).


Figure 1
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  		FIG. 1. The effect of exogenous maternal IGFs on maternal circulating IGF-I, IGF-II (A), and total IGFBP (B) concentrations and bioavailability of IGFs in the circulation indicated by IGF to IGFBP ratios (C) during treatment on d 35 of pregnancy. Data are from three to six mothers per treatment, and values are expressed as means ± SEM. Asterisks denote a statistically significant difference compared with the vehicle group, P < 0.034.

 
IGF receptor proteins are expressed by the guinea pig placenta during the treatment
To establish that IGF1R and IGF2R are expressed by the guinea pig placenta during the IGF treatment, immunolabeling was performed on guinea pig placenta recovered from vehicle-treated mothers killed on d 35 of pregnancy (Fig. 2Go). IGF1R and IGF2R were ubiquitously expressed by the guinea pig placenta, with profuse cytoplasmic staining observed in trophoblast and fetal endothelium of the labyrinth and trophoblast of the interlobium (Fig. 2Go, A and C). Both IGF receptor proteins were concentrated on the apical surface of trophoblast within large maternal blood sinusoids and within maternal blood spaces (Fig. 2Go, B and D).


Figure 2
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  		FIG. 2. Representative mid-sagittal serial sections of placentae on d 35 of pregnancy immunolabeled for the type 1 (A and B) and type 2 (C and D) IGF receptors. Representative negative control placental sections displayed (E and F). Two asterisks indicate maternal blood sinusoids and single asterisks indicate maternal blood spaces. Scale bars, 400 µm (A, C, and E) and 40 µm (B, D, and F).

 
Exogenous maternal IGF-II, but not IGF-I, enhances development of the placental exchange region (labyrinth)
IGF treatment in early to mid-pregnancy did not alter placental weight in late gestation (Table 1Go). However, there was a 17% difference in placental weight between IGF-I- and IGF-II-treated mothers (P = 0.039). Exogenous IGF-II increased placental labyrinthine cross-sectional area by 28% (P = 0.005) but not that of the interlobium (Fig. 3Go, A–C, and Table 1Go). The ratio of labyrinth to interlobium was increased by IGF-II (+37%, P = 0.054). IGF-II increased the proportion of the placenta comprised of labyrinth (+9%, P = 0.0003) and reduced that composed of the interlobium (–24%, P = 0.0003) (Table 1Go). IGF-II also increased the volume of placental labyrinth (+28%, P = 0.027) but did not alter that of the interlobium (Table 1Go). Maternal IGF-I treatment did not alter any placental parameter (Table 1Go).


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  		TABLE 1. Effect of maternal IGF treatment on placental structure near term

 

Figure 3
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  		FIG. 3. The effect of exogenous maternal IGF treatment on placental structure. Representative mid-sagittal sections of near-term placentae stained with Masson’s Trichrome to distinguish labyrinth and interlobium layers from mothers that had been treated with vehicle (A), IGF-I (B), or IGF-II (C) during early to mid-pregnancy. L, Labyrinth; i, interlobium. Scale bars, 400 µm. D, Representative mid-sagittal section of near-term placenta double-labeled and eosin stained to reveal structural components of the placental labyrinth, including fetal trophoblast (thin arrow), maternal blood spaces (asterisks), and fetal capillaries (broad arrows). Scale bar, 40 µm.

 
To examine placental labyrinthine development in response to earlier maternal IGFs in more detail, structural correlates of placental function were quantified. Maternal IGF treatment did not alter the proportions of the placental labyrinth composed of trophoblast, maternal blood spaces, or fetal capillaries (Fig. 4AGo). IGF-II increased the volume of trophoblast (+29%, P = 0.015) and that of maternal blood spaces (+46%, P = 0.035) within the placental labyrinth (Fig. 4BGo). The total surface area of trophoblast functioning in exchange was also increased by IGF-II (+39%, P = 0.037, Fig. 4CGo). There was no effect of IGF treatment on syncytiotrophoblast barrier thickness (vehicle, 4.7 ± 0.2 µm; IGF-I, 4.8 ± 0.2 µm; IGF-II, 4.4 ± 0.2 µm). Maternal IGF-I treatment did not affect any placental labyrinthine structural parameter measured.


Figure 4
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  		FIG. 4. The effect of exogenous maternal IGFs on structural correlates of placental exchange function near term. Proportions (A) and volumes (B) of fetal trophoblast, maternal blood spaces, and fetal capillaries in the placental labyrinth (exchange region), as well as the total surface area of syncytiotrophoblast for exchange (C). Data are from n = 1–3 placentae from each of seven to nine mothers per treatment. Values are expressed as means ± SEM. Asterisks denote a statistically significant difference compared with the vehicle group, P < 0.05. #, Positive correlation with fetal weight, r > 0.34 and P < 0.034.

