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DOHaD, nutrition and basic research
Full text linkDOHaD, NUTRITION AND BASIC RESEARCH
C. Mandò, I. Cetin; Department of Biomedical and Clinical Sciences Luigi Sacco University of Milan, Milan, Italy
The “DOHaD” (Developmental Origin of Health and Disease) theory describes how in utero exposure to environmental factors may have long-term effects on the structural and functional development of the fetus. Extensive retrospective studies, such as those on the Dutch famine of 1944, have reported correlations between maternal diet or nutritional status and the risk of pregnancy pathologies or to develop adverse conditions in the future adult. Indeed, macro- and micronutrients taken with the maternal diet can regulate the stability and expression of fetal/placental DNA and phenotype adaptations through epigenetic modifications, reversible mechanisms that occur without changes in the DNA sequence (DNA methylation, histone acetylation, microRNA) [1]. Recently, a large prospective longitudinal cohort study in humans (MANOE study) reported that maternal intake of methyl donors, especially during the periconceptional period, can affect the epigenoma of the offspring in genes related to obesity and diabetes. However, many observations on this issue are born from basic research studies performed on the placenta: placental epigenetic modifications are one of the main mechanisms through which nutritional and environmental factors affect fetal growth. Epigenetic regulation of placental phenotype and function has been extensively studied in the mouse. For example, “imprinted” placental genes (IGF2, H19) act as “nutritional sensors” by varying their methylation status based on environmental conditions. In our lab, we have recently reported lower functionality in the placenta of overweight/obese women with high gestational weight gain, with an important role in fetal sex [2]. Those placentas also exhibit alterations in mitochondrial content suggesting a bioenergetic placental imbalance resulting from an altered nutritional intake. Methylation of mitochondrial DNA may also be involved in these mechanisms [3]. Future research will allow to fully understand the underlying mechanisms of pregnancy pathologies in relation to maternal-fetal nutrition.
REFERENCES
[1] Vaiman D. Genes, epigenetics and miRNA regulation in the placenta. Placenta. 2017;52:127-33.
[2] Mandò C, Calabrese S, Mazzocco MI, Novielli C, Anelli GM, Antonazzo P, Cetin I. Sex specific adaptations in placental biometry of overweight and obese women. Placenta. 2016;38:1-7.
[3] Novielli C, Mandò C, Tabano S, Anelli GM, Fontana L, Antonazzo P, Miozzo M, Cetin I. Mitochondrial DNA content and methylation in fetal cord blood of pregnancies with placental insufficiency. Placenta. 2017;55:63-70
Placental phenotyping for a better approach to clinical therapies
No full textBACKGROUND.
Pregnancy pathologies with abnormal placental phenotype, namely Intrauterine Growth Restriction (IUGR) and Preeclampsia (PE), are well recognized to have a greater risk of neonatal mortality and morbidity and of cardiovascular and metabolic diseases in later life.
Intrauterine growth, which mainly depends on placental functionality, leads to the delivery (both at term and pre-term) of infants with sizes that may be normal or small for the infant genetic potential, leading to individuals with different neonatal nutritional and pharmacological needs. Nevertheless, the distinction between these two infant phenotypes is still difficult. A well-defined placental phenotype may help to optimize neonatal protocols.
Abnormalities of placental morphology have been reported in IUGR and severe PE, as well as defects in the oxygenation of the feto-placental unit. Moreover, several in vitro and in vivo studies also conducted by our group clearly showed changes in placental nutrient and micronutrient transport capacity.
We also recently demonstrated an increased mitochondrial (mt) DNA content in both IUGR placentas and maternal blood at delivery vs controls. We are currently investigating mt functionality in IUGR and PE placentas, for better understanding if possible mt abnormalities may represent specific oxidative markers of these pathologies.
Here we report our recent results on placental cells oxygen (O2) consumption and Respiratory Chain Complexes (RCC) gene expression in pregnancies complicated by IUGR or PE compared to normal pregnancies, to assess the respiratory phenotype of the placenta related to the pregnancy outcome.
METHODS.
Cytotrophoblast cells were isolated from 16 placentas (8 IUGR, 3 PE alone, 5 Controls -C-) of non-smoking women at elective caesarean section, and characterized by cytofluorimetry using cytokeratin-7 and anti-vimentin antibodies. mRNA levels of NDUFA9 (CI), ETFDH (CII), UQCRC1 (CIII) and COX4I1 (CIV) were quantified by Real Time (RT) PCR. O2 consumption, accounting for the mt functionality, was evaluated by High Resolution Respirometry (HRR), by administration of substrates and inhibitors of different RCC, thus allowing the measure of the global cell and of single complexes activity. Data were normalized by mtDNA content.
