Clinically Relevant Enhancement of Human Sperm Motility
Clinically Relevant Enhancement of Human Sperm Motility
Using a two-stage comprehensive approach, we have successfully identified several compounds that have robust and effective stimulation of sperm motility, are non-toxic, initiate a functional improvement as judged by Kremer testing and importantly have a positive response on a significant proportion of patient samples prepared under clinical conditions.
This study used a two-phase strategy. In Phase 1, 43 commercially available compounds with reported PDEI activity were screened for their effects on sperm motility using CASA. Pooled samples from three to four different donors were utilized to reduce variability and increase the number of cells available for simultaneous examination of multiple compounds (usually three to five in each run). Cells in the 40% fraction (those with poor motility) were used as putative surrogates for patient samples. Previous studies have suggested that these cells have a similar profile, in terms of motility, morphology and DNA status, to men with sperm dysfunction/male infertility (O'Connell et al., 2003; Glenn et al., 2007). The first screening was performed with NCM, as these are the conditions normally used for IUI (Bjorndahl et al., 2010). Moreover, an incubation time of 20 min was designed to fit with clinical procedures for sperm preparation. In general, consistent results on sperm motility were obtained. In Phase 1, the effects of the six leading compounds were determined using pooled samples. Experiments on individual samples then showed a similar profile of results to pooled samples, and, notably showed a significant increase in both total and progressive motility (Fig. 3A and B; Supplementary data, Fig. S1 http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1). Additionally, consistent stimulation of total and progressive motility were obtained when the cells were incubated under capacitating conditions (Fig. 3C).
The objective of Phase 1 was to allow a large number of compounds to be screened relatively efficiently in order to identify potential hit targets for further study. Phase 2 consisted of a more detailed assessment based around guidelines for the testing of compounds that can potentially be considered safe for clinical use (Mortimer et al., 2013). Phase 2 involved sperm function testing with a view to the use of the compounds in ART, e.g. IUI. Modified Kremer testing demonstrated that the stimulation in motility was also of functional benefit, i.e. higher numbers of cells penetrated the viscous media. Importantly, the compounds did not appear to have a significant negative effect as there was no significant induction of the AR (Supplementary data, Fig. S2 http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1) or PS exposure (Supplementary data, Fig. S2 http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1). This is consistent with the finding of motility maintenance over a significant time course, even with continuous incubation (Table I). Whilst the six key compounds selected for Phase 2 had positive effects, there were differences in the efficacy suggesting fewer lead candidates for future clinical use. For example, compounds #26, #37 and #38 had the most significant effect on Kremer testing which is broadly consistent, at least for compounds #37 and #38 with the positive effect on motility over time (Table I). Clinical use of the compounds would involve washing and effective removal prior to use. Table II demonstrates that stimulation of total motility was maintained over time; however, progressive motility was not consistently affected using compounds #1 and #30. Continual incubation (Table I) suggests that the positive effect on total motility of compounds #1 and #26 and #30 were not maintained throught incubation. For progressive motility, particular stimulation with compounds #38 and #37 was observed (Table I).
The fundamental clinical aim is to translate what happens in an experimental model to effects in patient samples. To address this, we tested a spectrum of diagnostic and treatment samples under both non-capacitating and capacitating conditions (Table III, Table IV and Table V). In general, in samples with good motility, e.g. IVF, there was a minimal effect on total motility but, in some cases, a noteable effect on progressive motility. In contrast, in samples with lower motility, there was a significant effect on both total and progressive motility. These clinical data give some indications as to the possible therapeutic use and effectiveness. Generally, there appears to be limited benefit for samples with good motility, as expected and consistent with previous data using PTX (Nassar et al., 1999). In cells incubated under non-capacitating conditions 15/23 and 17/23 of the samples responded to compound #26 with regard to total and progressive motility. From the limited data available, compounds #1 and #30 were less effective. In samples incubated under capacitating conditions (Table IV), compounds #37, #38 and #26 were the most effective in increasing the percentage total motile cells (~63% of samples) and, as for samples in NCM, the most significant effects were in samples with borderline/low motility. Only relatively few ICSI samples were examined (Table V); however, in the overwhelming majority of cases, cells incubated in compounds #26, #37 and #38 showed an increase in progressive motility and total motility.
