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Models of fetal growth restriction
European Journal of Obstetrics & Gynecology and Reproductive Biology 110 (2003) S29–S39
Models of fetal growth restriction Hobe J. Schro¨der* Abteilung fu¨r experimentelle Medizin, Klinik und Poliklinik fu¨r Frauenheilkunde und Geburtshilfe, Universita¨tsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
Abstract The growth of the fetus is determined by substrate supply mostly for mass accretion and energy gain, and by control systems. Experiments with whole animal models will face the following problems: (1) The fetus, like a three compartmental ‘‘Russian doll’’, is at the end of a long supply chain. There are interactions (e.g. hormones) and partitioning of substrates between the compartments. (2) The fetal organism is growing anddifferentiating at the same time and not in a steady-state. Experimental results thus dependon gestationalage. (3) About 75% of animal experiments on fetal growth restriction have been performed in rats and mice. The possible experimental methods and the results depend on the species which include sheep, pigs, rabbits, guinea pigs, horses and non-human primates. Many experiments have clearly shown that restriction of substrate supply will usually impair fetal growth. Less is known about growth control mechanisms but recent studies in gene mutant mice have opened a new approach to study the effects of systemic and local controlling factors. It appears that insulin-like growth factors and their binding proteins may play an important role for fetal growth. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Substrate supply; Control mechanisms; Gene mutants; Fetal growth
The use of animal models to explore fetal growth restriction has been extensively dealt with in excellent review articles [1–8] and books [9–14]. This brief overview focuses on general problems which are inherent to studies with whole pregnant animals, and on some recent results on the control of fetal growth.
1. Introduction As a first approach fetal growth can be compared to building a house. A plan (genome) is required as well as energy (metabolism), building materials (nutrition) and transportation (circulation). The accretion of material will have to follow a precise sequence in time and space because one cannot start with the top of the chimney, for example. However, this comparison between the house model and fetal growth is inappropriate in one important aspect: in the fetus growth and differentiation (which implies the maturation of functions) occur simultaneously. Although growth and differentiation appear to be inseparable, they actually may be controlled differently by growth-promoting and * Tel.: þ49-40-42803-3533; fax: þ49-40-42803-2395. E-mail address: [email protected] (H.J. Schro¨der).
by differentiation-promoting factors. This difference is hypothetical at present, but it may provide some day the key for the treatment of intrauterine growth restriction: if substrate supply is insufficient for both growth and differentiation, perhaps intrauterine growth can be halted and postponed until birth whereas differentiation and maturation can be made to continue successfully in utero. The useful message from the house model is that growth is determined by substrate supply, either for mass accretion or for energy gain, and by control systems, and this distinction will be followed to describe experimental approaches.
2. The Russian doll The schematic (Fig. 1) illustrates that the fetus is located inside the uterine-placental unit, which in turn is located inside the mother. This is the ‘‘Russian doll situation’’ which has, simple as it may be, far reaching consequences for the study of fetal growth restriction. As an example, we consider a study on the effects of reduced oxygen supply for growth and development of the fetal brain. Obviously, in this experiment fetal oxygen supply should somehow be diminished and Fig. 1 illustrates that the availability of oxygen may be restricted at various levels of the supply chain.
0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0301-2115(03)00170-2
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Fig. 1. The ‘‘Russian doll situation’’. Single headed arrows indicate flux of substrates with five possible positions (level 1 to level 5) for blocking substrate transfer (bars). Double headed arrows symbolize ‘‘partitioning’’ and mutual interactions between the three compartments (see text).
The first and most ‘‘upstream’’ level would be the reduction of maternal oxygen intake. Alternatively and hypothetically, it might be accomplished to reduce the amount of oxygen transferred to the uterine placental exchange unit (level 2), to restrict the transfer of oxygen to the fetus through the placenta (level 3) or inside the uterus via the umbilical vein (level 4), and finally to reduce oxygen supply to the fetal brain but not to other fetal regions (level 5). Even if it were experimentally possible to affect exclusively the supply of oxygen at each the above five levels (in fact, it is not), these were five different experiments. In general, the more upstream in the supply chain an experimental intervention is executed, the more global and possibly unspecific the downstream effects may be. For example, chronic maternal hypoxemia as in high altitude experiments (in growth restriction experiments interventions have to be long lasting and chronic) may affect the maternal cardiovascular system and thus maternal perfusion of the placenta, it may affect placental functions, it may affect the transfer capability of
the fetal cardiovascular system in general, and all these disturbances triggered along the supply chain may confound the effect of oxygen lack on the fetal brain, either aggravating it or alleviating it. From the ‘‘Russian doll’’ structure the problem of ‘‘interaction’’ between fetus, placenta and maternal tissues arises. Mother, the uterine-placental unit and the fetus do not act independently of each other, they rather cooperate, and the influence of placental hormones on maternal tissues is a long known example for this interaction. In metabolic studies, partitioning relates to the distribution of substrates between the three compartments. Experimental growth restriction affects partitioning, and experimental changes of partitioning may produce growth restricted fetuses (see below). Finally, maternal, utero-placental and fetal tissues are living tissues capable of adjustment and homeostatic regulations which do not simply subdue passively to imparted disturbances, and as the placenta and fetus are developing, they are certainly not in a steady-state situation. These problems are not unheard of in other whole-animal experiments, but they are especially concentrated and cumbersome in the study of fetal growth. The significance of these issues depend to some extent on the type of questions asked which can be divided into two broad categories. One is to imitate clinical situations which are known to be associated with growth restriction. Some ‘‘extrinsic’’ factors, that is extrinsic of the fetus, are listed in Table 1 which is abbreviated from a much longer list [5]. In this type of study the focus is on experimental interventions which imitate pathological human situations, and maternal, placental and fetal changes in form and functions may be observed. This may lead to diagnostic, therapeutic and pathophysiological insights. On the other side, in a reductionistic approach to
Table 1 Conditions associated with human fetal growth restriction Intrinsic
Extrinsic
Fetal factors
Environmental factors
Maternal factors
Intrauterine infections Chromosomal mosaicism Chromosomal anomalies Congenital malformations Inborn errors of metabolism Anaemia (Rh disease)
High altitude Variable nutrition Pollution Hyperthermia Irradiation
Undernutrition (including anorexia) Abnormalities Low maternal weight gain Chromosomal mosaicism Maternal age <16 Infarcts, focal lesions Drug use Abnormal placentation Smoking Alcohol Illicit drugs Low socio-economic status Medical complications Acute or chronic hypertension Antepartum haemorrhage Severe chronic infections Anaemia Disseminated lupus erythematosus Lupus obstetric syndrome Malignancy Abnormalities of the uterus Uterine fibroids
List of conditions associated with human fetal growth restriction. From [5].
Placental factors
Other factors Reproductive technologies
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unravel the complete, step-by-step cause-effect relationships in fetal growth restriction, the ‘‘Russian doll’’ situation with its many interdependencies poses much more of a problem. Of course, the questions asked in most studies will fall in between these two extremes.
3. Fetal growth restriction Normal fetal growth has been described as ‘‘the expression of the genetic potential to grow which is neither abnormally constrained nor promoted by internal or external factors’’. In this definition by Owens, Owens and Robinson [1] ‘‘internal’’ refers to fetal factors whereas ‘‘external’’ relates to anything else, that is placenta, uterus, mother and environment. Growth can be expressed as the gain in mass, or the gain in length, in area or in volume. With the advent of ultrasound B-scan, it has become possible in principle to continually follow the change of fetal dimensions during gestation, and based on these measurements, to follow the increase of (estimated) fetal weight. Total weight is the sum of all fetal organs and tissues, at birth especially of skeletal muscle, skeleton and fat, but single organs may follow their own growth trajectory which can be differently affected in growth restriction. This is the basis for the difference between proportionate and disproportionate growth with a reduced weight–length ratio in the second case. To establish growth restriction, abnormal deviation from the genetically determined growth trajectory has to be observed. The problem is that it is difficult to gain precise prospective knowledge of the undisturbed growth curve of an individual fetus, either in experimental animals, or in human fetuses. Perhaps volume measurements with three dimensional ultrasound B-scan may some day allow to predict individual fetal organ growth curves and their disturbances with sufficient precision not because the weight is less than some ‘‘normal’’ value, but because the growth trajectory has slowed down. At present, however, the diagnosis of growth restriction is based mostly on statistics, and usually growth curves with limits of what is considered normal are constructed. Growth restriction then frequently is defined as fetal weight below the 10th percentile at a given moment in gestation. Determining gestational age with accuracy may be difficult in the clinical situation, and this adds to the diagnostic uncertainty. There is, of course, no one normal growth curve which fits all, but number of fetuses, ethnic groups, socio-economic status and other parameters have to be considered. In animal experiments, gestational age should be known precisely and the genetically dependent variations may be minimized. It is typical then (Fig. 2) to observe two groups of growing fetuses, with a defined imposed disturbance in the experimental group, as in this example: when food intake of ewes is restricted starting at day 115 of gestation, the normal course of the crown-rump length (CRL) development
Fig. 2. Time course of a typical experiment on growth restriction. Food intake of maternal sheep is restricted at day 115 in the experimental group (term in sheep 145 days), and the increase of the CRL starts to slow down approximately at day 120. From [15], with permission.
is altered, and the difference becomes significant at some time point [15].
