2012: Intrauterine fetal growth restriction, adaptation and programming-a review Intrauterine fetal growth restriction, adaptation and programming–a review Ramanathan K. Kasimanickam,a Vanmathy R. Kasimancikam,a John P. Kastelicb aDepartment of Veterinary Clinical Sciences, Washington State University, Pullman, WA; bAgriculture and Agri-Food Canada, Lethbridge, AB Canada Abstract Intrauterine fetal growth restriction (IUGR) is fetal adaptation in response to a wide range of pathophysiological factors, including an inadequate supply of oxygen, nutrients, or both. In particular, low birth weight has been associated with subsequent disorders in adult life, including cardiovascular, neuro-developmental, metabolic, and reproductive diseases, independent of adult life style. Animal models are excellent tools to study the effects of genetic, molecular and cellular events that modulate intrauterine growth and development. Various experimental approaches have been used, involving alteration of numerous metabolic and physiologic events, with substantial variations among studies in both methods and species used. Intrauterine fetal growth restriction remains a challenging problem in humans and is a major concern in agricultural animal since it affects production parameters. The objective of this manuscript is to review animal models which explore fetal adaptation, including IUGR, and evaluate their outcomes, to suggest new opportunities for further research, and the potential for interventions to reduce or eliminate gestational events with deleterious effects on fetal, neonatal, and adult health. Keywords: Fetus, placenta; animal model; growth restriction Introduction Intrauterine fetal growth restriction results in unduly high perinatal morbidity and mortality.1-4 Intrauterine fetal growth restriction refers to a condition in which a fetus is unable to achieve its genetically determined potential size. The clinical description of IUGR is on the basis of birth weight below the tenth percentile, or fetal body weight less than two standard deviations from the mean of study population.5,6 Several pathophysiological factors can disturb or restrict fetal growth, including chromosomal abnormalities, gene defects, poor placental function, and maternal stress due to environment or nutrition. The physiological adaptations of the fetus to an adverse intrauterine environment in response to pathophysiological factors, including restrictions on its growth rate, depend on the nature, timing and intensity of extrauterine or intrauterine challenges. Adaptation of the fetus to a suboptimal intrauterine environment is of clinical importance in determining long-term, postnatal health.7 The physiological, neuroendocrine or metabolic adaptations that help the fetus to survive a period of intrauterine deprivation result in permanent programming of proliferation and differentiation events within key tissue and organ systems and pathological consequences in adult life.8,9 This is the so called Barker's hypothesis, after David J. P. Barker, who published the theory in 1997.7 The magnitude of fetal adaptations for both in utero survival and postnatal health outcomes provided the momentum for experimental studies to elucidate the nature and consequences of specific fetal adaptations to a compromised or poor intrauterine environment. This review summarizes animal model studies focused on the range of adaptations the fetus makes in response to a suboptimal uterine environment. Underlying mechanisms Normal growth has been described as, “the idiom of the genetic potential to grow which is neither abnormally constrained nor promoted by internal or external features”. In this definition, internal features relate to the fetus, whereas external features relate to the placenta, uterus, dam, and environment. However these internal and external characteristics, including the fetus, dam, and utero-placental unit, act collectively; therefore, the problem of interactions among the fetus, placenta and dam must be considered. Furthermore, the genetically determined growth course at each stage of gestation has to be understood before growth restriction can be imposed. However, due to inherent interactions among numerous components, it is very difficult to determine the physiologic growth curve of an individual fetus.10 In Clinical Theriogenology • Volume 4 Number 2 • June 2012133 addition a fetus can achieve normal fetal growth despite altered internal and/or external features, since the maternal, utero-placental and fetal tissues are capable of adapting to alterations. Furthermore, since the placenta and fetus are developing, they are not in a steady state. Consequently, the same experimental method may have different effects, depending on the stage of gestation when it is applied, emphasizing the need to carefully consider the timing of experimental alterations. Normal intrauterine growth takes place in phases–an embryonic and a fetal phase. The embryonic phase consists of proliferation, organization and differentiation of the embryo, whereas the fetal phase consists of continuing growth and functional maturation of the various tissues and organs. The fetal phase of intrauterine development depends on genetic, placental, and maternal factors. According to time of adverse intrauterine nutrition, a fetal growth restriction is classified as symmetric or asymmetric,11 as shown (Table 1). Table 1: Comparison of symmetric and asymmetric fetal growth Symmetric Asymmetric Occurs early in gestation Occurs late in gestation Entire fetus is proportionately small Fetus is normal length but reduced weight Multifactorial - genetic, infectious or toxic effects Due to placental insufficiency Postnatal make-up growth is rare Postnatal make-up growth possible Adapted from 11 Factors that cause fetal growth restriction Numerous factors can result in restriction of fetal growth, including: Maternal factors  Undernutrition  Maternal low birth weight  Maternal age  Chronic hypertension (gestational induced)  Pre-eclampsia  Diabetes Environmental factors  Toxicity  High altitude  Hyperthermia  Nutritional deficiencies Placental factors  Abnormal placentation  Chronic abruption, infarcts, focal lesions  Ischemia  Chronic inflammatory conditions (e.g. villitis or placentitis) Fetal factors  Chromosomal anomalies  Genetic conditions  Congenital malformations  Intrauterine infections  Multiple fetuses Impacts of fetal programming on postnatal life  Neurologic, endocrine and cardiac problems  Diabetes  Delayed puberty Clinical Theriogenology • Volume 4 Number 2 • June 2012 134  Poor ovarian follicular development  Fewer germ, Sertoli and Leydig cells  Poor placentation/fewer uterine carunlces  Poor uterine capacity  Reduced litter size  Poor postnatal growth  Poor meat quality Treatment modalities  Exogenous progesterone or synthetic progestins  Exogenous growth hormone  Dietary supplementation of energy, protein, or both  Antioxidants  Manipulations of arginine-nitric oxide/polyamine  