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The Journal of Clinical Endocrinology & Metabolism Vol. 82, No. 1 287-292
Copyright © 1997 by The Endocrine Society

Reproductive Endocrinology

The Maternal Insulin-Like Growth Factor (IGF) and IGF-Binding Protein Response to Trisomic Pregnancy during the First Trimester: A Possible Diagnostic Tool for Trisomy 18 Pregnancies

John P. Miell1, Katherine S. Langford, Jennifer S. Jones, Penny Noble, Melissa Westwood, Anne White and Kypros H. Nicolaides

Department of Medicine, King’s College School of Medicine and Dentistry (J.P.M., K.S.L., J.S.J.), London, United Kingdom SE5 9PJ; the Department of Fetal Medicine, Harris Birthright, King’s College Hospital (P.N., K.H.N.), London, United Kingdom SE5 9RS; and the Endocrine Sciences Group, Department of Medicine, University of Manchester (M.W., A.W.), Manchester, United Kingdom M13 9PT

Address all correspondence and requests for reprints to: Dr. John P. Miell, Department of Medicine, King’s College School of Medicine and Dentistry, Bessemer Road, London, United Kingdom SE5 9PJ.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Many lines of evidence point to an important role for the insulin-like growth factors (IGFs) in embryonic and fetal growth in human pregnancy. The bioavailability of IGFs is modulated by IGF-binding proteins (IGFBP-1 to -6), whose permissive or inhibitory actions are regulated in part by posttranslational modification. In second and third trimester pregnancies, maternal IGFBP-1 is elevated in preeclampsia and intrauterine growth retardation. In the first trimester, trisomic pregnancies result in derangement of maternal serum levels of peptides, including hCGß and pregnancy-associated plasma protein A. Trisomy 18 is characterized by growth failure in the first trimester, whereas trisomy 21 is not; thus, if maternal serum levels of IGFs and IGFBPs reflect fetal growth, changes specific to trisomy 18 may be expected.

We report maternal serum levels of IGF-I, IGF-II, and IGFBP-1, -2, and -3; IGFBP-1 phosphorylation; and IGFBP-3 proteolysis in pregnancies (n = 139) complicated by trisomy 18 or trisomy 21 compared with those in normal controls. Maternal IGF-I, IGF-II, and IGFBP-3 showed no significant difference between fetuses with a normal karyotype and those with trisomy 18 or 21. The mean IGFBP-1 level was significantly higher and the mean IGFBP-2 level was lower in fetuses with trisomy 18 compared with normal fetuses [108.8 ± 6.1 vs. 36.7 ± 1.9 µg/L (P = 0.0001) and 81.2 ± 5.5 vs. 206.1 ± 10.2 µg/L (P = 0.0001), respectively]. There was no significant difference between the trisomy 21 and normal groups. The reduction in IGFBP-2 was confirmed by Western ligand and immunoblotting, and there was no evidence of variation in lower mol wt products to suggest differential proteolysis. IGFBP-1 phosphoforms and IGFBP-3 proteolysis were not significantly different between groups. The finding of altered maternal serum levels of IGFBP-1 and IGFBP-2 specific to pregnancies complicated by trisomy 18 suggests that these binding proteins may be important mediators of fetal growth in the first trimester, and the clear differences in the ratio of IGFBP-1 to -2 may serve as an additional diagnostic marker for trisomy 18 pregnancies.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE INSULIN-LIKE growth factors (IGF-I and IGF-II) are mitogenic polypeptides that have been shown to be important determinants of fetal growth during human pregnancy (1, 2, 3, 4, 5). The actions of the IGFs are modulated by a series of at least six high affinity binding proteins that are ubiquitously expressed (IGFBP-1 through -6). Although initially thought to be inhibitory to the actions of IGFs, it is now becoming clear that in certain circumstances, IGFBPs may potentiate some or all of the mitogenic properties of IGFs. Posttranslational modification of IGFBPs by, for example, phosphorylation (IGFBP-1, -3, and -5), proteolysis (IGFBP-2 to -5), or glycosylation (IGFBP-3 to -6) may affect binding affinities and render IGFs more freely available for receptor interactions (6). The presence of integrin-binding domains and the ability of IGFBPs to associate with cell surface or extracellular matrix proteins may also be important in increasing local concentrations of IGFs or may result in direct, non-IGF-mediated effects (7).

