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Original Studies |
Departments of Reproductive Biology (A.H., F.L.) and Physiology of Nutrition (A.R.T., N.T., H.B.), Instituto Nacional de la Nutrición Salvador Zubirán, Mexico D.F., Mexico; and Unité Centre National de la Recherche Scientifique, UPR 1524, Endocrinologie, Métabolisme, et Développement, Hôpital Saint Vincent de Paul (M.G.), Paris, France
Address all correspondence and requests for reprints to: Dr. Ali Halhali, Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán, Vasco de Quiroga No. 15, Col. Tlalpan, C.P. 14000, Mexico D.F., Mexico.
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Abstract |
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Introduction |
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During preeclampsia, maternal serum IGF-I concentrations are lower compared with those in normal pregnant women (15, 16). In addition to its effects on fetal growth, it has been reported that IGF-I stimulates renal 1,25-dihydroxyvitamin D [1,25-(OH)2D] synthesis in nonpregnant humans and rodents (17, 18, 19, 20, 21, 22, 23, 24, 25, 26). Furthermore, previous results from this laboratory have showed that IGF-I stimulates human placental 1,25-(OH)2D production (27). Interestingly, maternal serum 1,25-(OH)2D concentrations are lower in preeclampsia than in normotensive pregnant women (15, 28, 29, 30).
During normal pregnancy, umbilical cord serum IGF-I and 1,25-(OH)2D levels are significantly lower than those in maternal serum, which are probably derived from placental and fetal tissues (31, 32, 33, 34, 35). As the umbilical cord serum concentrations of these two hormones have not been assessed during preeclampsia, the aim of the present work was to study maternal and umbilical cord serum concentrations of 1,25-(OH)2D and IGF-I, and other parameters involved in their regulation, as well as potential correlations between IGF-I and birth weight, and 1,25-(OH)2D levels in normal and preeclamptic pregnancies.
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Subjects and Methods |
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Maternal and umbilical cord blood samples were obtained from
patients in accordance with the guidelines of the Declaration of
Helsinki, and the protocol of this study was approved by the human
ethics committee of the Instituto Nacional de la Nutrición
Salvador Zubirán. The study was performed cross-sectionally at
delivery and included 24 preeclamptic (PE group) and 24 normotensive
(NT group) pregnant women and their respective newborns. The mean
gestational ages of the NT and PE groups were 40.3 ± 1.0 and
39.6 ± 1.2 weeks, respectively. All subjects signed a written
informed consent. Diagnosis of preeclampsia was based on the
simultaneous presence of hypertension (systolic blood pressure 
140 mm
Hg and/or diastolic blood pressure 90 mm Hg) and marked proteinuria
(at least 2+ on dipstick; >100 mg/dL) (1). Exclusion criteria were
known preexisting hypertension or previous preeclampsia; liver, renal,
heart, or endocrine disorders; and use of nutritional supplements
(calcium or vitamin D), diuretics, any kind of hormonal treatment, or
magnesium sulfate therapy. Only women giving birth to a clinically
healthy single infant were included in the study.
Newborns small for gestational age (below the 10th percentile) were classified according to the criteria of Lubchenco et al. (36). Maternal and umbilical cord blood samples were collected at delivery; after centrifugation, serum samples were aliquoted and frozen at -70 C until assayed.
Serum calcium, magnesium, and phosphorus determinations
Serum ionic calcium and magnesium were determined by ion-selective electrodes using a Nova 8 CRT electrolyte analyzer (Nova Biomedical, Waltham, MA). Serum total calcium and serum total magnesium were measured by atomic absorption spectrophometry (2380, Perkin-Elmer Corp., Norwalk, CT). Serum inorganic phosphorus concentrations were assessed by the Fiske and SubbaRow method (37).
Serum 1,25-(OH)2D, vitamin D-binding protein, IGFBP-3, PTH, and IGF-I quantification
Serum 1,25-(OH)2D concentrations were measured as previously described (38) using a commercial RRA kit (Nichols Institute Diagnostics, San Juan Capistrano, CA). The sensitivity was 2 pg/mL, and the intra- and interassay coefficients of variation were 5–11% and 9–15%, respectively. Serum vitamin D-binding protein (DBP) concentrations were assessed by the method of Bouillon et al. (39). Serum IGFBP-3 concentrations were measured using a commercial immunoradiometric assay kit (Diagnostics Systems Laboratories, Inc., Webster, TX). The sensitivity was 0.5 ng/mL, and the intra- and interassay coefficients of variation were less than 5%. Serum intact PTH concentrations were measured using a commercial immunoradiometric assay kit (Nichols Institute Diagnostics). The sensitivity was 1 pg/mL, and the intra- and interassay coefficients of variation were less than 5% and 10%, respectively. Serum IGF-I was quantified using a commercial RIA kit (Nichols Institute Diagnostics) after separation from its binding proteins, as previously described (40). The sensitivity was 60 pg/mL, and the intra- and interassay coefficients of variation were less than 5% and 10%, respectively.