Sildenafil citrate  Aspirin Experimental models of restriction of intrauterine fetal growth Various experimental approaches have been used to induce IUGR in several species, including mice, rats, guinea pig and sheep (it occurs spontaneously in virtually all species). Experimental methods to induce IUGR include a reduction in utero-placental blood flow by vascular occlusion or ligation, a reduction in materno-fetal contact by cornual ligation or removal, placental embolization, carunclectomy, genetic manipulation of the fetus, fetal infection, a reduction in maternal nutrient intake (energy, protein, or both), maternal overfeeding, and maternal hyperthermia (Figure 1).12-40 Figure 1: Experimental approaches to produce intrauterine fetal growth restriction (IUGR) in animals. Both nutritional and placental insufficiency models resulted in IUGR and postnatal maladies, including endothelial dysfunction, hypertension, insulin resistance, type 2 diabetes, reduced cognitive Clinical Theriogenology • Volume 4 Number 2 • June 2012135 development, and cardiac diseases. Fetal growth parameters and hormones in the ovine IUGR fetus relative to a control are shown (Table 2); that the IUGR fetus weighed less than the control validated the model. Table 2: Fetal growth parameters and hormones in the growth-restricted ovine fetus (relative to control) Carunclectomy Hyperthermia Maternal overfeeding Embolization Uterine artery ligation Birth weight ↓ NA ↓ ↓ NA Placental weight ↓ ↓ ↓ ↓ ↓ Fetal weight ↓ ↓ ↓ ↓ ↓ Relative organ weight ↑/= ↑/= ↑/= ↑/= ↑/= Fetal plasma ACTH ↑ ↑ Fetal plasma cortisol ↑ = females; ↑ males = ↑/= ↑ Noradrenaline ↑ ↑ ↑ Adapted from 40-42 Placental vascular development The placenta has a major role in regulation of fetal growth. The sow and mare have a diffuse placenta, with chorionic villi distributed over the entire surface of the chorion, whereas in ruminants the chrioallantois attaches to discrete sites on the uterine wall. Placental area of attachment continues to increase as gestation advances43,44 and vascular development of the placenta increases ~200% from mid- to late gestation.44 In the cow, cotyledonary growth progressively increases throughout gestation,45,46 whereas in the ewe, growth of the cotyledonary mass is exponential during the first 70–80 days of pregnancy, thereafter slowing markedly until lambing. Table 3: Comparison of percentage change in capillary vascularity from mid- to late pregnancy in sheep (Days 50 to 140) and cattle (Days 125 to 250) Placentomal tissue Vascularity parameter Sheep Cattle Caruncle CAD ↑ 214% ↓30% CND ↑ 37% ↑151% CSD ↑140% ↑32% Capillary size ↑45% ↓68% Cotyledon CAD ↑437 ↑186% CND ↑1093% ↑80% CSD ↑576% ↑172% Capillary size ↓ 25% ↑71% CAD - Capillary area density (a flow-related measure); CND - capillary number density (an angiogenesis-related measure); CSD - capillary surface density (a nutrient exchange-related measure). Studies on changes in placental vascularity in response to nutrient-restriction in ewes and cows are very limited, and appear to be largely lacking in sows. Seven days of fasting during mid-pregnancy in the ewe decreased vascular endothelial growth factor (VEGF) mRNA levels and placental weights on Day 90; however, placental weights were similar at lambing in nutrient-restricted and control ewes.47 In nutrient-restricted cows (Days 30 to 125 of gestation) there was a decrease in total placentome weight, including both the cotyledonary and caruncular portions, on Day 125 compared to control cows. This inhibition in total placentome weight persisted, even after realimentation until Day 250;46,48 however, only the weight of the cotyledonary tissue remained suppressed at Day 250.48 In contrast, in several sheep models of maternal nutrient restriction from early to mid-pregnancy followed by realimentation, there was significant compensatory growth of the entire placentome.46,49 Although maternal nutrient delivery Clinical Theriogenology • Volume 4 Number 2 • June 2012 136 during pregnancy has been shown to program fetal growth and development, both during pregnancy and later into adult life, it appears that maternal nutrition also programs placental development. In the cow, realimentation after ~90 days of nutrient restriction is the stimulus not only for altering placental vascularity and development, but also placental function.50,51 Animal models Several researchers used animals to study various aspects of fetal development. Those research scenarios could be used as animal models and compared with existing fetal growth restriction models to advance knowledge regarding mechanisms underlying IUGR. Some scenarios worthy of investigation include: 1. Large offspring syndrome in cows 2. Uteroplacental hypoxia in sheep 3. Embryonic/fetal adaptation in mares 4. Low birth weight piglets Nutritional status and environmental events can alter important physiological parameters of the embryo and fetus. This resetting is called as fetal or developmental programming and it can continue into childhood and adulthood to trigger disorders. The fetal cellular environment can modify the gene expression pattern in various tissues and organs, with long-term functional consequences in neonatal period and adulthood. Fetal programming involves primary alterations and secondary compensations. According to the concept of fetal programming, any detrimental circumstance during the critical period of in-utero development affects the subsequent pattern of growth and development of tissues and organs and may predispose the affected fetus to metabolic disorders later in life.7 The placental angio-architecture is vulnerable to any alterations in the maternal environment. Adverse uterine milieu produces an altered placental vascular phenotype and function. In utero, adverse events are critical factors in determining fetal growth, quality of life and overall health. Underlying mechanisms of fetal programming caused by various perturbations in the maternal compartment and how they are transmitted across the placenta remain to be completely established. Several vascular factors including bFGF, EGF, PDGF, angiopoietin 1 and 2, and the VEGF family, have been identified as important placental angiogenic regulators. Up-regulation of angiogenic factors VEGF, bFGF and eNOS have been documented in IUGR pregnancies. Differential expression of pro- and anti-angiogenic factors such as vascular endothelial growth factors (VEGF) and placental growth factors (PlGF) throughout gestation indicates that both play essential roles in placental angiogenesis.52-54 The imbalance of pro- and anti-angiogenic mediators during gestation results in impeded fetal growth and other complications. Clinical Theriogenology • Volume 4 Number 2 • June 2012137 Figure 2: Imbalance of pro- and anti-angiogenic mediators during gestation results in oxidative damage, placental ischemia, impeded fetal growth, and other complications. Large offspring syndrome in cattle In vitro production of bovine embryos often affects embryonic