RESULTS
IUGR presented significantly lower CIII and CIV mRNA levels vs C. On the contrary, both raw and normalized data in IUGR with or without PE (but not in PE without IUGR) showed significantly higher O2 consumption levels of RCC (altogether and singularly) vs C, particularly for CIV, suggesting a compensatory mechanism to their lower expression. These results shed new light into placental oxygenation in IUGR, suggesting that increased placental oxygen utilization may represent a limiting step in fetal growth restriction.
In conclusion, a detailed placental phenotyping may aid clinicians in the identification of babies at risk of short- and long-term consequences. This may help neonatologist in adopting early and more specific neonatal nutrition treatments or therapies by diversifying infants with small for gestational age size to infants with small size which did not reach their growth potential
Maternal predictors of intrauterine growth restriction
No full textPURPOSE OF REVIEW: Intrauterine growth restriction (IUGR) occurs when fetal growth rate falls below the genetic potential and affects a significant number of pregnancies, but still no therapy has been developed for this pregnancy disease. This article reviews the most recent findings concerning maternal characteristics and behaviours predisposing to IUGR as well as maternal early markers of the disease. A comprehensive understanding of factors associated with IUGR will help in providing important tools for preventing and understanding adverse outcomes.
RECENT FINDINGS: Maternal nutritional status, diet and exposure to environmental factors are increasingly acknowledged as potential factors affecting fetal growth both by altering nutrient availability to the fetus and by modulating placental gene expression, thus modifying placental function.
SUMMARY: Assessing nutritional and environmental factors associated with IUGR, and the molecular mechanisms by which they may have a role in the disease onset, is necessary to provide comprehensive and common guidelines for maternal care and recommended behaviours. Moreover, maternal genetic predispositions and early serum markers may allow a better and more specific monitoring of high risk pregnancies, optimizing the timing of delivery
Hematological and biochemical findings in fetal growth restriction and the relationship to hypoxia
No full textMany factors influence the process of intrauterine growth so that some fetuses do not follow their growth potential. Intrauterine growth restriction (IUGR) is a complex disease with different severity depending on placental compromise. A specific “insufficient” placental phenotype has been described in IUGR, characterized by defects in placental metabolism and nutrient transport, particularly amino acids and lipids, but also micronutrients such as iron and folate. These changes are independent of severity and likely responsible for intrauterine programming. Less severe IUGR (with no compromise in umbilical blood flows and fetal heart rate) do not exhibit alterations in oxygen, lactate and glucose concentrations in fetal blood. IUGR fetuses become progressively hypoxic and lactacidemic with severity, showing placental impairment of mitochondria biogenesis and function, and defects in cell production of energy together with oxidative stress. These severe conditions have to be carefully balanced with the burden of prematurity in the timing of delivery
Maternal micronutrients, placental growth and fetal outcome
No full textPregnancy can be regarded as a three-compartment model, with the mother, placenta, and fetus interacting to ensure proper fetal growth and development. Maternal health, along with maternal diet, body composition, metabolism, and placental nutrient supply, is the main fac- tor that can negatively or positively influence fetal development. Before reaching the fetus, nutrients from maternal diet are used by the placenta for its own metabolism. The quality and quantity of nutrients that reach the fetus are indeed influenced by placental shape, size, and characteristics. Placental growth and develgpment are influenced by the maternal diet itself.
This chapter aims to show how fetal and postnatal growth and development are strictly dependent on proper maternal nutritional intake before and during pregnancy and how over- supply, deficiency, or poor quality of nutrients may influence placental development and adversely affect pregnancy outcome and expression offetal genetic potential
Placental fatty acid transport in maternal obesity
No full textPregestational obesity is a significant risk factor for adverse pregnancy outcomes. Maternal obesity is associated with a specific proinflammatory,
endocrine and metabolic phenotype that may lead to higher supply of nutrients to the feto-placental unit and to excessive fetal fat
accumulation. In particular, obesity may influence placental fatty acid (FA) transport in several ways, leading to increased diffusion driving
force across the placenta, and to altered placental development, size and exchange surface area. Animal models show that maternal obesity is
associated with increased expression of specific FA carriers and inflammatory signaling molecules in placental cotyledonary tissue, resulting in
enhanced lipid transfer across the placenta, dislipidemia, fat accumulation and possibly altered development in fetuses. Cell culture experiments
confirmed that inflammatory molecules, adipokines and FA, all significantly altered in obesity, are important regulators of placental lipid
exchange. Expression studies in placentas of obese–diabetic women found a significant increase in FA binding protein-4 expression and in
cellular triglyceride content, resulting in increased triglyceride cord blood concentrations. The expression and activity of carriers involved in
placental lipid transport are influenced by the endocrine, inflammatory and metabolic milieu of obesity, and further studies are needed to
elucidate the strong association between maternal obesity and fetal overgrowth
Is the placenta an innocent bystander in perinatal programming?
