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University of Manchester, Academic Unit of Obstetrics and Gynecology, St. Mary’s Hospital for Women and Children (R.B., S.W.), Manchester, M13 OJH United Kingdom; Imperial College School of Medicine, Departments of Maternal and Fetal Medicine (S.R.S.) and Clinical Chemistry (M.H.), Chelsea and Westminster Hospital, London, SW 6 United Kingdom
Address all correspondence and requests for reprints to: Dr. Rekha Bajoria, M.D., Ph.D., St. Mary’s Hospital for Women and Children, Whitworth Park, Manchester, M13 OJH United Kingdom. E-mail: rbajoria{at}doctors.net.uk
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Abstract |
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In the growth-restricted (IUGR) twins, fetal concentrations of total essential (P < 0.01), nonessential (P < 0.01), and branched chain amino acids (P < 0.01) were lower than those in the appropriate for gestational age co-twins and concordant twin pairs. The IUGR twins had lower fetal concentrations of insulin (P < 0.001) and IGF-I (P < 0.05) and higher concentrations of IGFBP-1 (P < 0.01) than their appropriate for gestational age co-twins. In the discordant group, fetal IGFBP-1 had a negative association with fetal insulin (r = 0.71; P < 0.001), total essential amino acids (r = 0.78; P < 0.001), and branched chain amino acids (r = 0.64; P < 0.01). There was a positive correlation between total essential amino acids (r = 0.63; P < 0.001) and branched chain amino acids (r = 0.58; P < 0.01) and plasma insulin. However, there were no associations among fetal insulin, IGFBP-1 and nonessential amino acids.
These data demonstrate the link between the reduction in certain essential and nonessential amino acids and alterations in fetal circulating levels of insulin and IGFBP-1, in growth-restricted twins.
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Introduction |
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The logical approach to understand the individual impact of maternal nutrition and placental factors on fetal nutrition is to use twin pregnancy as a model. The major advantage of the twin model is that it optimizes for the effect of maternal confounding variables such as nutrition, hypertension, diabetes, and smoking on fetal growth, as these factors are common to both members of a twin pair. Furthermore, twins with birth weight discordance of more than 25% are associated with a 2.5-fold increase in perinatal death and disabilities in later childhood (18). A recent study in twins has shown that the low birth weight twin has higher blood pressure in childhood and adult life than its heavier co-twin (19, 20). The inverse association between size at birth and raised blood pressure in monozygotic (MZ)/dizygotic (DZ) twins cannot be explained by maternal nutrition or common genetic factors and instead suggests a link among placental dysfunction, IUGR, and fetal programming.
We, therefore, studied DZ/dichorionic (DC) twin pregnancies to evaluate the relationship among placental function, fetal nutrition, and the insulin-IGF axis. DZ twining is a powerful clinical model compared with MZ twins, because it obviates the problems of inter-twin vascular anastomosis unique to monochorionic placentation (21, 22). Although MZ twins with identical genome have a distinct advantage, the influence of inter-twin transfusion on fetal growth cannot be totally ruled out. Furthermore, DZ twins can be considered to be the closest match to singleton fetuses in at least two respects. Firstly, similar to singleton pregnancies, the DZ fetuses are unrelated in terms of genetic predisposition to low birth weight, type 2 diabetes, or cardiovascular disease. Secondly, each twin has a distinct placenta in a similar maternal environment.
To test the hypothesis that alteration in placental transport function may cause growth restriction of one of the DZ twins, we measured maternal and umbilical venous plasma concentrations of essential and nonessential amino acids in twins with or without discordant birth weights. An attempt was also made to establish the relationship among altered fetal nutrition, insulin, IGF-I, and IGF-binding protein-1 (IGFBP-1) levels at birth.
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Materials and Methods |
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Twenty DC twin pregnancies with (n = 10) or without (n = 10) discordance in birth weight were studied. Dichorionicity was established prenatally in the presence of discordant sex, two separate placental masses, intrafetal membrane thickness more than 2.0 mm, and twin peak sign and was confirmed at birth by histology (18).