gene expression and organogenesis, leading to large offspring syndrome (LOS). In cattle, the developmental competence of in vitro produced embryos is compromised compared to in vivo embryos. Embryos produced in vitro differ from those produced in vivo with regards to morphology, timing of development, embryonic metabolism, intercellular communication, cell number, and gene expression.55-61 A recent study compared three embryo production systems to identify genes affected by in vitro processes and associated with the LOS; 1) embryos produced in vitro (IVF), 2) embryos fertilized in vivo followed by in vitro culture (in vivo derived, IVD), and 3) embryos generated entirely in vivo by artificial insemination (AI-IVD).47 There were 306, 367, and 200 genes differentially expressed between the AI-IVD, IVF and IVD, and AI and IVF comparisons, respectively. Interestingly, 44 differentially expressed genes were identified between the AI embryos and both the in vitro and in vivo embryos, making these genes potential candidates for LOS. Comparing these genes in an IUGR model would provide additional insight into fetal programming. In another study, in vitro produced bovine embryos cultured in high concentrations of either serum or bovine serum albumin (BSA) had significantly more cells in Day 7 blastocysts, a larger blastocyst on Day 12, and differences in relative abundance of transcripts for heat shock protein 70.1, Cu/Zn-superoxide dismutase (SOD), glucose transporters-3 (Glut-3), Glut-4, bovine-fibroblast growth factor (bFGF), and insulin-like growth factor 1- receptor (IGF1-R) when compared with in vivo embryos; these differences provided evidence that the culture system had an important role in development.61 Furthermore, calves derived from in vitro embryos were significantly heavier than those derived from sheep oviduct culture, superovulation, or AI.62 Clinical Theriogenology • Volume 4 Number 2 • June 2012 138 Figure 3: Factors altering fetal and placental development and fetal programming (adapted from 47-49) Cloning by nuclear transfer of somatic cells (SCNT) is associated with high rates of embryonic and fetal mortality in various species.63,64 In bovine pregnancies with a fetus produced by SCNT, a poorly developed placenta with very few placentomes and limited vascularization has frequently been described during early pregnancy;63,64 that the extra-embryonic tissues fail to develop normally in SCNT clones causes abnormalities in implantation and placental development. In placentas of SCNT pregnancies examined between 180 and 280 days of gestation, there were indications that placental overgrowth preceded fetal overgrowth,64,65 perhaps due to alterations in placental genes during early development. For the fetus to survive, the placenta must overcome and compensate for developmental defects caused by programming errors; furthermore, in cloned offspring, adaptation to these changes may cause IUGR.65 Altered placental development due to SCNT-induced genomic perturbations would be consistent with such observations. It is apparent that the pre-implantation embryo is extremely sensitive to subtle changes in environment with long-term consequences for the health of the offspring. Genes that are implicated in these studies can be studied further using gene silencing or transfection models to determine their pathophysiology in the development of placentation and IUGR. Uteroplacental hypoxia in sheep Intrauterine growth restriction, often associated with functional placental insufficiency, increased perinatal mortality and morbidity. Although no animal model can fully simulate human pregnancy, the pregnant sheep has been used extensively to investigate maternal-fetal interactions. Embryo stress Heat shock Culture conditions Paternal and/or maternal Altered gene expression and imprinting Altered gene blastomere function Altered placental and fetal development Clinical Theriogenology • Volume 4 Number 2 • June 2012139 Figure 4: Placental oxidative stress and intrauterine growth restriction. Placental oxidative stress induced by generation of an abundance of oxidative free radicals cause lipid peroxidation and alters angiogenesis, which reduces nutrient delivery to the fetus, causing fetal growth restriction. Development of the placental vascular tree is regulated by several growth factor families. Based on differential expression of vascular endothelial growth factors (VEGF) and placental growth factors (PlGF) throughout gestation, both have essential roles in placental angiogenesis.66-68 The latter is divided into two phases, branching and non-branching.66-68 Branching angiogenesis, which occurs during the early phases of gestation under relatively hypoxic conditions, is responsible for expansion of the pre- existing vascular bed, and is driven by VEGF. Non-branching angiogenesis promotes elongation of pre- existing capillary loops and occurs later in gestation under the influence of PlGF. The balance of these two growth factors throughout gestation is responsible for formation of a normal placenta.67,68 The increasing transplacental oxygen gradient generates changes in angiogenic growth factors which may be associated with the underlying pathophysiology of post-placental hypoxic fetal growth restriction. An altered state of placental oxygenation is associated with aberrant villous morphology.69-71 A fetal growth restricted placenta that develops under maternal hypoxia (e.g. at a high altitude) is characterized by increased branching angiogenesis and reduced vascular impedance.72,73 In contrast, pregnancies with restricted fetal growth are characterized by the absence of end diastolic flow in the umbilical artery and increased vascular impedance, predominantly associated with non-branching angiogenesis. Placental oxidative stress induced by ischemic perfusion has been implicated in the pathophysiology of fetal growth restriction. Placental tissues from IUGR pregnancies have decreased placental anti-oxidant capacity, and lower mRNA expression of Cu/Zn superoxide dismutase (an important enzymatic antioxidant) and non-enzymatic antioxidant vitamin E.74-76 Based on several studies, decreased bioavailability of endothelium-derived NO, due to oxidative destruction of NO by ROS, may Intermittent perfusion of placental intervillous space Fluctuating oxygen concentrations Generation of oxygen free radicals (ROS/RNS) Placental oxidative stress Direct damaging effect • Altered angiogenesis • Lipid peroxidation • Cytokine synthesis Intrauterine growth restriction Clinical Theriogenology • Volume 4 Number 2 • June 2012 140 contribute to the impaired endothelium-dependent vasodilatory responses, for which treatment with antioxidant vitamins apparently have a beneficial role.74-76 Tocopherols are micronutrients which act as free radical scavengers, i.e. chain-breaking antioxidants. The major tocopherols in mammalian tissue are alpha and gamma. Under specific circumstances, gamma tocopherol might scavenge reactive species more