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Departments of Gynecology and Obstetrics (S.B., G.H.F., G.C., R.L.B., M.D., Y.E.-S., L.C.G.) and Pediatrics (D.M.W.), Stanford University Medical Center, Stanford, California 94305; Diagostics Systems Laboratories, Inc. (S.E.G.), Webster, Texas 77598; and Children’s Hospital Oakland (S.B.), Oakland, California 94609
Address all correspondence and requests for reprints to: Linda C. Giudice, Ph.D., M.D., Department of Gynecology and Obstetrics, Section of Reproductive Endocrinology and Infertility, Center for Research on Women’s Health and Reproductive Medicine, Stanford University School of Medicine, 300 Pasteur Drive, Room HH 333, MC 5317, Stanford, California 94305-5317. E-mail: . giudice{at}stanford.edu
Abstract
In cord blood and late gestation maternal serum, IGF-I is positively correlated with birth weight, whereas IGF-binding protein-1 (IGFBP-1) is inversely correlated with birth weight. Our goal was to determine whether maternal serum or amniotic fluid concentrations of IGF-I, IGFBP-1, or nonphosphorylated IGFBP-1 (npIGFBP-1) in early gestation predict later fetal growth abnormalities. Maternal serum was collected prospectively across gestation (5–40 wk) from 749 pregnant subjects. Amniotic fluid was collected after amniocentesis during wk 15–26 from 207 subjects. We compared median serum concentrations of IGF-I, IGFBP-1, and npIGFBP-1 in 38 subjects who delivered growth-restricted infants with the control group of 236 subjects with normal weight infants for each gestational age grouping, wk 5–12, 13–23, and 24–34. In the control group median IGF-I concentrations were 14.8, 11, and 15.6 nmol/liter for wk 5–12, 13–23, and 24–34, respectively, compared with 13.7, 14.3, and 10.6 nmol/liter in the intrauterine growth restriction (IUGR) group. Median IGFBP-1 concentrations were 8.5, 30.4, and 24.4 nmol/liter, respectively, in controls, compared with 11.4, 28.6, and 25.5 nmol/liter in the IUGR group. Median npIGFBP-1 concentrations were 6.9, 22, and 17.4 nmol/liter, respectively, in controls, compared with 5.0, 32.1, and 24.2 nmol/liter in the IUGR group. In the control group the median amniotic fluid IGFBP-1 level was 13,160 nmol/liter, and the median npIGFBP-1 level was 15,970 nmol/liter; in the IUGR group these levels were 13,440 and 18,440 nmol/liter, respectively. No clinically useful differences were found between the IUGR and control groups. Our results do not support the use of maternal serum IGF-I or IGFBP-1 or amniotic fluid IGFBP-1 or npIGFBP-1 early in gestation to predict later fetal growth restriction.
FETAL GROWTH restriction is a serious complication of pregnancy, leading to an increased risk of perinatal hypoxia, preterm delivery, and fetal demise. It is increasingly evident that the foundations of life-long health are in part built in utero. Recent studies show that for surviving intrauterine growth restriction (IUGR) infants, the long-term health risks continue. These infants grow into children and adults at risk for hypertension (1), dyslipidemia, obesity (2), diabetes, precocious adrenarche, and infertility (3). Considerable research on potential interventions for IUGR during pregnancy is being performed in animal models, including the use of nutritional adjuncts (4, 5), IGF-I and/or GH administration (6, 7), oxygen, and modulators of placental blood flow (8). To eventually direct such interventions toward women at high risk of fetal growth restriction, early identification of IUGR is necessary. Using current diagnostic methods, IUGR is identified late in pregnancy, when the fetal programming thought to lead to later health complications may already be immutable. Newer diagnostic modalities include advances in Doppler ultrasound measurement of placental and fetal organ blood flow, and fetal blood sampling by cordocentesis after administration of isotope-labeled nutrients to assess placental transfer of glucose and amino acids (8). However, these methods rely on expensive equipment, require extensive training of medical personnel, and, with cordocentesis, are highly invasive. A safe and inexpensive biochemical marker is needed to screen large numbers of pregnant women for the development of IUGR.
IGFs and their binding proteins (IGFBPs) are key regulators of fetal growth (9) and may provide such a marker for IUGR. IGF-I is a major growth promoter in the fetus. Of the six binding proteins, IGFBP-1 sequesters IGF-I and regulates the minute to minute availability of free IGF-I in the circulation (10, 11). At term, IGF-I concentrations in cord blood are positively correlated with birth weight, whereas IGFBP-1 concentrations are inversely correlated with birth weight (12, 13). Additionally, IGFBP-1 concentrations in fetal blood collected by cordocentesis in the third trimester are significantly higher in growth-restricted babies compared with normal weight babies (14, 15).
