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Endocrinology Vol. 138, No. 8 3349-3358
Copyright © 1997 by The Endocrine Society

ARTICLES

Direct Administration of Insulin-Like Growth Factor to Fetal Rhesus Monkeys (Macaca mulatta)1

Alice F. Tarantal, Maya K. Hunter and Sharron E. Gargosky2

California Regional Primate Research Center and the Department of Pediatrics, University of California (A.F.T.), Davis, California 95616; and the Department of Pediatrics, Oregon Health Sciences University (M.K.H., S.E.G.), Portland, Oregon 97201

Address all correspondence and requests for reprints to: Alice F. Tarantal, Ph.D., California Regional Primate Research Center, University of California, Davis, California 95616-8542. E-mail: aftarantal{at}ucdavis.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A potential treatment for the amelioration of fetal growth failure is insulin-like growth factor-I (IGF-I). To address concerns of safety and efficacy, IGF-I (80 µg/kg; GroPep Pty.) was administered ip to healthy rhesus monkey fetuses via ultrasound guidance every other day between gestational days (GD) 110–120 and 130–140 (third trimester; term = approximately GD 165 ± 10; n = 6). Pregnancies were monitored sonographically, and fetal/maternal blood samples were collected for complete blood counts, immunophenotyping, and biochemical analyses. Blood samples, external measures of the fetus and newborn, and tissue and organ weights were collected at fetal necropsy (GD 150; n = 2) or at term delivery of neonates (GD 160; n = 4). The results of these investigations have shown no evidence of hypoglycemia in the fetus or dam during the course of treatment. Circulating concentrations of fetal, but not maternal, IGF-I increased with treatment (~80 to ~1015 ng/ml), and there was no evidence of a change in serum IGF-II or an increase in IGF binding protein-3 compared with historical control values. Fetal lymphocytes and select red cell parameters increased, and a significant elevation in circulating B cells and CD4/CD8 ratios in fetal lymph nodes was shown. Although no changes were detected in body weights, increases in thymic, splenic, and kidney weights and small intestine lengths occurred. Thus, administration of IGF-I to the fetal monkey is safe and results in 1) transient increases in circulating IGF-I, 2) a significant effect on fetal hematopoietic and lymphoid tissues, and 3) an increase in select fetal organ weights and measures. These data suggest that IGF-I may represent a potential candidate for therapeutic treatment of growth-compromised human fetuses in utero.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INTRAUTERINE growth restriction (IUGR) is a significant pediatric problem and a major cause of perinatal morbidity and mortality, second only to prematurity (1). Some infants with IUGR may show catch-up growth postnatally, whereas others have irreversible growth perturbations and remain at high risk for life-long consequences (2, 3). Although numerous reports have identified etiological associations and common final pathways, such as nutrient transfer (4), substrate supply (3, 5, 6), as well as aberrations in cytokines (7, 8) and growth factors (6, 9, 10, 11), effective therapeutic treatment regimens have not been adequately developed.

A key modulator of somatic fetal growth is insulin-like growth factor (IGF) (6, 9, 11, 12, 13), which enhances glucose and amino acid uptake while inhibiting protein breakdown, and promotes the proliferation and differentiation of a variety of cells. Gene knock-out experiments (9, 11) have shown that loss of the IGF peptide or receptors may result in death of the conceptus in utero or postnatally, or in the severe IUGR associated with postnatal mortality. In the mammalian fetus, circulating concentrations of IGF increase during gestation (12, 14, 15), although little circulating IGF is free due to the IGF binding proteins (IGFBPs) (16). The IGFBPs display a high and specific affinity for IGF. To date, seven IGFBPs have been reported and shown to influence the bioavailability and bioactivity of IGF-I and -II (16, 17). The predominant serum IGFBP, IGFBP-3, may be either soluble or cell-associated, which influences its role as an inhibitory or potentiating IGFBP.

