ESPRESSIONE DI PROTEINE COINVOLTE NEL TRASPORTO PLACENTARE DEL FERRO IN GRAVIDANZE NORMALI E PATOLOGICHE

Background Intrauterine Growth Restriction (IUGR) and Pre-eclampsia (PE) are pregnancy pathologies associated with deficient placental function, leading to decreased nutrient and oxygen availability to the fetus. Iron (Fe) deficiency in pregnancy is associated with low birth weight and premature delivery. Nevertheless, Fe oversupply promotes the generation of free radicals and causes oxidative damage in the cells. A previous study performed in the lab where this thesis has been carried out demonstrated a significant decrease of the Fe cell-importer Transferrin Receptor (TfR1), located in the trophoblast cell (TC) microvillar membrane, in human Intrauterine Growth Restricted (IUGR) vs normal (N) placentas (Mandò C. et al. 2011). Ferroportin (FPN) is a trans-membrane protein located in the TC basal membrane that exports Fe towards the fetal circulation. Aim We hypothesized that TfR1 downregulation in IUGR placentas may be due to Fe intracellular accumulation. Thus, we measured FPN gene and protein expression in human IUGR vs N placentas. Then, we extended the Fe transporters investigation to PE placentas, by measuring TfR1 and FPN gene expression in human PE and PE associated with IUGR vs N placentas. Furthermore, we evaluated the relationship between Fe supplementation, food intake, haematologic parameters and pregnancy outcome in a cohort of Italian pregnant and healthy women. Materials and Methods Placentas were sampled at the time of elective cesarean section; villi were selected, washed and immediately frozen for following analysis. Umbilical venous and arterial blood was sampled for pH, oxygen measurements, Hb and lactate measurements, from a doubly clamped segment of the cord immediately after fetal extraction. All samples were collected in hepa- rinized syringes and kept on ice until the end of analysis. All the parameters were measured on a GEM Premier 3000 (Instrumentation Laboratory). FPN mRNA was quantified in a total of 50 N, 41 IUGR, 10 PE and 15 PE+IUGR placentas by Real Time PCR and FPN protein expression was quantified in 26 N and 14 IUGR by Enzyme-Linked ImmunoSorbent Assay. TfR1 mRNA was quantified in 28 N, 10 PE and 15 PE+IUGR placentas. For the study of Fe supplementation, 55 healthy Italian singleton pregnant women were randomized in 4 groups in relation to different doses and types of Fe supplementation (Controls, Fe Sulphate 30 mg, Fe liposomial 14 mg and Fe liposomial 28 mg). At 28-30 gestational weeks data about eating behavior were collected by food frequency questionnaires and hematologic parameters of Fe content (hemoglobin, ferritin, transferrin, serum Fe, folate, vitamin B12, homocystein) in maternal blood by biochemical analysis. Results and Discussion Fetuses from IUGR pregnancies, both with and without PE, have lower pO2 in umbilical vein, while there were no significant differences in fetal emoglobin (Hb) among controls, IUGR, PE and PE+IUGR groups. Both FPN mRNA and protein expression were not statistically different in IUGR compared to N placentas. FPN and TfR1 mRNA levels were not statistically different in PE and in PE+IUGR compared to N placentas. Our results showed no differences in FPN mRNA and protein placental levels between IUGR and N. This suggests that the Fe reaching IUGR fetuses may be decreased compared to normal pregnancies, as a consequence of TfR1 downregulation in the microvillar membranes. This could impair many cellular processes, since Fe is a very important element for enzyme functions and for a correct oxidative status in the cell. TfR1 and FPN mRNA levels were not statistically different in PE and in PE+IUGR vs. N placentas, suggesting that Fe transport is not affected in Preeclampsia. However, we aim at enlarging our analysis to reach definitive conclusions on the Fe transport system in PE placentas. No significant differences were found in the maternal hematologic parameters between the 4 groups of randomized pregnant women at 28-31 weeks, with the exception of hemoglobin, which was significantly higher in women supplemented with 28 mg of Fe liposomial compared to controls (p<0.01). The groups were homogeneous in relation to pregnancy outcomes: no differences were found in neonatal and placental weights, as well as in gestational age at delivery and in umbilical artery pH. The food frequency questionnaires analysis revealed that the control group assumed higher quantity of bioavailable iron (meat) compared to other groups. This may explain the absence of significant differences in the maternal iron status in the control group compared to the supplemented groups.