Despite the wealth of data on the IGF-IGFBP axis in human second and third trimester pregnancy, there is a relative paucity of data for the first trimester. Levels of IGFBP-1 and -2 in the extraembryonic coelomic fluid are higher than those in either maternal serum or amniotic fluid (8, 9), although amniotic fluid levels increase rapidly after about 10 weeks gestation (8). Although maternal IGFBP-1 levels tend to peak at about 12–14 weeks gestation (10), whereas IGF-I and -II levels continue to rise until term (11, 12), there is a negative correlation between maternal IGFBP-1 levels and birth weight in term and preterm pregnancies (13, 14). Consequently, increased maternal IGFBP-1 levels during the first trimester may be a marker of early intrauterine growth retardation. To test this hypothesis, we studied immunoreactive levels of IGF-I, IGFBP-1, IGFBP-2, and IGFBP-3 in maternal serum from normal pregnancies and pregnancies complicated by trisomy 18 or 21. Trisomy 18 is consistently associated with a reduction in fetal growth, which may be assessed in the first trimester by a reduction in crown-rump length from that expected for gestational age (15). This reduction in crown-rump length is not seen in fetuses with trisomy 21 (15), consequently allowing trisomy 21 fetuses to act as a normally growing, karyotypically abnormal control. As posttranslational modification may substantially alter the actions of binding proteins, we also assessed IGFBP-1 phosphorylation and IGFBP-3 proteolysis in each group.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

Maternal serum was obtained from 139 women immediately before chorionic villous sampling in the first trimester. The indication for karyotyping was maternal age or anxiety or an increased risk of trisomy indicated by nuchal translucency screening (16). Written informed consent was obtained from all women, and the study had the approval of the King’s College Hospital ethics committee. All samples were obtained from the antecubital fossa, centrifuged within 30 min of collection, and stored at -20 C until analysis. Once the karyotype was known, cases of trisomy 18 (n = 19), trisomy 21 (n = 18), and normal karyotype (n = 102) were identified. The gestational age in all cases was 11–13 weeks at the time of sampling, and the mean age did not differ significantly among groups.

Assays

Serum IGF-I was measured, after acid-ethanol extraction of its binding proteins, by RIA, using a polyclonal rabbit antiserum (R557A) raised against purified human IGF-I as previously described (5). The level of detection of this assay was 5 µg/L; the interassay coefficients of variation (CVs) were 9.0%, 4.5%, and 6.2% at analyte levels of 654, 231, and 78.4 µg/L, respectively, with an intraassay CV of 4% at 231 µg/L.

IGF-II was measured by RIA after acid-ethanol extraction of binding proteins and displacement of IGF-II from binding proteins with excess unlabeled IGF-I as previously described (5). The intra- and interassay CVs at 0.6 g/L were 3.6% and 12.2%, respectively, and the assay has 0.05% cross-reactivity with IGF-I and a sensitivity of 0.018 ng/tube.

IGFBP-3 was measured by immunoradiometric assay (IRMA), the reagents for which were purchased from DSL (Webster, TX). This assay has a sensitivity of 0.5 µg/L, and there was less than 0.01% cross-reactivity with IGFBP-2 or IGFBP-4. Neither IGF-I nor IGF-II at levels of up to 1.0 µg/tube interfered with the assay. The measurements are unaffected by the degree of proteolysis. The interassay CVs were 5.2% and 4.9% at analyte levels of 2.95 and 0.69 mg/L, respectively, with an intraassay CV of 2.9% at 3.37 mg/L.

IGFBP-2 was measured by RIA using reagents supplied by DSL. This assay has a sensitivity of 2.0 µg/L, with inter- and intraassay CVs of 10.6% and 4.1%, respectively, at 400 µg/L. There was no cross-reactivity with IGFBP-1, -3, or -4.

Total IGFBP-1 was measured by IRMA (DSL) with a sensitivity of 0.4 µg/L and interassay CVs of 2.5% at 10.0 and 6.9% at 123 µg/L, respectively. The intraassay CV was 2.3% at 9.8 µg/L. There was no cross-reactivity with IGFBP-2, -3, or -4.