In addition to developmental regulation and modulation by IGFBPs, the IGF peptides are nutritionally regulated (4, 6, 10) and themselves influence the distribution of nutrient supply. Studies in humans and animal models have shown that administration of IGF-I during catabolic and wasting states is a potent therapy for the amelioration of net tissue catabolism, the restoration of growth, the overall improvement in weight gain, and the prevention of weight loss (18, 19, 20, 21, 22, 23). Because IUGR is characterized by poor sc tissue turgor and a wasted appearance comparable to findings accompanying postnatal malnutrition, this suggests similar catabolic mechanisms (24, 25). Thus, IGF-I may be a potent method for reversing growth failure in utero; this is supported by studies in fetal sheep, where short term infusion of IGF-I resulted in rapid anabolic and anticatabolic effects on fetoplacental protein and carbohydrate metabolism (26).

To assess the potential therapeutic benefit of in vivo fetal treatment, IGF-I was administered directly to healthy rhesus monkey fetuses via ultrasound guidance during the third trimester. The monkey represents an excellent animal model for studying the effects of in utero IGF treatment because of the many physiological, reproductive, and developmental similarities when compared with the human. These similarities include rate of developmental stages, placental structure, extended period of gestation, growth characteristics, and the IGF axis (15, 27, 28, 29).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Normally cycling, adult female rhesus monkeys (Macaca mulatta) with a history of prior pregnancy were bred and identified as pregnant according to established methods (30). All procedures employed within the study conformed to the requirements of the Animal Welfare Act, and study protocols were approved before implementation by the institutional animal use and care committee at the University of California-Davis. Activities related to animal care (diet and housing) were performed according to standard California Regional Primate Research Center operating procedures.

Pregnancy in the rhesus macaque is divided into trimesters by 55-day increments, with gestational days (GD) 0–55 representing the first trimester, GD 56–110 representing the second trimester, and GD 111–165 representing the third trimester (term = approximately GD 165 ± 10) (15). Six fetuses were treated with IGF-I as described below; two were harvested on GD 150 for tissues, and four were delivered by cesarean section for newborns at term. Select parameters are presented and compared with those in control nontreated fetal and maternal monkeys of comparable gestational ages (n = 25–120, dependent upon parameter).

Fetal monitoring
All fetuses were sonographically evaluated to confirm normal growth and viability before initiating IGF-I treatment (29). The dams were sedated with ketamine hydrochloride (10 mg/kg) for these examinations. Standard sonographic measurements of the fetal head [biparietal (BPD) and occipitofrontal diameters, area, and circumference], abdomen (area and circumference), and limbs [humerus and femur lengths (FL)] in addition to gross anatomical evaluations (axial and appendicular skeleton, viscera, membranes, placenta, and amniotic fluid) were incorporated, as previously described, and all measures were compared with normative growth curves for rhesus fetuses (29, 30).

Fetal treatment and sample collection
Recombinant human IGF-I (80 µg/kg in 1 mg/ml BSA in 0.5 ml PBS; GroPep Pty., Adelaide, Australia) was administered ip to normally grown rhesus fetuses via ultrasound guidance (31) every other day from GD 110–120 and from GD 130–140 (see Fig. 1Go). Fetal blood [~2 ml; complete blood cell counts (CBC), CD4/CD8, total T and B cells, IGF-I, IGF-II, and IGFBP-3] was collected every 10 days from GD 110 (pretreatment) until harvest (GD 150 ± 2) or cesarean section (GD 160 ± 2) using standard ultrasound-guided techniques (28, 31). Fetal blood samples (100 µl) were also collected by cardiocentesis (28) to monitor glucose levels on GD 110, 114, 120, 130, 134, 140, and 150. All samples were collected immediately before IGF treatment; on GD 130 and GD 140 additional samples were obtained within 20 min of administering IGF-I ip. Maternal blood (~3 ml) was collected from a peripheral vessel within 5 min of fetal sample collection for CBCs, glucose levels, and analyses of the IGF axis (IGF-I, IGF-II, and IGFBP-3).



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  		Figure 1. Overall experimental design. IGF-I was administered directly to the fetus (ip) via ultrasound guidance over two 10-day treatment periods (see arrows), with a 10-day break in fetal administration (GD 120–130).

