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Original Articles |
University of Manchester, Academic Unit of Obstetrics and Gynecology (R.B., S.W.), Endocrine Sciences Research Group (M.J.G., M.W.), St. Mary’s Hospital, Manchester M13 0JH, United Kingdom; Imperial College School of Medicine, Department of Maternal and Fetal Medicine, Queen Charlotte’s and Chelsea and Westminster Hospital (R.B., S.R.S.), London W12, United Kingdom; and Department of Obstetrics and Gynecology, Liverpool Women’s Hospital (J.P.N.), Liverpool L69 3BX, United Kingdom
Address all correspondence and requests for reprints to: Dr. R. Bajoria, Department of Obstetrics and Gynecology, St. Mary’s Hospital, Whitworth Park, Manchester, United Kingdom M13 0JH. E-mail: rekha.bajoria{at}man.ac.uk
Abstract
To test the hypothesis that severe growth restriction (intrauterine growth retardation) in donor twins with chronic twin-twin transfusion syndrome (TTTS), a common complication of monochorionic twin pregnancy, is due to an aberration in the insulin-like growth factor (IGF) axis, we studied 25 sets of monochorionic twins with (n = 13) and without (n = 12) TTTS. Maternal and cord blood samples were collected at birth and analyzed for IGF-I, IGF-II, IGF-binding protein-1 (IGFBP-1), and IGFBP-1 phosphorylation status.
Fetal IGF-II levels in the recipient twins with TTTS were higher than those in the donor twins (829 ± 45 vs. 543 ± 60 ng/mL; P < 0.001), but were comparable with those in the non-TTTS twin pairs. IGF-I levels in recipient and donor twin pairs were similar. The total IGFBP-1 concentration was higher in the donor twins than in the recipients (1153 ± 296 vs. 419 ± 108 ng/mL; P < 0.001) and non-TTTS twin pairs (P < 0.01). The percent less phosphorylated IGFBP-1 was higher in the recipients than in the donor twins (P < 0.05). There were no differences in IGF-I, IGF-II, and IGFBP-1 levels between non-TTTS twin pairs. Maternal levels of IGFs were comparable in the two groups. In the TTTS group, fetal birth weight gave a positive correlation with serum IGF-II levels (y = 0.25x + 361.1; r = 0.47; P < 0.05), and a negative association with IGFBP-1 levels (y = -0.72x + 1593.6; r = 0.58; P < 0.01).
Our data argue against intertwin transfusion as the cause of intrauterine growth retardation in the donor twin and provide evidence that the placenta is the key regulator of the fetal IGF axis, especially when fetal genotype and maternal environments are similar.
THE PERINATAL LOSS of monochorionic (MC) twin pregnancies is 6-fold higher than that in dichorionic twin and singleton pregnancies (1). The predominant cause of the high fetal loss rate in MC twin pregnancies is due to chronic twin-twin transfusion syndrome (TTTS). With recent advancements in diagnostic and therapeutic modalities in fetal medicine, the survival rate of chronic TTTS has improved markedly from nil to 65% (2). Further improvement, however, in the clinical outcome is hindered because of our poor understanding of the pathophysiology of chronic TTTS.
Conventionally, it is believed that chronic TTTS occurs due to the transfer of blood along the unidirectional deep arterio-venous anastomotic channels present in the placenta (3, 4). This net flux of blood leads to growth restriction and severe oligohydramnios in one twin, called the donor, and volume overload, polyhydramnios, and polycythemia in the appropriate for gestational age cotwin, called the recipient (5). Recent data suggest that the pathophysiology of TTTS may not be this simple, and growth restriction of the donor twin may be attributed to impaired placental exchange of nutrients in one portion of the placenta (6). Several animal studies have suggested that the long-term effects of undernutrition in utero may be mediated via the insulin-like growth factor (IGF) axis (7, 8, 9, 10). It is therefore pertinent to establish how ongoing undernutrition may influence this axis in the donor twin, because this could be a causative factor for in utero programming of adult diseases (11).
