Elevated and Sustained Intracellular Calcium Signalling Is Necessary for Efficacious Induction of the Human Sperm Acrosome Reaction.

Progesterone and prostaglandin E1 are postulated to trigger the human sperm acrosome reaction (AR). However, their reported efficacy is very variable which likely, in part, reflects the plethora of experimental conditions and methodologies used to detect this physiologically relevant event. The purpose of this study was to develop an assay for the robust induction and objective measurement of the complete AR. Sperm from healthy volunteers or patients undertaking IVF were treated with a variety of ligands (progesterone, prostaglandin E1 or NH4Cl, alone or in combinations). AR, motility and intracellular calcium measurements were measured using flow cytometry, computer-assisted sperm analysis (CASA) and fluorimetry, respectively. The AR was significantly increased by the simultaneous application of progesterone, prostaglandin E1 and NH4Cl, following an elevated and sustained intracellular calcium concentration. However, we observed notable inter- and intra-donor sample heterogeneity of the AR induction. When studying the patient samples, we found no relationship between the IVF fertilization rate and the AR. We conclude that progesterone and prostaglandin E1 alone do not significantly increase the percentage of live acrosome-reacted sperm. This assay has utility for drug discovery and sperm toxicology studies but is not predictive for IVF success.

IVF or ICSI for fertility preservation?

In IVF, eggs and sperm are added together for fertilisation to occur whereas ICSI involves injecting a single sperm into each egg. ICSI is very effective where sperm count or swimming is poor (male infertility) but is slightly riskier than IVF in terms of health problems in children, although these risks are small. However, the risk of no eggs fertilising is higher for IVF compared to ICSI and couples undertaking fertility preservation, for example, before cancer treatment, usually only have time for one attempt. Using fertility preservation treatment cycle data reported to Human Fertilisation and Embryology Authority (HFEA), this study shows that ICSI results in higher number of fertilised eggs and embryos for storage or treatment compared to IVF. However, 19% of eggs are not used in ICSI treatment, so IVF appears to be better overall. Clinics should choose IVF or ICSI for fertility preservation depending on sperm characteristics rather than using ICSI for all.

Drug discovery for male subfertility using high-throughput screening: a new approach to an unsolved problem.

STUDY QUESTION: Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility? SUMMARY ANSWER: High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility. WHAT IS KNOWN ALREADY: There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform. STUDY DESIGN, SIZE, DURATION: Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016. PARTICIPANTS/MATERIALS, SETTING, METHODS: A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 µM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase in fluorescence in the primary screen were rescreened and evaluated further, resulting in 48 hit compounds that produced a concentration-dependent increase in [Ca2+]i. Sperm penetration studies confirmed in vitro exposure to two hit compounds (A and B) resulted in significant improvement in functional motility in spermatozoa from healthy volunteer donors (A: 1 cm penetration index 2.54, 2 cm penetration index 2.49; P < 0.005 and B: 1 cm penetration index 2.1, 2 cm penetration index 2.6; P < 0.005), but crucially, also in patient samples from those undergoing fertility treatment (A: 1 cm penetration index 2.4; P = 0.009, 2 cm penetration index 3.6; P = 0.02 and B: 1 cm penetration index 2.2; P = 0.0004, 2 cm penetration index 3.6; P = 0.002). This was primarily as a result of direct or indirect CatSper channel action, supported by evidence from electrophysiology studies of individual sperm. LIMITATIONS, REASONS FOR CAUTION: Increase and fluxes in [Ca2+]i are fundamental to the regulation of sperm motility and function, including acrosome reaction. The use of calcium signalling as a surrogate for sperm motility is acknowledged as a potential limitation in this study. WIDER IMPLICATIONS OF THE FINDINGS: We conclude that HTS can robustly, efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoa and functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility. STUDY FUNDING/COMPETING INTEREST(S): The majority of the data were obtained using funding from TENOVUS Scotland and Chief Scientist Office NRS Fellowship. Additional funding was provided by NHS Tayside, MRC project grants (MR/K013343/1, MR/012492/1) and University of Abertay. The authors declare that there is no conflict of interest. TRAIL REGISTRATION NUMBER: N/A.

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

STUDY QUESTION: 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? SUMMARY ANSWER: We 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 ALREADY: 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). STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTERESTS: 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.