effectively than alpha tocopherol.77 In that regard, gamma tocopherol was 35% more effective than alpha tocopherol at inhibiting systemic lipid peroxidation. Furthermore, gamma tocopherol is incorporated into the endothelial cells more rapidly than alpha tocopherol, and supplementation with gamma tocopherol was significantly more effective than alpha tocopherol at inhibiting iron-induced lipid peroxidation and occlusive thrombus formation.78 In addition to free radical scavenging properties, since gamma tocopherol has specific activities with key inflammatory enzymes, it may have an added advantage of suppressing inflammation during pregnancy. Supplementation of tocopherols may reduce utero-placental hypoxia and promote placental vascular development by reducing hypoxia. The sheep may be an appropriate model to study methods to overcome placental hypoxia, namely, the use of antioxidants to promote vasculogenesis. Embryonic/fetal adaptation in mares At the histological level, the exterior surface of the allantochorion in mares consists of highly complex microplacentomes, each packed with fetal and maternal capillaries, which enables materno-fetal exchange of nutritional, gaseous and waste products.79-84 A healthy and functional feto-maternal unit (placenta and uterus) is prerequisite for a successful pregnancy in the mare;84 anything affecting this fetomaternal attachment may result in abortion or weakness in the newborn foal. In that regard, intrauterine development of the fetus and fetal birth weight are closely related to the total area of contact of the allantochorion. In mares, placental compromise is evident in twinning, age-related endometrial degeneration, and maternal size. In twinning, allantochorions of the two conceptuses compete for the limited surface area of endometrium, whereas in endometrial degeneration, the area of placental exchange is reduced. Placental compromise related to maternal size has been well characterized by crossing large Shire horses with small Shetland ponies; the foal from the Shetland mare weighed only half that of its half sibling born from the Shire mare. Importantly, this difference persisted into adult life. Tischner created three pairs of sex- matched, full sibling Konik pony foals, whereby one of each pair had been transferred as a blastocyst to the uterus of a larger draft-type recipient mare, whereas the sibling was carried to term in its genetic pony mother.85 All three transferred foals were bigger at birth and grew faster while nursing their larger surrogate mothers.86 In other studies, embryos were transferred between thoroughbred (T) mares and pony (P) mares to establish four types of pregnancies: T-in-P, P-in-T, and, as controls, T-in-T and P-in-P. The T-in-T foals were heaviest and the P-in-P foals were lightest at birth.87 The P-in-T foals were heavier than their T-in-P counterparts. Further, differences in birth weight were paralleled by differences in placental mean weight, volume, total surface area and density of microcotyledons on the allantochorion and the total microscopic area of fetomaternal contact at the placental interface. Thus, intrauterine development of the fetus and the resulting size of the foal are closely related to the total area of contact of the allantochorion. The principal vasculogenic and angiogenic factor VEGF, and its two major receptor molecules, Flt and KDR, are localized throughout most of gestation on the two principal secretory cell types of the equine placenta, the glandular and lumenal epithelia of the maternal endometrium and the trophoblast of the fetal allantochorion.88 Perhaps, VEGF, Flt and KDR interact in the mare to facilitate development of maternal and fetal vascular networks for exchange of gases, nutrients and waste products throughout gestation. In this regard, mares could be used as model to study fetal adaptation during development. The use of T-P models will address both the restricted (T-P) and over-fed (P-T) scenarios. Questions addressed by these studies include: 1. What are differences in the placental vasculogenesis gene expression in restricted and over- weight maternal conditions? Clinical Theriogenology • Volume 4 Number 2 • June 2012141 2. What are differences in gene expression when there is fetal growth restriction in contrast to fetal adaptation in those conditions? Low birth weight in piglets In pigs, birth weight is a major determinant of postnatal survival. Since attempts to alleviate the detrimental effects of low birth weight after birth have achieved only limited success, it appears that the in utero environment during a critical stage of gestation can program fetal gene expression and metabolism, leading to low birth weight. The capacity of the placenta to transport nutrients has a direct effect on fetal growth. The placenta supplying the smallest fetus in the uterus is disproportionally lighter than those supplying average-sized fetuses in the same litter.89 Furthermore, there is a positive relationship between placental blood flow and fetal weight.90 Therefore, there is a generalized reduction in the ability of placentas supplying small fetuses to deliver nutrients. Supplementation of L-Arginine has been studied extensively studied using pig models. Arginine, a conditionally essential amino acid,91 is the nitrogenous precursor of nitric oxide (NO), a vasodilator and a cell signaling molecule.92,93 It has been established that NO and other products of arginine catabolism, e.g. proline and polyamines, are crucial for cell growth, migration, and proliferation, as well as angiogenesis.94 Gilts were supplemented with 0.0, 0.4, or 0.8% L-arginine between 0 and 25 d of gestation.95 Dietary supplementation with 0.4 or 0.8% L-arginine enhanced its concentrations in maternal plasma (64 and 98%, respectively) as well as the vascularity of chorionic and allantoic membranes, compared to the control group.95 Reproductive performance, including numbers of corpora lutea and fetuses, as well as placental and fetal weights, and embryonic mortality, did not differ between the 0.4% L-arginine and control groups. However, supplementation with 0.8% L-arginine between 0 and 25 d of gestation, increased placental vascularity, but adversely affected reproductive performance of gilts.92 Interestingly, giving rats 1.2% L-arginine for 7 d (immediately after breeding) reduced embryonic mortality by 30% .96 Similarly, supplementing 0.83% L-arginine to gilts between 30 and 114 d of gestation increased the number of live-born piglets by two per litter.97 The pig may be an appropriate model for experiments dealing with both birth weight and neonatal nutrition. There can be up to a 3-fold difference in body weight among littermates in normally fed sows, thus providing a natural form of intrauterine growth restriction.98,99 In addition, this model allows artificial rearing, which facilitates modulation and control of feed intake during the neonatal period. Moreover, comparisons of selection methods based on uterine capacity