4. The experimental window Because the growing fetus is not in a steady-state, the same experimental intervention may have different effects when applied at different gestational ages, and the placement and the characteristics of the ‘‘experimental window’’ have to be considered carefully (Fig. 3). A given disturbance of
Fig. 3. The experimental window in growth restriction experiments. Three hypothetical experiments (A: short dashed lines; B: dotted lines; C: long dashed lines) are indicated which vary by intensity and type of interventions (vertical arrows), by their onset and duration (horizontal arrows), and by their outcome. Experiment A is successful because the surviving fetus has a body weight distinctly below the 10% percentile for normal fetuses. In B, the intervention leads to fetal death (cross) with no clear impairment of growth. Experiment C demonstrates that the fetal growth trajectory has been affected, but fetal body weight is not reduced below the 10% percentile. The time course in C may reflect fetal and/or maternal adaptations (see text). Note that fetal body weight and gestational age are indicated as fractions of normal values.
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medium intensity (Fig. 3A) may last until delivery of a growth restricted fetus, and this will be a successful experiment. Some time later in gestation (Fig. 3B), a large disturbance of short duration is imposed, but this time the fetus dies before growth is clearly reduced, and this is an experimental failure. A long lasting (Fig. 3C), late gestational disturbance of small intensity may produce no detectable growth alteration at all. An example for this experimental outcome is a carefully conducted study with sheep on the effect of lowering the oxygen fraction in inspired maternal air for 3 weeks [16]. For 21 days fetal and maternal blood was monitored daily, and fetal arterial pO2 stayed reduced to 2/3 of normal (from 30 to about 22 mmHg) all the time, yet fetal weight was not changed. The main reason was that fetal arterial oxygen content was restored to normal values already at day 5 of the hypoxic period. This experimental outcome illustrates the adaptive mechanisms available for mother and fetus. However, when fetal arterial pO2 is reduced for 23 days to 12 mmHg by daily injections of microspheres into the fetal placental circulation [17], fetal body weight is distinctly reduced (see below).
5. Animal species in fetal growth restriction experiments
genetic studies. It is important to keep this ‘‘uneven distribution of methods’’ in mind because in reproductive sciences species differences are especially pronounced.
6. Experiments in the real world Many experimental approaches to restrict fetal growth have been designed as listed in Table 2 [5]. The sites of interventions are classified as maternal, placental and fetal with more specific manipulations at each level as indicated. This useful classification may be divided into experiments which manipulate the supply of substrates, and experiments which manipulate the control of growth.
7. Restriction of substrate supply 7.1. Maternal intake Restricting substrates is possible at various points of the supply chain (Fig. 1), and one way is manipulation of maternal intake which has the advantage that it does not require surgical intervention. This is very much ‘‘upstream’’
Various mammalian species have been used as models of fetal growth restriction, and Fig. 4 demonstrates that about three quarter of experiments have been performed in two rodent species, rats and mice. Naturally, the types of experiments are not equally divided between the species. If fetal blood samples are to be drawn daily, for example, one of the larger species for implanting chronic catheters will have to be used, and here the chronically instrumented fetal sheep is still one of the favorites. The small species are used frequently in studies which apply techniques based on morphology and on molecular biology like development of structures and weight, distribution of hormones and growth factors and their respective receptors in fetal tissues, and in
Fig. 4. Distribution of species in animal experiments on fetal growth restriction which have been published (numbers are number of articles). The search phrase in Medline83 was ‘‘animal name þ fet? þ growth þ retardation’’, search performed in August 2001.
Fig. 5. Typical changes in growth restriction experiments following undernutrition or hypobaric hypoxia of the mother (based on [1]). (a) Fetal body and organ weights, and of CRL, as percentages of control values. (b) Concentrations of substrates in arterial blood as percentages of control values.
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Table 2 Experimental restriction of fetal growth: interventions and sites of action Site of intervention Maternal Altered maternal substrate availability Maternal undernutrition Protein/caloric deficiency Selected nutrient deficiency Micronutrient deficiency Maternal hypoxaemia Altitude Hypobaric hypoxaemia Normobaric hypoxaemia Reduced maternal fractional inspired oxygen content Intratracheal nitrogen Carbon monoxide Anaemia Methhaemoglobinaemia Maternal hypertension
Species
Sheep, pig, rat, mouse
Sheep, rat, guinea pig
Baboon, sheep, rat
Placental Reduction/restriction of placental growth Sheep Reduction in number of implantation sites (uterine endometrial caruncles) prior to pregnancy by hemihysterectomy or carunclectomy Maternal hyperthermia Separation of placentomes during pregnancy Interference with uteroplacental vasculature Sheep, monkey, guinea pig, rabbit, rat Partial occlusion of uterine artery Embolization of uteroplacental circulation Ligation of interplacental vessels Ligation of uterine artery Interference with fetoplacental circulation Sheep Embolization via umbilical artery Fetal Viral infection Organ/gland ablation Hypophysectomie, hypothalamic–pituitary disconnection Thyroidectomy, radioactive iodine Pancreatectomy (surgical, chemical: streptozotocin) Nephrectomy Other Banding ascending aorta Fistula Gene deletion Insulin-like growth factor axis IGF-I, IGF-II, type 1 receptor, type 2 receptor, IGFBPs, c-jun
Mouse Sheep, monkey
Sheep
Mouse
List of experimental procedures to generate fetal growth restriction. From [5].
and the ‘‘Russian doll’’ structure may cause difficulties in interpretation of the results, but the restriction can be very specific down to the level of one atom or molecule when exclusively the availability of oxygen is reduced, or when one essential amino acid in food is omitted, for example. In addition, this type of animal experiment can be close to what may happen to human patients. The effects chronic undernutrition (that is reduced caloric intake of an otherwise normally balanced food) and chronic lack of oxygen of the mother may have on fetal growth have frequently been investigated, and some of the results obtained in fetal sheep are summarized in Fig. 5 (based on [1]). Undernutrition and lack of oxygen both may restrict fetal and placental growth
and the growth of single fetal organs, but there are differences (Fig. 5a). Typically, brain weight is least affected whereas liver weight suffers especially with low nutritional intake. Hypoxemia, on the other hand, may even increase growth of an organ like the adrenals which reflects the high levels of plasma catecholamines typical for this situation. The concentrations of some substrates in fetal arterial blood may be affected also (Fig. 5b). Undernutrition especially generates fetal hypoglycemia, with resultant hypoinsulinemia not shown here, whereas maternal hypoxia generates fetal hypoxemia, increase of lactate and the increase of certain amino acids like alanine, or the decrease of others as well. It is emphasized that these are possible changes, and
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depending on the exact experimental protocol and species, variations are considerable. Basically, inside the mother (Fig. 1) substrate supply to the fetus can be restricted at three sites: at maternal vessels which supply the uterine-placental unit (level 2), at the placenta itself (level 3), and at umbilical vessels (level 4). These approaches are further ‘‘downstream’’ in the supply chain than the restriction of maternal uptake (level 1), and they are in one sense more specific because, for example, ligating one uterine artery should not affect too much other maternal tissues. On the other hand, reducing blood flow through the placenta, or reducing placental mass may affect the supply and transfer of all substances (and heat [18]) that need to be exchanged between mother and fetus, and the effects will be rather global therefore. A surgical intervention usually is necessary to reduce blood flow and placental mass though Alexander has shown years ago in sheep that chronic maternal hyperthermia generates fetal growth restriction [19,20] because maternal placental blood flow is reduced [21]. As surgery may directly affect the fetal growth trajectory, the surgical intervention itself is another confounding variable in growth restriction studies and the use of sham operated animals as controls is typical. 7.2. Utero-placental circulation In small polytocous mammals like guinea pigs the maternal side of the placentas are supplied with blood from an arcade between the uterine and ovarian artery. Ligating the uterine artery at this point reduces the supply to placentae and fetuses which are now at the end of the ovarian pipe line.
This technique has been applied especially in the guinea pig and reliably reduces fetal and placental weight [22,23]. It is, however, an all or nothing approach. In large species like the sheep, an adjustable occluder with exteriorized controller has been placed around the maternal common iliac artery which allows resistance changes as required [24]. In combination with flow probes placed on both uterine arteries, the experimentalist has complete quantitative control over maternal placental blood supply. 7.3. Reduction of placental mass Two approaches are in use to reduce placental mass and exchange, and both are typically applied to fetal sheep: embolization and carunclectomy. 7.4. Embolization With embolization, placental exchange area as well as placental blood flow may be reduced by injecting microspheres of 15–30 mm diameter into the fetal or maternal placental vascular bed. Some quantification of the embolization procedure is possible, and many experiments have demonstrated that fetal and placental weight can effectively be reduced [25–28,17]. 7.5. Carunclectomy Carunclectomy is possible in sheep which have a cotyledonary placenta. In this species, the blastocyst grows to a size of about 10 cm which fills the uterine cavity before
Fig. 6. Relationship between function and growth in the sheep placenta in late gestation. Some sheep were subjected to restriction of implantation by surgical removal of most placental implantation sites from the uterus prior to pregnancy. From [5], with permission.