For practical purposes, three concentrations were adopted in Phase 1 using doses of 1, 10 and 100 µM. The objective was to determine which concentration was the most effective under these conditions (non-capacitating conditions with cells in the 40% fraction). Concentrations of 1 and 10 µM did have pro-motility effects, in some cases, but it was very much less (data not shown) and as such Phase 2 only used PDEIs at a concentration of 100 µM. For some of the compounds tested, 100 µM is much greater than the reported IC50 (e.g compound #1), whilst for others (e.g. compound #26) it is comparable (see Supplementary data, Table SII http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1). What is interesting is that the reported IC50 for a number of compounds varies remarkably perhaps because some are generated using purified recombinant enzymes and others on a wide variety of different cell types (Supplementary data, Table SII http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1). For spermatozoa there are no available data; there is no information on what concentration of compound enters the sperm cytoplasm, whether there are pumps effectively making high external concentrations necessary, the specificity of the sperm PDE complex(s) or which complexes are present (see below). Preliminary experiments on the three most promosing candidates (compounds #26, #37 and #38) utilized concentrations from 0.5 to 100 µM to examine a potential concentration effect on motility and kinematic parameters. For compound #26, progressive motility was significantly stimulated at 20–100 µM (in keeping with the IC50 for other cells), whereas progressive motility was signfiicantly stimulated at 1–100 µM for compound #37 and at 0.5–100 µM for compound #38, both of which are within the ranges of the IC50 for other cell types (see above and Supplementary data, Figs S3–S5 http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1, respectively).
Surprisingly, in view of the plethora of information available on PDEs in other cells, there is a remarkable paucity of studies on the identity, location and nature of PDEs in the human spermatozoon (Conti and Beavo, 2007; Houslay, 2009). The measurement of sperm PDE activity in the presence of inhibitors for PDE-1 (8-MeO-IBMX) (Fisch et al., 1998), PDE-4 (RS 25344) (Fisch et al., 1998), PDE-3 (milrinone) (Lefièvre et al., 2002), PDE-5 (sildenafil) and stimulators for PDE-1 (calcium/CaM) (Lefièvre et al., 2000, 2002) suggests that these PDEs are present in human spermatozoa, although PDE-5 is present at very low levels. mRNA transcripts of PDEs have been detected (Richter et al., 1999) but very few studies examine localization (Lefièvre et al., 2002) and there are minimal data on protein expression. In fact, proteomic studies of human sperm reveal a paucity of PDE in spermatozoa (Baker et al., 2013; Wang et al., 2013). There are no studies examining the role of defective PDEs in sperm dysfunction, e.g. aberrant expression. In view of the high concentrations of compounds used in this study the specificity of effect on PDEs is also uncertain. Other biochemical pathways could be affected and as such we do not know if the biological effect is via PDE and/or another mechanism. Notwithstanding the clinical end-point is real: there are significant changes in movement without an adverse effect on sperm function; however, more detailed biochemical studies are required to ascertain the mechanism(s) of action.
In view of the above it is perhaps not surprising that of the six key compounds identified as potential clincial candidates (dipyridamole, ibudilast, 8-MeO-IBMX, etazolate hydrochloride, papaverine and tofisopam) there is a noteable lack of data on human sperm. No information is available on dipyridamole, ibudilast or tofisopam. Etazolate hydrochloride, which is reported as a selective PDE-4 inhibitor (as SQ20009), increases cAMP in hamster sperm (Mrsny et al., 1984) and phosphorylation of membrane proteins (presumably as part of capacitation) in humans (Huacuja et al., 1977), although there are no data on motility. 8-MeO-IBMX, reported as a specific inhibitor of calmodulin-sensitive cyclic GMP PDE, has been used in mice fertilization studies (Baxendale and Fraser, 2005) but there are no reports of effects on the motility of human spermatozoa. Papaverine, reported as a PDE-10A inhibitor, has been used at a concentration of 500 µM to increase cyclic nucleotides and mimic the effects of capacitation in human sperm. After 5 min of incubation, there was an increased calcium response to progesterone (Torres-Flores et al., 2008). Papaverine has also been used to mimic capacitation changes by modulating the cAMP pathway in boars (Harrison, 2004) and guinea pig sperm (Hunnicutt et al., 2008).
In conclusion, we have successfully identified several compounds that have robust and effective stimulation of sperm motility, are non-toxic to the cells, initiate a functional improvement as judged by Kremer testing and importantly have a positive response on a significant proportion of patient samples under clinical conditions of treatment. Ibudilast, papaverine and tofisopam appear to be very promising candidates but further experiments are still necessary to establish safety and clinical effectiveness, e.g. IVF and/IUI trials. There are significant challenges with screening for the effects of a large number of compounds on human spermatozoa. CASA is not well suited to traditionally high-throughput screening. In the long term, if significant progress is to be made in understanding sperm function, there is a genuine need to develop a high-throughput assay which would enable the rapid screening of thousands of compounds.
