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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 to 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 mM) of an ion channel-focused 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 (LC-MS). Hit compounds were grouped by structure activity relationships (SARs) and five representative compounds then further investigated fordirect effects on spermatozoa, using computer assisted sperm assessment (CASA), sperm penetration assay and whole cell patch clamping.
Main results and 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 3x median absolute deviation (MAD) above the baseline. 74 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, 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; 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; 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, and efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoaand functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility.
- sperm function
- male infertitlity
- drug discovery
- high-throughput screening
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- 3 Finished
1/03/14 → 31/08/15
Physiological Studies of Sperm from Normal and Sub Fertile Men (joint with Universities of Birmingham and Durham)
Barratt, C. & Wilson, S.
1/04/13 → 1/04/16
1/01/13 → 30/06/14