title: OS048 Mitochondrial content and function in placental cells and tissuesof preeclampsia and IUGR.
abstract: BACKGROUND
  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].
  
  
  OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Clinical Sciences L. Sacco, Unit Obst and Gynec, University of Milan, Milan, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Mando'
authors.affiliation: Clinical Sciences L. Sacco, Unit Obst and Gynec, University of Milan, Milan, Italy.
authors.forename: M A
authors.initials: MA
authors.lastname: Marino
authors.affiliation: Clinical Sciences L. Sacco, Unit Obst and Gynec, University of Milan, Milan, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Miriam
authors.affiliation: Clinical Sciences L. Sacco, Unit pharmacology, University of Milan, Milan, Italy.
authors.forename: C De
authors.initials: CD
authors.lastname: Palma
authors.affiliation: Clinical Sciences L. Sacco, Unit Immunology, University of Milan, Milan, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Borelli
authors.affiliation: Clinical Sciences L. Sacco, Unit Immunology, University of Milan, Milan, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Trabattoni
authors.affiliation: Unit Obstet and Gynecol, Children Hospital V.Buzzi, Milan, Italy.
authors.forename: T
authors.initials: T
authors.lastname: Stampalija
authors.affiliation: Unit Obstet and Gynecol, Children Hospital V.Buzzi, Milan, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Ferrazzi
authors.affiliation: Clinical Sciences L. Sacco, Unit pharmacology, University of Milan, Milan, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Clementi
authors.affiliation: Clinical Sciences L. Sacco, Unit Obst and Gynec, University of Milan, Milan, Italy.
authors.forename: I
authors.initials: I
authors.lastname: Cetin