Discussion
Using a two-stage comprehensive approach, we have successfully identified several compounds that have robust and effective stimulation of sperm motility, are non-toxic, initiate a functional improvement as judged by Kremer testing and importantly have a positive response on a significant proportion of patient samples prepared under clinical conditions.
This study used a two-phase strategy. In Phase 1, 43 commercially available compounds with reported PDEI activity were screened for their effects on sperm motility using CASA. Pooled samples from three to four different donors were utilized to reduce variability and increase the number of cells available for simultaneous examination of multiple compounds (usually three to five in each run). Cells in the 40% fraction (those with poor motility) were used as putative surrogates for patient samples. Previous studies have suggested that these cells have a similar profile, in terms of motility, morphology and DNA status, to men with sperm dysfunction/male infertility (O'Connell et al., 2003; Glenn et al., 2007). The first screening was performed with NCM, as these are the conditions normally used for IUI (Bjorndahl et al., 2010). Moreover, an incubation time of 20 min was designed to fit with clinical procedures for sperm preparation. In general, consistent results on sperm motility were obtained. In Phase 1, the effects of the six leading compounds were determined using pooled samples. Experiments on individual samples then showed a similar profile of results to pooled samples, and, notably showed a significant increase in both total and progressive motility (Fig. 3A and B; Supplementary data, Fig. S1 http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1). Additionally, consistent stimulation of total and progressive motility were obtained when the cells were incubated under capacitating conditions (Fig. 3C).
The objective of Phase 1 was to allow a large number of compounds to be screened relatively efficiently in order to identify potential hit targets for further study. Phase 2 consisted of a more detailed assessment based around guidelines for the testing of compounds that can potentially be considered safe for clinical use (Mortimer et al., 2013). Phase 2 involved sperm function testing with a view to the use of the compounds in ART, e.g. IUI. Modified Kremer testing demonstrated that the stimulation in motility was also of functional benefit, i.e. higher numbers of cells penetrated the viscous media. Importantly, the compounds did not appear to have a significant negative effect as there was no significant induction of the AR (Supplementary data, Fig. S2 http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1) or PS exposure (Supplementary data, Fig. S2 http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1). This is consistent with the finding of motility maintenance over a significant time course, even with continuous incubation (Table I). Whilst the six key compounds selected for Phase 2 had positive effects, there were differences in the efficacy suggesting fewer lead candidates for future clinical use. For example, compounds #26, #37 and #38 had the most significant effect on Kremer testing which is broadly consistent, at least for compounds #37 and #38 with the positive effect on motility over time (Table I). Clinical use of the compounds would involve washing and effective removal prior to use. Table II demonstrates that stimulation of total motility was maintained over time; however, progressive motility was not consistently affected using compounds #1 and #30. Continual incubation (Table I) suggests that the positive effect on total motility of compounds #1 and #26 and #30 were not maintained throught incubation. For progressive motility, particular stimulation with compounds #38 and #37 was observed (Table I).
The fundamental clinical aim is to translate what happens in an experimental model to effects in patient samples. To address this, we tested a spectrum of diagnostic and treatment samples under both non-capacitating and capacitating conditions (Table III, Table IV and Table V). In general, in samples with good motility, e.g. IVF, there was a minimal effect on total motility but, in some cases, a noteable effect on progressive motility. In contrast, in samples with lower motility, there was a significant effect on both total and progressive motility. These clinical data give some indications as to the possible therapeutic use and effectiveness. Generally, there appears to be limited benefit for samples with good motility, as expected and consistent with previous data using PTX (Nassar et al., 1999). In cells incubated under non-capacitating conditions 15/23 and 17/23 of the samples responded to compound #26 with regard to total and progressive motility. From the limited data available, compounds #1 and #30 were less effective. In samples incubated under capacitating conditions (Table IV), compounds #37, #38 and #26 were the most effective in increasing the percentage total motile cells (~63% of samples) and, as for samples in NCM, the most significant effects were in samples with borderline/low motility. Only relatively few ICSI samples were examined (Table V); however, in the overwhelming majority of cases, cells incubated in compounds #26, #37 and #38 showed an increase in progressive motility and total motility.