 
Exogenous maternal IGFs increase fetal survival
Maternal IGF treatment did not affect total litter size (Table 2Go). However, the number of resorptions was reduced by IGF-I (–77%, P = 0.009) and IGF-II (–60%, P = 0.01), while IGF-II also increased the number of viable fetuses (+25%, P = 0.034) near term (Table 2Go). Maternal IGFs did not alter the ratio of females to males (Table 2Go).


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  		TABLE 2. Effect of maternal IGF treatment on litter composition and fetal dimensions near term

 
Exogenous maternal IGFs increase fetal growth with IGF-specific effects on fetal body composition
Maternal IGF-I and IGF-II treatment in early to mid-pregnancy increased fetal weight near term by 17% (P = 0.002) and 11% (P = 0.042), respectively (Table 3Go). Both maternal IGF treatments significantly skewed the fetal weight distribution to the right (both P < 0.0005; Fig. 5AGo). The percentage of fetuses heavier than 81 g was 5% in controls, 37% in IGF-I, and 19% in IGF-II-treated animals (Fig. 5AGo). IGF-I treatment increased fetal crown-to-rump length by 9% (P = 0.014), as well as abdominal circumference by 10% (P = 0.05). IGF-I increased the fetal weight to placental weight ratio by 29% (vehicle, 14.82 ± 0.86; IGF-I, 19.14 ± 0.73; IGF-II, 16.18 ± 0.65; P < 0.01). Fetal weight correlated positively with placental weight across all treatments (r = 0.27, P = 0.026) and within each of the IGF-I and IGF-II treatment groups (r = 0.44, P = 0.042 and r = 0.40, P = 0.038, respectively) but not in vehicle-treated dams alone (Fig. 5BGo). Overall, fetal weight correlated positively with both the mid-sagittal cross-sectional area and the estimated total volume of the placental labyrinth (r = 0.58, P = 0.009 and r = 0.43, P = 0.006, respectively), as well as the volume of trophoblast and fetal capillaries in the placental labyrinth (r = 0.34, P = 0.034 and r = 0.62, P < 0.001, respectively).


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  		TABLE 3. Effect of maternal IGF treatment on fetal weight and body composition near term

 

Figure 5
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  		FIG. 5. The effect of exogenous maternal IGF treatment on fetal weight distribution (A) and on the association of fetal weights with placental weights (B). Each fetus from seven to nine mothers per treatment is represented.

 
Maternal IGF-I treatment increased fetal carcass weight (+19%, P = 0.002), increased the combined weights of fetal kidneys (+20%, P = 0.028), caecum (+24%, P = 0.027), total gastrointestinal tract (+13.5%, P = 0.049), and the combined fetal fat depots (+16%, P = 0.028) (Table 3Go). Conversely, IGF-I reduced the fractional weights of the fetal spleen (–24%, P = 0.001), liver (–12.5%, P = 0.002), and brain (–18.5%, P = 0.004) (Table 3Go). Both IGF-I and IGF-II increased the weights of the fetal retroperitoneal fat (+24%, P = 0.004; +18%, P = 0.031, respectively) and combined fetal muscle mass (+22%, P = 0.008; +19%, P = 0.024, respectively; Table 3Go). IGF-I and IGF-II also increased the fetal triceps absolute (+29%, P = 0.001; +24%, P = 0.01, respectively) and relative weights (both +16%, P < 0.03, Table 3Go). Body composition of male and female fetuses was similar and was similarly affected by maternal IGF treatment (data not shown).

Exogenous maternal IGFs increase concentrations of amino acids in the fetal circulation
Maternal IGF-I and IGF-II treatment increased fetal circulating amino acid concentrations (+196%, P = 0.026 and +137%, P = 0.029, respectively) and maternal IGF-I reduced fetal circulating cholesterol concentrations (–30%, P = 0.049) near term (Fig. 6AGo). There was no effect of treatment on fetal plasma glucose, triglyceride, or free fatty acid concentrations (Fig. 6AGo).


