Role of second generation phosphodiesterase inhibitors on mammalian sperm mobility
Over three decades ago, W.H.O. declared infertility as a public health issue; due to its impact on millions of people worldwide. While cases of infertility could be multifactorial (affecting both male and female), 50% of cases are due to male factor infertility and this is mostly characterised by reduced sperm motility (asthenozoospermia). Assisted Reproduction Technology (ART) is the only treatment option available for this condition. Over 20 years ago, non-selective phosphodiesterase inhibitors (PDEi), such as pentoxifylline, were shown to enhance motility of human spermatozoa; however, contradictory results and stimulation of premature acrosome reaction has precluded their clinical use. Advancement in our knowledge have now made it clear that human sperm express several different PDEs and these are compartmentalised at different regions of the cells. By using type-specific phosphodiesterase inhibitors, differential modulation of sperm motility can be achieved without affecting other sperm function such as acrosome reaction. Additionally, by enhancing sperm function through PDE inhibition, there is a possibility of increasing IVF rates. The objective of this thesis is to: (1) examine the effect of phosphodiesterase inhibitors on spermatozoa in order to identify compounds that have clinically relevant enhancement of human sperm motility; (2) identify the signalling pathway(s) involved in the motility enhancing effects of identified compounds by targeting the modulator and mediator of cyclic nucleotides; (3) develop an animal IVF model to assess effects of Ibudilast on fertilization; and (4) optimise high performance liquid chromatography (HPLC) techniques for routine detection of cyclic nucleotides in sperm cells. A two phase drug screening approach was used to systematically and comprehensively screen series of compounds in order to identify those that have clinically relevant enhancement of human sperm motility. In phase 1, 6 compounds (out of 43 compounds) were found to have strong effects on poor motility samples, with magnitude of response ≥60% increase in percentage total motility. Additionally, these compounds significantly enhanced sperm penetration into cervical mucus substitute (p≤0.05), and they did not affect sperm acrosomal integrity nor cause externalisation of phosphatidylserine (p=0.6 respectively). 63% of IVF samples treated with compounds #26, #37 and #38 had significant increase in percentage total motility. For ICSI samples, compounds #26, #37 and #38 were the most effective. In respect to total motility, 88%, 81% and 79% of samples treated with these compounds showed significant increases in total motility, and 94%, 93% and 81% of samples showed significant increases in percentage of progressive cells, respectively. Analysis of the signalling pathways, using PKA, sGC and PKG inhibitors, showed that chosen PDE inhibitors were working predominantly through PKA signalling pathways. Additionally, this study revealed that this pathway is needed for the maintenance of basal progressive motility and hyperactivation in human sperm. Animal IVF studies showed that addition of Ibudilast (compound #26) during sperm-oocyte incubation leads to higher IVF rates. Lastly, this study used an HPLC system to detect cAMP in boar sperm. This was done to explore if HPLC system can be used for high throughput detection of cyclic nucleotides in mammalian sperm.
Sperm, Male fertility, Phosphodiesterase inhibitors, Enhancement of sperm motility, Drug discovery, In vitro fertilization
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