SDS-PAGE, Western ligand blotting (WLB), and Western immunoblotting (WIB)
Analysis of serum IGFBPs by WLB and WIB was performed as previously described (41, 42, 43). A 20-µL aliquot of a serum pool from each group was diluted in 280 µL buffer (2.5% SDS, 10% glycerol, and 0.02% bromophenol in 0.06 mol/L Tris-HCl, pH 6.8). An aliquot of 50 µL of each sample was applied to 11% SDS-PAGE in the absence of a reducing agent and run overnight. Proteins were then electroblotted onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc., Hercules, CA). The nitrocellulose membrane was blocked at 4 C with 1.5 mol/L NaCl in 100 mmol/L Tris base, pH 7.4 (TBS)/0.5% gelatin, washed with TBS/0.1% Tween-20, and incubated for 48 h with 600,000 cpm [125I]IGF-I (Amersham Pharmacia Biotech, Aylesbury, UK) at 4 C. Membranes were washed twice with TBS/0.1% Tween-20 and three times with TBS. The concentrations of IGFBPs in nitrocellulose membranes were determined using electronic autoradiography with an Instant Imager (Packard Instrument Co, Downers Grove, NJ). Membranes were also exposed to Extascan film (Eastman Kodak Co., Rochester, NY) at -80 C with an intensifying screen. For WIB analysis, nitrocellulose membranes were incubated overnight at 4 C with a polyclonal anti-IGFBP-3 (Upstate Biotechnology, Inc., Lake Placid, NY). After thorough washing, membranes were incubated for 1 h with horseradish peroxidase-conjugated antirabbit IgG (Bio-Rad Laboratories, Inc.). Peroxidase-conjugated antibody was detected with diaminobenzidine as substrate.
Statistical analysis
Results are presented as the mean ± SD.
Analysis of statistical differences between the NT and PE groups was
performed by the Mann-Whitney U test, and comparisons of frequencies of
newborns small for gestational age were made using
2 test. Associations between variables were
tested using Spearman rank correlation test. Differences were
considered statistically significant at P < 0.05.
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Results |
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Table 1
summarizes the clinical
characteristics of mothers and their newborns belonging to NT and PE
groups. Maternal and gestational ages and newborn birth lengths were
similar in both groups. Birth weights of newborns were significantly
lower in the PE group (P < 0.05), and the frequency of
newborns small for gestational age (SGA) newborns was higher in the PE
group (P < 0.05).
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Maternal circulating levels of total calcium,
1,25-(OH)2D, and IGF-I levels were significantly
lower (P < 0.001) in the PE group than in the NT group
(Table 2). No differences were found in
maternal circulating levels of ionic calcium, total and ionic
magnesium, inorganic phosphorus, DBP, IGFBP-3, and intact PTH between
the two groups studied. In umbilical cord blood (Table 3), significantly lower serum
concentrations of total calcium (P < 0.001), IGF-I
(P < 0.001), and 1,25-(OH)2D
(P < 0.05) were observed in the PE group compared with
the NT group.
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. As depicted in Fig. 1A, WLB analysis
of serum umbilical cord showed a group of different bands with
molecular masses between 24–43 kDa. These bands corresponded to
IGFBP-3 (38–43 kDa), IGFBP-2 (34 kDa), IGFBP-1 (30 kDa), and
IGFBP-4 (24 kDa), respectively. In umbilical cord serum of the PE
group, WLB analysis showed that the amount of IGFBP-3 was 64% lower
than that in the NT group. In contrast, the amount of IGFBP-1 was
2.9-fold higher in umbilical cord blood of the PE group compared with
the NT group. WIB analysis of umbilical cord serum using a specific
anti-IGFBP-3 antiserum revealed two bands that corresponded to
IGFBP-3 and to one of the proteolytic immunoreactive fragments of this
protein (Fig. 1B). In maternal serum, WLB analysis showed the complete
disappearance of IGFBP-3 in both groups. Also, this analysis revealed
an increment of 7-fold in IGFBP-1 in the PE group (Fig. 1A).