Figure 6
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  		FIG. 6. The effect of exogenous maternal IGFs on circulating metabolites in the fetus (A) and mother (B) near term and estradiol (C) and progesterone (D) in the mother on d 35 and 62 of pregnancy. Fetal data are from all fetuses of six to eight mothers per treatment, and values are expressed as estimated marginal means adjusted for the number of viable pups ± SEM. Maternal data are from six to eight mothers per treatment, and values are expressed as means ± SEM. AA, Amino acids; Chol, cholesterol; d35, d 35 of pregnancy; d62, d 62 of pregnancy; FFA, free fatty acids; Gluc, glucose; Trig, triglycerides. Asterisks denote a statistically significant difference compared with the vehicle group, P < 0.049.

 
Exogenous maternal IGF-I, but not IGF-II, alters maternal body composition
Weight gain and body composition analyses were performed to determine whether exogenous IGFs affected the mother. Both exogenous maternal IGF-I and IGF-II did not alter maternal weight gain during or after IGF treatment (Fig. 7Go), nor total body and lean body mass near term (Table 4Go). IGF-I reduced maternal interscapular fat depot weight (–25%, P = 0.028) and the fractional weights of the perirenal (–32%, P = 0.05), retroperitoneal (–33%, P = 0.037), and interscapular fat (–28%, P = 0.01; Table 4Go). IGF-I reduced the absolute and fractional weights of the combined adipose depot weights in the mother by approximately 30%, (P = 0.016 and P = 0.007, respectively). IGF-II did not alter the absolute or relative weights of any maternal organ or tissue examined.


Figure 7
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  		FIG. 7. The effect of exogenous IGFs on maternal weight gain during pregnancy. Female guinea pigs were weighed three times weekly during the study to determine an average weekly weight, from 1 wk before mating and during pregnancy up until kill. Minipumps were inserted on d 20 of pregnancy to deliver vehicle, IGF-I, or IGF-II for 18 d. Term, which is approximately 67–70 d of pregnancy, is denoted on the graph. Data are from seven to nine dams per treatment, and values are expressed as means ± SEM.

 

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  		TABLE 4. Effect of maternal IGF treatment on maternal adipose tissue weights near term

 
Exogenous maternal IGF treatment does not alter maternal circulating metabolite concentrations
Maternal IGF treatment did not alter circulating concentrations of glucose, free fatty acids, amino acids, triglycerides, or cholesterol in the mother near term (Fig. 6BGo).

Exogenous maternal IGF treatment and maternal circulating hormone concentrations
To determine whether treatment of the mother during early to mid-pregnancy with IGFs altered maternal circulating estradiol (Fig. 7CGo) and progesterone (Fig. 7DGo), their concentrations were determined on d 35 of pregnancy in the additional cohort of guinea pigs in which the plasma IGF and IGFBP concentrations were determined as described above. Treating the mother during early to mid-pregnancy with IGF-I doubled circulating maternal estradiol concentrations in late pregnancy, although this was not quite significant (P = 0.078). IGF-I treatment did not alter mid or late pregnancy circulating progesterone concentrations. Exogenous maternal IGF-II during early to mid-pregnancy increased circulating estradiol concentrations (+150%) in mid-pregnancy and progesterone concentrations in mid (+53%) and late (+83%) pregnancy in the mother; however, these also did not reach statistical significance (P > 0.08) (Fig. 7Go, C and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates for the first time that administration of IGF-II to the mother in early to mid-pregnancy increases placental structural and functional capacity by increasing the volume and surface area of the exchange region of the placenta near term, whereas IGF-I has no effect on the placenta. IGF-I, in contrast, reduced maternal adiposity late in pregnancy, whereas IGF-II did not affect maternal body composition. Importantly, however, maternal treatment with either IGF in early to mid-pregnancy substantially reduced fetal resorptions, increased fetal weight, and increased fetal circulating amino acid concentrations near term. Furthermore, administration of IGF-II also increased fetal viability in late pregnancy. This suggests that maternal IGF abundance, particularly that of IGF-II, during the period of early placental growth and development may determine in part the margin of safety between placental capacity to deliver, and fetal demand for, substrates throughout pregnancy.

Specifically, in the current study, administration of 1 mg/kg·d IGFs increased the abundance of maternal circulating IGF-II and IGF-I by 2.5- to 3.4-fold, during early to mid-pregnancy. The concentration of free IGF to IGFBP ratio in the maternal plasma, and hence bioavailable IGF, was also substantially increased. Similar IGF treatment of guinea pigs during early to mid-pregnancy increased placental weight at mid-gestation (41), which was not sustained to near term in the current study. Importantly, however, the functional capacity of the placenta, as indicated by the mid-sagittal cross-sectional area, proportion and volume of the region devoted to exchange (labyrinth) were increased late in gestation, by prior maternal IGF-II treatment. Furthermore, although the composition of this exchange region of the placenta was unaltered by earlier maternal IGF treatment, the total volume of trophoblast and maternal blood spaces, as well as the total surface area of placenta functioning in exchange were increased by IGF-II. As the labyrinth expands at the expense of the interlobium in the second half of pregnancy in the guinea pig (30, 34, 51), together these changes in the structure of the placenta as a result of earlier exogenous maternal IGF-II are suggestive of a more mature placenta and would be expected to increase placental transport capacity. In contrast, maternal exogenous IGF-I had no effect on placental structural development.