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Fig. 7. Effect of restricting placental growth on oxygen delivery to (*) and consumption by (*) the gravid uterus and fetus in sheep in late gestation. Placental growth was restricted as described in Fig. 6. From [5], with permission.
cotyledons are formed. These develop only where caruncles are located in the uterine epithelium. Caruncles can be seen as dark spots when the uterus is opened, and they can be removed or destroyed before the animals conceive [29,30]. The number of caruncles left determines the number of cotyledons and thus largely placental mass later in gestation. Placental weight can thus be made to vary widely, and this clearly affects fetal weight (Fig. 6, Fig. 7, based on [1]). This is caused by reduction of placental exchange functions as seen from the antipyrine clearance. Maternal and fetal placental blood flow also depend on placental weight, and likely the reduction of substrate supply is caused both by reduction of exchange area and by blood flow. However, other, more specific placental functions like hormone production may contribute also to the effects. It is interesting to note (Fig. 7) that as far as oxygen supply is concerned, there appears to be a wide safety margin when oxygen delivery decreases because fetal oxygen uptake and consumption appears to decrease only when placental mass is reduced to about half of normal.
restriction has been induced by ligating one umbilical artery. It appears, however, that this can be survived by fetal sheep only when accomplished at midgestation and not later [33]. It has been demonstrated recently [34] that a controlled reduction (30%) of umbilical artery blood flow for 3 days can be tolerated by the ovine fetus (gestational age 125 days). It is not known whether this technique can be used to provoke fetal growth restriction. It is possible also to affect the supply chain inside the fetus (Fig. 1, level 5), and blood flow through the fetal liver can be changed without altering placental blood flow rate [35]. When the ductus venosus in fetal sheep is closed by an embolization coil, all the blood returning from the
7.6. Partitioning With placental and fetal growth restriction, there is also a change of partitioning. The results from typical experiments ([5]; c.f. also [31,32]) demonstrate (Fig. 8) that normally 57% of the oxygen taken up by the pregnant uterus is consumed by the placenta. In this example, reduction of placental mass reduces placental oxygen consumption from 3.2 mmol/min and kg placental weight to 1.9 mmol/min and kg. Thus oxygen delivery to the fetus is relatively increased and oxygen consumption per kg body weight of the normal and of the growth restricted fetus is not different. It is typical also that in this type of growth restriction amino acids instead of flowing into the fetus may enter placental tissues from fetal protein metabolism for rescue of the placenta. 7.7. Fetal circulation The final member of the supply chain outside the fetal body is umbilical flow (Fig. 1, level 4), and fetal growth
Fig. 8. Partitioning of substrates between placenta and fetus: effects of restriction of placental and fetal growth. Rates of flux of substrates in mmol/min between mother, placenta and fetus in control sheep and in sheep with restricted placental and fetal growth in late gestation. Placental growth was restricted as described in Fig. 6. From [5], with permission.
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Fig. 9. (a) Total fetal placental mass at term, (b) relationship between placental mass and lamb birth weight, (c) fetal cotyledon no. and (d) mean fetal cotyledon weight in adolescent dams over nourished (open bars or circles, n ¼ 37) or moderately-fed (closed bars or circles, n ¼ 34) throughout their entire pregnancy. Values for a, c, and d are means with their standard errors represented by vertical bars. Mean values were significantly different from those for moderately-fed dams: P < 0:001. Data from [63], with permission.
placenta is forced to flow through the liver, and this increases liver weight and the proliferation rate of hepatocytes within several days [36]. Inversely, when the ductus venosus is dilated by implanting a stent, blood flow through the liver and liver proliferation rate appear to be reduced [37]. 7.8. Effects of over-nourishment in adolescent pregnant sheep Paradoxically, when adolescent sheep (60% of adult weight) are over-nourished during pregnancy, fetal and placental weight are distinctly reduced by about 1/3 [38]. The number of cotyledons as well as mean cotyledonary weight are decreased (Fig. 9). The fetal-placenta-weight ratio is maintained, and neonatal mortality and the frequency of still-birth are increased. It is proposed that the main disturbance here is the reduction of placental growth with subsequent fetal growth restriction. The high concentrations of insulin, IGF-I and leptin [39] in maternal plasma typical for over-nourishing perhaps provide a sustained anabolic stimulus for the deposition of maternal tissues (especially lipids), and the stimulus is so powerful that it shifts nutrient supply away from the placenta in spite of increased maternal uptake.
8. Alterations of growth control Increasingly, animal experiments are performed which intend to influence the control mechanisms of growth. From a large number of factors which may be involved very few are addressed here. In principle, the effects of growth controlling substances may be established by decreasing or increasing their concentration in fetal tissues, and this can
Fig. 10. A pair of 6 day old and a pair of 2-month-old littermates, wild type (w and W) and IGF-I (/) mutants (i and I). From [58], with permission.
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Table 3 Summary of mutant phenotypes in mice Knockout
I II I þ II 1R 2R 1R þ 2R I þ 1R II þ 1R I þ 2R II þ 2R II þ 1R þ 2R
Birth weight (% normal)
60 60 30 45 130 100 45 30 74 30
Placental size
Neonatal lethality
Growth rate Embryonic
Postnatal
Normal Restricted ? Normal Normal ? ? Restricted
Increased Normal Always Always Always (if maternal) Variable Always Always
Restricted Restricted Restricted Restricted Increased Normal Restricted Restricted
Restricted Normal
? ?
Normal Always
Restricted Restricted
Normal
Restricted/normal
List of gene ‘‘knockouts’’ of the IGF-axis in mice. Based on [47,59]. Abbreviations: I: IGF-I; II: IGF-II; 1R: type 1 receptor; 2R: type 2 receptor.
be done in several ways. In chronically catheterized fetal sheep, for example, it is possible to infuse selected substances for days or weeks into the fetal circulation. This has been done with variable success with IGF-I [40], catecholamines [41,42] or insulin [43] among other substances. Hormones may be removed by destroying or removing the respective glands, and this has been accomplished with the pancreas [44], the thyroid [45] and the pituitary [46]. Evidence from human fetuses [47–49] and from animal experiments suggest that IGF-I and IGF-II, the IGF receptors, and the six IGF-binding proteins [50–55] may be important to control fetal growth as opposed to embryonic growth (cf. contribution of VKM Han, this issue). With the advent of transgenic and knock-out mice [56–61] it became very clear how important the IGF-system is for fetal growth. For example, Fig. 10 demonstrates wild-type and IGF-Inull-mutant mice at the age of 6 days, and at the age of 2 months [58]. At birth, the IGF I mutant weighs 60% of normal fetuses but the placenta has normal weight, and this growth reduction appears to be maintained during postnatal growth. The knock-out technique has been applied to other genes and gene combinations in the IGF-system, and some of the results are summarized Table 3 (based on [47–59]). Deleting the IGF-II gene has an effect on birth weight comparable to IGF-I deletion but lethality of the neonates is zero, and postnatally, the animals grow to normal size and are fertile. If both IGF-I and IGF-II are knocked out, the fetuses weigh only 30% of normal at birth, and they cannot survive. When the type-1 IGF-receptor is not expressed, fetal growth also is strongly restricted, but with knock-out of the type-2 receptor, fetal growth is increased by one-third. A possible explanation is that the type-2 receptor binds IGFs and effectively functions as an inhibiting growth factor sink. When both receptors are deleted, fetal growth is normal. This surprising observation hints at effects of the IGFs which are not mediated by the type-1/type-2 receptors, and as the result of an additional knock-out of IGF-II indicates, it may be IGF-II which is a ligand for this unknown receptor.
In the future, much more will be learnt from these exciting experiments, especially if it becomes possible to repeat them in other species, and to target the gene knock-out or gene over-expression to defined tissues, and perhaps to defined time-points during embryonic and fetal development (cf. VKM Han, this issue). In summary, to explore the intricacies of fetal growth restriction, whole animal models are valuable and indispensable tools. They have yielded many useful insights into the basic mechanisms underlying fetal growth, and they are necessary both for the advancement of science, and for the benefit of patients, either in utero as fetus, or later in life as adults [62].
References [1] Owens JA, Owens PC, Robinson JS. Experimental fetal growth retardation: metabolic and endocrine aspects. In: Gluckman PD, Johnston BM, Nathanielsz PW, editors. Research in perinatal medicine (VIII). Advances in fetal physiology: reviews in honor of G.C. Liggins. Ithaca (New York): Perinatology Press; 1989. p. 263–86. [2] Carter AM. Placental blood flow and fetal oxygen supply in animal models of intra-uterine growth retardation. In: Soma H, editor. Placenta: basic research for clinical application. Basel: Karger; 1991. p. 23–31. [3] Owens JA. Endocrine and substrate control of fetal growth— placental and maternal influences and insulin-like growth factors. Reprod Fertil Dev 1991;3:501–17. [4] Carter AM. Animal models in fetal physiology. In: Svendsen P, Hau J, editors. Handbook of laboratory animal science, vol. 2. London: CRC Press; 1994. p. 77–92. [5] Owens JA, Owens PC, Robinson JS. Experimental restriction of fetal growth. In: Hanson MA, Spencer JAD, Rodeck CH, editors. Fetus and neonate: physiology and clinical applications. Growth, vol. 3. Cambridge: Cambridge University Press; 1995. p. 139–75. [6] Robinson JS, Owens JA. Pathophysiology of intrauterine growth failure. In: Gluckman PD, Heyman MA, editors. Pediatrics and perinatology. The scientific basis. 2nd ed. London: Edward Arnold; 1996. p. 290–97. [7] Sparks JW, Ross JC, Cetin I. Intrauterine growth and nutrition. In: Polin RA, Fox WW, editors. Fetal and neonatal physiology. 2nd ed., vol. 1. Philadelphia: Saunders; 1998. p. 267–89.
