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Center for Human Growth and Maturation at the London Center for Pediatric Endocrinology and Metabolism, Department of Obstetrics and Gynecology, University College (C.H.R.), London, United Kingdom W1T 3AA; and Program in Development and Fetal Health, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto (J.C.P.K.), Toronto, Canada
Address all correspondence and requests for reprints to: Dr. P. C. Hindmarsh, Center for Human Growth and Maturation, Cobbold Laboratories, Middlesex Hospital, Mortimer Street, London, United Kingdom W1T 3AA. E-mail: p.hindmarsh{at}ucl.ac.uk.
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Abstract |
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Introduction |
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The neonate secretes large amounts of GH, and the secretory profile is similar to that observed in acromegaly, with a persistent elevation of trough concentrations (13, 14). Despite these elevated GH concentrations, serum IGF-I concentrations at birth are lower than those in childhood, suggesting other factors that regulate it. The relationship between GH and birth size is unclear, with groups demonstrating no relationship (15) to inverse relationships (16, 17). These differences probably relate to sample size issues and to the observation that growth in humans is not fully GH dependent until after 6 months of postnatal life (18, 19). IGF-I and -II are important for fetal growth, as evidenced by mouse knockout studies (20) and numerous clinical studies relating birth weight to the cord concentrations of IGF-I (21, 22, 23, 24, 25) and, to a much lesser extent, IGF-II (22, 23, 24, 25).
In the presentation of data relating cord GH-IGF concentrations with birth weight many studies have polarized data between normal and small individuals, not controlled for confounding factors such as parity and not differentiated between term and preterm birth (25, 26, 27). Furthermore, small sample sizes may have failed to determine biological effects (15). Although the general concept that the IGF axis relates to birth size is accepted, there remains the question of whether the sexual dimorphism in birth size is explicable in terms of a similar dimorphism in the circulating concentrations of the GH-IGF axis. To address this question it is necessary to study a large cohort of normal pregnancies to provide sufficient power to assess the relative importance of variables contributing to this relationship. Accordingly, we have studied cord concentrations of GH, IGF-I, IGF-II, and IGF-binding protein 3 (IGFBP-3) in the offspring from 1650 singleton pregnancies and related values to measures of birth size.
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Subjects and Methods |
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The cohort has been described in detail previously (12, 28). In brief, patients were consecutive mothers booked at the Department of Obstetrics and Gynecology at University College London Hospitals between April 1996 and July 1997; 1790 mothers fulfilled the entry criteria, and 1650 (92%) agreed to participate. They did not differ in terms of clinical or pregnancy outcomes from those who refused. Inclusion criteria were first prenatal visit before 20 wk, ultrasound examination demonstrating a structurally normal single fetus, and a Caucasian mother. The exclusion criteria were increased nuchal translucency or evidence of a major malformation in the ultrasound scan (n = 6) or maternal steroid use for chronic inflammatory or thrombotic disorders (n = 16). Menstrual dates were used to assign gestational age unless the first ultrasound measurement (crown rump length before 12 wk, biparietal diameter for 12–20 wk) differed by more than 7 d. Current cigarette consumption was categorized as nonsmoking or less than 10, 10–20, or more than 20 cigarettes/d. Pregnancy complications were noted along with mode of delivery.
Complete perinatal outcome and neonatal anthropometric data were available for 1485 babies. Birth weight was measured using electronic self-calibrating scales (Seca, Birmingham, UK), length by Infantometer (Child Growth Foundation, London, UK), and head circumference with a metal tape (Holtain, Crymych, UK). Three separate measurements were taken, and the mean recorded. The coefficient of variation of the measurement error for length was 0.15% based on 10 infants, each measured 5 times by 3 observers.
After the birth of the baby and before completion of the third stage of labor, blood was collected into EDTA from the umbilical cord vein. This was centrifuged and separated, and plasma was stored immediately at -20 C. The assays for IGF-I, IGF-II, IGFBP-3, and GH were performed in batches within 3 months of collection.
The study was approved by the research ethics committee of University College London Hospitals, and written informed consent for participation was obtained from the mother for herself at the commencement of the study and for her newborn child after delivery.