 
Hematology/immunology
CBCs were performed on maternal, fetal, and neonatal blood samples (0.25 ml), which were placed directly into Microtainer tubes with EDTA (Becton Dickinson Co., Rutherford, NJ) and evaluated with a Serono Baker Diagnostic System (Allentown, PA), as previously described (28). Parameters assessed included total white (WBC) and red blood cell counts (RBC), packed cell volume, hemoglobin (HgB), hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte and platelet counts, total plasma protein, plasma fibrinogen, and a differential cell count. All electronic counts were verified by a manual differential. Plasma protein determination and evaluation of RBC morphology were also performed. Slides were stained with Wright-Giemsa for morphological evaluation.

Fetal blood (50 µl) and tissues (liver, spleen, thymus, bone marrow, and axillary, inguinal, and mesenteric lymph nodes) were collected and phenotypically analyzed by surface immunofluorescence (fluorescence-activated cell sorter analysis), as previously described (32). Specimens were incubated with T and B lymphocyte-specific antibodies, with erythrocytes cleared using the Coulter Q-Prep (Coulter, Hialeah, FL). The following mouse antihuman lymphocyte monoclonal antibodies, cross-reactive with rhesus lymphocytes, were used according to the manufacturer’s instructions: anti-CD2 [Leu 5b-fluorescein isothiocyanate (FITC), Becton Dickinson; or anti-T11-FITC, Coulter], anti-CD19 (Leu16-phycoerythrin, Becton Dickinson Co., Mountain View, CA), anti-CD4 (Leu3a-phycoerythrin, Becton Dickinson), and anti-CD8 (Leu2a-FITC, Becton Dickinson). The cells were fixed in 1% paraformaldehyde and analyzed by dual laser flow cytometry (FACScan, Becton Dickinson Co.). Lymphocytes were gated orthorhombically by characteristic forward and side-scatter dimensions that do not exclude contaminating nucleated RBCs.

Peptides and antiserum
Recombinant human IGF-I for assay purposes was obtained from Bachem (Torrance, CA), and recombinant human IGF-II was provided by Eli Lilly Research Laboratories (Indianapolis, IN). A specific polyclonal antiserum generated against Chinese hamster ovary-derived, glycosylated IGFBP-3 was designated {alpha}IGFBP-3g1. All RIAs (IGF-I, IGF-II, and IGFBP-3) were performed as previously described, using small aliquots of serum (250 µl) (15). IGF-I and IGF-II were iodinated by a modification of the chloramine-T method to specific activities of 350–500 µCi/µg (15).

Western ligand blot (WLB) analysis
Samples were subjected to WLB analysis as previously described (15). Briefly, 2 µl normal human serum or 5 µl fetal monkey serum were diluted with nonreducing SDS-dissociation buffer (0.5 M Tris, pH 6.8; 69% glycerol; and 4% SDS), then loaded onto a 1-mm discontinuous SDS-polyacrylamide gel and electrophoresed through a 4% stacking gel and a 10% separating gel at 50 V overnight. Mol wt markers were electrophoresed in SDS-dissociation buffer containing 1 mM dithiothreitol as a reductant. Proteins were then electrotransferred to 0.45-µm nitrocellulose (Schleicher and Schuell, Keene, NH) at 0.2 A for 1 h with a Hoefer Semidry Transphor unit (Hoefer Scientific Instruments, San Francisco, CA). The filter-immobilized proteins were then treated sequentially with Nonidet P-40 (3%, vol/vol) for 30 min, BSA (1%, wt/vol) for 2 h, and Tween-20 (0.1%, vol/vol) for 10 min. Each treatment was carried out at 4 C and in buffer comprised of 0.1 M Tris containing 0.15 M NaCl at pH 7.4. The treated nitrocellulose filters were probed with radiolabeled IGF-II overnight. The nitrocellulose sheets were washed extensively in Tween-20 (1%, vol/vol), dried, and exposed to x-ray film (Kodak X-Omat AR, Eastman Kodak, Rochester, NY) in the presence of Cronex Hi-plus Intensifying Screens (DuPont-New England Nuclear Research Products, Boston, MA.) for 10 days at -70 C.