Furthermore, WIB showed three proteolytic immunoreactive fragments
of IGFBP-3 with molecular masses of 29, 18, and 15 kDa (Fig. 1B).
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, maternal and umbilical cord
serum concentrations of IGF-I and maternal serum
1,25-(OH)2D were significantly (P
< 0.01) lower in the PE group compared to the NT group despite an
adequate newborn birth weight. In the PE group with newborns SGA, both
IGF-I and 1,25-(OH)2D were significantly
(P < 0.05) lower in maternal and umbilical cord
compartments compared with those in the NT group.
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The association between 1,25-(OH)2D and
IGF-I concentrations was analyzed (Fig. 2). Maternal serum
1,25-(OH)2D concentrations correlated
significantly with IGF-I (
= 0.69; P = 0.001)
only in the NT group (Fig. 2A). A similar
result was obtained when intact PTH concentrations were positively
correlated with 1,25-(OH)2D serum levels in the
maternal compartment of the NT group ( = 0.74;
P = 0.0005). In contrast, in the umbilical cord serum,
a significant association between 1,25-(OH)2D and
IGF-I was observed only in the PE group ( = 0.41;
P < 0.05; Fig. 2B). In this group, the association
between umbilical 1,25-(OH)2D and IGF-I was seen
in newborns AGA ( = 0.65; P = 0.01), but not in
newborns SGA.
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, maternal and
umbilical cord serum IGF-I concentrations correlated significantly with
newborn birth weight and length in the PE group ( = 0.59;
P = 0.005 and = 0.48; P =
0.02, respectively). These associations were maintained in both
compartments of the SGA PE group, whereas in the AGA PE group these
associations were of significance only in the umbilical cord
compartment.
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Discussion |
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As previously observed (15, 16), maternal serum IGF-I concentrations were significantly lower in the PE group than in the NT group. In postnatal life, IGF-I is synthesized mainly in the liver and is primarily regulated by GH (45), whereas during pregnancy the synthesis of this growth factor also occurs in placental and fetal tissues (31, 32, 33, 34). The increase in serum IGF-I levels during pregnancy (3, 46) may not be associated with an elevation in pituitary GH production, as circulating levels of this hormone do not increase during this physiological stage (47). Therefore, stimulation of IGF-I synthesis during normal pregnancy may be associated with an increase in GH production by the placenta (48), and the reduced circulating IGF-I levels in the PE group could probably be attributed to a decrease in placental GH synthesis (16). In addition, the finding in this study of similar concentrations of IGFBP-3 in both NT and PE groups may indicate the low bioavailability of IGF-I during preeclampsia. During pregnancy, serum IGFBP-3 concentrations decreased markedly, and this decrease has been attributed to an endogenous pregnancy-related serum IGFBP-3 proteolytic activity (33, 42, 46, 49). In this regard, IGFBP-3 is functionally different during pregnancy, and this protease-induced alteration of IGFBP-3 is considered to be a fundamental mechanism in regulating IGF-I bioavailability (50). In the present study, the data obtained by WLB analysis showed a complete disappearance of IGBP-3 in maternal serum of both groups. Also, this analysis revealed an increment in IGFBP-1 in the PE group, which was consistent with previous studies (16, 51, 52, 53). On the other hand, WIB analysis of IGFBP-3 in maternal serum showed the presence of three bands with molecular masses of 29, 18, and 15 kDa, which might correspond to the proteolytic immunoreactive fragments of IGFBP-3. These results may also indicate a low IGF-I bioavailability in preeclampsia.
We observed that maternal serum 1,25-(OH)2D concentrations were lower in PE than in NT pregnant women. This decrease was not associated with changes in serum calcium, phosphate, or PTH concentrations. Although IGF-I is considered a stimulatory factor of renal and placental 1,25-(OH)2D synthesis (17, 18, 19, 20, 21, 22, 23, 24, 25, 26), these two hormones were only significantly associated in NT group. The finding of a similar association of PTH and 1,25-(OH)2D in the maternal compartment of the NT group may indicate that IGF-I is also involved in 1,25-(OH)2D synthesis during normal pregnancy. In the PE group, the lack of association between these two hormones in the maternal compartment suggests an IGF-I resistance condition similar to that observed with insulin during preeclampsia (54). This condition could be independent of fetal growth, as no association was found between 1,25-(OH)2D and IGF-I even when newborns were AGA. Even though it has been speculated that low 1,25-(OH)2D levels impair intestinal calcium absorption leading to hypocalciuria during preeclampsia (28, 29), fractional intestinal absorption of calcium is not reduced in preeclamptic women despite low serum 1,25-(OH)2D levels and low urinary calcium excretion (30). Thus, the effects of low 1,25-(OH)2D levels on calcium metabolism during preeclampsia need to be further investigated.