The diagnosis of discordant growth in DC twins was made when the difference in birth weight was 20% or more with normal amniotic fluid volume in the larger twin’s sac; and the smaller twin had an abdominal circumference at the fifth percentile or below with abnormal umbilical artery Doppler waveforms. Pregnancies complicated by fetal structural abnormalities, aneuploidy and single intrauterine death, embryo reduction, and selective fetocide were excluded. There were 16 sets of twins with unlike-sex and 4 with like-sex pairing. In like-sex pairs, dizygosity was assigned by DNA analysis. Clinical details of some of these pregnancies have been reported previously (23).
DC twins with differences in birth weight of 10% or less and normal amniotic fluid volumes in both sacs constituted the concordant/control group. All pregnancies were monitored by serial ultrasound scans for fetal growth, amniotic fluid volume, and umbilical artery Doppler waveforms.
Collection of blood samples
Maternal blood samples were obtained from the antecubital vein. Umbilical venous blood was collected at birth from each twin from a segment of clamped cord. Blood samples were collected into tubes containing EDTA. The samples were centrifuged immediately after collection in a laboratory located on the delivery suite, and the plasma was stored at -70 C until batch assay was performed. Additional umbilical arterial and venous samples from a segment of cord clamped at both ends were also obtained immediately after delivery of the twins for determination of hemoglobin and acid base status according to routine clinical practice of the unit. The acid-base status and hemoglobin level was then determined within minutes of collection of the samples on a gas and hemoglobin analyzer instrument located in the delivery unit. The study was approved by the hospital research ethics committee, and informed consent was obtained from all women.
Measurement of amino acids
The plasma amino acid concentrations were determined in 3.5% (wt/vol) sulfosalicylic acid deproteinized samples using the System 6300 high performance analyzer (Beckman Coulter, Inc., Beckman, CA) and eluted with a three-buffer system (lithium buffers A, B, and C; Beckman Coulter, Inc.). Peaks were detected after reaction with ninhydrin and integrated using the 406 analog to digital converter and System Gold software (Beckman Coulter, Inc.). A series of amino acid solutions from Beckman Coulter, Inc., was used as reference amino acid standards.
Measurements of insulin, IGF-I, and IGFBP-1
The plasma concentration of insulin was determined by RIA using commercially available kits (Pharmacia \|[amp ]\| Upjohn, Inc., Uppsala, Sweden) with intra- and interassay coefficients of variation ranging from 3–6% and from 5–7%, respectively. Plasma IGF-I and IGFBP-1 concentrations were determined by RIA as described previously (23). The intra- and interassay coefficients of variation for IGF-I ranged from 4.0–5.7% and from 5.2–7.4%, respectively, and those for IGFBP-1 were 8% and 6.8%, respectively. Some of the IGF data have been published previously (23).
Data analysis
Clinical data are expressed as the median and range, whereas amino acid, insulin, IGF, and IGFBP-1 concentrations are expressed as the mean ± 95% confidence interval (CI). Delta values (
) indicate differences between AGA and IUGR twins in the discordant growth group, and between twin 1 and twin 2 in the concordant growth group. For parametric data, the paired t test was used to compare values between twin pairs and the t test was used to compare data between groups. For nonparametric data, comparisons between groups were performed using the Mann-Whitney test. The percent growth discordance was defined as the difference in birth weights expressed as a proportion of the weight of the larger twin. In the control group the heavier twin was labeled twin 1, and the lighter twin 2.
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Results |
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. The gestational age at delivery was comparable between the two groups. In the discordant growth group, seven women had cesarean section for fetal compromise (absent end diastolic flow in the IUGR twin). In nine cases, the IUGR twin was born second; one in the vaginal delivery group and eight in cesarean section group. In the concordant growth group, four women required cesarean section for nonvertex presentation of the first twin (n = 2) or previous cesarean section (n = 2). The birth weight of the AGA twin in the discordant growth group was comparable with that of the concordant twin pairs. The IUGR twin in the discordant growth group was significantly more hypoxic (P < 0.01) and acidotic (P < 0.01) than the AGA co-twins. Similarly, IUGR twins were more hypoxic (P < 0.001) and acidotic (P < 0.01) than the concordant twin pairs. No such differences were found between AGA discordant twins and concordant twin pairs (Table 1).
