Placental mitochondrial content and function in intrauterine growth restriction and preeclampsia.

Intrauterine growth restriction (IUGR) and pregnancy hypertensive disorders such as preeclampsia (PE) associated with IUGR share a common placental phenotype called "placental insufficiency", originating in early gestation when high availability of energy is required. Here, we assess mitochondrial content and the expression and activity of respiratory chain complexes (RCC) in placental cells of these pathologies. We measured mitochondrial (mt)DNA and nuclear respiratory factor 1 (NRF1) expression in placental tissue and cytotrophoblast cells, gene and protein expressions of RCC (real-time PCR and Western blotting) and their oxygen consumption, using the innovative technique of high-resolution respirometry. We analyzed eight IUGR, six PE, and eight uncomplicated human pregnancies delivered by elective cesarean section. We found lower mRNA levels of complex II, III, and IV in IUGR cytotrophoblast cells but no differences at the protein level, suggesting a posttranscriptional compensatory regulation. mtDNA was increased in IUGR placentas. Both mtDNA and NRF1 expression were instead significantly lower in their isolated cytotrophoblast cells. Finally, cytotrophoblast RCC activity was significantly increased in placentas of IUGR fetuses. No significant differences were found in PE placentas. This study provides genuine new data into the complex physiology of placental oxygenation in IUGR fetuses. The higher mitochondrial content in IUGR placental tissue is reversed in cytotrophoblast cells, which instead present higher mitochondrial functionality. This suggests different mitochondrial content and activity depending on the placental cell lineage. Increased placental oxygen consumption might represent a limiting step in fetal growth restriction, preventing adequate oxygen delivery to the fetus.

Mitochondrial content and hepcidin are increased in obese pregnant mothers.

OBJECTIVE: Maternal obesity is characterized by systemic low-grade inflammation and oxidative stress (OxS) with the contribution of fetal sex dimorphism. We recently described increased mitochondrial content (mtDNA) in placentas of obese pregnancies. Here, we quantify mtDNA and hepcidin as indexes of OxS and systemic inflammation in the obese maternal circulation. METHODS: Forty-one pregnant women were enrolled at elective cesarean section: 16 were normal weight (NW) and 25 were obese (OB). Obese women were further classified according to the presence/absence of maternal gestational diabetes mellitus (GDM); [OB/GDM(-)]: n = 15, [OB/GDM(+)]: n = 10. mtDNA and hepcidin were evaluated in blood (real-time PCR) and plasma (ELISA). RESULTS: mtDNA and hepcidin levels were significantly increased in OB/GDM(-) versus NW, significantly correlating with pregestational BMI. Male/female (M/F) ratio was equal in study groups, and overall F-carrying pregnancies showed significantly higher mtDNA and hepcidin levels than M-carrying pregnancies both in obese and normal weight mothers. CONCLUSIONS: Our results indicate a potential compensatory mechanism to increased obesity-related OxS and inflammation, indicated by the higher hepcidin levels found in obese mothers. Increased placental mitochondrial biogenesis, due to lipotoxic environment, may account for the greater mtDNA amount released in maternal circulation. This increase is namely related to F-carrying pregnancies, suggesting a gender-specific placental response.

Placental fatty acid transport in maternal obesity.

Pregestational 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.

OS048 Mitochondrial content and function in placental cells and tissuesof preeclampsia and IUGR.

INTRODUCTION: Early onset placenta Preeclampsia (ePE) with Intrauterine Growth Restriction (IUGR) is associated with insufficient placental function, leading to decreased nutrient and oxygen (O2) availability for the fetus [1]. Mitochondria (mt) are the cell energy producers. Mt dysfunctions could be involved in altered placental metabolism leading to ePE and IUGR. We previously demonstrated higher levels of mtDNA in human IUGR placentas [2]. OBJECTIVES: Here we investigate mtDNA levels in ePE and PE without IUGR placentas, and we present an innovative technique, High Resolution Respirometry (HRR), on cytotrophoblast cells (CTC) from PE, IUGR and control placentas (C), measuring cell O2 consumption which represents respiratory chain efficiency. METHODS: mtDNA was measured by Real-Time PCR in 20 PE placentas, with (n=14) or without (n=6) IUGR, and 45 C. CTC were isolated from 4 PE, 4 IUGR and 6 C and characterized by flow cytometry, staining samples with anti-cytokeratin-7 and anti-vimentin antibodies. Cells were located in chambers with atmospheric O2levels; 2 different protocols were used, with or without digitonin permeabilization, allowing to measure the O2 consumption of the respiratory chain complexes singularly or all together. Substrates and inhibitors of different respiratory chain complexes were sequentially administered (succinate, ADP, oligomycin, FCCP, rotenone, antimycin A, glutamate, malate, myxothiazol, TMPD, ascorbate, pyruvate, cytochrome C, differently combined depending on the protocol) and O2 consumption levels were recorded. Data were normalized by Citrate Synthase (CS) activity and CTC mtDNA content. RESULTS: PE placentas: mtDNA content was significantly increased in ePE+IUGR (p=0.02) vs C; opposite to this, mtDNA was decreased in PE without IUGR (p=0.03). CTC: single mt O2 consumption (obtained by normalizing data both by CS activity and mtDNA) was slightly increased both in PE and IUGR. The global cell respiration was increased, though not significantly. The trend towards higher O2 consumption studied on permeabilized cells  was confirmed for all the respiratory chain complexes. CONCLUSION: Our study showed that mtDNA is increased also in ePE with IUGR and added the novel observation that mtDNA is decreased in PE without IUGR. In both conditions placental mitochondria present an altered respiratory chain activity, with a trend to a higher respiratory capacity. This could lead to higher ATP production likely as an attempt to compensate for other aspects of placental disease due to small or inefficient exchange capabilities. Further data are needed to confirm these preliminary results, together with specific enzymatic assays to asses the respiratory chain complexes functionality. Supported by Fondazione Giorgio Pardi, Associazione Studio Malformazion(ASM) and by a Grant COFIN (Italian Ministry of Research) on: New markers for preterm deliveries.

Transferrin receptor gene and protein expression and localization in human IUGR and normal term placentas.

Iron (Fe) deficiency in pregnancy is associated to low birth weight and premature delivery while in adults it can result in increased blood pressure and cardiovascular disease. Cellular Fe uptake is mediated by the Transferrin Receptor 1 (TFRC), located in the trophoblast membranes. Here, we measured TFRC mRNA expression (Real Time PCR) and TFRC protein expression and localization (Western Blotting and immunohistochemistry) in IUGR compared to control placentas. A total of 50 IUGR and 56 control placentas were studied at the time of elective cesarean section. IUGR was defined by ultrasound in utero, and confirmed by birth weight <10th percentile. Three different severity groups were identified depending on the umbilical artery pulsatility index and fetal heart rate. TFRC mRNA expression was significantly lower in IUGR placentas compared to controls (p < 0.05), and this was confirmed for TFRC protein levels. In both experiments the most severe IUGR group presented lower expression compared to the other groups, and this was also related to umbilical venous oxygen levels. TFRC protein localization in the villous trophoblast did not differ in the groups, and was predominantly present in the syncytiotrophoblast. In conclusion, these are the first observations about TFRC expression in human IUGR placentas, demonstrating its significant decrease in IUGR vs controls. Thus, Fe transport could be limited in IUGR placentas. Further studies are needed to study components of the placental Fe transport system and to clarify the regulation mechanisms involved in TFRC expression, possibly altered in IUGR placentas.