Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity

Steve Tardif, Oladipo Madamidola, Sean G. Brown, Lorna Frame, Linda Lefièvre, Paul G. Wyatt, Christopher L. R. Barratt, Sarah J. Martins da Silva

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    Abstract

    STUDY QUESTIONCan 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?

    SUMMARY ANSWERWe have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples.

    WHAT IS KNOWN ALREADYFor >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).

    STUDY DESIGN, SIZE, DURATIONThis 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.

    PARTICIPANTS/MATERIALS, SETTING, METHODSAll 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.

    MAIN RESULTS AND THE ROLE OF CHANCEIn 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.

    LIMITATIONS, REASONS FOR CAUTIONThis 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.
    WIDER IMPLICATIONS OF THE FINDINGSWe 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.
    STUDY FUNDING/COMPETING INTEREST(S)

    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.

    Original languageEnglish
    Pages (from-to)2123–2135
    Number of pages13
    JournalHuman Reproduction
    Volume29
    Issue number10
    Early online date14 Aug 2014
    DOIs
    Publication statusPublished - 10 Oct 2014

    Fingerprint

    Phosphodiesterase Inhibitors
    Sperm Motility
    Spermatozoa
    Intracytoplasmic Sperm Injections
    Acrosome Reaction
    Hospital Units
    Tissue Donors
    Biomedical Research
    Asthenozoospermia
    Andrology
    Cervix Mucus
    Pentoxifylline
    Acrosome
    Assisted Reproductive Techniques
    Preclinical Drug Evaluations
    Methylcellulose
    Semen Analysis
    Fluorescein-5-isothiocyanate
    Annexin A5
    Phosphatidylserines

    Cite this

    Tardif, S., Madamidola, O., Brown, S. G., Frame, L., Lefièvre, L., Wyatt, P. G., ... Martins da Silva, S. J. (2014). Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity. Human Reproduction, 29(10), 2123–2135. https://doi.org/10.1093/humrep/deu196
    Tardif, Steve ; Madamidola, Oladipo ; Brown, Sean G. ; Frame, Lorna ; Lefièvre, Linda ; Wyatt, Paul G. ; Barratt, Christopher L. R. ; Martins da Silva, Sarah J. / Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity. In: Human Reproduction. 2014 ; Vol. 29, No. 10. pp. 2123–2135.
    @article{25da09730e9a4e8f9eb180ba02ca8a18,
    title = "Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity",
    abstract = "STUDY QUESTIONCan 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?SUMMARY ANSWERWe have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples.WHAT IS KNOWN ALREADYFor >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).STUDY DESIGN, SIZE, DURATIONThis 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.PARTICIPANTS/MATERIALS, SETTING, METHODSAll 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.MAIN RESULTS AND THE ROLE OF CHANCEIn 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.LIMITATIONS, REASONS FOR CAUTIONThis 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.WIDER IMPLICATIONS OF THE FINDINGSWe 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.STUDY FUNDING/COMPETING INTEREST(S)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.",
    author = "Steve Tardif and Oladipo Madamidola and Brown, {Sean G.} and Lorna Frame and Linda Lefi{\`e}vre and Wyatt, {Paul G.} and Barratt, {Christopher L. R.} and {Martins da Silva}, {Sarah J.}",
    year = "2014",
    month = "10",
    day = "10",
    doi = "10.1093/humrep/deu196",
    language = "English",
    volume = "29",
    pages = "2123–2135",
    journal = "Human Reproduction",
    issn = "0268-1161",
    publisher = "Oxford University Press",
    number = "10",

    }

    Tardif, S, Madamidola, O, Brown, SG, Frame, L, Lefièvre, L, Wyatt, PG, Barratt, CLR & Martins da Silva, SJ 2014, 'Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity', Human Reproduction, vol. 29, no. 10, pp. 2123–2135. https://doi.org/10.1093/humrep/deu196

    Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity. / Tardif, Steve; Madamidola, Oladipo; Brown, Sean G.; Frame, Lorna; Lefièvre, Linda; Wyatt, Paul G.; Barratt, Christopher L. R.; Martins da Silva, Sarah J.

    In: Human Reproduction, Vol. 29, No. 10, 10.10.2014, p. 2123–2135.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Clinically relevant enhancement of human sperm motility using compounds with reported phosphodiesterase inhibitor activity

    AU - Tardif, Steve

    AU - Madamidola, Oladipo

    AU - Brown, Sean G.

    AU - Frame, Lorna

    AU - Lefièvre, Linda

    AU - Wyatt, Paul G.

    AU - Barratt, Christopher L. R.

    AU - Martins da Silva, Sarah J.

    PY - 2014/10/10

    Y1 - 2014/10/10

    N2 - STUDY QUESTIONCan 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?SUMMARY ANSWERWe have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples.WHAT IS KNOWN ALREADYFor >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).STUDY DESIGN, SIZE, DURATIONThis 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.PARTICIPANTS/MATERIALS, SETTING, METHODSAll 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.MAIN RESULTS AND THE ROLE OF CHANCEIn 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.LIMITATIONS, REASONS FOR CAUTIONThis 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.WIDER IMPLICATIONS OF THE FINDINGSWe 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.STUDY FUNDING/COMPETING INTEREST(S)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.

    AB - STUDY QUESTIONCan 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?SUMMARY ANSWERWe have identified several compounds that produce robust and effective stimulation of sperm motility and, importantly, have a positive response on patient samples.WHAT IS KNOWN ALREADYFor >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).STUDY DESIGN, SIZE, DURATIONThis 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.PARTICIPANTS/MATERIALS, SETTING, METHODSAll 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.MAIN RESULTS AND THE ROLE OF CHANCEIn 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.LIMITATIONS, REASONS FOR CAUTIONThis 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.WIDER IMPLICATIONS OF THE FINDINGSWe 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.STUDY FUNDING/COMPETING INTEREST(S)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.

    U2 - 10.1093/humrep/deu196

    DO - 10.1093/humrep/deu196

    M3 - Article

    VL - 29

    SP - 2123

    EP - 2135

    JO - Human Reproduction

    JF - Human Reproduction

    SN - 0268-1161

    IS - 10

    ER -