No full textDuring pregnancy, a well functioning placenta is needed to ensure appropriate growth and development of the fetus [1]. Indeed, a malfunctioning or “insufficient” placenta has been recognized as the “cause” of Intrauterine Growth Restriction (IUGR) [2], leading to decreased oxygen delivery as well as altered placental transport of nutrients, mainly amino acids and lipids, but also micronutrients such as iron and folate. A number of previous studies from our lab support this hypothesis, demonstrating a specific placental phenotype of IUGR [3], recently confirmed with decreased levels of placental Transferrin Receptor (TFRC – mediating cellular iron uptake) or of Sodium-coupled Neutral Amino acid transporter 2 (SNAT2) in IUGR versus controls [4, 5] (summarized in Tab. 1). Maternal nutritional status, diet and exposure to environmental factors are increasingly acknowledged as potentially affecting placental gene expression, thus modifying placental function. These epigenetic associations link intrauterine environment to adverse perinatal outcomes reprogramming the fetal epigenome with several mechanisms, such as methylation or miRNA, thus affecting gene expression and activity in preeclamptic (PE) and IUGR tissues [6]. Changes in miRNA expression pattern have been observed in placental tissue and associated with several pregnancy pathologies as preeclampsia (DOWN miR-21, UP miR-155, DOWN miR-223), GDM (DOWN miR-132), IUGR (DOWN miR-21, DOWN miR-210) and preterm birth (UP miR-493, UP miR-338) [7]. In this context, an active placental metabolism is crucial to support both trophoblast invasion and placentation [8]. Alterations in early implantation may lead to mismatches in oxygen (O2) delivery to different areas of the placenta, with less O2 exchange between the uterine and the umbilical circulations [9]. Mitochondrial DNA (mtDNA) copy number is positively correlated with the number of mitochondria. We have previously demonstrated altered mitochondrial content in IUGR placentas [10], with higher mtDNA levels in IUGR maternal blood [11]. Moreover, we measured the functionality of the respiratory complexes (RCC) by high-resolution respirometry (HRR), in order to assess potential alterations in placental energy metabolism [12] (summarized in Tab. 1). Preliminary observations suggest similar changes in placental mitochondria, DNA content and function of obese pregnant women. These pregnancies are characterized by low-grade inflammation and oxidative stress [13]. Moreover, dysregulated mt genes methylation (D-loop and CO1 hypomethylation) might expand our findings of higher mtDNA content in fetal cord blood of
IUGR and PE [14]. These preliminary data may indeed suggest a compensatory attempt of fetuses to increase energy production through higher mtDNA content and RCC (CO1) expression, representing a further link between epigenetic
changes and perinatal programming of diseases. Another issue is related to the placental hormonal function. The placenta as a source of a wide array of hypothalamic or pituitary hormones was a hot topic in the 60-70s, then neglected because of the radioactive techniques needed at that time. Steroid hormones, and in particular estrogens, are important for uterine/placental vascular adaptations to pregnancy, but also essential for trophoblast cells syncytialization in placenta. During pregnancy, the feto-placental unit is a source of estrogens through its aromatase enzyme Cytochrome P450 (CYP19) involved in estradiol (E2) production [15]. Interestingly, CYP19 levels appeared signi%cantly higher in IUGR placentas that we recently analyzed. We might speculate that the CYP19 alterations have an estrogenrelated protective action in more severe IUGR placentas, which we showed to be characterized by increased mtDNA [16]. Ongoing analyses will evaluate if these placental molecular alterations result in E2 hormone altered production. Placental mesenchymal stromal cells (p-MSCs) may also represent an interesting point to evaluate in order to understand normal and abnormal placental development. In IUGR pregnancies, p-MSCs have lower proliferation rate with earlier shift towards homogeneity than in controls. In vitro findings also demonstrate that multipotency of IUGR derived p-MSCs is restricted, as their capacity for adipocyte differentiation is increased, whereas their differentiation ability towards endothelial cell lineage is decreased (Fig. 1) [17]. These findings are indicative of changes that may also be reflected in the developing fetus (summarized in Tab. 1).
The potential role for p-MSCs in pregnancy pathologies, as well as the striking mitochondrial changes involved in energy production, open new perspectives for understanding the development of the diseases and potential routes of prevention and treatment
Hypoxia and mitochondrial DNA : primary human trophoblast culture as a tool to investigate mechanisms behind placental insufficiency pathologies
No full textThe placenta is an active organ between mother and fetus. Therefore it is essential to maintain the homeostasis for the developing fetus environment: adverse influences during intrauterine life may contribute to develop adult diseases, according to the fetal programming theory (Barker, MolMedToday 1995). Defects in placentation may lead to obstetric pathologies like Intrauterine Growth Restriction (IUGR) and Preeclampsia (PE), both associated with placental insufficiency (Levy et al, Am J Obstet Gynecol. 2002). PE and IUGR arise in a low oxygen microenvironment, while placenta hypoperfusion and hypoxia, occurring later in pregnancy, induce trophoblast injury (Oh et al, Placenta 2011).