over treatment modalities could also be evaluated. Fetal programming Epigenetic refers to the study of changes in gene function that may be mitotically and/or meiotically inherited, but are not related to changes in DNA sequence.100 Several mechanisms of epigenetics have been reported, including DNA methylation, modification of histones and modification of gene expression by non-coding small interfering or long RNAs.101 Interestingly, miRNAs down-regulate or weaken several target genes; and the key mechanisms underlying this process are DNA methylation and histone acetylation.100 These molecular mechanisms effectively determine if gene transcription or gene silencing occurs. In brief, histone acetylation and deacetylation are associated with gene transcription and silencing respectively. The histone tails can also be methylated and can activate or silence gene expression. Other examples of epigenetic modifications include phosphorylation, ubiquitinization and sumoylation.102 Epigenetic mechanisms are important in imprinting where one gene allele derived from one parent is silenced, whereas the allele of the same gene but inherited from the other parent is expressed. Embryonic development is characterised by two periods of epigenetic programming, which occur during gametogenesis and the pre-implantation period of pregnancy, respectively. The pre-implantation embryo is extremely sensitive to subtle changes in the environment, with long-term consequences for the health of the offspring. Although underlying mechanisms of epigenetic perturbations are unknown, there are indications of altered epigenetic programming of embryo and endometrium, particularly at the Clinical Theriogenology • Volume 4 Number 2 • June 2012 142 maternal-fetal interface. Suboptimal physiology of the endometrium can subtly or substantially affect embryo development before implantation, with visible and sometimes severe consequences on placentation, fetal development and pregnancy outcome. Thus, whereas pregnancy outcome relies on embryo quality, it appears that the developmental course of the embryo is also epigenetically driven by endometrial mechanisms from the very earliest stages of pregnancy.103 In the context of assisted reproductive technologies, developing diagnostic and prognostic tools based on endometrial factors will be valuable, with objectives to estimate the reproductive capacity of the mother, to correct suboptimal endometrial functionality and to assess the developmental potential of the embryo. On a more fundamental basis, an essential issue will be to carefully analyse situations in which embryos can overcome the control exerted by the endometrium.104 In ruminants, a major difficulty in elucidating pregnancy-related gene functions at the materno-fetal interface is the number of genes to be analyzed. To bypass this problem, alternative options have been published. In that regard, lentiviral infection of blastocyst-stage embryos is an effective approach to study trophoblast gene function.105-107 As mentioned above, fetal programming may be achieved through several mechanisms. At a cellular level, programming affects aspects of cell cycle dynamics and apoptosis, whereas at the subcellular level, telomerase shortening and altered function of various enzyme systems have been documented. These are important mechanisms during development and differentiation, allowing various tissues and organs to develop from the same original genotype. The consequence of methylation in nutritional programming has been ably demonstrated in animal models. A sheep model with methionine deficiency (induced by dietary deficiency of vitamin B12 and folate; Figure 5) around the time of conception, demonstrated that male offspring were adipose and insulin resistant, and that these changes were associated with methylation of several genes.108,109 These experiments highlighted an important role in nutritional programming. Perhaps reductions in dietary methionine and folate during the periconceptional period will cause widespread epigenetic alterations to DNA methylation in offspring, and modify adult health-related phenotypes.110 Furthermore, genes which increase susceptibility to diabetes also reduce birth weight by influencing insulin-mediated growth, i.e. low birth weight and type 2 diabetes are both phenotypes of the same genotype.109,111 It was observed that the rare mutation in glucokinase gene (GCK) reduces insulin secretion in β-cells. If the GCK mutation was inherited paternally, birth weight was reduced by 500 gm, provided the mother was normal. However, if the baby inherited the GCK mutation maternally, birthweight was normal (since maternal diabetes negated the effect of mutation) or it was 500 g above average if the mutation was not inherited.112 Maternal under- nutrition and lower socio-economic status remain the most important causes of low birth weight worldwide. Numerous animal models of under-nutrition and micronutrient supplementation have confirmed the role of nutritional fetal programming. Imprinted genes have diverse functions, notably including regulation of fetal growth.113,114 Much attention has been directed to the IGF signaling pathway, which has a major influence on fetal size. This pathway contains two components encoded by oppositely imprinted genes, namely, IGF2 (a growth promoting factor from the paternal allele) and IGF2R (a growth inhibitory factor from the maternal allele). These genes fit the parent-offspring conflict hypothesis for the evolution of genomic imprinting.115,116 It appears that not all components of growth regulatory pathways are encoded by imprinted genes; perhaps within a pathway, the influence of a single gene by each of the parental genomes may be sufficient for the parent-offspring conflict to be enacted. Several imprinted genes are known to influence energy homeostasis. In that regard, some, including IGF2 and growth factor receptor- bound protein 10 (GRB10), may coordinate growth via glucose-regulated metabolism.117-119 Since perturbation of fetal growth can be associated with metabolic disorders in adulthood, these imprinted genes are considered candidates for involvement in this phenomenon of fetal programming. Clinical Theriogenology • Volume 4 Number 2 • June 2012143 Figure 5: Nutrition and DNA methylation. Factors during pregnancy influencing development of complex traits in adult life should be expanded to include epigenetic factors, e.g. DNA methylation, which occurs in utero. Epigenetic factors regulate gene expression and thereby cell differentiation and organogenesis. The process of epigenotype establishment is sensitive to environmental conditions, with nutrition being one of the most important factors. For example, DNA methylation depends on availability of several nutrients, including methionine and vitamins B6, B12, and folate. Conclusion In summary, poor fetal growth can result from a myriad of fetal, placental, and maternal conditions. Based on numerous studies in various populations, there