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[8] Han VK, Carter AM. Control of growth and development of the fetoplacental unit. Curr Opin Pharmacol 2001;1:632–40. [9] Ciba Foundation Symposium 27. Size at Birth. Ciba Foundation Symposia. Amsterdam: Associated Scientific Publishers; 1974. [10] Sharp F, Fraser RB, Milner RDG, editors. Fetal Growth. London: Springer; 1989. [11] Gluckman PD, Johnston BM, Nathanielsz PW, editors. Research in perinatal medicine (VIII). Advances in fetal physiology: reviews in honor of G.C. Liggins. Ithaca, New York: Perinatology Press; 1989. [12] Hanson MA, Spencer JAD, Rodeck CH, editors. Fetus and neonate: physiology and clinical applications. Growth, vol. 3. Cambridge: Cambridge University Press; 1995. [13] Gluckman PD, Heymann MA, editors. Pediatrics and perinatology: the scientific basis. 2nd ed. London: Edward Arnold; 1996. [14] Harding R, Bocking AD, editors. Fetal growth and development. Cambridge: Cambridge University Press; 2001. [15] Fowden AL. Growth and metabolism. In: Harding R, Bocking AD, editors. Fetal growth and development. Cambridge: Cambridge University Press; 2001. p. 44–69. [16] Kitanaka T, Gilbert RD, Longo LD. Maternal and fetal responses to long-term hypoxemia in sheep. In: Ku¨ nzel W, Jensen A, editors. The endocrine control of the fetus: physiologic and pathophysiologic aspects. Berlin: Springer; 1988. p. 38–63. [17] Pennell CE, Smyth JP, Turner AJ, Coughtrey H, Murray HG, Newnham JP. The effects of growth restriction on the fetal heart rate patterns and blood pressure responses to repeated cord occlusion in the ovine fetus. J Soc Gynecol Invest 2002;9(Suppl):64A [abstract]. [18] Schro¨ der HJ, Power GG. Engine and radiator: fetal and placental interactions for heat dissipation. Exp Physiol 1997;82:403–14. [19] Alexander G, Williams D. Heat stress and development of the conceptus in the domestic sheep. J Agricult Sci (Cambridge) 1971;76:53–72. [20] Ross JC, Fennessey PV, Wilkening RB, Battaglia FC, Meschia G. Placental transport and fetal utilization of leucine in a model of fetal growth retardation. Am J Physiol 1996;270:E491–503. [21] Alexander G, Hales JRS, Stevens D, Donnelly JB. Effects of acute and prolonged exposure to heat on regional blood flows in pregnant sheep. J Dev Physiol 1987;9:1–15. [22] van Martens EV, Harel S, Zamenhof S. Experimental intrauterine growth retardation. Biol Neonate 1975;26:221–31. [23] Carter AM. Current topic: restriction of placental and fetal growth in the guinea-pig. Placenta 1993;14:125–35. [24] Clark KE, Durnwald M, Austin JE. A model for studying chronic reduction in uterine blood flow in pregnant sheep. Am J Physiol 1982;242:H297–301. [25] Creasy RK, Barrett CT, de Swiet M, Kahanpaa KV, Rudolph AM. Experimental intra-uterine growth retardation in the sheep. Am J Obstet Gynecol 1972;112:566–73. [26] Charlton V, Johengen M. Fetal intravenous nutritional supplementation ameliorates the development of embolization-induced growth retardation in the sheep. Pediatr Res 1987;22:55–61. [27] Trudinger BJ, Stevens D, Connelly A, et al. Umbilical artery flow velocity waveforms and placental resistance: The effects of embolization of the umbilical circulation. Am J Obstet Gynecol 1987;157:1443–8. [28] Gagnon R, Challis J, Johnston L, Fraher L. Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus. Am J Obstet Gynecol 1994;170:929–38. [29] Alexander G. Studies on the placenta of the sheep (ovies aries L.). Effect of surgical reduction in the number of caruncles. J Reprod Fertil 1964;7:307–22. [30] Robinson JS, Kingston EJ, Jones CT, Thorburn GD. Studies on experimental growth retardation in sheep. The effect of removal of endometrial caruncles on fetal size and metabolism. J Dev Physiol 1979;1:379–98. [31] Cetin I, Marconi AM, Bozzetti P, et al. Umbilical amino acid concentrations in appropriate and small-for-gestational age infants: a
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42] [43] [44]
[45]
[46]
[47]
[48]
[49]
[50]
[51] [52]
biochemical difference present in utero. Am J Obstet Gynecol 1988;158:120–6. Cetin I. Amino acid interconversions in the fetal-placental unit: the animal model and human studies in vivo. Pediatr Res 2001;49: 148–54. Emmanouilides GC, Townsend DE, Bauer RA. Effects of single umbilical artery ligation in the lamb fetus. Pediatrics 1968;42: 919–27. Gardner DS, Fletcher AJ, Fowden AL, Giussani DA. A novel method for controlled and reversible long term compression of the umbilical cord in fetal sheep. J Physiol 2001;535:217–29. Tchirikov M, Schro¨ der HJ. Single case report: late gestational fetal sheep may survive blockage of the ductus venosus for 1 week. Placenta 1998;19:333–6. Tchirikov M, Kertschanska S, Schro¨ der HJ. Obstruction of ductus venosus stimulates cell proliferation in organs of fetal sheep. Placenta 2001;22:24–31. Tchirikov M, Kertschanska S, Stu¨ renberg HJ, Schro¨ der HJ. Liver blood perfusion as a possible instrument for fetal growth regulation. Troph Res 2002;16:S153–8. Wallace JM. Nutrient partitioning during pregnancy: adverse gestational outcome in overnourished adolescent dams. Proc Nutr Soc 2000;59:107–17. Thomas L, Wallace JM, Aitken RP, Mercer JG, Trayhurn P, Hoggard N. Circulating leptin during ovine pregnancy in relation to maternal nutrition, body composition and pregnancy outcome. J Endocrinol 2001;169:465–76. Lok F, Owens JA, Mundy L, Robinson JS, Owens PC. Insulin-like growth factor I promotes growth selectively in fetal sheep in late gestation. Am J Physiol 1996;270:R1148–55. Bassett JM. Glucose and fetal growth derangement. In: Hanson MA, Spencer JAD, Rodeck CH, editors. Fetus and neonate: physiology and clinical applications. Growth, vol. 3. Cambridge: Cambridge University Press; 1995. p. 223–54. Milley JR. Ovine fetal metabolism during norepinephrine infusion. Am J Physiol 1997;273:E336–47. Milley JR. The effect of chronic hyprinsulinemia on ovine fetal growth. Growth 1986;50:390–401. Fowden AL, Mao XZ, Comline RS. Effects of pancreatectomy on the growth and metabolite concentrations of the sheep fetus. J Endocrinol 1986;110:225–31. Thorburn GD. The role of the thyroid gland and kidneys in fetal growth. Ciba Foundation Symposium 27: size at birth. Amsterdam: Associated Scientific Publishers; 1974. p. 185–214. Mesiano S, Young IR, Baxter RC, Hintz RL, Browne CA, Thorburn GD. Effect of hypophysectomy with and without thyroxine replacement on growth and circulating concentrations of insulin-like growth factors I and II in the fetal lamb. Endocrinology 1987;120:1821–30. Gluckman P.D., Insulin-like growth factors and their binding proteins. In: Hanson MA, Spencer JAD, Rodeck CH, editors. Fetus and neonate: physiology and clinical applications. Growth, vol. 3. Cambridge: Cambridge University Press; 1995. p. 97–115. Giudice LC, DeZegher F, Gargosky SE, et al. Insulin-like growth factors and their binding proteins in the term and preterm human fetus and neonate with normal and extremes of intrauterine growth. J Clin Endocrinol Metab 1995;80:1548–55. Christou H, Connors JM, Ziotopoulou M, et al. Cord blood leptin and insulin-like growth factor levels are independent predictors of fetal growth. J Clin Endocrinol Metab 2001;86:935–8. Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocrinol Rev 1989;10:68–91. Rotwein P. Structure, evolution, expression and regulation of insulinlike growth factors I and II. Growth Factors 1991;5:3–18. Kelley KM, Youngman OH, Gargosky SE, et al. Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics. Int J Biochem Cell Biol 1996;28:619–37.
H.J. Schro¨ der / European Journal of Obstetrics & Gynecology and Reproductive Biology 110 (2003) S29–S39 [53] Rajaram S, Baylink DJ, Mohan S. Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocrinol Rev 1997;18:801–31. [54] Reinecke M, Collet C. The phylogeny of the insulin-like growth factors. Int Rev Cytol 1998;183:1–94. [55] Hwa V, Youngman OH, Rosenfeld RG. The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocrinol Rev 1999; 20:761–87. [56] DeChiara TM, Efstratiadis A, Robertson EJ. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 1990;345:78–80. [57] Liu J-P, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 1993;75:59–72. [58] Baker J, Liu J-P, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993;75:73–82.
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[59] Efstratiadis A. Genetics of growth: developmental roles of IGF and insulin receptors. Exp Clin Endocrinol Diabetes 1996;104: 4–6. [60] Eggenschwiler J, Ludwig T, Fisher P, Leighton PA, Tilghman SM, Efstratiadis A. Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith–Wiedemann and Simpson–Golabi–Behmel syndromes. Genes Dev 1997;11: 3128–42. [61] Schneider MR, Lahm H, Wu M, Hoeflich A, Wolf E. Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins. FASEB J 2000;14:629–40. [62] Barker DJP, Fall CHD. Fetal and infant origins of cardiovascular disease. Arch Dis Child 1993;68:797–9. [63] Wallace JM, Bourke DA, Aitken RP. Nutrition and fetal growth: paradoxical effects in the overnourished adolescent sheep. J Reprod Fertil 1999;54(Suppl):385–99.