Fetal/neonatal evaluations
Fetuses were delivered by hysterotomy on GD 150 ± 2 (n = 2), and a complete tissue harvest/necropsy was performed. Included were assessments of growth (body and organ weights; placental weight; morphometrics consisting of hand, foot, humerus, and femur lengths; biparietal and occipitofrontal diameters; head, arm, and chest circumferences; and crown-rump length) and collection of amniotic fluid, fetal blood, and multiple fetal tissues. The following organ weights were obtained: brain, thymus, spleen, liver, right and left kidneys, right and left adrenals, and small and large intestines. The gut was weighed and measured according to the method of Read et al. (22). Sections of all tissues collected were immersed in 10% neutral buffered formalin. Portions of selected specimens were obtained for histopathological evaluation (sectioned at 6 µm) and stained with hematoxylin and eosin.

The remaining four animals were delivered by cesarean section on GD 160 ± 2, with collection of maternal and fetal (umbilical artery) blood at the time of delivery. Simian Apgar scores were assessed (33), and body and placental weights and morphometric evaluations were conducted. Infants were placed in the nursery and hand-reared for postnatal studies. Postnatal observations included daily monitoring of health, food intake, and body weight as well as weekly morphometric assessments (hand, foot, humerus, and femur lengths; biparietal and occipitofrontal diameters; head, arm, and chest circumferences; crown-rump length; and skinfold thicknesses (Harpenden skinfold calipers)] (Quinton Instruments, Seattle, WA) (33).

Data analysis
Means and SDs or SEMs were calculated using Apple Macintosh systems with statistical software (StatView 512+, Brainpower, Calabasas, CA). Statistical significance (P < 0.05) was assessed by ANOVA or Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fetal growth in utero
Sonographic assessments of fetal growth included standardized measures of the head, abdomen, and limbs. Parameters evaluated before, during, and after the treatment periods did not reveal any statistically significant differences compared with historical control fetuses (29). Fetal BPDs, which rapidly increase in control fetuses during the early to midthird trimester (GD 110–140; 39.3 ± 0.5 to 47.3 ± 0.3 mm, mean ± SD) (29), showed comparable changes in IGF-treated fetuses (38.2 ± 0.7 to 47.0 ± 0.8 mm; Fig. 2aGo). The reduction in cranial growth acceleration during the latter stages of the third trimester was comparable in IGF-treated and nontreated fetuses. FLs showed a similar overall growth pattern in both groups (controls: GD 110, 27.7 ± 0.6 mm; GD 150, 40.6 ± 1.0 mm; IGF-treated: GD 110, 27.6 ± 1.3 mm; GD 150, 41.3 ± 1.8 mm), although it is interesting to note that IGF-treated fetuses showed a transient increase in FLs on GD 140 compared with historical controls (Fig. 2bGo). The other parameters assessed (amniotic fluid volume and placental, skeletal, and organ development) did not reveal any differences when comparing IGF-treated to nontreated fetuses.



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  		Figure 2. Sonographic measures of fetal growth did not reveal any significant differences in BPD (a), abdominal circumferences (data not shown), or FLs (b) as a result of direct fetal administration of IGF-I (mean ± SD). A transient rise in FLs was noted during GD 130–140. Arrows indicate the periods during which IGF-I was administered.