Rapid placental structural differentiation and growth occurs in early to mid gestation in all eutherian mammals. In humans and guinea pigs, trophoblasts invade deep within the uterus and its arterioles, extensively remodeling them, to permit increased maternal blood flow to the placenta (32, 52, 53). This ensures delivery of oxygen and nutrients to the placenta, and subsequently to the fetus. The sustained effects of maternal IGF-II supplementation in early to mid-pregnancy on the placenta reported here are the converse of those observed after specific deletion of IGF-II within the placenta. IGF-II is abundantly expressed by invasive trophoblasts of human (54), mouse (55), rat (56), and guinea pig placenta (57). Ablation of placenta-specific Igf2 gene expression (P0 transcript) in mice reduced the surface area for exchange, increased the exchange barrier thickness and also impaired nutrient transport capacity of the placenta (16, 17).

Reduced maternal circulating IGF-II in mid-pregnancy, as a result of undernutrition in guinea pigs (36), is associated with similar consequences to those of placental Igf2 gene deletion (17), with a delay and impairment in the functional maturation of the placenta and with reduced fetal growth in both mid and late gestation (37). Together these findings indicate that maternal circulating IGF-II may act in an endocrine fashion to modulate placental development, in addition to any autocrine/paracrine effects of locally produced IGF-II. We suggest that exposure to increased circulating maternal IGF-II in early to mid-pregnancy may provide a foundation of increased placental trophoblast proliferation and invasion of the uterus and its vasculature, which leads to increased volumes of both trophoblast and maternal blood spaces in the placental labyrinth in late gestation. This would be expected to increase maternal blood flow to the placenta and enhance growth of the area devoted to exchange improving placental transfer of oxygen and nutrients to the fetus from the mother. This was consistent with increased circulating fetal amino acid concentrations with earlier maternal IGF treatment, near term. Hence, maternal IGF-II supplementation presumably increased fetal growth and viability predominantly by these actions on the placenta. Current studies in our laboratory are focused on determining whether early maternal IGF treatment increases placental transport of nonmetabolizable analogs of glucose and amino acids in the fetal circulation and tissues and whether treatment affects nutrient partitioning in the mother.

Supplementing the mother during early to mid-pregnancy with either IGF had a sustained positive effect on fetal weight, length, and girth near term, which is consistent with the anabolic effects on the fetus seen at mid-pregnancy after similar treatment in the guinea pig (41). The increased fetal weight observed with maternal IGF treatment appears to be substantially due to increased muscle mass overall and proportionately for selected muscles and perhaps enhanced fetal bone growth as indicated by increased carcass weights. This may be metabolically beneficial in later life because muscle is an important site for insulin-induced glucose uptake. Indeed, fetal growth restriction in the guinea pig, induced by maternal food restriction and accompanied by reductions in circulating maternal IGF concentrations (36), is characterized by deficits in muscle mass, increased adiposity in the fetus near term (58) and with increased blood pressure and impaired glucose and cholesterol homeostasis in adult offspring (59, 60, 61).

The present study suggests that increased maternal IGF-I and IGF-II abundances during early to mid-pregnancy promote fetal growth and viability near term by multiple mechanisms. In addition to direct effects of IGF-II on placental structural development, which in the current study were positively associated with fetal weights, the IGFs may increase nutrient transporter expression (20, 21, 22) and/or placental vasodilation (26), which would allow for more substrate to be delivered to the fetus for its growth. The IGFs may also influence placental metabolism and function, which, in turn, may drive major physiological adaptations to pregnancy in the mother, including the development of insulin resistance to divert nutrients to the conceptus (62, 63, 64). This has been attributed to placental production of hormones including estrogen, progesterone, and placental lactogen (64, 65) that reduce maternal insulin secretion (64, 66) and antagonize the effects of insulin on maternal tissues, including fat deposition (65). Treatment of the mother with IGF-II enhanced placental weight in mid-pregnancy (41) and is accompanied by elevated maternal circulating estradiol and progesterone concentrations, although these were not significant. This would be expected to amplify insulin resistance and other adaptations such as fat deposition in the mother. Consistent with this, exogenous IGF-II during early to mid-pregnancy in guinea pigs increased maternal interscapular adiposity at mid-pregnancy (41) and there was a trend to raised maternal circulating glucose concentrations near term. These increased maternal adipose stores were depleted to normal by late pregnancy in the current study, which may have further enhanced nutrient availability for the fetus, either directly or indirectly. This suggests that IGF-II acts on the placenta to increase fetal growth, by sustainedly promoting placental development, but additionally may enhance maternal physiological adaptation to pregnancy.