Models of fetal growth restriction Hobe J. Schro¨der* Abteilung fu¨r experimentelle Medizin, Klinik und Poliklinik fu¨r Frauenheilkunde und Geburtshilfe, Universita¨tsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany
Abstract The growth of the fetus is determined by substrate supply mostly for mass accretion and energy gain, and by control systems. Experiments with whole animal models will face the following problems: (1) The fetus, like a three compartmental ‘‘Russian doll’’, is at the end of a long supply chain. There are interactions (e.g. hormones) and partitioning of substrates between the compartments. (2) The fetal organism is growing anddifferentiating at the same time and not in a steady-state. Experimental results thus dependon gestationalage. (3) About 75% of animal experiments on fetal growth restriction have been performed in rats and mice. The possible experimental methods and the results depend on the species which include sheep, pigs, rabbits, guinea pigs, horses and non-human primates. Many experiments have clearly shown that restriction of substrate supply will usually impair fetal growth. Less is known about growth control mechanisms but recent studies in gene mutant mice have opened a new approach to study the effects of systemic and local controlling factors. It appears that insulin-like growth factors and their binding proteins may play an important role for fetal growth. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Substrate supply; Control mechanisms; Gene mutants; Fetal growth
The use of animal models to explore fetal growth restriction has been extensively dealt with in excellent review articles [1–8] and books [9–14]. This brief overview focuses on general problems which are inherent to studies with whole pregnant animals, and on some recent results on the control of fetal growth.
1. Introduction As a first approach fetal growth can be compared to building a house. A plan (genome) is required as well as energy (metabolism), building materials (nutrition) and transportation (circulation). The accretion of material will have to follow a precise sequence in time and space because one cannot start with the top of the chimney, for example. However, this comparison between the house model and fetal growth is inappropriate in one important aspect: in the fetus growth and differentiation (which implies the maturation of functions) occur simultaneously. Although growth and differentiation appear to be inseparable, they actually may be controlled differently by growth-promoting and * Tel.: þ49-40-42803-3533; fax: þ49-40-42803-2395. E-mail address: [email protected] (H.J. Schro¨der).
by differentiation-promoting factors. This difference is hypothetical at present, but it may provide some day the key for the treatment of intrauterine growth restriction: if substrate supply is insufficient for both growth and differentiation, perhaps intrauterine growth can be halted and postponed until birth whereas differentiation and maturation can be made to continue successfully in utero. The useful message from the house model is that growth is determined by substrate supply, either for mass accretion or for energy gain, and by control systems, and this distinction will be followed to describe experimental approaches.
2. The Russian doll The schematic (Fig. 1) illustrates that the fetus is located inside the uterine-placental unit, which in turn is located inside the mother. This is the ‘‘Russian doll situation’’ which has, simple as it may be, far reaching consequences for the study of fetal growth restriction. As an example, we consider a study on the effects of reduced oxygen supply for growth and development of the fetal brain. Obviously, in this experiment fetal oxygen supply should somehow be diminished and Fig. 1 illustrates that the availability of oxygen may be restricted at various levels of the supply chain.
0301-2115/$ – see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0301-2115(03)00170-2
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Fig. 1. The ‘‘Russian doll situation’’. Single headed arrows indicate flux of substrates with five possible positions (level 1 to level 5) for blocking substrate transfer (bars). Double headed arrows symbolize ‘‘partitioning’’ and mutual interactions between the three compartments (see text).
The first and most ‘‘upstream’’ level would be the reduction of maternal oxygen intake. Alternatively and hypothetically, it might be accomplished to reduce the amount of oxygen transferred to the uterine placental exchange unit (level 2), to restrict the transfer of oxygen to the fetus through the placenta (level 3) or inside the uterus via the umbilical vein (level 4), and finally to reduce oxygen supply to the fetal brain but not to other fetal regions (level 5). Even if it were experimentally possible to affect exclusively the supply of oxygen at each the above five levels (in fact, it is not), these were five different experiments. In general, the more upstream in the supply chain an experimental intervention is executed, the more global and possibly unspecific the downstream effects may be. For example, chronic maternal hypoxemia as in high altitude experiments (in growth restriction experiments interventions have to be long lasting and chronic) may affect the maternal cardiovascular system and thus maternal perfusion of the placenta, it may affect placental functions, it may affect the transfer capability of
the fetal cardiovascular system in general, and all these disturbances triggered along the supply chain may confound the effect of oxygen lack on the fetal brain, either aggravating it or alleviating it. From the ‘‘Russian doll’’ structure the problem of ‘‘interaction’’ between fetus, placenta and maternal tissues arises. Mother, the uterine-placental unit and the fetus do not act independently of each other, they rather cooperate, and the influence of placental hormones on maternal tissues is a long known example for this interaction. In metabolic studies, partitioning relates to the distribution of substrates between the three compartments. Experimental growth restriction affects partitioning, and experimental changes of partitioning may produce growth restricted fetuses (see below). Finally, maternal, utero-placental and fetal tissues are living tissues capable of adjustment and homeostatic regulations which do not simply subdue passively to imparted disturbances, and as the placenta and fetus are developing, they are certainly not in a steady-state situation. These problems are not unheard of in other whole-animal experiments, but they are especially concentrated and cumbersome in the study of fetal growth. The significance of these issues depend to some extent on the type of questions asked which can be divided into two broad categories. One is to imitate clinical situations which are known to be associated with growth restriction. Some ‘‘extrinsic’’ factors, that is extrinsic of the fetus, are listed in Table 1 which is abbreviated from a much longer list [5]. In this type of study the focus is on experimental interventions which imitate pathological human situations, and maternal, placental and fetal changes in form and functions may be observed. This may lead to diagnostic, therapeutic and pathophysiological insights. On the other side, in a reductionistic approach to
Table 1 Conditions associated with human fetal growth restriction Intrinsic
Extrinsic
Fetal factors
Environmental factors
Maternal factors
Intrauterine infections Chromosomal mosaicism Chromosomal anomalies Congenital malformations Inborn errors of metabolism Anaemia (Rh disease)
High altitude Variable nutrition Pollution Hyperthermia Irradiation
Undernutrition (including anorexia) Abnormalities Low maternal weight gain Chromosomal mosaicism Maternal age <16 Infarcts, focal lesions Drug use Abnormal placentation Smoking Alcohol Illicit drugs Low socio-economic status Medical complications Acute or chronic hypertension Antepartum haemorrhage Severe chronic infections Anaemia Disseminated lupus erythematosus Lupus obstetric syndrome Malignancy Abnormalities of the uterus Uterine fibroids
List of conditions associated with human fetal growth restriction. From [5].
Placental factors
Other factors Reproductive technologies
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unravel the complete, step-by-step cause-effect relationships in fetal growth restriction, the ‘‘Russian doll’’ situation with its many interdependencies poses much more of a problem. Of course, the questions asked in most studies will fall in between these two extremes.
3. Fetal growth restriction Normal fetal growth has been described as ‘‘the expression of the genetic potential to grow which is neither abnormally constrained nor promoted by internal or external factors’’. In this definition by Owens, Owens and Robinson [1] ‘‘internal’’ refers to fetal factors whereas ‘‘external’’ relates to anything else, that is placenta, uterus, mother and environment. Growth can be expressed as the gain in mass, or the gain in length, in area or in volume. With the advent of ultrasound B-scan, it has become possible in principle to continually follow the change of fetal dimensions during gestation, and based on these measurements, to follow the increase of (estimated) fetal weight. Total weight is the sum of all fetal organs and tissues, at birth especially of skeletal muscle, skeleton and fat, but single organs may follow their own growth trajectory which can be differently affected in growth restriction. This is the basis for the difference between proportionate and disproportionate growth with a reduced weight–length ratio in the second case. To establish growth restriction, abnormal deviation from the genetically determined growth trajectory has to be observed. The problem is that it is difficult to gain precise prospective knowledge of the undisturbed growth curve of an individual fetus, either in experimental animals, or in human fetuses. Perhaps volume measurements with three dimensional ultrasound B-scan may some day allow to predict individual fetal organ growth curves and their disturbances with sufficient precision not because the weight is less than some ‘‘normal’’ value, but because the growth trajectory has slowed down. At present, however, the diagnosis of growth restriction is based mostly on statistics, and usually growth curves with limits of what is considered normal are constructed. Growth restriction then frequently is defined as fetal weight below the 10th percentile at a given moment in gestation. Determining gestational age with accuracy may be difficult in the clinical situation, and this adds to the diagnostic uncertainty. There is, of course, no one normal growth curve which fits all, but number of fetuses, ethnic groups, socio-economic status and other parameters have to be considered. In animal experiments, gestational age should be known precisely and the genetically dependent variations may be minimized. It is typical then (Fig. 2) to observe two groups of growing fetuses, with a defined imposed disturbance in the experimental group, as in this example: when food intake of ewes is restricted starting at day 115 of gestation, the normal course of the crown-rump length (CRL) development
Fig. 2. Time course of a typical experiment on growth restriction. Food intake of maternal sheep is restricted at day 115 in the experimental group (term in sheep 145 days), and the increase of the CRL starts to slow down approximately at day 120. From [15], with permission.
is altered, and the difference becomes significant at some time point [15].
4. The experimental window Because the growing fetus is not in a steady-state, the same experimental intervention may have different effects when applied at different gestational ages, and the placement and the characteristics of the ‘‘experimental window’’ have to be considered carefully (Fig. 3). A given disturbance of
Fig. 3. The experimental window in growth restriction experiments. Three hypothetical experiments (A: short dashed lines; B: dotted lines; C: long dashed lines) are indicated which vary by intensity and type of interventions (vertical arrows), by their onset and duration (horizontal arrows), and by their outcome. Experiment A is successful because the surviving fetus has a body weight distinctly below the 10% percentile for normal fetuses. In B, the intervention leads to fetal death (cross) with no clear impairment of growth. Experiment C demonstrates that the fetal growth trajectory has been affected, but fetal body weight is not reduced below the 10% percentile. The time course in C may reflect fetal and/or maternal adaptations (see text). Note that fetal body weight and gestational age are indicated as fractions of normal values.