 
Hematology/immunology
Analysis of fetal CBCs revealed significant differences in WBCs (Fig. 3aGo) and RBCs (Fig. 3bGo) in IGF-treated fetuses at defined time periods compared with those in control fetuses of comparable gestational ages. Typically, total WBCs in the fetal monkey increase during the third trimester, with a predominance of lymphocytes and a marked elevation of neutrophils in the late third trimester (28), similar to findings in the human fetus (34, 35). IGF-treated fetuses showed an elevation in WBCs at the end of the initial 10-day treatment period (GD 120; IGF, 4.64 ± 0.58 x 103/µl; controls, 2.78 ± 0.16 x 103/µl, mean ± SEM), which was due to an increase in circulating lymphocytes (GD 120; IGF, 3579.4 ± 315.5 cells/µl; controls, 2476.8 ± 140.4 cells/µl; Fig. 3dGo). A slight, nonsignificant elevation in fetal neutrophils was noted during this same time period (Fig. 3cGo). All values returned to baseline levels on GD 130, which was 10 days after the first 10-day ip treatment period (GD 110–120). This was followed by a modest elevation by GD 140, 10 days into the second period of IGF therapy (GD 130–140). It is of note that a significant rise in lymphocyte counts occurred on GD 150 (P < 0.05), 10 days after the final ip injection of IGF-I (IGF = 8471.0 ± 2777.0 cells/µl; controls, 3104.1 ± 270.9 cells/µl). Increases in monocytes (IGF, 518.0 ± 518 cells/µl; controls, 244.1 ± 93.5 cells/µl) and eosinophils (IGF, 221.0 ± 75.0 cells/µl; controls, 13.3 ± 8.7 cells/µl) were also observed at this point (GD 150).



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  		Figure 3. Fetal blood samples were collected in utero via ultrasound-guided cardiocentesis (28). An increase in WBCs was noted during the initial 10-day treatment period (GD 110–120), which declined to baseline levels 10 days later (GD 130), then increased significantly on GD 150, 10 days after the last ip injection of IGF-I (a). Total RBCs showed a mild elevation on GD 140 and a marked rise on GD 150 (b), similar to WBCs. The rise in WBC counts was primarily attributed to an elevation in circulating lymphocytes (d); little change was detected in segmented neutrophils (c). All values represented are the mean ± SEM.

 
In addition to changes in leukocyte populations, fetal administration of IGF-I had a significant effect on select fetal RBC parameters at similar gestational points. Whereas no differences were noted when comparing IGF-treated to nontreated fetuses on GD 110, 120, or 130, elevations in RBCs (Fig. 3bGo) and HgB (Table 1Go) were observed on GD 140, and marked rises in RBCs, HgB, hematocrit, and platelet and reticulocyte counts were noted on GD 150 (Table 1Go). Findings in the peripheral circulation correlated with enhanced hepatic erythropoiesis; erythropoietic islands are typically not observed in the fetal liver at this advanced stage of gestation when the bone marrow has taken on the primary hematopoietic role (28) (Tarantal, A. F., unpublished observation).


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  		Table 1. Select hematologic parameters for fetuses administered IGF-I in utero compared to historical controls

 
There were no changes observed in maternal CBCs as a result of fetal administration of IGF-I (data not shown).

To further assess effects on the fetal immune system, immunophenotyping (CD4/CD8; total T and B cells) was performed on fetal blood and tissues (32). Fetal blood CD4/CD8 ratios for IGF-treated fetuses were unchanged during and after both treatment periods compared with nontreated control values (Fig. 4aGo), although total B cells were substantially elevated on GD 150 (P < 0.05; Fig. 5Go, a and b). Similarly, there were no significant changes in the CD4/CD8 ratios for most fetal tissues evaluated (liver, spleen, thymus, and bone marrow; data not shown), although a marked elevation in the CD4/CD8 ratio in all fetal lymph nodes was observed (inguinal, axillary, and mesenteric) at this point. The control mean CD4/CD8 ratio on GD 150 is 5.47 ± 1.34 for axillary lymph nodes, 4.91 ± 0.81 for inguinal lymph nodes, and 4.79 ± 1.19 for mesenteric lymph nodes (mean ± SEM), with an overall mean ratio of approximately 5. IGF-treated fetuses had lymph node CD4/CD8 ratios that were roughly double or triple the control ratio (axillary lymph nodes, 7.50 ± 3.78; mesenteric lymph nodes, 7.54 ± 1.73; inguinal lymph nodes, 14.55 ± 5.20; P < 0.05; Fig. 4bGo). These findings correlated with enlarged lymph nodes in all locations, with an increased density of cortical lymphocytes.