Nonphosphorylated IGFBP-1 was measured by IRMA (DSL). This assay recognizes primarily nonphosphorylated IGFBP-1 with rapid rises in recorded levels after dephosphorylation of IGFBP-1 in vitro with alkaline phosphatase (17). The sensitivity is 0.2 µg/L, with inter- and intraassay CVs of 4.5% and 2.2%, respectively, at an analyte level of 12.0 µg./L

Immunoblotting

Diluted serum was supplemented with sample buffer and heated at 100 C for 5 min before being subjected to SDS-PAGE at constant voltage (50 V) for 16 h. The proteins were then electroblotted onto a nitrocellulose sheet (0.35 amperes for 4 h). After transfer, the nitrocellulose was blocked in TBS (Tris-buffered saline, 0.15 mol/L NaCl, and 0.01 mol/L Tris-HCl, pH 7.4) with 0.1% Tween-20 and 1% BSA for 6 h. Subsequently, the nitrocellulose was incubated for 1 h with either anti-IGFBP-3 antibody diluted 1:8000 (Celtrix, Richmond, CA) or anti-IGFBP-2 antibody diluted 1:3000 (Upstate Biotechnology., Lake Placid, NY). After washing in TBS with 0.1% Tween-20, the nitrocellulose was incubated with goat antirabbit IgG conjugated with horseradish peroxidase (Sigma, Poole, UK) for 1 h and then exposed to ECL reagents (Amersham, Aylesbury, UK) for 1 min before autoradiography.

IGFBP-3 protease assay

The level of proteolytic activity directed against recombinant IGFBP-3 was assessed by the method of Lamson et al. (18). Pooled serum samples were diluted 1:10 with 0.1 mol/L Tris-HCl, pH 7.4. Thirty microliters of dilute sample were incubated for 5 h with 10 µl [125I]IGFBP-3 (30,000 cpm) at 37 C, and the reaction was quenched with 30 µl sample buffer. The samples were subjected to SDS-PAGE, and the resultant gels were dried and autoradiographed.

IGFBP-1 phosphoforms

The degree of phosphorylation of IGFBP-1 was assessed by two methods. 1) Analysis of phosphorylated isoforms of IGFBP-1 was performed by immunoprecipitation, non-SDS-PAGE, and Western ligand blot as previously described (19). Briefly, samples were incubated overnight with antibody (Mab 6303, kindly provided by Medix Biochemica, Helsinki, Finland) before the addition of precipitating antibody. Samples were further incubated for 1 h at 37 C, and bound antibody was separated by centrifugation at 2800 rpm for 10 min. After three washes in phosphate-buffered saline with 0.25% BSA and 0.1% Tween-20, pellets were resuspended in gel loading buffer [170 mmol/L Tris-HPO4, pH 5.5; 90 mmol/L n-octyl glucoside (Sigma, Poole, UK); 40% glycerol; and 0.008% bromophenol blue] and electrophoresed as previously described. After transfer, nitrocellulose membranes were hybridized with [125I]IGF-I (150,000 cpm/mL) and autoradiographed, and the resultant bands were assessed by densitometry (SW2000, Ultra-Violet Products, Cambridge, UK).

2) In addition, samples were assayed using a specific IRMA for nonphosphorylated IGFBP-1 (see above). Subtracting levels obtained in this assay from those derived from the total IGFBP-1 IRMA allowed determination of the percentage of heavily and lesser phosphorylated isoforms of IGFBP-1.

Statistical analysis

Results are given as the mean ± SEM. Values for the three groups were compared by ANOVA, and at P < 0.05, the calculation was completed with Fisher’s least significant difference test.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Mean gestational age was the same for all groups. Immunoreactive IGF-I and IGFBP-3 did not differ significantly between any of the groups (Table 1Go).


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  		Table 1. Gestational age and maternal serum levels of IGF-I and -II, total IGFBP-1, IGFBP-2, and -3 in control pregnancies and those complicated by trisomy 18 and trisomy 21.

 
The IGFBP-1 level was elevated in the maternal circulation in cases of trisomy 18, and the IGFBP-2 level was lower in cases of trisomy 18 compared with normal control values [IGFBP-1, 108.8 ± 6.1 vs. 36.7 ± 1.9 (P = 0.0001); IGFBP-2, 81.2 ± 5.5 vs. 206.1 ± 10.2 (P = 0.0001)]. There were no differences between the trisomy 21 group and normal controls (IGFBP-1, 38.2 ± 6.2 vs. 36.7 ± 1.9 µg/L; and IGFB-2, 197.3 ± 21.1 vs. 206.1 ± 10.2 µg/L). The ratio of IGFBP-1 to IGFBP-2 was significantly higher in trisomy 18 than in trisomy 21 or normal controls (Fig. 1Go). Despite the wide range of values obtained for this ratio, there was little or no overlap between trisomy 18 and the other two groups.



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  		Figure 1. Individual levels of total IGFBP-1 and IGFBP-2 and the ratio of IGFBP-1 to -2 in first trimester maternal serum from normal pregnancies (n = 102) and those complicated by trisomy 18 (n = 19) or trisomy 21 (n = 18).