Data from animal and human studies show that the IGF system and hypoxia, a result of uteroplacental insufficiency (a major cause of IUGR), are interrelated. In animal models of growth restriction, including uterine artery ligation and maternal hypoxia, elevated IGFBP-1 concentrations are associated with growth restriction (16, 17, 18). Chronic hypoxia in normal birth weight infants is associated with elevation of IGFBP-1 concentrations in cord blood (19). Additionally, IGFBP-1 levels are inversely correlated with cord blood pO2 (20).
Maternal serum concentrations of IGFBP-1 reflect contributions from maternal liver, the decidua, and the fetus (21) and correlate inversely with fetal size estimation by ultrasound at 20–24 and 30–34 wk gestation (22). Although nonpregnant serum IGFBP-1 consists mainly of a highly phosphorylated isoform produced by the liver, during pregnancy many lesser and nonphosphorylated isoforms are also found. In amniotic fluid, IGFBP-1 made by decidua is found in exceedingly high levels in the nonphosphorylated isoform (21, 23), and serum levels of nonphosphorylated IGFBP-1 (npIGFBP-1) could reflect changes in decidually produced IGFBP-1.
We undertook this study to test the hypothesis that maternal serum IGFBP-1 is a marker for IUGR. We focused our attention on IGFBP-1 because of a plethora of data showing elevation of IGFBP-1 late in gestation in growth-restricted pregnancies and in the setting of hypoxia. Additionally, in vitro studies (24, 25) demonstrating the important role of IGFBP-1 in placental invasion and differentiation support the theory that early abnormalities in placentation could cause later fetal growth restriction. In particular, npIGFBP-1, reflecting decidual production of IGFBP-1, may be a better marker of placental dysfunction leading to IUGR. The goal of this study was to determine whether maternal serum concentrations of IGF-I, IGFBP-1, or npIGFBP-1 in early gestation predict later fetal growth abnormalities. An additional goal was to determine whether IGFBP-1 or npIGFBP-1 concentrations in amniotic fluid obtained after clinically indicated amniocentesis predict later fetal growth restriction.
Subjects and Methods
Subjects
Pregnant women attending the Johnson Center for Pregnancy and Newborn Services at Lucile Packard Children’s Hospital (Stanford, CA) were enrolled in this study. The study was approved by the Stanford University Administrative Panel on Human Subjects in Medical Research, and written informed consent was obtained from all subjects. Of 1256 women approached, 749 enrolled and provided samples during the course of this study. Samples included maternal serum and amniotic fluid. Maternal serum was collected at selected routine clinic visits throughout gestation. Amniotic fluid samples were obtained at the time of clinically indicated prenatal amniocentesis, usually performed between gestational wk 15–20. None of the subjects in either the IUGR or normal group had infants identified with chromosomal abnormalities. A database was established for clinical data, which were collected on all subjects, including maternal age and parity, last menstrual period, and past medical history of previous hypertension, diabetes, or previous complicated pregnancies. Pregnancy outcome data were collected after delivery and included birth weight, cord gases (if obtained), gestational age, and presence and severity of any complications, in particular, IUGR. Growth restriction was defined as birth weight equal to or less than the 10th percentile for gestational age at delivery (26). Gestational age was defined using the last menstrual period or first or second trimester ultrasound. Other identified complications included preeclampsia and gestational diabetes.
Gestational weeks were grouped as follows: wk 5–12, 13–23, and 24–34. Samples in the 24–34 wk group were obtained 1 h after a 50-g oral glucose load. For the remainder of the samples, the fed or fasting state was not defined.
Of the 749 subjects, 187 had a pregnancy complication, including 38 with growth-restricted infants. Two hundred and thirty-six gestational age-matched normal controls were selected, with a preference for subjects with multiple longitudinal samples, to bring to a total more than 1000 samples assayed.