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medium intensity (Fig. 3A) may last until delivery of a growth restricted fetus, and this will be a successful experiment. Some time later in gestation (Fig. 3B), a large disturbance of short duration is imposed, but this time the fetus dies before growth is clearly reduced, and this is an experimental failure. A long lasting (Fig. 3C), late gestational disturbance of small intensity may produce no detectable growth alteration at all. An example for this experimental outcome is a carefully conducted study with sheep on the effect of lowering the oxygen fraction in inspired maternal air for 3 weeks [16]. For 21 days fetal and maternal blood was monitored daily, and fetal arterial pO2 stayed reduced to 2/3 of normal (from 30 to about 22 mmHg) all the time, yet fetal weight was not changed. The main reason was that fetal arterial oxygen content was restored to normal values already at day 5 of the hypoxic period. This experimental outcome illustrates the adaptive mechanisms available for mother and fetus. However, when fetal arterial pO2 is reduced for 23 days to 12 mmHg by daily injections of microspheres into the fetal placental circulation [17], fetal body weight is distinctly reduced (see below).
5. Animal species in fetal growth restriction experiments
genetic studies. It is important to keep this ‘‘uneven distribution of methods’’ in mind because in reproductive sciences species differences are especially pronounced.
6. Experiments in the real world Many experimental approaches to restrict fetal growth have been designed as listed in Table 2 [5]. The sites of interventions are classified as maternal, placental and fetal with more specific manipulations at each level as indicated. This useful classification may be divided into experiments which manipulate the supply of substrates, and experiments which manipulate the control of growth.
7. Restriction of substrate supply 7.1. Maternal intake Restricting substrates is possible at various points of the supply chain (Fig. 1), and one way is manipulation of maternal intake which has the advantage that it does not require surgical intervention. This is very much ‘‘upstream’’
Various mammalian species have been used as models of fetal growth restriction, and Fig. 4 demonstrates that about three quarter of experiments have been performed in two rodent species, rats and mice. Naturally, the types of experiments are not equally divided between the species. If fetal blood samples are to be drawn daily, for example, one of the larger species for implanting chronic catheters will have to be used, and here the chronically instrumented fetal sheep is still one of the favorites. The small species are used frequently in studies which apply techniques based on morphology and on molecular biology like development of structures and weight, distribution of hormones and growth factors and their respective receptors in fetal tissues, and in
Fig. 4. Distribution of species in animal experiments on fetal growth restriction which have been published (numbers are number of articles). The search phrase in Medline83 was ‘‘animal name þ fet? þ growth þ retardation’’, search performed in August 2001.
Fig. 5. Typical changes in growth restriction experiments following undernutrition or hypobaric hypoxia of the mother (based on [1]). (a) Fetal body and organ weights, and of CRL, as percentages of control values. (b) Concentrations of substrates in arterial blood as percentages of control values.
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Table 2 Experimental restriction of fetal growth: interventions and sites of action Site of intervention Maternal Altered maternal substrate availability Maternal undernutrition Protein/caloric deficiency Selected nutrient deficiency Micronutrient deficiency Maternal hypoxaemia Altitude Hypobaric hypoxaemia Normobaric hypoxaemia Reduced maternal fractional inspired oxygen content Intratracheal nitrogen Carbon monoxide Anaemia Methhaemoglobinaemia Maternal hypertension
Species
Sheep, pig, rat, mouse
Sheep, rat, guinea pig
Baboon, sheep, rat
Placental Reduction/restriction of placental growth Sheep Reduction in number of implantation sites (uterine endometrial caruncles) prior to pregnancy by hemihysterectomy or carunclectomy Maternal hyperthermia Separation of placentomes during pregnancy Interference with uteroplacental vasculature Sheep, monkey, guinea pig, rabbit, rat Partial occlusion of uterine artery Embolization of uteroplacental circulation Ligation of interplacental vessels Ligation of uterine artery Interference with fetoplacental circulation Sheep Embolization via umbilical artery Fetal Viral infection Organ/gland ablation Hypophysectomie, hypothalamic–pituitary disconnection Thyroidectomy, radioactive iodine Pancreatectomy (surgical, chemical: streptozotocin) Nephrectomy Other Banding ascending aorta Fistula Gene deletion Insulin-like growth factor axis IGF-I, IGF-II, type 1 receptor, type 2 receptor, IGFBPs, c-jun
Mouse Sheep, monkey
Sheep
Mouse
List of experimental procedures to generate fetal growth restriction. From [5].
and the ‘‘Russian doll’’ structure may cause difficulties in interpretation of the results, but the restriction can be very specific down to the level of one atom or molecule when exclusively the availability of oxygen is reduced, or when one essential amino acid in food is omitted, for example. In addition, this type of animal experiment can be close to what may happen to human patients. The effects chronic undernutrition (that is reduced caloric intake of an otherwise normally balanced food) and chronic lack of oxygen of the mother may have on fetal growth have frequently been investigated, and some of the results obtained in fetal sheep are summarized in Fig. 5 (based on [1]). Undernutrition and lack of oxygen both may restrict fetal and placental growth
and the growth of single fetal organs, but there are differences (Fig. 5a). Typically, brain weight is least affected whereas liver weight suffers especially with low nutritional intake. Hypoxemia, on the other hand, may even increase growth of an organ like the adrenals which reflects the high levels of plasma catecholamines typical for this situation. The concentrations of some substrates in fetal arterial blood may be affected also (Fig. 5b). Undernutrition especially generates fetal hypoglycemia, with resultant hypoinsulinemia not shown here, whereas maternal hypoxia generates fetal hypoxemia, increase of lactate and the increase of certain amino acids like alanine, or the decrease of others as well. It is emphasized that these are possible changes, and
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depending on the exact experimental protocol and species, variations are considerable. Basically, inside the mother (Fig. 1) substrate supply to the fetus can be restricted at three sites: at maternal vessels which supply the uterine-placental unit (level 2), at the placenta itself (level 3), and at umbilical vessels (level 4). These approaches are further ‘‘downstream’’ in the supply chain than the restriction of maternal uptake (level 1), and they are in one sense more specific because, for example, ligating one uterine artery should not affect too much other maternal tissues. On the other hand, reducing blood flow through the placenta, or reducing placental mass may affect the supply and transfer of all substances (and heat [18]) that need to be exchanged between mother and fetus, and the effects will be rather global therefore. A surgical intervention usually is necessary to reduce blood flow and placental mass though Alexander has shown years ago in sheep that chronic maternal hyperthermia generates fetal growth restriction [19,20] because maternal placental blood flow is reduced [21]. As surgery may directly affect the fetal growth trajectory, the surgical intervention itself is another confounding variable in growth restriction studies and the use of sham operated animals as controls is typical. 7.2. Utero-placental circulation In small polytocous mammals like guinea pigs the maternal side of the placentas are supplied with blood from an arcade between the uterine and ovarian artery. Ligating the uterine artery at this point reduces the supply to placentae and fetuses which are now at the end of the ovarian pipe line.
This technique has been applied especially in the guinea pig and reliably reduces fetal and placental weight [22,23]. It is, however, an all or nothing approach. In large species like the sheep, an adjustable occluder with exteriorized controller has been placed around the maternal common iliac artery which allows resistance changes as required [24]. In combination with flow probes placed on both uterine arteries, the experimentalist has complete quantitative control over maternal placental blood supply. 7.3. Reduction of placental mass Two approaches are in use to reduce placental mass and exchange, and both are typically applied to fetal sheep: embolization and carunclectomy. 7.4. Embolization With embolization, placental exchange area as well as placental blood flow may be reduced by injecting microspheres of 15–30 mm diameter into the fetal or maternal placental vascular bed. Some quantification of the embolization procedure is possible, and many experiments have demonstrated that fetal and placental weight can effectively be reduced [25–28,17]. 7.5. Carunclectomy Carunclectomy is possible in sheep which have a cotyledonary placenta. In this species, the blastocyst grows to a size of about 10 cm which fills the uterine cavity before
Fig. 6. Relationship between function and growth in the sheep placenta in late gestation. Some sheep were subjected to restriction of implantation by surgical removal of most placental implantation sites from the uterus prior to pregnancy. From [5], with permission.