The IGF system is important in cell growth and differentiation and plays a pivotal role as a mediator of fetal growth (12, 13, 14, 15). Studies in transgenic mice confirm that IGF factors are essential for embryonic and fetal growth. Fetal mice homozygous for a targeted disruption of the IGF-I or IGF-II gene are 40% smaller than their normal littermates (16, 17). The IGF-II-deficient mice also had reduced placental growth, but survived normally, whereas the mice lacking IGF-I showed increased neonatal death. However, the literature relating to humans is conflicting. Most studies have reported reduced IGF-I levels in intrauterine growth-retarded (IUGR) fetuses with no association between IGF-II and birth weight (18, 19, 20, 21, 22). In contrast, studies have also shown normal IGF-I levels (23, 24) and reduced IGF-II levels in singleton IUGR fetuses (19, 21). Although the reason for this discrepancy is not obvious, it is plausible that the synthesis of these growth-promoting hormones is regulated by genetics (fetus), or extrinsic (maternal/placental) factors may influence growth in utero (25, 26). Little is known of their relative contribution and importance for intrauterine growth, as singleton studies are limited in this regard.
This issue can only be resolved by undertaking a systematic study in MC twin pregnancies with interpair variation in birth weight. TTTS provides an excellent alternative to an animal experimental model to study the interaction between genetic and environmental factors on fetal growth. However, little information is available on IGF axes in twin pregnancies with discordant growth. Studies in normal twin pairs suggest that fetal circulating IGF-I levels may be genetically determined (27). This raises the possibility that unlike singleton pregnancies, growth restriction in the donor twin may not be due to reduced IGF-I concentrations and that placental pathology may override the genetic determinants of endogenous production of IGF-I by altering the concentrations of IGF-binding proteins (IGFBPs), clearance rate (28), and placental secretion (29). We therefore conducted a study in MC twin pregnancies to establish whether placental factors can influence the IGF axis and thereby cause growth restriction in the donor twin with severe TTTS.
Subjects and Methods
Patients
Twenty-five women with MC twin pregnancies with (n = 13) or
without (n = 12) TTTS were studied. Monochorionicity was
established prenatally in the presence of concordant genitalia,
intrafetal membrane thickness less than 2.0 mm, and single placental
mass and was confirmed at birth by histology. The diagnosis of TTTS was
made on the basis of serial ultrasound scan criteria of growth
discordance of more than 20% with polyhydramnios (amniotic fluid
index, >40 cm) in the larger twin and anhydramnios or oligohydramnios
(single deepest pool, <2 cm) in the smaller twin (3). The
uncomplicated MC twins (n = 13) with growth discordance of less
than 10% and normal amniotic fluid volumes in both sacs (amniotic
fluid index, 
24 cm) constituted the control group. Pregnancies
complicated by fetal structural abnormalities, aneuploidy, intrauterine
demise of one twin, embryo reduction, and selective fetocide were all
excluded. All pregnancies were monitored by serial ultrasound scans for
fetal growth, amniotic fluid volume, and umbilical artery Doppler
waveforms.
Collection of samples
Maternal blood samples were obtained from the antecubital vein and umbilical venous blood from a clamped segment of cord at the birth of each twin. No maternal samples were available from seven women. The samples were centrifuged, and serum was stored at -70 C until a batch assay was performed. All samples were collected by R. Bajoria at Hammersmith Trust Hospitals (n = 16), St. Mary’s Hospital, Manchester (n = 6), and Liverpool Women’s Hospital, Liverpool (n = 3). Informed consent for collection of maternal samples was obtained as instructed by the hospital research ethics committee.
Immunoassays
Plasma IGF-I was determined by an IGF-II-blocked RIA (30), with intra- and interassay coefficients of variation (CV) of 4.0–5.7% and 5.2–7.4%, respectively.
Plasma IGF-II was measured after acid-ethanol extraction using our previously reported two-site immunoradiometric assay (31). This assay has a sensitivity of 30 µg/L and a CV of less than 10% between 200-4500 ng/mL, both within and between assays.