is a clear association between intrauterine growth restriction and the occurrence of obesity, type 2 diabetes mellitus, cardiovascular, neurological disorders, and reproductive disorders later in life. Animal studies have confirmed that these associations are independent from postnatal influences. However, mechanisms of intrauterine programming and the critical time(s) during pregnancy when an insult causes fetal problems have not been completely elucidated. Although there are treatment modalities available, large-scale prospective studies are needed before therapeutic interventions, given either during pregnancy or after birth, to prevent adverse effects of fetal programming, are scientifically justified. Animal models such as large offspring syndrome, fetal adaptation in mares, placental hypoxic condition in sheep, and nutritional management of IUGR in sheep and pigs, can be used to understand schemes involved in mechanisms and timing of fetal programming, thereby providing new opportunities to reduce or eliminate gestational events which result in fetal, neonatal and adult health shortcomings. References 1. Blair E, Stanley F: Intrauterine growth and spastic cerebral palsy. I. Association with birth weight for gestational age. Am J Obstet Gynecol 1990;162:229-237. 2. Barker DJP, Gluckman PD, Godfrey KM, et al: Fetal nutrition and cardiovascular disease in adult life. Lancet 1993;341:938-941. 3. Casey BM, Lucas MJ, McIntire DD, et al: Pregnancy outcomes in women with gestational diabetes compared with the general obstetric population. Obstet Gynecol 1997;90:869-873. 4. Brodsky D, Christou H: Current concepts in intrauterine growth restriction. J Intensive Care Med 2004;19:307-319. 5. Pardi G, Cetin I, Marconi AM, et al: Diagnostic value of blood sampling in fetuses with growth retardation. N Engl J Med 1993;328:692-696. Clinical Theriogenology • Volume 4 Number 2 • June 2012 144 6. Usher R, McLean F: Intrauterine growth of live-born Caucasian infants at sea level: standards obtained from measurements in 7 dimensions of infants born between 25 and 44 weeks of gestation. J Pediatr 1969;74:901-910. 7. Barker DJP. Maternal nutrition, fetal nutrition, and disease in later life. Nutrition 1997;13:807-813. 8. Pardi G, Marconi AM, Cetin I: Placental-fetal interrelationship in IUGR fetuses–a review. Trophoblast Res 2002;23:S136–141. 9. McMillen IC, Adams MB, Ross JT, et al: Fetal growth restriction: adaptations and consequences. Reproduction 2001;122:195-204. 10. Reeves SD, Bernstein IM: Optimal growth modeling. Semin Perinatol 2008;32:148-153. 11. Dashe JS, McIntire DD, Lucas MJ, et al: Effects of symmetric and asymmetric fetal growth on pregnancy outcomes. Obstet Gynecol 2000;96:321-327. 12. Lafeber HN, Rolph TP, Jones CT: Studies on the growth of the fetal guinea pig. The effects of ligation of the uterine artery on organ growth and development. J Develop Physiol 1985;6:441-459. 13. Boyle DW, Lecklitner S, Liechty EA: Effect of prolonged uterine blood flow reduction on fetal growth in sheep. Am J Physiol 1996;270:R246–R253. 14. Clapp JF, Szeto HH, Larrow R, et al: Fetal metabolic response to experimental placental vascular damage. Am J Obstet Gynecol 1981;140:446-451. 15. Block BS, Schlafer DH, Wentworth RA, et al: Regional blood flow distribution in fetal sheep with intrauterine growth retardation produced by decreased umbilical placental perfusion. J Dev Physiol 1990;13:81-85. 16. Murotsuki J, Challis JRG, Han VKM, et al: Chronic fetal placental embolization and hypoxaemia cause hypertension and myocardial hypertrophy in fetal sheep. Am J Physiol 1997;272:R201-R207. 17. Robinson JS, Kingston EJ, Jones CT, et al: Studies on experimental growth retardation in sheep: the effect of removal of endometrial caruncles on fetal size and metabolism J Develop Physiol 1979;1:379-398. 18. Ergaz Z, Avgil M, Ornoy A: Intrauterine growth restriction-etiology and consequences: what do we know about the human situation and experimental animal models? Reprod Toxicol 2005;20:301-322. 19. Edwards LJ, McMillen IC: Periconceptional nutrition programs development of the cardiovascular system in the fetal sheep. Am J Physiol Regul Integr Comp Physiol 2002;283:R669-R679. 20. Hawkins P, Steyn C, McGarrigle HH, et al: Cardiovascular and hypothalamic-pituitary-adrenal axis development in late gestation fetal sheep and young lambs following modest maternal nutrient restriction in early gestation. Reprod Fertil Dev 2000;12:443-456. 21. Ozaki T, Hawkins P, Nishina H, et al: Effects of undernutrition in early pregnancy on systemic small artery function in late-gestation fetal sheep. Am J Obstet Gynecol 2000;183:1301-1307. 22. Kind KL, Clifton PM, Grant PA, et al: Effect of maternal feed restriction during pregnancy on glucose tolerance in the adult guinea pig. Am J Physiol Regul Integr Comp Physiol 2003;284:R140-R152. 23. Ozaki T, Nishina H, Hanson MA, et al: Dietary restriction in pregnant rats causes gender-related hypertension and vascular dysfunction in offspring. J Physiol 2001;530:141-452. 24. Langley SC, Jackson AA: Increased systolic blood pressure in adult rats induced by fetal exposure to maternal low protein diets. Clin Sci (Lond) 1994;86:217-222; discussion 121. 25. Nishina H, Green LR, McGarrigle HH, et al: Effect of nutritional restriction in early pregnancy on isolated femoral artery function in mid-gestation fetal sheep. J Physiol 2003;553:637-647. 26. Munoz G, Bongiorni-Malave I: Influence of dietary protein restriction on ovulation, fertilization rates and pre- implantation embryonic development in mice. J Exp Zool 1979;210:253-257. 27. Kind KL, Clifton PM, Grant PA, et al: Effect of maternal feed restriction during pregnancy on glucose tolerance in the adult guinea pig. Am J Physiol Regul Integr Comp Physiol 2003;284:R140-R152. 28. Contreras RJ, Wong DL, Henderson R, et al: High dietary NaCl early in development enhances mean arterial pressure of adult rats. Physiol Behav 2000;71:173-181. 29. Vickers MH, Breier BH, Cutfield WS, et al: Fetal origins of hyperphagia, obesity, and hypertension and postnatal amplification by hypercaloric nutrition. Am J Physiol Endocrinol Metab 2000;279:E83-E87. 30. Wallace JM, Luther JS, Milne JS, et al: Nutritional modulation of adolescent pregnancy outcome– a review. Placenta 2006;27(Suppl A):S61-S68. 31. Ogata E, Bussey M, Finley S: Altered gas exchange, limited glucose, branched chain amino acids, and hypoinsulinism retard fetal growth in the rat. Metabolism 1986;35:970-977. 32. Louey S, Cock ML, Stevenson KM, et al: Placental insufficiency and fetal growth restriction lead to postnatal hypotension and altered postnatal growth in sheep. Pediatr Res 2000;48:808-814. 33. Creasy RK, Barrett CT, de Swiet M, et al: Experimental intrauterine growth retardation in the sheep. Am J Obstet Gynecol 1972;112:566-573. 34. Gagnon R, Challis J, Johnston L, et al: Fetal endocrine responses to chronic placental embolization in the late-gestation ovine fetus. Am J Obstet Gynecol 1994;170:929-938. 35. 