Hormone assays
IGF-I. IGF-I was measured by a commercial immunoradiometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). This is a nonextraction method where IGFBPs are separated from IGF-I by acidification of the sample, and excess IGF-II is added to block binding proteins from recombining with IGF-I. The within-assay coefficients of variance (CV) were 4.6% and 3.3% at 61.0 and 292.5 µg/liter, respectively. The between-assay CV were 15.5% and 11.3% at 88.6 and 240.4 µg/liter, respectively. The standards were prepared from recombinant IGF-I and were calibrated against WHO First International Reference Preparation (87/518). The minimum detection limit of the assay was 6 µg/liter.
IGF-II. IGF-II was measured by a commercial coated tube immunoradiometric assay (Diagnostic Science Laboratories, Webster, TX). This is a nonextraction method where IGFBPs are separated from IGF-II by dilution and acidification of the sample. The within-assay CV were 6.5%, 3.4%, and 4.7% at 245, 409, and 1432 µg/liter, respectively. The between-assay CV were 14.5% and 7.2% at 273 and 785 µg/liter, respectively. The standards were calibrated against a preparation of recombinant IGF-II. The minimum detection limit of the assay was 12 µg/liter.
IGFBP-3. IGFBP-3 was measured by a commercial coated tube immunoradiometric assay (Diagnostic Science Laboratories). Samples were analyzed at a 1:100 dilution. The within-assay CV were 3.9%, 3.2%, and 1.8% at 7.35, 27.53, and 82.72 µg/liter, respectively. The between-assay CV were 7.6% and 4.2% at 5.43 and 27.15 µg/liter, respectively. The standards were calibrated against recombinant nonglycosylated IGFBP-3. The minimum detection limit of the assay was 0.5 µg/liter.
GH. GH was measured by a commercial immunoradiometric assay (Hybritech Europe, Liege, Belgium). The within-assay CV were 10.6%, 4.9%, 5.2%, 4.9%, and 5.0% at 1.4, 3.5, 14.4, 26.4, and 99.4 mU/liter, respectively. The between-assay CV were 15.4%, 9.7%, 8.0%, and 6.3% at 4.1, 8.1, 13.6, and 35.8 mU/liter, respectively. The assay was standardized in micrograms per liter against HS2243E (NIH) and has been recalibrated to milliunits per liter against the WHO First International Standard (80/505). The minimum detection limit of the assay was 0.5 mU/liter. At concentrations of PRL and human placental lactogen in excess of 2000 mU/liter, the cross-reactivities were less than 0.5% and less than 0.01%, respectively.
Statistics
All data were explored for the normality of their distribution and log-transformed where appropriate. Values for birth weight, length, and head circumference were expressed as SD scores (SDS) using the 1990 British growth reference (29). Relationships between hormonal parameters and anthropometric measures and pregnancy parameters were estimated using Pearson’s correlation coefficient. Multiple linear regression analysis was used to explore the effects of mode of delivery, gestational age at delivery, and parity on cord serum hormone concentrations and to explore the relationship of sex and cord hormone concentrations on birth size. One-way ANOVA with the Student-Newman-Keuls post hoc test was used to determine differences between the mean values of several groups. The t test was used to determine differences between the sexes in the anthropometric and biochemical measures made. 
2 analysis was used to determine the distribution of factors, such as parity and mode of delivery, between the sexes. The sample size of the study was constructed in part on the basis of determining differences between the sexes in the IGF axis. In determining sample size it was assumed that the mean difference between male and female birth weights is approximately 200 g, and the data from previous studies (where males and females were grouped together) suggested that IGF-I increased by approximately 20 ng/ml for every 1-kg increase in birth weight. Given the estimated SD of IGF-I in previous studies as 20 ng/ml, then a sample size of 800 in total would be required to detect a difference between the sexes of approximately 4 ng/ml at the 5% level of significance and with 90% power.