Assays
All serum and amniotic fluid samples from subjects with IUGR-complicated pregnancies and from controls were assayed for IGF-I, total IGFBP-1, npIGFBP-1, and insulin. Serum or plasma was frozen immediately at -20 C until assays were performed. IGF-I, total IGFBP-1, and npIGFBP-1 were measured by coated tube two-site immunoradiometric assay (Diagostics Systems Laboratories, Inc., Webster, TX). Insulin was measured by RIA (Diagostics Systems Laboratories, Inc.). All samples were assayed in duplicate, and measurements were performed according to the manufacturer’s instructions. The assay for IGF-I included an extraction step. The minimum detection limit for IGF-I was 0.65 nmol/liter (5 ng/ml), and the intra- and interassay coefficients of variation were 3.4% and 8.2%, respectively. The minimum detection limit for total IGFBP-1 was 0.11 nmol/liter (0.5 ng/ml), and the intra- and interassay coefficients of variation were 5.2% and 6.0%, respectively. The minimum detection limit for npIGFBP-1 was 0.11 nmol/liter (0.5 ng/ml), and the intra- and interassay coefficients of variation were 5.2% and 6.0%, respectively. The minimum detection limit for insulin was 46 pmol/liter (6 µU/ml), and the intra- and interassay coefficients of variation were 8.3% and 12.2%, respectively. Results are reported in System International units.
Statistical analysis
Concentrations of IGF-I, IGBBP-1, and npIGFBP-1 were analyzed using the SAS 8.0 TS-00M0 (SAS Institute, Inc., Cary, NC) by a biostatistician (R.L.B.). Preliminary Shapiro-Wilkes tests revealed marked departures from normal in the sample distribution of all parameters, and therefore, the Wilcoxon rank-sum test, a nonparametric counterpart of the two-sample t test, was used to compare levels of each analyte in sera from subjects vs. controls. A z-score of infant birth weight was calculated for each subject using a U.S. national standard for birth weight (26). Results are reported as the median value and interquartile range for each analyte in each gestational age grouping. P < 0.05 was considered significant.
Results
IGF-I, IGFBP-1, npIGFBP-1, and insulin in maternal serum: IUGR vs. control
Data were grouped according to first trimester (wk 5–12) and second trimester (wk 13–23). Third trimester (wk 24–34) samples were grouped as such because these were obtained after glucose challenge. Median serum results with interquartile range for IGF-I, total and nonphosphorylated IGFBP-1, and insulin for subjects with pregnancies complicated by IUGR vs. the control group of subjects with normal pregnancies for each gestational age group are shown in Table 1
. In the control group, median IGF-I was higher late in gestation. IGFBP-1 and npIGFBP-1 increased from early to midgestation and then declined in wk 24–34, probably related to the known effect of insulin on IGFBP-1 (10).
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Results of assays for IGF-I, IGFBP-1, npIGFBP-1, and insulin (Fig. 1, A–D, respectively) are plotted as a function of gestational age in weeks for the IUGR and control groups. Median npIGFBP-1 tended to be higher in the IUGR group compared with the control group in wk 24–34, but did not reach statistical significance due to the small number of samples in the third trimester. However, the ranges overlapped considerably, reducing the clinical utility of these differences in the methods used to obtain samples in this study.
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Maternal serum levels of IGF-I (Fig. 2) and npIGFBP-1 (Fig. 3) vs. birth weight z-score are plotted for gestational wk 5–12 and 13–23. Although statistically significant differences between the IUGR and control groups (P = 0.03) were observed in IGF-I in wk 13–23, there was considerable overlap between the groups.
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In the control group median amniotic fluid IGFBP-1 was 13,160 nmol/liter, and npIGFBP-1 was 15,970 nmol/liter; in the IUGR group the levels were 13,440 and 18,440 nmol/liter, respectively. No significant difference was found between the IUGR and control groups.
Discussion
Considerable data demonstrate elevation of maternal serum IGFBP-1 late in gestation in IUGR-complicated pregnancies and link utero-placental insufficiency and hypoxia (causes of IUGR) with elevated IGFBP-1. Thus, our goal was to determine whether maternal serum concentrations of IGF-I, IGFBP-1, and npIGFBP-1 in early gestation predict later fetal growth abnormalities. To test our hypothesis we prospectively collected serum samples from a large number of pregnant subjects throughout gestation and compared the concentrations of IGF-I, total IGFBP-1, npIGFBP-1, and insulin for those subjects who delivered growth-restricted infants with those for a control group who delivered normal weight infants.