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Fig. 7. Effect of restricting placental growth on oxygen delivery to (*) and consumption by (*) the gravid uterus and fetus in sheep in late gestation. Placental growth was restricted as described in Fig. 6. From [5], with permission.
cotyledons are formed. These develop only where caruncles are located in the uterine epithelium. Caruncles can be seen as dark spots when the uterus is opened, and they can be removed or destroyed before the animals conceive [29,30]. The number of caruncles left determines the number of cotyledons and thus largely placental mass later in gestation. Placental weight can thus be made to vary widely, and this clearly affects fetal weight (Fig. 6, Fig. 7, based on [1]). This is caused by reduction of placental exchange functions as seen from the antipyrine clearance. Maternal and fetal placental blood flow also depend on placental weight, and likely the reduction of substrate supply is caused both by reduction of exchange area and by blood flow. However, other, more specific placental functions like hormone production may contribute also to the effects. It is interesting to note (Fig. 7) that as far as oxygen supply is concerned, there appears to be a wide safety margin when oxygen delivery decreases because fetal oxygen uptake and consumption appears to decrease only when placental mass is reduced to about half of normal.
restriction has been induced by ligating one umbilical artery. It appears, however, that this can be survived by fetal sheep only when accomplished at midgestation and not later [33]. It has been demonstrated recently [34] that a controlled reduction (30%) of umbilical artery blood flow for 3 days can be tolerated by the ovine fetus (gestational age 125 days). It is not known whether this technique can be used to provoke fetal growth restriction. It is possible also to affect the supply chain inside the fetus (Fig. 1, level 5), and blood flow through the fetal liver can be changed without altering placental blood flow rate [35]. When the ductus venosus in fetal sheep is closed by an embolization coil, all the blood returning from the
7.6. Partitioning With placental and fetal growth restriction, there is also a change of partitioning. The results from typical experiments ([5]; c.f. also [31,32]) demonstrate (Fig. 8) that normally 57% of the oxygen taken up by the pregnant uterus is consumed by the placenta. In this example, reduction of placental mass reduces placental oxygen consumption from 3.2 mmol/min and kg placental weight to 1.9 mmol/min and kg. Thus oxygen delivery to the fetus is relatively increased and oxygen consumption per kg body weight of the normal and of the growth restricted fetus is not different. It is typical also that in this type of growth restriction amino acids instead of flowing into the fetus may enter placental tissues from fetal protein metabolism for rescue of the placenta. 7.7. Fetal circulation The final member of the supply chain outside the fetal body is umbilical flow (Fig. 1, level 4), and fetal growth
Fig. 8. Partitioning of substrates between placenta and fetus: effects of restriction of placental and fetal growth. Rates of flux of substrates in mmol/min between mother, placenta and fetus in control sheep and in sheep with restricted placental and fetal growth in late gestation. Placental growth was restricted as described in Fig. 6. From [5], with permission.
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Fig. 9. (a) Total fetal placental mass at term, (b) relationship between placental mass and lamb birth weight, (c) fetal cotyledon no. and (d) mean fetal cotyledon weight in adolescent dams over nourished (open bars or circles, n ¼ 37) or moderately-fed (closed bars or circles, n ¼ 34) throughout their entire pregnancy. Values for a, c, and d are means with their standard errors represented by vertical bars. Mean values were significantly different from those for moderately-fed dams: P < 0:001. Data from [63], with permission.
placenta is forced to flow through the liver, and this increases liver weight and the proliferation rate of hepatocytes within several days [36]. Inversely, when the ductus venosus is dilated by implanting a stent, blood flow through the liver and liver proliferation rate appear to be reduced [37]. 7.8. Effects of over-nourishment in adolescent pregnant sheep Paradoxically, when adolescent sheep (60% of adult weight) are over-nourished during pregnancy, fetal and placental weight are distinctly reduced by about 1/3 [38]. The number of cotyledons as well as mean cotyledonary weight are decreased (Fig. 9). The fetal-placenta-weight ratio is maintained, and neonatal mortality and the frequency of still-birth are increased. It is proposed that the main disturbance here is the reduction of placental growth with subsequent fetal growth restriction. The high concentrations of insulin, IGF-I and leptin [39] in maternal plasma typical for over-nourishing perhaps provide a sustained anabolic stimulus for the deposition of maternal tissues (especially lipids), and the stimulus is so powerful that it shifts nutrient supply away from the placenta in spite of increased maternal uptake.
8. Alterations of growth control Increasingly, animal experiments are performed which intend to influence the control mechanisms of growth. From a large number of factors which may be involved very few are addressed here. In principle, the effects of growth controlling substances may be established by decreasing or increasing their concentration in fetal tissues, and this can
Fig. 10. A pair of 6 day old and a pair of 2-month-old littermates, wild type (w and W) and IGF-I (/) mutants (i and I). From [58], with permission.
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Table 3 Summary of mutant phenotypes in mice Knockout
I II I þ II 1R 2R 1R þ 2R I þ 1R II þ 1R I þ 2R II þ 2R II þ 1R þ 2R
Birth weight (% normal)
60 60 30 45 130 100 45 30 74 30
Placental size
Neonatal lethality
Growth rate Embryonic
Postnatal
Normal Restricted ? Normal Normal ? ? Restricted
Increased Normal Always Always Always (if maternal) Variable Always Always
Restricted Restricted Restricted Restricted Increased Normal Restricted Restricted
Restricted Normal
? ?
Normal Always
Restricted Restricted
Normal
Restricted/normal
List of gene ‘‘knockouts’’ of the IGF-axis in mice. Based on [47,59]. Abbreviations: I: IGF-I; II: IGF-II; 1R: type 1 receptor; 2R: type 2 receptor.
be done in several ways. In chronically catheterized fetal sheep, for example, it is possible to infuse selected substances for days or weeks into the fetal circulation. This has been done with variable success with IGF-I [40], catecholamines [41,42] or insulin [43] among other substances. Hormones may be removed by destroying or removing the respective glands, and this has been accomplished with the pancreas [44], the thyroid [45] and the pituitary [46]. Evidence from human fetuses [47–49] and from animal experiments suggest that IGF-I and IGF-II, the IGF receptors, and the six IGF-binding proteins [50–55] may be important to control fetal growth as opposed to embryonic growth (cf. contribution of VKM Han, this issue). With the advent of transgenic and knock-out mice [56–61] it became very clear how important the IGF-system is for fetal growth. For example, Fig. 10 demonstrates wild-type and IGF-Inull-mutant mice at the age of 6 days, and at the age of 2 months [58]. At birth, the IGF I mutant weighs 60% of normal fetuses but the placenta has normal weight, and this growth reduction appears to be maintained during postnatal growth. The knock-out technique has been applied to other genes and gene combinations in the IGF-system, and some of the results are summarized Table 3 (based on [47–59]). Deleting the IGF-II gene has an effect on birth weight comparable to IGF-I deletion but lethality of the neonates is zero, and postnatally, the animals grow to normal size and are fertile. If both IGF-I and IGF-II are knocked out, the fetuses weigh only 30% of normal at birth, and they cannot survive. When the type-1 IGF-receptor is not expressed, fetal growth also is strongly restricted, but with knock-out of the type-2 receptor, fetal growth is increased by one-third. A possible explanation is that the type-2 receptor binds IGFs and effectively functions as an inhibiting growth factor sink. When both receptors are deleted, fetal growth is normal. This surprising observation hints at effects of the IGFs which are not mediated by the type-1/type-2 receptors, and as the result of an additional knock-out of IGF-II indicates, it may be IGF-II which is a ligand for this unknown receptor.
In the future, much more will be learnt from these exciting experiments, especially if it becomes possible to repeat them in other species, and to target the gene knock-out or gene over-expression to defined tissues, and perhaps to defined time-points during embryonic and fetal development (cf. VKM Han, this issue). In summary, to explore the intricacies of fetal growth restriction, whole animal models are valuable and indispensable tools. They have yielded many useful insights into the basic mechanisms underlying fetal growth, and they are necessary both for the advancement of science, and for the benefit of patients, either in utero as fetus, or later in life as adults [62].
References [1] Owens JA, Owens PC, Robinson JS. Experimental fetal growth retardation: metabolic and endocrine aspects. In: Gluckman PD, Johnston BM, Nathanielsz PW, editors. Research in perinatal medicine (VIII). Advances in fetal physiology: reviews in honor of G.C. Liggins. Ithaca (New York): Perinatology Press; 1989. p. 263–86. [2] Carter AM. Placental blood flow and fetal oxygen supply in animal models of intra-uterine growth retardation. In: Soma H, editor. Placenta: basic research for clinical application. Basel: Karger; 1991. p. 23–31. [3] Owens JA. Endocrine and substrate control of fetal growth— placental and maternal influences and insulin-like growth factors. Reprod Fertil Dev 1991;3:501–17. [4] Carter AM. Animal models in fetal physiology. In: Svendsen P, Hau J, editors. Handbook of laboratory animal science, vol. 2. London: CRC Press; 1994. p. 77–92. [5] Owens JA, Owens PC, Robinson JS. Experimental restriction of fetal growth. In: Hanson MA, Spencer JAD, Rodeck CH, editors. Fetus and neonate: physiology and clinical applications. Growth, vol. 3. Cambridge: Cambridge University Press; 1995. p. 139–75. [6] Robinson JS, Owens JA. Pathophysiology of intrauterine growth failure. In: Gluckman PD, Heyman MA, editors. Pediatrics and perinatology. The scientific basis. 2nd ed. London: Edward Arnold; 1996. p. 290–97. [7] Sparks JW, Ross JC, Cetin I. Intrauterine growth and nutrition. In: Polin RA, Fox WW, editors. Fetal and neonatal physiology. 2nd ed., vol. 1. Philadelphia: Saunders; 1998. p. 267–89.