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  		Figure 4. Fetal blood samples collected for immunophenotyping did not indicate any differences in CD4/CD8 ratios for IGF-treated fetuses compared with nontreated controls (mean ± SEM; a). Although most fetal tissues (liver, spleen, thymus, and bone marrow) did not show any differences, there was a significant elevation in CD4/CD8 ratios in all fetal lymph nodes evaluated (axillary, inguinal, and mesenteric; inguinal lymph node is shown; P < 0.05; b). These findings correlated with an increased density of lymphocytes in the cortical region of all lymph nodes assessed histologically.

 


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  		Figure 5. Assessment of fetal blood samples on GD 150 showed an increase in total T cells (a) and a statistically significant rise (P < 0.05) in total B cells (b) when comparing historical controls to IGF-I-treated fetuses.

 
Glucose
Because exogenous administration of IGF-I can result in hypoglycemia, fetal and maternal glucose levels during and after the treatment period were assessed. Overall, there was no indication of any significant change in circulating fetal (Fig. 6Go) or maternal (data not shown) glucose after direct fetal administration of IGF-I. Fetal glucose levels remained relatively constant from GD 110–150 and typically ranged from roughly 34–45 mg/dl. Glucose levels normally increase at term, with a mean (±SEM) of 60.0 ± 6.2 mg/dl at birth. Glucose in IGF-treated fetuses showed a transient nonsignificant decline on GD 140, with a rise to control values on GD 150. Thus, there was no evidence of fetal hypoglycemia as a result of direct IGF-I administration.



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  		Figure 6. Overall, fetal glucose levels in IGF-treated fetuses roughly paralleled those in age-matched, nontreated control fetuses.

 
IGF analysis
Exogenous IGF-I administered to the fetus cannot be distinguished from the endogenous peptide with current assay methodologies; therefore, total circulating IGF-I was assessed by RIA. In historical controls, serum IGF-I concentrations have shown a developmental increase from GD 100–150, which remains relatively constant until term (15). The IGF-treated fetuses were within the range of control values before IGF-I administration. With administration of IGF-I peptide, a dramatic and significant increase in fetal circulating concentrations was observed (Fig. 7aGo). Fetal blood samples collected within 20 min of IGF-I administration showed a rapid rise in circulating IGF-I from roughly 80 ng/ml to approximately 1015 ng/ml. A return to baseline values was noted on GD 150, 10 days after the last in utero ip treatment. Of note is the fact that IGF-II concentrations were unaffected by IGF-I administration (data not shown) even when IGF-I peaked in serum. No effects on maternal IGF-I or IGF-II levels were measured when assessed throughout gestation or immediately after the administration of peptide (data not shown).



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  		Figure 7. Fetal ip administration of exogenous IGF-I significantly increased fetal circulating levels of IGF-I (a; two IGF-treated fetuses shown). Asterisks indicate fetal blood samples collected within 20 min of ip IGF-I administration. In contrast to findings with IGF-I, no significant effects were noted on IGF-II levels (data not shown) or circulating IGFBP-3 (b).

 
As IGFBP-3 is the major serum carrier of IGF-I, the inability of IGF-I to be maintained in the circulation may be reflected in the circulating levels of IGFBP-3. Thus, IGFBP-3 was assessed by WLB (Fig. 8Go) and RIA (Fig. 7bGo). In normal human serum, WLB has shown a 45/40-kDa IGFBP-3 doublet with smaller IGFBPs at 34, 29, and 24 kDa. In contrast, as previously reported, fetal monkey serum shows a faint 45/40-kDa doublet representing IGFBP-3 and a predominant 29-kDa IGFBP that may represent IGFBP-1 (15). Throughout the course of gestation, the intensity of the 45/40-kDa IGFBP-3 doublet increases. Although no effects on IGFBP-3 were detected with IGF-I administration, it is of note that the 29-kDa IGFBP was influenced by the administration of IGF-I, which was particularly evident when assessing the 20 min post-IGF administration samples (Fig. 8Go).



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  		Figure 8. WLB of sera from two IGF-treated fetuses (945–0029 and 945-0039) showed intact 44-kDa IGFBP-3, which was relatively unchanged during the treatment period. Note the transient effect of IGF treatment on the 29-kDa IGFBP when assessed in fetal blood samples collected before and 20 min post-IGF administration (GD 130 and GD 140).

