title: A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels.
abstract: OBJECTIVE
  Could drugs targeting ATP-sensitive K(+) (K(ATP)) channels prevent any spontaneous increase in intracellular Ca(2+) that may occur in human metaphase II (MII) oocytes under in vitro conditions?
  
  
  CONCLUSIONS
  Pinacidil, a K(ATP) channel opener, and glibenclamide, a K(ATP) channel blocker, prevent a spontaneous increase in intracellular Ca(2+) in human MII oocytes.
  
  
  BACKGROUND
  The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca(2+) homeostasis is crucial for cell wellbeing and increased intracellular Ca(2+) levels is a well-established indicator of cell stress.
  
  
  METHODS
  Supernumerary human oocytes (n = 102) collected during IVF/ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca(2+) levels were monitored in oocytes in the following experimental groups: (i) Control, (ii) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP.
  Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean ± SD) age 34.1 ± 0.6 years, n = 41. Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca(2+)-sensitive dye, and monitored by laser confocal microscopy for 2 h at 10 min intervals. Time between oocyte collection and start of Ca(2+) monitoring was 80.4 ± 2.1 h.
  
  
  RESULTS
  Intracellular levels of Ca(2+) increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0 ± 11.8 AU (AU = arbitrary units; n = 23) to 91.8 ± 14.0 AU (n = 19; P < 0.001) after 2 h of monitoring. Pinacidil (100 µM) inhibited this increase in Ca(2+) (85.3 ± 12.3 AU at the beginning of the experiment, 81.7 ± 11.0 AU at the end of the experiment; n = 13; P = 0.616). Glibenclamide (100 µM) also inhibited the increase in Ca(2+) (74.7 ± 10.6 AU at the beginning and 71.8 ± 10.9 AU at the end of the experiment; n = 13; P = 0.851. DNP (100 mM) induced an increase in intracellular Ca(2+) that was inhibited by glibenclamide (100 µM; n = 9) but not by pinacidil (100 µM; n = 5).
  
  
  CONCLUSIONS
  Owing to clinical and ethical considerations, it was not possible to monitor Ca(2+) in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4 ± 2.1 h (n = 102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilized, it is possible that they may have been abnormal with impaired Ca(2+) homeostasis and, furthermore, the altered Ca(2+) homeostasis might have been associated solely with the protracted incubation.
  
  
  CONCLUSIONS
  These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca(2+), which can be counteracted by drugs targeting K(ATP) channels. As Ca(2+) homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that K(ATP) channel openers and blockers should be tested as drugs for improving success rates of ART.
  
  
  BACKGROUND
  University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest.
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK.
authors.forename: Gonçalo
authors.initials: G
authors.lastname: Fernandes
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK.
authors.forename: Navin
authors.initials: N
authors.lastname: Dasai
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK.
authors.forename: Natalia
authors.initials: N
authors.lastname: Kozlova
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK Department of Anatomy and Clinical Embryology, King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: Albaraa
authors.initials: A
authors.lastname: Mojadadi
authors.affiliation: Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee.
authors.forename: Mandy
authors.initials: M
authors.lastname: Gall
authors.affiliation: Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee.
authors.forename: Ellen
authors.initials: E
authors.lastname: Drew
authors.affiliation: Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Barratt
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee University of Abertay, Dundee, UK.
authors.forename: Oladipo A
authors.initials: OA
authors.lastname: Madamidola
authors.affiliation: University of Abertay, Dundee, UK.
authors.forename: Sean G
authors.initials: SG
authors.lastname: Brown
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK.
authors.forename: Alison M
authors.initials: AM
authors.lastname: Milne
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Martins da Silva
authors.affiliation: Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Whalley
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee.
authors.forename: Christopher L R
authors.initials: CL
authors.lastname: Barratt
authors.affiliation: Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK a.jovanovic@dundee.ac.uk.
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Jovanović
mesh_terms: D002118:Calcium / Q000378:metabolism*; D046149:Embryo Culture Techniques; D006706:Homeostasis; D059471:In Vitro Oocyte Maturation Techniques / Q000379:methods*; D049990:Membrane Transport Modulators / Q000494:pharmacology*; D008954:Models, Biological; D009865:Oocytes / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D020110:Pinacidil / Q000494:pharmacology*; D013312:Stress, Physiological
chemical_list: D049990:Membrane Transport Modulators; D020110:Pinacidil; D002118:Calcium
keywords: ATP-sensitive K+ channels; Ca2+; glibenclamide; human oocytes; pinacidil