In confirmation of previous work measuring IGF-I in normal pregnancy (27), our data in the control group show a general increase in IGF-I from early to late gestation. IGFBP-1 increased from early to midgestation, but was lower in wk 24–34, when all samples were collected after glucose challenge, probably a reflection of the negative regulation of insulin on total IGFBP-1 (10, 22). Samples collected during wk 5–12 and 13–23 were not controlled for fasting or fed states, a potential cause of increased variability in our study. This effect was mitigated, however, by the measurement of npIGFBP-1, as circulating npIGFBP-1 derives from the decidua. Although IGFBP-1 is insulin dependent in endometrial stromal cells in vitro (24), it is unlikely that minute to minute changes in insulin affect decidual IGFBP-1 in vivo.
In the IUGR group, our data did not show elevation of IGFBP-1 late in gestation, as shown in previous work by others (22). However, our study suffered in having a lower number of samples from IUGR subjects in late gestation. Despite this, a trend toward a higher npIGFBP-1 was demonstrated late in gestation, supporting our contention that serum npIGFBP-1 is a better marker of decidually produced IGFBP-1. There was no difference in either IGFBP-1 or npIGFBP-1 in wk 5–12 or 13–23, for both of which a larger number of samples were obtained, when comparing the IUGR and normal groups. Although the median IGF-I concentration is higher (P = 0.03) in wk 13–23 for the IUGR group, there is significant overlap of the ranges of these values, making clinical prediction of IUGR based on IGF-I values impossible. These data refute the use of maternal serum IGF-I, IGFBP-1, or npIGFBP-1 early in gestation as predictors of later fetal growth restriction.
An additional goal of our study was to determine whether IGFBP-1 or npIGFBP-1 concentrations in second trimester amniotic fluid predict later fetal growth restriction. In amniotic fluid, concentrations of IGFBP-1 are 100-fold higher than those in maternal serum and consist mainly of the nonphosphorylated and less highly phosphorylated isoforms of IGFBP-1 produced by the decidua (21, 23). Previous reports of amniotic fluid IGFBP-1 levels have found conflicting evidence of correlations with birth weight. Although elevation of amniotic fluid IGFBP-1 in the second trimester has been reported in association with growth restriction (28, 29, 30), more recently, Verhaeghe et al. (31) did not establish such a correlation. Our data confirm that amniotic fluid IGFBP-1 and npIGFBP-1 concentrations are 100-fold higher than maternal serum levels, with npIGFBP-1 comprising the majority of the phosphoisoforms seen. However, there was no difference in median IGFBP-1 or npIGFBP-1 between the IUGR and control groups. From our data we conclude that amniotic fluid IGFBP-1 and npIGFBP-1 early in gestation do not predict later IUGR.
Considerable interest in the use of IGFBP-1 as a marker of IUGR is apparent in review of the literature. Both in vivo and in vitro evidence point to the importance of IGFBP-1 in normal placental implantation, differentiation, and growth. An association of IGFBP-1 in maternal (22) and fetal cord serum (14, 15) late in gestation with abnormalities of fetal growth has been demonstrated by several groups. We undertook this study to evaluate prospectively the utility of IGFBP-1 or npIGFBP-1 early in gestation to predict fetal growth abnormalities. Our data show clearly that there is no correlation between early gestation maternal serum or amniotic fluid IGFBP-1 or npIGFBP-1 with later development of fetal growth restriction.
Acknowledgments
Footnotes
This work was supported by Genentech Center for Clinical Research and Education Fellowship Grant (to S.B.) and NIH Grant HD-25220-09 (to L.C.G.). This work was presented in part at IGFBP2000, Terrigal, Australia, 2000.
Abbreviations: IGFBP-1, IGF-binding protein-1; IUGR, intrauterine growth restriction; npIGFBP-1, nonphosphorylated IGF-binding protein-1.
Received July 11, 2001.
Accepted January 11, 2002.
References
5ß1 integrin and inhibits cytotrophoblast invasion into decidualized endometrial stromal cultures. Growth Horm IGF Res 8:21–31[Medline]
5ß1 integrin via mitogen-activated protein kinase pathway. J Clin Endocrinol Metab 86:2484–2493This article has been cited by other articles:
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  V. E. Murphy, R. Smith, W. B. Giles, and V. L. Clifton Endocrine Regulation of Human Fetal Growth: The Role of the Mother, Placenta, and Fetus Endocr. Rev., April 1, 2006; 27(2): 141 - 169. [Abstract] [Full Text] [PDF]
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 D. K. Tisi, J. J. Emard, and K. G. Koski Total Protein Concentration in Human Amniotic Fluid Is Negatively Associated with Infant Birth Weight J. Nutr., July 1, 2004; 134(7): 1754 - 1758. [Abstract] [Full Text]
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