 
In historical controls, serum IGFBP-3 levels, as assessed by RIA, have been shown to increase modestly during the second and third trimesters (15). In contrast to IGF-I (Fig. 7aGo), IGFBP-3 was relatively constant during IGF-I administration, maintaining concentrations within 2 SD of age-matched control values (Fig. 7bGo). Thus, findings by WLB and RIA are consistent with the fact that IGFBP-3 reflected serum IGF-I concentrations, as evidenced by the lack of an increase in IGFBP capacity, which resulted in an inability to maintain circulating concentrations of IGF-I after treatment.

Fetal/neonatal evaluations
No statistically significant differences were detected for overall body weights when comparing control to IGF-treated fetuses (Table 2Go) or to term neonates (Table 3Go). There was a nonsignificant increase in thymic, splenic, and kidney weights for harvested fetuses, and a statistically significant increase in small intestine length was detected (P < 0.05; Table 2Go). No changes in any external measures (Table 3Go) or placental weights (data not shown) were noted as a result of IGF-I administration.


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  		Table 2. Body and select organ weights (grams) and measures (millimeters) for fetuses administered IGF-I in utero and harvested on GD 150

 

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  		Table 3. Select measures for fetuses administered IGF-I in utero and delivered at term (GD 160)

 
All simian Apgar scores in IGF-treated newborns were within the normal range for newborn monkeys (33), and there was no evidence of compromise in any infant at birth as a result of in utero IGF-I treatment. All infants remained healthy during the postnatal observation period and displayed normal growth patterns during the 4 months they were evaluated (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of these investigations have shown that IGF-I (80 µg/kg) can be safely administered to the rhesus fetus during the third trimester, with delivery of healthy viable neonates at term. Additionally, these studies indicate that direct fetal administration of exogenous IGF-I 1) transiently increases circulating levels of IGF-I, as measured by RIA, 2) has a significant effect on fetal hematopoiesis and select hematopoietic/lymphoid tissues, and 3) results in increases in select fetal organ weights and measures. These effects were observed as a result of direct intermittent fetal bolus injection of IGF-I ip during the third trimester.

The results of studies in the monkey are comparable to those reported in fetal sheep in which IGF-I was chronically infused (26 ± 3 µg/h·kg for 10 days; third trimester) and resulted in an elevation in serum IGF-I and select organ weights (liver, lung, heart, kidneys, spleen, pituitary, and adrenals) (36). The IGF axis was analyzed in greater detail in the monkey by assessing the changes in IGFBPs by WLB and specifically evaluated the dominant IGFBP, IGFBP-3, by RIA. Consistent with studies in humans (37), the administration of IGF-I did not increase circulating IGFBPs, particularly IGFBP-3. IGFBP-3 is considered to reflect the circulating concentrations of IGF-I and to stabilize and maintain an endocrine store of this peptide (16). The inability of exogenous IGF-I to maintain elevated concentrations of circulating IGF-I levels posttreatment is consistent with the fact that exogenous administration does not increase circulating concentrations of IGFBP-3. This finding is viewed as favorable because the clinical application of IGF-I would have immediate and short lived anabolic effects and thus avoid the potential for hypoglycemia and for exceeding the body’s capacity to clear and process this protein.

The effects of IGF-I on hematopoiesis (38, 39, 40, 41, 42) and the immune system (40, 43, 44) have been well documented. IGF-I is known to have trophic effects on hematopoietic cell development, with its mitogenic action proposed to be mediated through the type I IGF receptor (IGF-IR) (39, 40). IGF-IR has been characterized on immunocompetent cells and shown to act as a paracrine/autocrine factor on the immune system (40). Further, IGF-I has been shown to enhance erythroid maturation, support limited erythroid proliferation, inhibit apoptosis, and stimulate heme synthesis (45). Stuart et al. (46) reported that 97% of monocytes and 88% of B lymphocytes possess IGF-IR, whereas only 2% of T lymphocytes show expression.