Mitochondria (mt), key players of cellular respiration, may act as a possible link between oxygenation defects of placental cells and these pregnancy complications. Our group previously demonstrated significantly higher mitochondrial DNA content (mtDNA) in IUGR placentas versus controls (Lattuada et al, Placenta 2008). Moreover, a correlation between PE placentas and mitochondrial dysfunction has already been demonstrated and linked to oxidative stress (Myatt, Histochem Cell Biol. 2004; Hung and Burton, Taiwan J Obstet Gynecol. 2006). Other studies have shown the influence of acute and chronic hypoxia on mtDNA content in murine models (Widshwendter, MolMedToday 1998; Gutsaeva et al, J.Neurosci. 2008). These data highlight the necessity to better understand the oxygenation pathways in placental physiological cells and the effect of low oxygen in this system.
Our proposal is thus to study primary physiological trophoblast cells, an in vitro model already used in other studies, coltured under different oxygen conditions, and investigating the effects on mitochondria copy number.
Primary trophoblast cells were isolated as previously described (Kliman et al, Endocrinology 1986) from human placentas of term singleton uncomplicated pregnancies (non-smoking women with appropriately grown fetuses and no maternal/fetal pathologies). Trophoblasts were maintained in standard conditions (20% oxygen -O2-; 5% CO2; 37C°). After 4 hours (T0) cells were incubated in the following conditions: 20%O2; 20%O2 with 0,2mM Cobalt Chloride (CoCl2, activator of HIF1α) in the medium; 8%O2 (which mimics O2 conditions of 2nd-3rd trimester placentas); 0.1%O2. Cells were coltured for 72 hours and freezed at T24, T48, T72. A further condition was represented by shifting cells from 20% to 8 or 0.1% of O2 at T48, to investigate low O2 effects on already syncytialized cells. Total DNA was extracted by Trizol from cells freezed at T24, T48, T72. mtDNA was analyzed by real-time PCR (2-Ct method) using Cytocrome B as mt target gene and the nuclear RNasi P as endogenous gene.
At T24, T48, T72, all cells coltured at low O2 (8-0.1%) present a trend, though not significant, towards higher mtDNA levels compared to standard conditions, confirming a possible role of low O2 levels in up-regulating mtDNA levels. The same tendency is observed in cells shifted to 8 or 0.1% O2 at T48, thus showing a similar response in syncytiotrophoblast cells, and in 20%O2-CoCl2 conditions, possibly indicating a mediation by HIF1α. Data clustered by O2 concentration show no significant variations between different time intervals. These are preliminary data: we intend to increase the sample number and to reduce the variability in the experimental procedure. Moreover, we propose to standardize the number of cells plated and to extend the adhesion interval from 4 to 12 hours, improving the amount and quality of mtDNA. To enrich our data, we want to study further aspects of mt functionality, for example the possible alterations of respiratory chain complexes caused by lack of O2
PLACENTAL IRON TRANSPORT AND MATERNAL ABSORPTION
No full textThe iron need in pregnancy is significantly higher in comparison to that in the nonpregnant state. The iron absorbed during pregnancy is used for expansion of the maternal erythrocyte mass, to fulfill the fetus's iron needs, to create placenta, and to cope with blood loss at delivery. Term neonates have a total body store of about 1 g of iron, all derived from the mother. Despite the overall increase in nutritional requirements, biochemical, metabolic, and physiological adjustments of the maternal organism happen in order to meet the extra demands and to support the homeostasis of iron. In all healthy pregnant women with sufficient iron stores, the increased iron absorption is coupled with the mobilization of iron stores. Unfortunately, iron deficiency during pregnancy is alarmingly common. The function of placental transport determines the composition of umbilical cord blood providing nutrients and oxygen to the fetus to ensure appropriate fetal growth. Iron in the developing fetus is accumulated against a concentration gradient and, in the case of maternal iron deficiency, the placenta can protect the fetus significantly through the increased expression of placental transferrin receptor together with a rise in divalent metal transporter 1 (DMT1). Despite the resistance of the fetus to maternal deficiency, any stress that alters placental development or function may have consequences for the developing fetus. Despite its central importance in fetal development, little is known about the mechanism of iron transfer across the placenta. Consequently, it is crucial to understand the molecular basis of placental iron transport in order to optimize the iron intake recommendation, reducing adverse pregnancy outcomes for both the mother and her child
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