IGFBP-1 was determined by two RIAs (RIA 6303 and RIA 6305) to allow assessment of IGFBP-1 phosphorylation status as well as a measurement of levels (32). The assays use human recombinant IGFBP-1 for standards and radiolabel and either monoclonal antibody 6303 or 6305 (provided by Medix Biochemica, Kauniainen, Finland). RIA 6303 recognizes all isoforms of IGFBP-1, including the phosphoform characteristic of normal plasma, whereas the 6305 RIA only detects the non- and lesser phosphorylated isoforms. RIA 6303 has a detection limit of 5 µg/L, whereas that of RIA 6305 is 2 µg/L. The interassay CVs are 8% and 10%, respectively. The intraassay CVs were 6.8% and 7.6%, respectively.
Data analysis
Clinical data are expressed as medians and ranges, whereas IGFs concentrations are expressed as the mean ± SEM. For parametric data, the paired t test was used to compare values within twin pairs and Student’s t test between groups. Fisher’s exact test was used for blocked comparisons. For nonparametric data, comparison between groups were performed by the Mann-Whitney test. The percent growth discordance was defined as the difference in birth weight expressed as a proportion of the birth weight of the larger twin. In the control group, the heavier twin was labeled as twin 1, and the lighter as twin 2.
Results
The clinical parameters of the two groups are given in Table 1
.
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Levels of IGF-I (311 ± 47 vs. 261 ± 46 ng/mL), IGF-II (982 ± 156 vs. 605 ± 149 ng/mL; P = NS), total IGFBP-1 (338 ± 46 vs. 484 ± 163 ng/mL; P = NS), and less phosphorylated (lp) IGFBP-1 (124 ± 34 vs. 348 ± 88 ng/mL; P = NS) were comparable between TTTS and non-TTTS groups, respectively.
Fetal
Fetal IGF-I levels in recipient and donor twins in TTTS group (52 ± 8 vs. 55 ± 6; P = NS) and non-TTTS group (P = NS) were comparable.
Fetal IGF-II levels in recipient twins were higher than those in donor
twins (829 ± 45 vs. 543 ± 60 ng/mL;
P < 0.001), but were similar to twin 1 (721 ±
69; P < 0.01) and twin 2 (631 ± 46 ng/mL;
P < 0.01) of the non-TTTS group (Fig. 1). IGF-II levels in donor twins were
markedly lower than twin 1 (P < 0.01) and twin 2
(P < 0.01).
|
). No differences in total IGFBP-1
(366 ± 98 vs. 338 ± 114 ng/mL; P
= NS) and lpIGFBP-1 levels (148 ± 45 vs. 124 ±
34; P = NS) were found between the control twin
pairs.
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In non-TTTS twins, the IGF-I, and total IGFBP-1 levels within each pair
are positively correlated (Table 2).
However no association was found between birth weight and the IGF-I,
IGF-II, and total and lpIGFBP-1 levels in the non-TTTS group. In
contrast, only total IGFBP-1 levels within each TTTS twin pair is
positively correlated. Furthermore, in the TTTS group, there was a
negative correlation between birth weight and total IGFBP-1 levels
(y = -0.72x + 1593.6; r = 0.58;
P < 0.01), whereas a positive association was present
between birth weight and IGF-II levels (y =
0.25x + 361.1; r = 0.47; P < 0.05;
Fig. 3).
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The results of this study support the hypothesis that the placenta is the key controller of genotype-phenotype interaction of the fetal IGF axis, especially when fetal genotype and maternal environments are similar. There were no significant differences in IGF-I levels between recipient and donor twins despite birth weight differences of more than 20%. This was unexpected and is at variance with data from singleton pregnancies where IUGR fetuses have reduced IGF-I levels, with comparable IGF-II concentrations (12, 13, 14, 15).
The difference between the IGF axis of donor and recipient twins may be a consequence of twin to twin transfusion. All TTTS placentas have vascular anastomoses that facilitate transfusion of blood from the donor to the recipient twin (3), which could be the explanation for increased IGF-II levels in the recipient twin. It is possible that IGF-I is also transferred in this way, but that some compensatory mechanism results in the two infants having similar levels. The placenta can apparently clear IGF-I from the fetal circulation (28), which may be occurring in the recipient twin. There is also limited evidence of placental IGF-I secretion into the fetal compartment (29), although this peptide is not abundantly produced at this site. However, decreased IGFBP-1 levels in the recipient twin cannot be accounted for by this model of intertwin transfusion.