Many investigators have reported that IGF-I stimulates lymphopoiesis both in vitro and in vivo (44, 47). Clark et al. (44) showed that chronic administration of 100 µg recombinant human IGF-I for 7 days to adult mice increased lymphocyte counts in thymus, spleen, and lymph nodes, with an elevation in the number of splenic CD4+ and CD8+ B cells. Increases in thymic and splenic weights were attributed to increased populations of lymphocytes in these organs. Consistent with these findings, we have shown that intermittent bolus injection of IGF-I to the monkey fetus in the third trimester results in elevated peripheral blood lymphocyte counts and an increase in B cells as well as increased populations in all lymph nodes assessed. Studies in the fetal monkey have shown that under in vivo conditions, exogenous administration of IGF-I can affect fetal hematopoiesis, with actions primarily on the lymphoid and erythroid populations. It is currently unclear if these effects are due to enhanced cellular proliferation or maturation or are the results of a reduction in apoptosis and, hence, an enhancement of cell survival. These are all known actions of IGF-I in vivo and in vitro, although few studies have assessed the hematopoietic effects of IGF-I under the developmental conditions described in this study. Further investigations will be required to determine the underlying mechanism for these changes and to assess whether a more chronic treatment period, particularly during the latter stages of gestation, would enhance these effects. Based on these and other studies it is clear that the IGF axis plays an important role in the maintenance of the immune system. These are important findings because aberrations in the IGF axis have been reported in human immunodeficiency virus-infected children with growth abnormalities and failure to thrive (48) as well as in our animal model of pediatric acquired immunodeficiency syndrome (simian immunodeficiency virus-infected fetal monkeys), in which significant IUGR and wasting have been shown to occur (32).

In the fetal monkey, intermittent administration of IGF-I significantly affected fetal organ growth, although overall effects on body weight were not shown to occur. These results are in agreement with studies in fetal sheep, in which the effects of chronic infusion of IGF-I were studied (36). It has been proposed that the effects of IGF-I on defined organs may be due to direct actions mediated by the IGF-IR or to a synergistic effect with other growth-promoting factors (16, 36). Although a greater effect on fetal organ weights was anticipated, it is likely that the effects would be greater under conditions where normal growth processes are disrupted. Infectious (32) and noninfectious (Tarantal, A. F., and S. E. Gargosky, unpublished observations) fetal monkey models of IUGR have been developed in which aberrations of the IGF axis have been shown to occur. Both models have shown similar effects on fetal/neonatal growth and the IGF axis, and suggest that comparable growth-restricting mechanisms may be involved. Thus, these models provide a unique setting for studying the role of the IGF axis in fetal growth control as well as novel in utero growth-regulating therapies such as IGF-I. Because of the complexity of growth-regulating mechanisms and the need to test novel therapeutic interventions, relevant animal models that can address specific questions that cannot ethically be explored in the human are essential. The monkey is an excellent choice because of similarities in developmental ontogeny with respect to prenatal and postnatal growth patterns, hematopoiesis, as well as characteristic features of the IGF axis biochemically and molecularly (15, 27, 28, 29, 30, 32, 33) (Tarantal, A. F., unpublished observations) compared with the human.

In summary, because of the relationship between fetal growth, the IGF axis, and the anabolic, hematopoietic, immune system, and growth-related effects associated with IGF administration, IGF-I is proposed as an effective method for improving fetal growth and health in utero. The increase in free IGF-I in the absence of altered IGFBP-3 should prove advantageous to the growth-compromised fetus, as more IGF will be bioavailable to essential tissues during critical stages of development. The effects of IGF-I on fetal hematopoiesis suggest that immuno- or hematopoietically compromised fetuses can benefit from in utero treatment. Hence, further studies with the fetal monkey will provide essential evidence concerning the role of the IGF axis in fetal health and disease and provide a method for studying novel growth-regulating and immunomodulatory therapies in vivo that will be directly applicable to the compromised human fetus.


   Footnotes
 
1 This work was supported by NIH Grants DK-49317 and RR-00169. Back

2 Current address: Pharmacia & Upjohn, Kalamazoo, Michigan 49001-0199. Back

Received February 11, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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