 
Phosphorylation of IGFBP-1 was assessed by both n-octylglucoside PAGE with subsequent ligand blotting, autoradiography, and analysis of visualized bands by densitometry (n-OG gels; Fig. 2Go) and comparison of data obtained from assays specific for nonphosphorylated IGFBP-1 and total IGFBP-1. There were no consistent differences in the degree of IGFBP-1 phosphorylation among the three groups determined by either method. Densitometric analysis of autoradiograms derived from the n-OG gels suggested that the most heavily phosphorylated isoform (and, therefore, that showing the greatest electrophoretic mobility) accounted for 51 ± 4%, 49 ± 7.7%, and 57.5 ± 5.2% of the total IGFBP-1 density in trisomy 18, trisomy 21, and control pregnancies, respectively. After subtraction of nonphosphorylated from total isoforms measured by IRMA, phosphorylated isoforms accounted for 54.6 ± 4.4%, 55.9 ± 3.2%, and 53.0 ± 4.9% of the total IGFBP-1 in trisomy 18, trisomy 21, and control pregnancies, respectively.



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  		Figure 2. Differently phosphorylated isoforms of IGFBP-1 in first trimester maternal serum from control normal pregnancies and those complicated by trisomy 18 and 21. Samples were immunoprecipitated with MAb 6303 and then analyzed by non-SDS-PAGE and ligand blotting (see Materials and Methods). Lane 1, Human recombinant IGFBP-1 (nonphosphorylated); lane 2, amniotic fluid taken at term; lane 3, a pool of normal human sera. Lanes 4–7 are samples from normal pregnancies; lanes 8–11 and 12–15 are from trisomy 18 and -21 pregnancies, respectively.

 
Western immunoblotting confirmed the reduction seen in IGFBP-2 levels in trisomy 18 when measured by RIA (data not shown). Furthermore, the distribution of IGFBP-2 isoforms was similar in the three groups, suggesting that proteolytic modification of IGFBP-2 probably did not account for the reduction in immunoreactivity.

There was no difference in proteolytic activity or distribution of mol wt isoforms of IGFBP-3 in any of the three groups, as assessed by immunoblotting with specific anti-IGFBP-3 antiserum or protease gels using labeled recombinant glycosylated IGFBP-3 as substrate (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We have demonstrated increased levels of IGFBP-1 and decreased levels of IGFBP-2 in maternal serum from pregnancies complicated by trisomy 18 compared with those in normal and trisomy 21 pregnancies. The lack of difference between trisomy 21 and normal pregnancies suggests that these changes are specific to trisomy 18 and do not simply reflect chromosomal abnormality per se.

Abnormalities in maternal serum biochemistry have been used extensively in screening for fetal aneuploidy, and over a decade ago it was reported that trisomy 18 pregnancies were associated with reduced maternal serum alphafetoprotein (20). However, invasive testing for chromosomal abnormalities by chorion villous sampling has only recently been considered safe during the first trimester of human pregnancy, and a number of the earlier tests were designed to be applied during the second trimester. Nevertheless, there are data to suggest specific maternal biochemical abnormalities within the first trimester in pregnancies complicated by trisomy. Pregnancy specific ß1-glycoprotein is low in trisomy 21 (21), as is pregnancy-associated plasma protein A in both trisomy 18 and 21 (22, 23). Maternal AFP has been reportedly low in both trisomy 18 and 21 in a number of studies (24, 25, 26), although other studies have not duplicated these data (27, 28). Both free hCGß and total hCG have been reported to be lower in trisomy 18 than in control pregnancies in many studies (29, 30, 31). Although biochemical data may slightly increase the efficacy of relative risk calculation for chromosomally abnormal fetuses, the false positive rates and sensitivities are not appreciably better than those using maternal age alone and do not approach those using biochemical screening in the second trimester (sensitivity, 50–70%; false positives, 5%) (32, 33, 34). Consequently, any biochemical test in the first trimester that may be both sensitive and specific would add greatly to ultrasound findings of growth impairment, increased nuchal translucency, or other markers of chromosomal abnormality, particularly in less experienced hands.

The IGFs and their binding proteins are thought to be important determinants of embryonic growth in the first trimester (2, 3, 4, 9). Fetuses with trisomy 18 are known to be small for gestational age in the first trimester, whereas fetuses with trisomy 21 exhibit normal growth at this stage of pregnancy. As the changes in this study are specific for trisomy 18, they may be specifically related to fetal growth failure during the first trimester.