In singleton IUGR babies the reported abnormalities in the IGF axis may reflect maternal maladaptation to pregnancy. Maternal nutrition (26, 33), hypertension (34), and diabetes (35) are known to alter the maternal IGF axis, such that placental nutrient transfer and ultimately the fetal IGF axis and prenatal growth are reduced (8, 9, 36, 37). However, maternal environment is obviously not the explanation for growth restriction in only one infant of a twin pair, suggesting that placental factors may underlie the findings of our study.
Alternatively, comparable IGF-I levels may simply be a reflection of the identical genetic makeup of the MC twins. Rosen et al. (38) have also shown that a polymorphic microsatellite within the IGF-I gene is associated with differences in serum IGF-I levels in several cohorts, even after correction for age and sex. Genes encoding somatostatin, GHRH, GH, and the receptors for these hormones might be subjected to polymorphic variation, and these could also influence the IGF-I level (39). Variation in IGF-I levels in fetuses, children, and adults have been shown to be almost completely of genetic origin (27, 40, 41). IGF-II and IGFBP-1, however, are not so genetically regulated (27, 40), and thus, their levels may be subject to maternal or placental factors.
The lack of correlation between IGF-I and fetal weight may be due to a relative, rather than absolute, deficiency in IGF-I levels. IGF expression and levels must be considered in concert with the IGFBP milieu, because these ultimately control IGF activity. IGFBP-1 is thought to be the most important binding protein during fetal life and regulates IGF-I dissociation and thereby availability at the receptors (42). In keeping with studies on singleton IUGR fetuses (43), we report a negative association between birth weight and IGFBP-1, the predominant IGFBP at the maternal/fetal interface, with increased IGFBP-1 levels in the growth-restricted donor twin. We, however, have extended these findings by examining IGFBP-1 phosphorylation status, which also impacts IGF activity. The highly phosphorylated isoform of IGFBP-1 has a high affinity for IGF and can therefore sequester IGF away from its cell surface receptors. The non- and lesser phosphorylated isoforms have a relatively lower affinity and have been reported to enhance IGF action (42). We found that not only did the donor twin have increased IGFBP-1 levels, but that a greater proportion of the IGFBP-1 was present as the inhibitory phosphorylated isoform. Our recent study on growth abnormalities in association with diabetic pregnancy also revealed perturbations in IGFBP-1 phosphorylation status (35), and we speculate that the relative increase in the highly phosphorylated isoform may reflect failure of the placental unit to generate non- and lesser phosphorylated IGFBP-1.
Increased IGFBP-1 may have implications for fetal growth through its effect on trophoblast migration and placental development, although its role in this regard is unclear, as it has been reported to both augment (44, 45) and inhibit (46) trophoblast invasion. It may also detrimentally influence the mitogenic activity of IGF and IGF-stimulated transfer of nutrients across the placenta. Thus, the finding that fetal birth weight and total IGFBP-1 levels are strongly correlated supports the proposition that up-regulation of the binding protein may cause fetal growth restriction by creating an IGF-I-deficient state. Secondly, an increased concentration of phosphorylated IGFBP-1 may inhibit IGF-I-stimulated trophoblast uptake of amino acids and perpetuate fetal undernutrition (47). Thus, increased IGFBP-1 levels in the donor twin, who is known to have a coexisting placental pathology and impaired placental transfer of certain essential amino acids (6), further supports the hypothesis that IGFBP-1 is regulated by intrauterine factors rather than maternal and/or genetic factors (40).