The mechanism by which increased maternal IGF-I abundance in early to mid-pregnancy sustainedly promotes fetal growth is less clear. The enhanced placental weight at mid-gestation by prior maternal IGF-I treatment (41), which is no longer apparent in late gestation, may have had persistent effects on the fetus that increased fetal growth near term. In addition, unlike IGF-II, IGF-I did not increase maternal fat deposition in mid-pregnancy (41) and in fact reduced fat depot weights near term. Reduced perirenal fat weight was associated with increased maternal circulating progesterone. Reduced adiposity may reflect increased mobilization and/or reduced deposition during pregnancy, which may have increased substrate availability in the maternal circulation for fetal growth. This has been observed in growth hormone-treated pigs where maternal circulating IGF-I concentration was elevated and associated with reductions in weight of maternal backfat depots (67). Another possible explanation is that larger fetuses of IGF-I-treated dams may signal to the mother via nutrient sensors in the fetal circulation (such as IGFs and insulin), to influence placental metabolism and increase mobilization of maternal adipose tissue stores late in pregnancy.

These differential IGF effects may reflect their distinct interactions with various receptors, because IGF-I binds with high affinity to the IGF1R but negligibly to IGF2R. In contrast, IGF-II binds to both these receptors, as well as to the insulin receptor. In the current study, during mid-pregnancy, the guinea pig placenta ubiquitously expressed both IGF receptor proteins. More importantly, however, at the time of IGF treatment, IGF1R and IGF2R were localized to the apical surface of trophoblasts, within large maternal blood vessels and blood spaces of the labyrinth. In addition, insulin binding sites have preciously been identified in trophoblast of the guinea pig placenta (68, 69, 70). This pattern of expression is consistent with the localization of all three receptors to placental trophoblasts in humans and rats (56, 71, 72, 73, 74, 75, 76, 77) and abundant expression of IGF1R and IGF2R in invasive trophoblast populations within the human decidua and its vasculature (75).

The specific effects of IGF-II on the placenta, which were not evident in IGF-I-treated animals, suggest that IGF-II actions on the placenta may be mediated by the insulin receptor, which has been implicated in mediating IGF-II effects on fetal growth (78) or by the IGF2R, which it binds with much greater affinity than the IGF1R. There is evidence to suggest that IGF-II acts through IGF2R to promote trophoblast migration and invasion (79), and placental angiogenesis and vascular remodeling (80). IGF-II then, indirectly at least, may enhance placental function by increasing blood supply to the placenta. In contrast, the effects of maternal IGF-I treatment are likely to have been mediated by the IGF1R, particularly because this treatment also reduced IGF-II in the mother.

In conclusion, increased maternal IGF-II in early pregnancy sustainedly promotes placental structural and functional capacity and fetal growth and viability, whereas IGF-I appears to act through the mother to enhance fetal growth to near term. This suggests sustained major and complementary roles in placental and fetal growth for increased circulating IGFs in the mother in early pregnancy.


   Acknowledgments
 
We thank GroPep Pty. Ltd. for supplying recombinant human IGFs. We thank Jasper Button, Carly Burgstad, and Cherise Fletcher for their assistance in the guinea pig postmortems. We thank Dr. Carolyn Scott, Kolling Institute of Medical Research, for her kind gift of IGF2R antibodies. We acknowledge the technical assistance of Natasha Campbell, Pat Grant, and Dr. Kathy Gatford in the analysis of plasma IGFs.


   Footnotes
 
This work was supported by a National Health and Medical Research Council project grant to C.T.R. and a Channel 7 Children’s Research Foundation grant to J.A.O. and C.T.R.

Disclosure: A.S.-P., K.P., J.O., J.R., and C.R. have nothing to declare.

First Published Online March 23, 2006

Abbreviation: IGFBP, IGF binding protein.

Received October 19, 2005.

Accepted for publication March 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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