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;157:1443-1448. 36. Charlton V, Johengen M: Fetal intravenous nutritional supplementation ameliorates the development of embolization- induced growth retardation in sheep. Pediatr Res 1987;22:55-61. Clinical Theriogenology • Volume 4 Number 2 • June 2012145 37. Jones CT, Gu W, Harding JE, et al: Studies on the growth of the fetal sheep. Effects of surgical reduction in placental size, or experimental manipulation of uterine blood flow on plasma sulphation promoting activity and on the concentration of insulin-like growth factors I and II. J Dev Physiol 1988;10:179-189. 38. Robinson JS, Kingston EJ, Jones CT, et al: Studies on experimental growth retardation in sheep. The effect of removal of endometrial caruncles on fetal size and metabolism. J Dev Physiol 1979;1:379-398. 39. Butler TG, Schwartz J, McMillen IC: Differential effects of the early and late intrauterine environment on corticotrophic cell development. J Clin Invest 2002;110:783-791. 40. Vuguin PM: Animal models for small for gestational age and fetal programming of adult disease. Horm Res 2007;68:113-123. 41. Wallace JM, Reganault TRH, Limesand SW, et al: Investigating the causes of low birth weight in contrasting ovine paradigms. J Physiol 2005;565:19-26. 42. Morrison JL: Sheep models of intrauterine growth restriction: Fetal adaptations and consequences. Clin Exp Pharmacol Physiol 2008:35:730-743. 43. Knight JW, Bazer FW, Thatcher WW, et al: Conceptus development in intact and unilaterally hysterectomized- ovariectomized gilts: interrelations among hormonal status, placental development, fetal fluids and fetal growth. J Anim Sci 1977;44: 620-637. 44. Vonnahme KA, Wilson ME, Ford SP: Relationship between placental vascular endothelial growth factor expression and placental/endometrial vascularity in the pig. Biol Reprod 2001;64:1821-1825. 45. Reynolds LP, Ferrell CL, Robertson DA, et al: Growth hormone, insulin and glucose concentrations in bovine fetal and maternal plasmas at several stages of gestation. J Anim Sci 1990;68:725-733. 46. Vonnahme KA, Zhu MJ, Borowicz PP, et al: Effect of early gestational undernutrition on angiogenic factor expression and vascularity in the bovine placentome. J Anim Sci 2007;85:2464-2472. 47. McMullen S, Osgerby JC, Milne JS, et al: The effects of acute nutrient restriction in the mid-gestational ewe on maternal and fetal nutrient status, the expression of placental growth factors and fetal growth. Placenta 2005:26:25-33. 48. Zhu MJ, Du M, Hess BW, et al: Maternal nutrient restriction upregulates growth signaling pathway in the cotyledonary artery of cow placentomes. Placenta 2007;28:361-368. 49. Heasman, L., Clarke, L., Firth, K., et al: Influence of restricted maternal nutrition in early to mid gestation on placental and fetal development at term in sheep. Pediatr Res 1998;44:546-551. 50. Vonnahme KA, Ford SP, Nijland MJ, et al: Alteration in cotyledonary (COT) vascular responsiveness to angiotensin II (ANG II) in beef cows undernourished during early pregnancy. Biol Reprod 2004;70:110. 51. Vonnahme KA, Reynolds LP, Nijland MJ, et al: Impacts of undernutrition during early to mid gestation on basal vascular tone of the cotyledonary and caruncular arterial beds in the bovine placentome. J Soc Gynecol Investig 2004;11:222A. 52. Zygmunt M, Herr F, Munstedt K, et al: Angiogenesis and vasculogenesis in pregnancy. Eur J Obstet Gynecol Reprod Biol 2003;110:S10-S18. 53. Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27-31. 54. Kasimanickam RK, Kasimanickam VR, Rodriguez JS, et al: Tocopherol induced angiogenesis in placental vascular network in late pregnant ewes. Reprod Biol Endocrinol 2010;8:86. 55. Bertolini M, Mason JB, Beam SW, et al: Morphology and morphometry of in vivo- and in vitro-produced bovine concepti from early pregnancy to term and association with high birth weights. Theriogenology 2002;58: 973-994. 56. Van Soom A, Boerjan ML, Bols PEJ, et al: Timing of compaction and inner cell allocation in bovine embryos produced in vivo after superovulation. Biol Reprod 1997;57:1041-1049. 57. Leibo SP, Loskutoff NM: Cryobiology of in vitro-derived bovine embryos. Theriogenology 1993;43:81-94. 58. Khurana NK, Niemann H: Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo. Biol Reprod 200;62:847-856. 59. Niemann H, Wrenzycki C: Alteration of expression of developmentally important genes in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent development. Theriogenology 2000;53:21-34. 60. Everts RE, Chavatte-Palmer P, Razzak A, et al: Aberrant gene expression patterns in placentomes are associated with phenotypically normal and abnormal cattle cloned by somatic cell nuclear transfer. Physiol Genomics 2008;33:65-77. 61. Lazzari G, Wrenzycki C, Herrmann D, et al: Cellular and molecular deviations in bovine in vitro-produced embryos are related to the large offspring syndrome. Biol Reprod. 2002;67:767-775. 62. Gutierrez-Adan A, Rizos D, Lonergan P, et al: Differential gene expression in bovine blastocysts produced in different culture systems. Theriogenology 2002;57:639. 63. Constant F, Guillomot M, Heyman Y, et al: Large offspring or large placenta syndrome? Morphometric analysis of late gestation bovine placentomes from somatic nuclear transfer pregnancies complicated by hydrallantois. Biol Reprod 2006;75:122-130. 64. Hill JR, Burghardt RC, Jones K, et al: Evidence for placental abnormality as the major cause of mortality in first- trimester somatic cell cloned bovine fetuses. Biol Reprod 2000;63:787-794. 65. Beyhan Z, Forsberg EJ, Eilertsen KJ, et al: Gene expression in bovine nuclear transfer embryos in relation to donor cell efficiency in producing live offspring. Mol Reprod Dev 2007;74:18-27. Clinical Theriogenology • Volume 4 Number 2 • June 2012 146 66. Park JE, Chen HH, Winer J, et al: Placenta growth factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. J Biol Chem 1994:269:25646-25654. 67. Cheung CY, Singh M, Ebaugh MJ, et al: Vascular endothelial growth factor gene expression in ovine placenta and fetal membranes. Am J Obstet Gynecol 1995;173:753-759. 68. de Vrijer B, Davidsen ML, Wilkening RB, et al: Altered placental and fetal expression of IGFs and IGF-binding proteins associated with intrauterine growth restriction in fetal sheep during early and mid-pregnancy. Pediatr Res 2006;60:507-512. 69. Reynolds LP, Redmer DA: Utero-placental vascular development and placental function. J Anim Sci 1995;73:1839- 1851. 70. Reynolds LP, Borowicz PP, Vonnahme KA, et al: Animal models of placental angiogenesis. Placenta 2005;26:689- 708. 71. Reynolds LP, Borowicz PP, Vonnahme KA, et al: Placental angiogenesis in sheep models of compromised pregnancy. J Physiol 2005;15:43-58. 72. Mayhew TM: Changes in fetal capillaries during preplacental hypoxia: growth, shape remodelling and villous capillarization in placentae from high-altitude pregnancies. Placenta 2003;24:191-198. 73. Wheeler T, Elcock CL, Anthony FW: Angiogenesis and the placental environment. Placenta 1995;16:289-296. 