Elevated maternal and fetal serum levels of IGFBP-1 have previously been reported in pregnancies complicated by fetal growth failure in the late second and third trimesters, possibly as a result of poor placentation (5, 10). Elevated first trimester maternal serum IGFBP-1 in association with trisomy 18 concurs with these findings and may indicate that whatever processes lead to elevation of IGFBP-1 and fetal growth failure in later pregnancy are also operating in the first trimester. The rise in maternal IGFBP-1 levels during the first trimester of normal pregnancies has been attributed to increases derived from the decidualized endometrium, although the liver may remain a significant source of IGFBP-1 production, with the increases mediated in part by increases in estrogen and progesterone levels.

During the first trimester, extraembryonic coelomic fluid contains high levels of IGFBP-1 (2), the majority of which exists in the nonphosphorylated form (9). In contrast, first trimester maternal serum contains predominantly the more heavily phosphorylated species. It seems likely, therefore, that either the sites of production of IGFBP-1 or the sites and extent of its posttranslational modification are different on the maternal and fetal sides of the circulation. Whether the elevation in maternal IGFBP-1 levels in response to trisomy 18 reflects an increase in IGFBP-1 production by the feto-placental unit, impaired placentation, or a maternal response to a growth-retarded fetus remains to be determined.

In contrast to IGFBP-1, maternal serum levels of IGFBP-2 were reduced in trisomy 18. IGFBP-2 levels are generally lower in pregnancy than in nonpregnant subjects (35, 36), but in the late second and third trimesters, maternal serum IGFBP-2 is unchanged in the presence of a severely growth-retarded fetus despite marked elevations in fetal IGFBP-2 (5). The reason for the reduction in maternal IGFBP-2 levels in pregnancy is unclear. IGFBP-2 is thought to be regulated by IGF-II; levels are extremely high in cases of nonislet cell tumor hypoglycemia associated with high levels of big IGF-II (37) and transgenic mice overexpressing human IGF-II (38). Adults with GH deficiency have high IGFBP-2 levels that normalize on GH replacement, and some studies have shown low levels in acromegaly (39), suggesting an inverse relationship between GH and IGFBP-2. Pituitary GH is replaced by GH variant (hGH-V) in pregnancy (40), and it is conceivable that this protein has a greater inhibitory effect on IGFBP-2 expression and/or transcription than normal GH. In addition, the expression of IGFBP-2 messenger ribonucleic acid in rat Leydig cells is markedly reduced in a dose-dependent manner by culture in the presence of hCG (41), which is, of course, present in the maternal circulation. Endometrial cells are known to synthesize and secrete IGFBP-2 under the partial regulation of the sex steroids estradiol and progesterone (42). Blockade of the progesterone receptor with RU486 results in a reduction of the stimulatory effect of progesterone, but there is as yet no evidence of impairment of progesterone activity or receptor function in association with trisomy 18. As the sites of production of IGFBPs measured in maternal serum in the first trimester are unknown, the functional significance of these reported differences is unclear. Although IGFBP-2 is known to be present within the fetoplacental unit (43), production here may not influence maternal serum levels.

We have previously demonstrated differences in maternal IGFBP-3 proteolysis in multiple pregnancies in the first trimester and pregnancies affected by uteroplacental insufficiency in the second trimester (36). In the current study, protease activity assessed by both proteolysis of intact labeled IGFBP-3 and immunoblotting with specific anti-IGFBP-3 antiserum showed no differences, suggesting that the regulation of maternal protease activity is different in the case of trisomic pregnancies from that when intrauterine growth is restricted as a result of fetal starvation or multiple pregnancy.

The finding of altered maternal serum levels of IGFBP-1 and IGFBP-2 specific to pregnancies complicated by trisomy 18 suggests that these binding proteins may be important mediators of fetal growth in the first trimester. The data reported here do not make it possible to determine whether the changes seen underlie the growth failure or are a result of the process causing it. Further work is needed to study changes in gene expression in the placenta and fetus, but this is necessarily limited by ethical issues in obtaining human samples for analysis. Nevertheless, if these preliminary data are confirmed in larger studies, the ratio of IGFBP-1 to -2 may be a useful diagnostic adjunct in the screening process for chromosomal abnormalities during the first trimester of human pregnancy.


   Footnotes
 
1 Recipient of a grant from the Wellcome Trust. Back

Received July 10, 1996.

Revised September 9, 1996.

Accepted September 16, 1996.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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M. S. Boyne, M. Thame, F. I. Bennett, C. Osmond, J. P. Miell, and T. E. Forrester
The Relationship among Circulating Insulin-Like Growth Factor (IGF)-I, IGF-Binding Proteins-1 and -2, and Birth Anthropometry: A Prospective Study
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