Low circulating IGF-II levels in the growth-restricted donor twin suggests that IGF-II may be a more important regulator of fetal growth. Although these results are consistent with reduced IGF-II levels in singleton IUGR fetuses (19, 21), they are at odds with the findings of other investigators, who failed to show any differences in IGF-II levels in singleton IUGR and appropriate for gestational age human fetuses (12, 14). Most members of the IGF axis are produced at the fetal/maternal interface, although two peptides were found to be aberrant in our study, namely IGF-II and IGFBP-1. IGF-II is present in placental chorionic villi and fetal membranes from as early as 6 weeks gestation (48, 49), whereas IGFBP-1 is a major secretory product of decidualized endometrium (48, 49). Placenta does not clear IGF-II from the fetal circulation (50); thus, it is likely that the reduced IGF-II levels seen in growth-restricted donor twins is due to placental pathology resulting in decreased IGF-II production. These fetuses have reduced placental masses (3, 4), nonphysiological trophoblast invasion of spiral arteries (51), and diminished microvasculature (52). Indeed, studies in which placental weight is reduced as a result of uterine artery ligation (53) or nutritional depravation (7, 8) also report reduced fetal IGF-II levels. Furthermore, placental growth is reduced in IGF-II, but not IGF-I, knockout mice (16, 17). IGF-II stimulates trophoblast migration (45, 46). Thus, inadequate placental development as a consequence of reduced IGF-II levels could exacerbate the effect on nutrient transfer of a decrease in IGF-II. Excess IGF-II has been linked with the fetal overgrowth observed in the human genetic disorder Beckwith-Wiedemann syndrome (54). In this condition IGF-II levels are increased as a result of biallelic expression of the normally imprinted IGF-II gene. Deletion of the type 2 IGF/mannose-6-phosphate receptor, which is thought to have a role in clearing IGF-II from the circulation, also leads to increased IGF-II levels and excessive fetal somatic overgrowth (55).
The findings of this study, therefore, argue against intertwin transfusion as the cause of TTTS and supports the alternative theory of discordant placental development as the primary determinant of severe growth restriction in the donor twin. This information is pertinent to understand the pathology of chronic TTTS. In particular, our work highlights the importance of alteration of the IGF axis and thereby reduced availability of the nutrients in the pathogenesis of growth restriction of the donor twin. As a consequence of growth restriction, feto-placental resistance is higher in the donor twin (56). This then initiates transfer of blood from donor to the recipient twin (3, 4), which, in turn, perpetuates the clinical manifestation of oligohydramnios-polyhydramnios sequalae (57). The more severe the placental dysfunction, the smaller the donor twin, the larger the intertwin disparity in size, and the poorer the prognosis. In keeping with this concept, we also found that fetal IGF-II and IGFBP-1 levels, which are produced predominantly by the placenta, correlate directly with the differences in birth weight between recipient and donor twins.
The observation that discordant growth in TTTS is independent of intertwin transfusion but results from placental dysfunction may be relevant to the clinical management of this enigmatic condition. Therefore, it may be reasonable to conclude that laser ablation of placental anastomoses is unlikely to address the underlying pathology of chronic TTTS (58) and improve fetal growth. High rates of intrauterine death of the donor twin occurring within 24 h of laser ablation support this hypothesis (59). Alternatively, therapeutic amnioreduction by reducing amniotic fluid pressure may reverse the underlying pathophysiology of TTTS by improving placental perfusion (60) and thereby fetal growth. Anecdotal case reports have shown that amnioreduction is frequently accompanied by rapid resolution of discordant bladder dynamics, catch-up growth in the donor twin, and reversal of hydrops in the recipient twin (1).
In conclusion, this study shows that donor twins with chronic TTTS have reduced IGF-II levels with increased IGFBP-1 compared with the recipient and normal MC twin pairs. Increased IGFBP by modulating IGF activity may play a major role in the pathology of fetal growth restriction and the metabolic status of the donor twin, which in part reflects the efficacy of placental perfusion and transfer of nutrients from mother to fetus.
Footnotes
1 This work was supported by The Royal Society and the Research
Graduate Support Unit, University of Manchester. 
Received June 5, 2000.
Revised September 27, 2000.
Accepted March 6, 2001.
References
5ß1 integrin and
inhibits cytotrophoblast invasion into decidualised endometrial stromal
cultures. GH IGF Res. 8:21–31.
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