74. Irani, K: Oxidant signaling in vascular cell growth, death and survival: a review of the roles of reactive oxygen species in smooth muscle and endothelial cell mitogenic and apoptic signaling. Circ Res 2000;87:179-183. 75. Poston L, Raijmakers TM: Trophoblast oxidative stress, antioxidants, and pregnancy outcome–a review. Placenta (suppl. Trophoblast Research) 2004;25:S72-S78. 76. Wang Y, Walsh SW: Placental mitochondria as a source of oxidative stress in pre-eclampsia Placenta 1998;19:581- 586. 77. Hensley K, Benaksas EJ, Bolli R, et al: New perspectives on vitamin E: -tocopherol and carboxyethylhydroxychroman metabolites in biology and medicine, Free Rad Bio Med 2003;13:1-15. 78. Saldeen T, Li D, Mehta JL: Differential effects of alpha and gamma tocopherol on low density lipoprotein oxidation, superoxide activity, platelet aggregation and arterial thrombogenesis. J Am Coll Cardiol 1999;34:1208-1215. 79. Steven DH: Placentation in the mare. J Reprod Fertil (Suppl) 1982;31:41-55. 80. Allen WR, Moor RM: The origin of the equine endometrial cups. I. Production of PMSG by fetal trophoblast cells. J Reprod Fertil 1972;29,313-316. 81. Allen WR, Hamilton DW, Moor RM: The origin of equine endometrial cups. II. Invasion of the endometrium by trophoblast. Anat Rec 1973;117,475-501. 82. Urwin VE, Allen WR: Pituitary and chorionic gonadotrophin control of ovarian function during early pregnancy in equids. J Reprod Fertil (Suppl) 1982;32:371-382. 83. Holtan DW, Nett TM, Estergreen, VL: Plasma progesterone in pregnant mares. J Reprod Fertil (Suppl) 1975;23:419- 424. 84. Steven DH, Samuel CA: Anatomy of the placental barrier in the mare. J Reprod Fertil (Suppl) 1975;23:579-582. 85. Tischner M: Embryo recovery from Polish-pony mares and preliminary observations on foal size after transfer of embryos to large mares. Equine Vet J (Suppl) 1985;3:96-98. 86. Tischner M, Klimczak M: The development of Polish ponies born after embryo transfer to large recipients. Equine Vet J (Suppl) 1989;8:62-63. 87. Allen WR, Wilsher S, Turbull C, et al: Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero. Reproduction 2002;123:445-453. 88. Allen WR, Gower S, Wilsher S: Immunohistochemical localization of vascular endothelial growth factor (VEGF) and its two receptors (Flt-1 and KDR) in the endometrium and placenta of the mare during the oestrous cycle and pregnancy. Reprod Domest Anim 2007;42:516-526. 89. Ashworth CJ, Finch AM, Page KR, et al: Causes and consequences of fetal growth retardation in pigs. Reproduction (Suppl) 2001;58:233-246. 90. Wootton R, MacFadyen IR, Cooper JE: Measurement of placental blood flow in the pig and its relationship to placental and fetal weight. Biol Neonate 1977;31:333-339. 91. Wu G, Bazer FW, Davis TA, et al: Arginine metabolism and nutrition in growth, health and disease. Amino Acids 2009;37:153-168. 92. Li X, Bazer FW, Gao H, et al: Amino acids and gaseous signaling. Amino Acids 2009;37:65-78. 93. Maul H, Longo M, Saade GR, et al: Nitric oxide and its role during pregnancy: from ovulation to delivery. Curr Pharm Des. 2003;9:359-580. 94. Wu G. Amino acids: metabolism, functions, and nutrition. Amino Acids 2009;37:1-17. 95. Li X, Bazer FW, Johnson GA, et al: Dietary supplementation with 0.8% L-arginine between days 0 and 25 of gestation reduces litter size in gilts. J Nutr 2010 Jun;140:1111-6. Epub 2010 Apr 14. 96. Zeng X, Wang F, Fan X, et al: Dietary arginine supplementation during early pregnancy enhances embryonic survival in rats. J Nutr 2008;138:1421-1425. 97. Mateo RD, Wu G, Bazer FW, et al: Dietary L-arginine supplementation enhances the reproductive performance of gilts. J Nutr 2007;137:652-656. Clinical Theriogenology • Volume 4 Number 2 • June 2012147 98. Morise A, Louveau I, Le Hue¨rou-Luron I: Growth and development of adipose tissue and gut and related endocrine status during early growth in the pig: impact on low birth weight. Animal 2008;2:73-83. 99. Poore KR, Fowden AL: The effects of birth weight and postnatal growth patterns on fat depth and plasma leptin concentrations in juvenile and adult pigs. J Physiol 2004;558:295-304. 100. Palini S, De Stefani S, Scala V, et al: Epigenetic regulatory mechanisms during preimplantation embryo development. Ann NY Acad Sci 2011:1221:54-60. 101. Zama AM, Uzumcu M. Epigenetic effects of endocrine-disrupting chemicals on female reproduction: an ovarian perspective. Front Neuroendocrinol 2010;31:420-439. 102. Jammes H, Junien C, Chavatte-Palmer P. Epigenetic control of development and expression of quantitative traits. Reprod Fertil Dev 2011;23:64-74. 103. Patrizio P, Bianchi V, Lalioti MD, et al: High rate of biological loss in assisted reproduction: it is in the seed, not in the soil. Reprod Biomed Online 2007;14:92-95. 104. Quenby S, Vince G, Farquharson R, et al: Recurrent miscarriage: a defect in nature’s quality control? Hum Reprod 2002;17:1959-1963. 105. Okada Y, Ueshin Y, Isotani A, et al: Complementation of placental defects and embryonic lethality by trophoblast- specific lentiviral gene transfer. Nat Biotech 2007; 25:233-237. 106. Georgiades P, Cox B, Gertsenstein M, et al: Trophoblast specific gene manipulation using lentivirus-based vectors. Biotechniques 2007; 42:317-324. 107. Purcell SH, Cantlon JD, Wright CD, et al: The involvement of proline-rich 15 in early conceptus development in sheep. Biol Reprod 2009;81:1112-1121. 108. Sinclair KD, Allegrucci C, Singh R, et al: DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status. Proc Natl Acad Sci USA 2007;104:19351- 19356. 109. Ridderstråle M, Groop L: Genetic dissection of type 2 diabetes. Mol Cell Endocrinol 2009;297:10‐17.  110. Simmons RA: Developmental origins of adult disease. Pediatr Clin North Am 2009;56:449-466. 111. Spyer G, Macleod KM, Shepherd M, et al: Pregnancy outcome in patients with raised blood glucose due to a heterozygous glucokinase gene mutation. Diabet Med 2009;26:14-18. 112. Hattersley AT, Beards F, Ballantyne E, et al: Mutations in the glucokinase gene of the fetus result in reduced birth weight. Nat Genet 1998;19:268-270. 113. Diplas AI, Lambertini L, Lee MJ, et al: Differential expression of imprinted genes in normal and IUGR human placentas. Epigenetics 2009;14:4. 114. Amor DJ: Genomic imprinting, small babies and assisted reproduction. Eur J Hum Genet 2009;17:1-2. 115. Deng T, Kuang Y, Zhang D, et al: Disruption of imprinting and aberrant embryo development in completely inbred embryonic stem cell-derived mice. Dev Growth Differ 2007;49:603-610. 116. Jiang L, Jobst P, Lai L, et al: Expression levels of growth-regulating imprinted genes in cloned piglets. Cloning Stem Cells 2007;9:97-106. 117. Jaenisch R: DNA methylation and imprinting: why bother? Trends Genet 1997;13:323-329. 118. Holliday R, Grigg GW: DNA methylation and mutation. Mutat Res 1993;285:61-67. 119. Zeisel SH: Epigenetic mechanisms for nutrition determinants of later health outcomes. Am J Clin Nutr 2009;89:1488S- 1493S. Clinical Theriogenology • Volume 4 Number 2 • June 2012 148