C Mando'1, M A Marino1, F Miriam1, C De Palma2, M Borelli3, D Trabattoni3, T Stampalija4, E Ferrazzi4, E Clementi2, I Cetin1. 1. Clinical Sciences L. Sacco, Unit Obst and Gynec, University of Milan, Milan, Italy. 2. Clinical Sciences L. Sacco, Unit pharmacology, University of Milan, Milan, Italy. 3. Clinical Sciences L. Sacco, Unit Immunology, University of Milan, Milan, Italy. 4. Unit Obstet and Gynecol, Children Hospital V.Buzzi, Milan, Italy.
Abstract
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.
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.
Authors: Evangeline Deer; V Ramana Vaka; Kristen M McMaster; Kedra Wallace; Denise C Cornelius; Lorena M Amaral; Mark W Cunningham; Babbette LaMarca Journal: Am J Obstet Gynecol MFM Date: 2020-10-27
Authors: Ramana Vaka; Evangeline Deer; Mark Cunningham; Kristen M McMaster; Kedra Wallace; Denise C Cornelius; Lorena M Amaral; Babbette LaMarca Journal: J Clin Med Date: 2021-10-29 Impact factor: 4.964