In this and other studies (44), the body weights of newborns from PE women were lower than those of newborns from NT pregnant women. Low birth weight could be due to insufficient fetal nutrient supply as a consequence of reduced uteroplacental blood flow in preeclampsia (1, 2). The alteration in fetal nutrient supply could also explain the low umbilical cord serum IGF-I levels observed in the PE group. Indeed, low IGF-I levels have been observed in newborns SGA (6, 7, 8, 9, 10, 11, 12, 13, 14). In our study, the proportion of newborns SGA was higher in the PE than in the NT group, and serum IGF-I levels correlated positively with birth weight and birth length only in the PE group. The significant association between IGF-I and birth weight and length observed in the PE group suggests that IGF-I is an important regulatory factor of fetal growth, particularly in newborns SGA. It has been shown that IGF-I binding to erythrocytes increases in preeclamptic women compared with that in normotensive pregnant women (55), suggesting that the number of IGF-I erythrocyte receptors increases in preeclampsia. Whether this observation could take place in other IGF-I targets is unknown and deserves to be further investigated. However, in our study this mechanism could occur, because in the AGA PE group low maternal and umbilical cord IGF-I serum concentrations were observed despite adequate birth weight. Interestingly, this hypothetical compensatory effect of low IGF-I and high number of IGF-I receptors appeared to be insufficient to correct the low newborn birth weight in the SGA PE group. The umbilical cord serum IGFBP profile observed in the PE group revealed lower IGFBP-3 and higher IGFBP-1 amounts compared with those in the NT group. The finding that low umbilical cord serum IGFBP-3 levels did not correlate with a concomitant increase in immunoreactive fragments of IGFPB-3 in the PE group may indicate the absence of proteolysis of this protein in the umbilical cord, as previously demonstrated (8). This observation suggests a decrease in IGFBP-3 synthesis rather than proteolysis of this binding protein in preeclampsia.
High amounts of IGFBP-1 have been associated with intrauterine growth retardation (8, 11, 12). Furthermore, high IGFBP-1 is found during hypoxia (3, 56), and it has been suggested that hypoxia regulation of IGFBP-1 is a mechanism operating in the human fetus to restrict IGF-I-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability (56). Hypoxia and a possible reduced fetal availability of substrate due to decreased uteroplacental blood flow are conditions often observed during preeclampsia (1, 2). In fact, it has been suggested that levels of IGFBP-1 could be a sensitive indicator of fetal nutrition and the short or long term response to reduced fetal nutrient supply (3). Studies in rodents show that nutritional status modulates the expression of IGF-I and IGFBPs (57, 58, 59), giving additional evidence that a high IGFBP-1/IGFBP-3 ratio and low IGF-I levels are involved in intrauterine growth retardation.
A low umbilical cord 1,25-(OH)2D serum concentration was observed in the PE group. Although synthesis of 1,25-(OH)2D occurs in placenta and fetal kidney, its role in fetal calcium metabolism has not yet been well defined (35). Although IGF-I stimulates renal and placental 1,25-(OH)2D synthesis (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27), association between these two hormones was observed only in the maternal compartment and the umbilical cord serum of the NT and PE groups, respectively. These observations suggested a differential regulation of 1,25-(OH)2D synthesis by IGF-I in the maternal and fetal compartments depending on the presence or absence of preeclampsia. However, whether preeclampsia is a condition affecting IGF-I-mediated effects on 1,25-(OH)2D synthesis deserves to be further investigated.
In summary, maternal and umbilical cord serum IGF-I and 1,25-(OH)2D levels were low in preeclampsia, and umbilical cord serum IGF-I, IGFBP-1 and IGFBP-3 concentrations were associated with low newborn birth weights. Thus, low IGF-I and 1,25-(OH)2D in umbilical cord serum observed in preeclampsia may be implicated in fetal growth and skeletal mineralization, particularly in newborns SGA.
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Footnotes |
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Received September 8, 1999.
Revised December 15, 1999.
Accepted December 29, 1999.
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