title: Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity.
abstract: OBJECTIVE
  Can we identify compound(s) with reported phosphodiesterase inhibitor (PDEI) activity that could be added to human spermatozoa in vitro to enhance their motility without compromising other sperm functions?
  
  
  CONCLUSIONS
  We have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples.
  
  
  BACKGROUND
  For >20 years, the use of non-selective PDEIs, such as pentoxifylline, has been known to influence the motility of human spermatozoa; however, conflicting results have been obtained. It is now clear that human sperm express several different phosphodiesterases and these are compartmentalized at different regions of the cells. By using type-specific PDEIs, differential modulation of sperm motility may be achieved without adversely affecting other functions such as the acrosome reaction (AR).
  
  
  METHODS
  This was a basic medical research study examining sperm samples from normozoospermic donors and subfertile patients attending the Assisted Conception Unit (ACU), Ninewells Hospital Dundee for diagnostic semen analysis, IVF and ICSI. Phase 1 screened 43 commercially available compounds with reported PDEI activity to identify lead compounds that stimulate sperm motility. Samples were exposed (20 min) to three concentrations (1, 10 and 100 µM) of compound, and selected candidates (n = 6) progressed to Phase 2, which provided a more comprehensive assessment using a battery of in vitro sperm function tests.
  
  
  METHODS
  All healthy donors and subfertile patients were recruited at the Medical Research Institute, University of Dundee and ACU, Ninewells Hospital Dundee (ethical approval 08/S1402/6). In Phase 1, poor motility cells recovered from the 40% interface of the discontinuous density gradient were used as surrogates for patient samples. Pooled samples from three to four different donors were utilized in order to reduce variability and increase the number of cells available for simultaneous examination of multiple compounds. During Phase 2 testing, semen samples from 23 patients attending for either routine diagnostic andrology assessment or IVF/ICSI were prepared and exposed to selected compounds. Additionally, 48 aliquots of prepared samples, surplus to clinical use, were examined from IVF (n = 32) and ICSI (n = 16) patients to further determine the effects of selected compounds under clinical conditions of treatment. Effects of compounds on sperm motility were assessed by computer-assisted sperm analysis. A modified Kremer test using methyl cellulose was used to assess sperm functional ability to penetrate into viscous media. Sperm acrosome integrity and induction of apoptosis were assessed using the acrosomal content marker PSA-FITC and annexin V kit, respectively.
  
  
  RESULTS
  In Phase 1, six compounds were found to have a strong effect on poor motility samples with a magnitude of response of ≥ 60% increase in percentage total motility. Under capacitating and non-capacitating conditions, these compounds significantly (P ≤ 0.05) increased the percentage of total and progressive motility. Furthermore, these compounds enhanced penetration into a cervical mucus substitute (P ≤ 0.05). Finally, the AR was not significantly induced and these compounds did not significantly increase the externalization of phosphatidylserine (P = 0.6, respectively). In general, the six compounds maintained the stimulation of motility over long periods of time (180 min) and their effects were still observed after their removal. In examinations of clinical samples, there was a general observation of a more significant stimulation of sperm motility in samples with lower baseline motility. In ICSI samples, compounds #26, #37 and #38 were the most effective at significantly increasing total motility (88, 81 and 79% of samples, respectively) and progressive motility (94, 93 and 81% of samples, respectively). In conclusion, using a two-phased drug discovery screening approach including the examination of clinical samples, 3/43 compounds were identified as promising candidates for further study.
  
  
  CONCLUSIONS
  This is an in vitro study and caution must be taken when extrapolating the results. Data for patients were from one assessment and thus the robustness of responses needs to be established. The n values for ICSI samples were relatively small.
  
  
  CONCLUSIONS
  We have systematically screened and identified several compounds that have robust and effective stimulation (i.e. functional significance with longevity and no toxicity) of total and progressive motility under clinical conditions of treatment. These compounds could be clinical candidates with possibilities in terms of assisted reproductive technology options for current or future patients affected by asthenozoospermia or oligoasthenozoospermia.
  
  
  BACKGROUND
  This study was funded primarily by the MRC (DPFS) but with additional funding from the Wellcome Trust, Tenovus (Scotland), University of Dundee, NHS Tayside and Scottish Enterprise. The authors have no competing interests. A patent (#WO2013054111A1) has been published containing some of the information presented in this manuscript.
authors.affiliation: Reproductive and Developmental Biology, Medical School, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, UK Present address: International Center for Biotechnology, MOFA Global, Mount Horeb, WI 53572, USA.
authors.forename: Steve
authors.initials: S
authors.lastname: Tardif
authors.affiliation: Reproductive and Developmental Biology, Medical School, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, UK Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee DD1 9SY, UK University of Abertay, Dundee DD11HG, UK.
authors.forename: Oladipo A
authors.initials: OA
authors.lastname: Madamidola
authors.affiliation: University of Abertay, Dundee DD11HG, UK.
authors.forename: Sean G
authors.initials: SG
authors.lastname: Brown
authors.affiliation: Reproductive and Developmental Biology, Medical School, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, UK.
authors.forename: Lorna
authors.initials: L
authors.lastname: Frame
authors.affiliation: Medical School, University of Birmingham, Birmingham B152TG, UK.
authors.forename: Linda
authors.initials: L
authors.lastname: Lefièvre
authors.affiliation: Drug Discovery Unit, College of Life Sciences, University of Dundee, Dundee, UK.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Wyatt
authors.affiliation: Reproductive and Developmental Biology, Medical School, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, UK Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee DD1 9SY, UK c.barratt@dundee.ac.uk.
authors.forename: Christopher L R
authors.initials: CL
authors.lastname: Barratt
authors.affiliation: Reproductive and Developmental Biology, Medical School, Ninewells Hospital, University of Dundee, Dundee DD1 9SY, UK Assisted Conception Unit, NHS Tayside, Ninewells Hospital, Dundee DD1 9SY, UK.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Martins Da Silva
mesh_terms: D000177:Acrosome / Q000187:drug effects; D017209:Apoptosis / Q000187:drug effects; D006801:Humans; D008297:Male; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D013081:Sperm Motility / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D010726:Phosphodiesterase Inhibitors
keywords: drug discovery; male fertility; phosphodiesterase inhibitors; sperm; sperm motility