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Maternal.
The plasma concentrations of essential and nonessential amino acids were similar between discordant and concordant birth weight groups (Table 2).
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and Table 4). Similarly, in the discordant DC twins the fetal concentrations and female/male ratios of nonessential amino acids such as taurine (P < 0.05), serine (P < 0.05), tyrosine (P < 0.01), glycine (P < 001), aspartic acid (P < 0.05), and glutamic acid (P < 0.05) were reduced in IUGR twins compared with those in AGA co-twins (Table 3
). Fetal concentrations of alanine, glutamic acid, ornithine, hydroxyproline, and proline were similar in discordant DC twins (Table 3). In contrast, fetal concentrations and female/male ratios of all essential and nonessential amino acid were comparable between concordant DC twin pairs (Table 4). The interpair differences in amino acids in both discordant and concordant MC twins were independent of the mode of delivery.
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In the discordant group, fetal insulin concentrations in IUGR twins were significantly lower than those in AGA co-twins (, 5.4; CI, 3.5–7.3 µU/ml; P < 0.001) and the concordant twin pairs (mean, 2.7; CI, 1.8–3.5 vs. mean 8.1; CI, 4.2–12 µU/ml; P = NS; Fig. 2). The fetal insulin levels were comparable between concordant twin pairs (, 1; CI, -0.5 to 2.1 µU/ml; P = NS). Similarly, fetal IGF-I levels in the IUGR twins were lower than those in the AGA co-twins (, 105; CI, -112 to 323 ng/ml; P < 0.05) and concordant twin pairs (mean, 29; CI, 20–39 vs. mean 66; CI, 47–85 ng/ml; P < 0.01). Fetal IGFBP-1 levels in the IUGR twins were higher than the AGA co-twins (, 510; CI, 220–800 ng/ml; P < 0.01) and concordant twin pairs (mean, 863; CI, 598-1128 vs. , 289; CI, 160–418 ng/ml; P < 0.01; Table 5). There were no differences in IGF-I (, 7.1; CI, -4.1 to 18.3 ng/ml) or IGFBP-1 (, 105; CI, -112 to 323 ng/ml) levels between concordant twin pairs.
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In the discordant twins, fetal IGFBP-1 had a negative association with fetal insulin (y = -1007.5log(x) + 1246.8; r = 0.71; P < 0.001; n = 20), total essential amino acids (y = -3585.5log(x) + 11755.9; r = 0.78; P < 0.001; n = 20), and branched chain amino acids (y = -2565.8log(x) + 7259.8; r = 0.64; P < 0.001; n = 20; Fig. 3). A significant positive correlation was found among total essential amino acids (y = 580.2log(x) + 947.7; r = 0.63; P < 0.01; n = 20), branched chain amino acids (y = 206.1log(x) + 273.4; r = 0.58, P < 0.05, n = 20), and fetal insulin (Fig. 4). There was no association among IGF-I, IGFBP-1, insulin, and nonessential amino acids in the fetal circulation. In the concordant growth group, there was no association among insulin, IGFBP-1, and amino acids. In both groups, no correlation was found among IGF-I, fetal insulin, and amino acids in twins with or without discordant growth.
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Discussion |
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Selective, rather than global, differences in amino acids between the discordant twins are highly suggestive of impaired placental transport function. Amino acids are transported from the maternal to the fetal circulation by specific amino acid transporters located on the microvillous and basal plasma membranes of the human placenta (26). Activities of systems A, L, and ß amino acid transporters are impaired in singleton IUGR placentas (6, 7, 8, 9, 10, 11). We also found a marked reduction in the concentrations of amino acids that are transported by system L (valine, leucine, isoleucine, phenylalanine, and tyrosine) and system ß (taurine) in the growth-restricted twins. In addition, lower fetal concentrations of phenylalanine and tyrosine may be a consequence of increased fetal utilization as a part of the "brain-sparing" effect in growth-restricted babies (27, 28)
The reduction of fetal concentrations of glycine and glutamine in the IUGR twins suggests a disturbance of placental utilization and metabolism as the cause of fetal undernutrition. The predominant source of circulating fetal glycine is derived from placental conversion of maternal serine (29). Similarly, lower fetal glutamine levels may be a consequence of reduced placental oxidation, followed by its release into the fetal circulation (30). Lower fetal circulating levels of serine in the IUGR twins perhaps reflect disturbances of the fetal hepatic synthesis from amino acids such as glycine, glutamine, and alanine.