For practical purposes, three concentrations were adopted in Phase 1 using doses of 1, 10 and 100 µM. The objective was to determine which concentration was the most effective under these conditions (non-capacitating conditions with cells in the 40% fraction). Concentrations of 1 and 10 µM did have pro-motility effects, in some cases, but it was very much less (data not shown) and as such Phase 2 only used PDEIs at a concentration of 100 µM. For some of the compounds tested, 100 µM is much greater than the reported IC50 (e.g compound #1), whilst for others (e.g. compound #26) it is comparable (see Supplementary data, Table SII http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1). What is interesting is that the reported IC50 for a number of compounds varies remarkably perhaps because some are generated using purified recombinant enzymes and others on a wide variety of different cell types (Supplementary data, Table SII http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1). For spermatozoa there are no available data; there is no information on what concentration of compound enters the sperm cytoplasm, whether there are pumps effectively making high external concentrations necessary, the specificity of the sperm PDE complex(s) or which complexes are present (see below). Preliminary experiments on the three most promosing candidates (compounds #26, #37 and #38) utilized concentrations from 0.5 to 100 µM to examine a potential concentration effect on motility and kinematic parameters. For compound #26, progressive motility was significantly stimulated at 20–100 µM (in keeping with the IC50 for other cells), whereas progressive motility was signfiicantly stimulated at 1–100 µM for compound #37 and at 0.5–100 µM for compound #38, both of which are within the ranges of the IC50 for other cell types (see above and Supplementary data, Figs S3–S5 http://humrep.oxfordjournals.org/content/early/2014/08/13/humrep.deu196/suppl/DC1, respectively).
Surprisingly, in view of the plethora of information available on PDEs in other cells, there is a remarkable paucity of studies on the identity, location and nature of PDEs in the human spermatozoon (Conti and Beavo, 2007; Houslay, 2009). The measurement of sperm PDE activity in the presence of inhibitors for PDE-1 (8-MeO-IBMX) (Fisch et al., 1998), PDE-4 (RS 25344) (Fisch et al., 1998), PDE-3 (milrinone) (Lefièvre et al., 2002), PDE-5 (sildenafil) and stimulators for PDE-1 (calcium/CaM) (Lefièvre et al., 2000, 2002) suggests that these PDEs are present in human spermatozoa, although PDE-5 is present at very low levels. mRNA transcripts of PDEs have been detected (Richter et al., 1999) but very few studies examine localization (Lefièvre et al., 2002) and there are minimal data on protein expression. In fact, proteomic studies of human sperm reveal a paucity of PDE in spermatozoa (Baker et al., 2013; Wang et al., 2013). There are no studies examining the role of defective PDEs in sperm dysfunction, e.g. aberrant expression. In view of the high concentrations of compounds used in this study the specificity of effect on PDEs is also uncertain. Other biochemical pathways could be affected and as such we do not know if the biological effect is via PDE and/or another mechanism. Notwithstanding the clinical end-point is real: there are significant changes in movement without an adverse effect on sperm function; however, more detailed biochemical studies are required to ascertain the mechanism(s) of action.
In view of the above it is perhaps not surprising that of the six key compounds identified as potential clincial candidates (dipyridamole, ibudilast, 8-MeO-IBMX, etazolate hydrochloride, papaverine and tofisopam) there is a noteable lack of data on human sperm. No information is available on dipyridamole, ibudilast or tofisopam. Etazolate hydrochloride, which is reported as a selective PDE-4 inhibitor (as SQ20009), increases cAMP in hamster sperm (Mrsny et al., 1984) and phosphorylation of membrane proteins (presumably as part of capacitation) in humans (Huacuja et al., 1977), although there are no data on motility. 8-MeO-IBMX, reported as a specific inhibitor of calmodulin-sensitive cyclic GMP PDE, has been used in mice fertilization studies (Baxendale and Fraser, 2005) but there are no reports of effects on the motility of human spermatozoa. Papaverine, reported as a PDE-10A inhibitor, has been used at a concentration of 500 µM to increase cyclic nucleotides and mimic the effects of capacitation in human sperm. After 5 min of incubation, there was an increased calcium response to progesterone (Torres-Flores et al., 2008). Papaverine has also been used to mimic capacitation changes by modulating the cAMP pathway in boars (Harrison, 2004) and guinea pig sperm (Hunnicutt et al., 2008).
In conclusion, we have successfully identified several compounds that have robust and effective stimulation of sperm motility, are non-toxic to the cells, initiate a functional improvement as judged by Kremer testing and importantly have a positive response on a significant proportion of patient samples under clinical conditions of treatment. Ibudilast, papaverine and tofisopam appear to be very promising candidates but further experiments are still necessary to establish safety and clinical effectiveness, e.g. IVF and/IUI trials. There are significant challenges with screening for the effects of a large number of compounds on human spermatozoa. CASA is not well suited to traditionally high-throughput screening. In the long term, if significant progress is to be made in understanding sperm function, there is a genuine need to develop a high-throughput assay which would enable the rapid screening of thousands of compounds.