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Results |
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A total of 1650 women entered the study, and 1484 delivered a live infant, of whom 70 were preterm deliveries, and 196 developed complications of pregnancy. Of the 166 who did not complete the study, 127 had moved away or were lost to follow-up, 28 had either a miscarriage or termination of pregnancy, and 11 withdrew. As many of the previous studies of the GH-IGF axis have included preterm infants, pregnancy complications, or conditions known to influence fetal growth (e.g. smoking), analysis in this study was confined to those pregnancies with a normal course delivering at term (gestation of 37 wk or greater). Pregnancies complicated by antepartum hemorrhage, pregnancy-induced hypertension, preeclampsia (30), or gestational diabetes and where cigarette smoking continued were excluded. The resulting cohort for study was 987, or 67% of those delivered. The clinical and anthropometric details of the mothers and babies are shown in Table 1
. Anthropometric measures were no different from the United Kingdom population values.
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There was a significant decrease in cord plasma IGF-I concentration with gestational age (37–42 wk), so that for every week advance in pregnancy, the cord serum IGF-I concentration decreased by 4 µg/liter (r = -0.20; P < 0.001). The cord plasma IGFBP-3 concentration showed a less significant effect (r = -0.08; P = 0.02). Plasma concentrations of IGF-II and GH showed no change with gestational age. Birth weight SDS also showed a slight decrease with gestational age of -0.19 SDS (r = -0.25; P < 0.001), or approximately 90 g for each week advance in pregnancy. These effects were not dependent on parity, as gestational age at delivery (39 wk) was similar in para 0 offspring compared with paras 1, 2, and 3+.
Figure 1 shows the relationship of parity with birth weight SDS and the cord hormonal concentrations. Birth weight SDS was significantly greater with para 1 and above (by one-way ANOVA: F = 16.59; P < 0.001) compared with para 0 (by Student-Newman-Keuls post hoc test: P < 0.05). Cord plasma IGF-I (by one-way ANOVA: F = 13.28; P < 0.001) and, to a lesser extent, cord IGFBP-3 (by one-way ANOVA: F = 3.11; P = 0.03) concentrations paralleled the birth weight changes, but cord plasma IGF-II and GH concentrations did not. These effects remained unchanged after adjusting for gestational length at delivery.
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Table 2 depicts the contributions of these variables to the hormone concentrations measured in cord blood. Some 10.2% of the variability in cord plasma IGF-I concentrations was explained predominantly by the sex of the offspring and parity. A similar situation was observed for cord plasma IGFBP-3 concentrations, although the adjusted r2 was 2.7%. None of the factors, apart from maternal height, appeared to influence cord plasma IGF-II concentrations, and the adjusted r2 was only 1%. Sex of the baby, mode of delivery, and parity influenced cord plasma GH concentrations, but the percentage of the variability explained was 2.6%.
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There was no difference in age of gestation at delivery between the sexes (t = 1.29; P = 0.20), parity (2 = 3.04; P = 0.39), and mode of delivery (2 = 6.45; P = 0.09). Figure 2 shows the effect of sex of the baby on measurements of birth size expressed in absolute terms and cord plasma hormone concentrations. Birth weight, length, and head circumference measurements were greater in males than females (P < 0.001). Cord plasma concentrations of IGF-I (P < 0.001) and IGFBP-3 (P < 0.001) were significantly lower in males than females. Cord plasma GH concentrations were higher in males than females (P = 0.05), but no difference was noted between the sexes for IGF-II. Despite females having a 140-g difference in birth weight compared with males (3.9%), males had an 8.1 µg/liter (10.9%) lower cord plasma IGF-I concentration compared with females.
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Table 3 shows the contribution of the cord hormone concentrations to anthropometric measures at birth after adjustment for gestational age, parity, and maternal height. In all instances the cord plasma concentrations of IGF-I and IGFBP-3 were major determinants of the anthropometric measures. Neither GH nor IGF-II cord plasma concentrations contributed to explaining the variability of the anthropometric measures. In all instances, sex of the individual acted as an individual predictor of birth size. In the case of birth weight, the contribution was similar to that of IGF-I, whereas for length and head circumference sex was a greater contributor to the explanation of the anthropometric measures variability. The combination of IGF-I, IGFBP-3, and sex explained 38.0% of the variability in birth weight, 25.0% in birth length, and 22.7% in head circumference.