The underlying mechanism that regulates placental transport of amino acids remains unclear. There is now increasing evidence that placental transport can be modified by the fetal hormonal milieu (31, 32, 33). Our data also suggest that fetal circulating insulin levels are substantially lower in the IUGR than the AGA co-twins, with a positive association with total essential and branched chain fetal amino acid concentrations. Similarly, in this cohort IGF-I levels were reduced, and IGFBP-1 levels were raised in the IUGR twins. Studies in sheep also suggest that fetal nutrition may be altered by IGF-I (34). Although we did not find any association between fetal IGF-I levels and amino acids, the elevated IGFBP-1 levels in IUGR twins probably suggest that IGF-I influences fetal growth through IGFBP-1. Hepatic transcripts of IGFBP-1 are increased in the growth-restricted fetal rat (35). Our findings of a negative association among IGFBP-1, total essential amino acids, and branched chain amino acids is consistent with the suggestion that fetal IGFBP-1 may alter the uptake and transport of amino acids across the placenta. Indeed, IGFBP-1 inhibits IGF-I-stimulated [3H]
-amino isobutyric acid uptake by human trophoblast cells (36). These data, therefore, suggest that nutrient insufficiency may be the primary factor that alters the fetal production of insulin and IGFs, the key hormones involved in the regulation of fetal growth. We appreciate that statistical association between amino acids and fetal levels of IGFBP-1 and insulin does not necessarily indicate causation or effect. However, the possibility remains that alteration in the fetal somatotropic axis may be the primary event, which, in turn, influences the placental transport function of amino acids.
Alternatively, the functional status of various pathways of placental transport of nutrients to the fetus may be altered by fetal hypoxia directly or indirectly. In accordance with this proposition, the IUGR twins were more hypoxic and acidotic than their AGA co-twins. In addition, most of the IUGR twins had absent end-diastolic flow velocitometry of the umbilical artery, which was further indicative of fetal hypoxia. Fetal hypoxia is considered to be the most potent stimulus for the production of IGFBP-1 by the fetal/maternal unit (37, 38). Recent experimental studies also suggest that hypoxic stress may cause suppression of fetal insulin secretion, thereby influencing fetal growth potential (39). In addition, hypoinsulinemia, acting independently or coordinately with hypoxia, may further increase the production of IGFBP-1, thereby inhibiting the interaction between IGF and its receptor. We also found a negative association between fetal insulin and IGFBP-1 levels. Accordingly, we postulate that fetal hypoxia may be the primary event in the pathogenesis of IUGR, which then influences fetal growth through its action on nutrient uptake and utilization through hypoinsulinemia and a trophoblast-driven paracrine mechanism of increased production of IGFBP-1 at the fetal/maternal unit.
In conclusion, we have shown that a disturbance in the pathways of placental transport of amino acids causes an alteration in the fetal insulin-IGF axis, which then causes IUGR in one of the DC twins. This information is pertinent for fetal origin hypothesis and challenges the recent proposal that by changing maternal diet, fetal growth restriction can be prevented (40). Further studies are warranted to evaluate the effect of hypoxia on IGF-mediated amino acid transporter activities in the DC placenta in relation to fetal growth.
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Acknowledgments |
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Footnotes |
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Abbreviations: AGA, Appropriate for gestational age; CI, confidence interval; DC, dichorionic; DZ, dizygotic; IGFBP, IGF-binding protein; IUGR, intrauterine growth-restricted; MZ, monozygotic.
Received February 26, 2001.
Accepted October 11, 2001.
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, AGA twin; 
, IUGR twin. The lower panel compares the umbilical venous concentrations of essential (C) and nonessential (D) amino acids between concordant DC twin pairs. 
