title: Depolarization of sperm membrane potential is a common feature of men with subfertility and is associated with low fertilization rate at IVF.
abstract: Are significant abnormalities in outward (K(+)) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success?
  
  
  
  Negligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF.
  
  
  
  Sperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K(+) channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K(+) channels in human spermatozoa or the incidence and functional consequences of K(+) channel defects.
  
  
  
  Spermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study.
  
  
  
  Samples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca(2+) influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K(+) signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities.
  
  
  
  Patch clamp electrophysiology was used to assess outward (K(+)) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca(2+)]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function.
  
  
  
  For practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects.
  
  
  
  These data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized.
  
  
  
  The majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest.
  
  
  
  Not applicable.
authors.affiliation: School of Science, Engineering and Technology, Abertay University, Dundee DD11HG, UK.
authors.forename: Sean G
authors.initials: SG
authors.lastname: Brown
authors.affiliation: School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Publicover
authors.affiliation: Reproductive and Developmental Biology, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Mansell
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
authors.forename: Polina V
authors.initials: PV
authors.lastname: Lishko
authors.affiliation: Reproductive and Developmental Biology, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK.
authors.forename: Hannah L
authors.initials: HL
authors.lastname: Williams
authors.affiliation: Reproductive and Developmental Biology, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK.
authors.forename: Mythili
authors.initials: M
authors.lastname: Ramalingam
authors.affiliation: Wolfson Research Institute, School of Medicine, Pharmacy and Health, University of Durham, Queen's Campus, Stockton on Tees TS17 6BH, UK.
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Wilson
authors.affiliation: Reproductive and Developmental Biology, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK c.barratt@dundee.ac.uk.
authors.forename: Christopher L R
authors.initials: CL
authors.lastname: Barratt
authors.affiliation: Reproductive and Developmental Biology, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Sutton
authors.affiliation: Reproductive and Developmental Biology, School of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee DD19SY, UK.
authors.forename: Sarah Martins
authors.initials: SM
authors.lastname: Da Silva
mesh_terms: D020013:Calcium Signaling; D005260:Female; D005306:Fertilization / Q000502:physiology; D005307:Fertilization in Vitro; D006801:Humans; D007248:Infertility, Male / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000235:genetics*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013081:Sperm Motility; D013094:Spermatozoa / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015221:Potassium Channels
keywords: CatSper; IVF; Slo1; Slo3; male infertility; patch clamp electrophysiology; potassium channel; sperm dysfunction; spermatozoa