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Discussion |
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A sex difference in the cord plasma IGF-I concentration has been reported recently in singleton pregnancies (31) and for IGF-II in a study by Gluckman et al. (21). Our data confirm and extend these observations by demonstrating, in multiple linear regression analysis, that the anthropometric measures of birth weight, length, and head circumference are related to cord plasma concentrations of IGF-I and IGFBP-3 after adjusting for gestational length, parity, and maternal height and, independently, the sex of the individual. Males have a greater birth weight, length, and head circumference and reduced skinfold thickness compared with females, and differences in head size can be observed as early as 20 wk gestation (12). These observations suggest that size at birth is a composite of factors determined by the gender of the individual acting directly or indirectly through modulation of factors known to influence growth in utero. The question remains of whether the IGF effects are truly independent, as suggested by the regression analysis, or merely a secondary phenomenon reflecting differences in size of the individual. Evidence for the former comes from the observation that circulating IGF-I concentrations are low in low birth weight individuals (16, 17, 23, 24, 25, 26, 27) and results in this study showing that the proportion of birth weight explained by the IGF axis (35%) is similar to the reduction in birth size observed in IGF-I knockout mice (32).
The direction of the sexual dimorphism in the IGF family with higher cord serum concentrations in females than males is the reverse of that observed in the elderly, but similar to the urinary excretion patterns of IGF-I in childhood (9, 10). This may be explained in part by an alteration in the GH-IGF-I axis over time from a continuous mode of secretion in the neonate (13, 14) to a more sexually dimorphic pulsatile pattern in adulthood (5, 6, 9). It is generally accepted that neonates are relatively insensitive to GH despite GH receptor expression in various tissues, to a lesser extent in liver, from 8 wk of fetal age (33, 34). The sexual dimorphism either reflects differential GH sensitivity between the sexes or altered non-GH-dependent synthesis and metabolism of IGF-I. The feedback effect of IGF-I on GH appears to be operative in the newborn, as high cord plasma IGF-I concentrations were associated with lower cord plasma GH concentrations. GH secretion in females appeared to be more sensitive (significantly different regression slopes for males and females) to the effects of IGF-I. However, this greater sensitivity in females to IGF-I was not reflected in more general measures such as body size, where despite a 10% difference in cord plasma IGF-I concentrations between the sexes, females had an almost 4% lower birth weight than males.
Cord plasma IGF-II and GH concentrations did not impact on neonatal anthropometric measures in this type of regression analysis. The lack of effect of GH is consistent with the clinical observation of normal birth weights of children with GH deficiency. The IGF-II data would tend to support the overall concept that despite the higher cord plasma concentrations of IGF-II compared with IGF-I, it is IGF-I in the latter part of gestation that is the important factor in determining fetal growth (21, 35).
In this study we have obtained sufficient power to quantify the precise roles for IGF-I and IGFBP-3 in the determination of birth size and to demonstrate sexual dimorphism of these peptides. Unlike previous studies, we confined analysis to the offspring of singleton Caucasian pregnancies and excluded from analysis factors known to influence intrauterine growth, such as preeclampsia, pregnancy-induced hypertension, and cigarette smoking. We did not include in the analysis cord plasma concentrations of insulin and IGFBP-1, as we were not able to control for variable glucose infusions that the mothers might have received and the additional confounding factors of insulin and acute hypoxia impacting on the circulating concentration of IGFBP-1 (36). These would need to be addressed in a prospective study directed at controlling for these factors. However, we have avoided many of the problems associated with previous reports, namely the polarization of studies in which subjects with intrauterine growth restriction of multiple etiologies were compared with appropriately grown babies. Furthermore, we adjusted for confounding factors known to influence birth size, gestational length, parity, and maternal height. Our results can therefore, be reviewed as a more robust assessment of the interaction of the IGF family with other factors, such as sex, in determining birth size.
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Acknowledgments |
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Footnotes |
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Abbreviations: CV, Coefficients of variance; IGFBP-3, IGF-binding protein 3; SDS, SD score.
Received December 26, 2002.
Accepted April 18, 2003.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) and females (
). Data are shown as the mean ± SEM. Numbers in each group shown in parentheses.