S38
H.J. Schro¨ der / European Journal of Obstetrics & Gynecology and Reproductive Biology 110 (2003) S29–S39
[8] Han VK, Carter AM. Control of growth and development of the fetoplacental unit. Curr Opin Pharmacol 2001;1:632–40. [9] Ciba Foundation Symposium 27. Size at Birth. Ciba Foundation Symposia. Amsterdam: Associated Scientific Publishers; 1974. [10] Sharp F, Fraser RB, Milner RDG, editors. Fetal Growth. London: Springer; 1989. [11] Gluckman PD, Johnston BM, Nathanielsz PW, editors. Research in perinatal medicine (VIII). Advances in fetal physiology: reviews in honor of G.C. Liggins. Ithaca, New York: Perinatology Press; 1989. [12] Hanson MA, Spencer JAD, Rodeck CH, editors. Fetus and neonate: physiology and clinical applications. Growth, vol. 3. Cambridge: Cambridge University Press; 1995. [13] Gluckman PD, Heymann MA, editors. Pediatrics and perinatology: the scientific basis. 2nd ed. London: Edward Arnold; 1996. [14] Harding R, Bocking AD, editors. Fetal growth and development. Cambridge: Cambridge University Press; 2001. [15] Fowden AL. Growth and metabolism. In: Harding R, Bocking AD, editors. Fetal growth and development. Cambridge: Cambridge University Press; 2001. p. 44–69. [16] Kitanaka T, Gilbert RD, Longo LD. Maternal and fetal responses to long-term hypoxemia in sheep. In: Ku¨ nzel W, Jensen A, editors. The endocrine control of the fetus: physiologic and pathophysiologic aspects. Berlin: Springer; 1988. p. 38–63. [17] Pennell CE, Smyth JP, Turner AJ, Coughtrey H, Murray HG, Newnham JP. The effects of growth restriction on the fetal heart rate patterns and blood pressure responses to repeated cord occlusion in the ovine fetus. J Soc Gynecol Invest 2002;9(Suppl):64A [abstract]. [18] Schro¨ der HJ, Power GG. Engine and radiator: fetal and placental interactions for heat dissipation. Exp Physiol 1997;82:403–14. [19] Alexander G, Williams D. Heat stress and development of the conceptus in the domestic sheep. J Agricult Sci (Cambridge) 1971;76:53–72. [20] Ross JC, Fennessey PV, Wilkening RB, Battaglia FC, Meschia G. Placental transport and fetal utilization of leucine in a model of fetal growth retardation. Am J Physiol 1996;270:E491–503. [21] Alexander G, Hales JRS, Stevens D, Donnelly JB. Effects of acute and prolonged exposure to heat on regional blood flows in pregnant sheep. J Dev Physiol 1987;9:1–15. [22] van Martens EV, Harel S, Zamenhof S. Experimental intrauterine growth retardation. Biol Neonate 1975;26:221–31. [23] Carter AM. Current topic: restriction of placental and fetal growth in the guinea-pig. Placenta 1993;14:125–35. [24] Clark KE, Durnwald M, Austin JE. A model for studying chronic reduction in uterine blood flow in pregnant sheep. Am J Physiol 1982;242:H297–301. [25] Creasy RK, Barrett CT, de Swiet M, Kahanpaa KV, Rudolph AM. Experimental intra-uterine growth retardation in the sheep. Am J Obstet Gynecol 1972;112:566–73. [26] Charlton V, Johengen M. Fetal intravenous nutritional supplementation ameliorates the development of embolization-induced growth retardation in the sheep. Pediatr Res 1987;22:55–61. [27] Trudinger BJ, Stevens D, Connelly A, et al. Umbilical artery flow velocity waveforms and placental resistance: The effects of embolization of the umbilical circulation. Am J Obstet Gynecol 1987;157:1443–8. [28] Gagnon R, Challis J, Johnston L, Fraher L. Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus. Am J Obstet Gynecol 1994;170:929–38. [29] Alexander G. Studies on the placenta of the sheep (ovies aries L.). Effect of surgical reduction in the number of caruncles. J Reprod Fertil 1964;7:307–22. [30] Robinson JS, Kingston EJ, Jones CT, Thorburn GD. Studies on experimental growth retardation in sheep. The effect of removal of endometrial caruncles on fetal size and metabolism. J Dev Physiol 1979;1:379–98. [31] Cetin I, Marconi AM, Bozzetti P, et al. Umbilical amino acid concentrations in appropriate and small-for-gestational age infants: a
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42] [43] [44]
[45]
[46]
[47]
[48]
[49]
[50]
[51] [52]
biochemical difference present in utero. Am J Obstet Gynecol 1988;158:120–6. Cetin I. Amino acid interconversions in the fetal-placental unit: the animal model and human studies in vivo. Pediatr Res 2001;49: 148–54. Emmanouilides GC, Townsend DE, Bauer RA. Effects of single umbilical artery ligation in the lamb fetus. Pediatrics 1968;42: 919–27. Gardner DS, Fletcher AJ, Fowden AL, Giussani DA. A novel method for controlled and reversible long term compression of the umbilical cord in fetal sheep. J Physiol 2001;535:217–29. Tchirikov M, Schro¨ der HJ. Single case report: late gestational fetal sheep may survive blockage of the ductus venosus for 1 week. Placenta 1998;19:333–6. Tchirikov M, Kertschanska S, Schro¨ der HJ. Obstruction of ductus venosus stimulates cell proliferation in organs of fetal sheep. Placenta 2001;22:24–31. Tchirikov M, Kertschanska S, Stu¨ renberg HJ, Schro¨ der HJ. Liver blood perfusion as a possible instrument for fetal growth regulation. Troph Res 2002;16:S153–8. Wallace JM. Nutrient partitioning during pregnancy: adverse gestational outcome in overnourished adolescent dams. Proc Nutr Soc 2000;59:107–17. Thomas L, Wallace JM, Aitken RP, Mercer JG, Trayhurn P, Hoggard N. Circulating leptin during ovine pregnancy in relation to maternal nutrition, body composition and pregnancy outcome. J Endocrinol 2001;169:465–76. Lok F, Owens JA, Mundy L, Robinson JS, Owens PC. Insulin-like growth factor I promotes growth selectively in fetal sheep in late gestation. Am J Physiol 1996;270:R1148–55. Bassett JM. Glucose and fetal growth derangement. In: Hanson MA, Spencer JAD, Rodeck CH, editors. Fetus and neonate: physiology and clinical applications. Growth, vol. 3. Cambridge: Cambridge University Press; 1995. p. 223–54. Milley JR. Ovine fetal metabolism during norepinephrine infusion. Am J Physiol 1997;273:E336–47. Milley JR. The effect of chronic hyprinsulinemia on ovine fetal growth. Growth 1986;50:390–401. Fowden AL, Mao XZ, Comline RS. Effects of pancreatectomy on the growth and metabolite concentrations of the sheep fetus. J Endocrinol 1986;110:225–31. Thorburn GD. The role of the thyroid gland and kidneys in fetal growth. Ciba Foundation Symposium 27: size at birth. Amsterdam: Associated Scientific Publishers; 1974. p. 185–214. Mesiano S, Young IR, Baxter RC, Hintz RL, Browne CA, Thorburn GD. Effect of hypophysectomy with and without thyroxine replacement on growth and circulating concentrations of insulin-like growth factors I and II in the fetal lamb. Endocrinology 1987;120:1821–30. Gluckman P.D., Insulin-like growth factors and their binding proteins. In: Hanson MA, Spencer JAD, Rodeck CH, editors. Fetus and neonate: physiology and clinical applications. Growth, vol. 3. Cambridge: Cambridge University Press; 1995. p. 97–115. Giudice LC, DeZegher F, Gargosky SE, et al. Insulin-like growth factors and their binding proteins in the term and preterm human fetus and neonate with normal and extremes of intrauterine growth. J Clin Endocrinol Metab 1995;80:1548–55. Christou H, Connors JM, Ziotopoulou M, et al. Cord blood leptin and insulin-like growth factor levels are independent predictors of fetal growth. J Clin Endocrinol Metab 2001;86:935–8. Daughaday WH, Rotwein P. Insulin-like growth factors I and II. Peptide, messenger ribonucleic acid and gene structures, serum, and tissue concentrations. Endocrinol Rev 1989;10:68–91. Rotwein P. Structure, evolution, expression and regulation of insulinlike growth factors I and II. Growth Factors 1991;5:3–18. Kelley KM, Youngman OH, Gargosky SE, et al. Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics. Int J Biochem Cell Biol 1996;28:619–37.
H.J. Schro¨ der / European Journal of Obstetrics & Gynecology and Reproductive Biology 110 (2003) S29–S39 [53] Rajaram S, Baylink DJ, Mohan S. Insulin-like growth factor-binding proteins in serum and other biological fluids: regulation and functions. Endocrinol Rev 1997;18:801–31. [54] Reinecke M, Collet C. The phylogeny of the insulin-like growth factors. Int Rev Cytol 1998;183:1–94. [55] Hwa V, Youngman OH, Rosenfeld RG. The insulin-like growth factor-binding protein (IGFBP) superfamily. Endocrinol Rev 1999; 20:761–87. [56] DeChiara TM, Efstratiadis A, Robertson EJ. A growth-deficiency phenotype in heterozygous mice carrying an insulin-like growth factor II gene disrupted by targeting. Nature 1990;345:78–80. [57] Liu J-P, Baker J, Perkins AS, Robertson EJ, Efstratiadis A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 1993;75:59–72. [58] Baker J, Liu J-P, Robertson EJ, Efstratiadis A. Role of insulin-like growth factors in embryonic and postnatal growth. Cell 1993;75:73–82.
S39
[59] Efstratiadis A. Genetics of growth: developmental roles of IGF and insulin receptors. Exp Clin Endocrinol Diabetes 1996;104: 4–6. [60] Eggenschwiler J, Ludwig T, Fisher P, Leighton PA, Tilghman SM, Efstratiadis A. Mouse mutant embryos overexpressing IGF-II exhibit phenotypic features of the Beckwith–Wiedemann and Simpson–Golabi–Behmel syndromes. Genes Dev 1997;11: 3128–42. [61] Schneider MR, Lahm H, Wu M, Hoeflich A, Wolf E. Transgenic mouse models for studying the functions of insulin-like growth factor-binding proteins. FASEB J 2000;14:629–40. [62] Barker DJP, Fall CHD. Fetal and infant origins of cardiovascular disease. Arch Dis Child 1993;68:797–9. [63] Wallace JM, Bourke DA, Aitken RP. Nutrition and fetal growth: paradoxical effects in the overnourished adolescent sheep. J Reprod Fertil 1999;54(Suppl):385–99.