Investigating Human Sperm Function for Fertilisation using novel Phenotypic Screening Strategies

Intracellular Ca2+ signalling is essential for sperm function. However, the molecular rationale underpinning Ca2+ regulation in human sperm physiology is insufficiently described. To induce capacitation, Ca2+ influx into sperm via the CatSper channel is required. CatSper is sensitive to oviductal ligands and pH; P4, and PGE1 potently activate CatSper via unique mechanisms of action. Human sperm devoid of CatSper are unable to fertilise in vivo or in vitro as they fail to respond to P4-evoked calcium influx. There is a known relationship between P4-induced intracellular Ca2+ activity and fertilisation outcomes, singling CatSper as a promising biomarker for sperm fertilisation potential. Population fluorometric analysis of sperm cells from patients undergoing assisted conception unveiled higher instances of abnormal PGE1-induced intracellular Ca2+ compared to donor sperm, which may be indicative of subfertility. No significant correlations between agonist-induced Ca2+ response and fertilisation outcome were observed, though the assay did show a promising ability to predict fertilisation outcomes which is worth further exploration and refinement. PLCζ represents the elusive sperm factor responsible for mobilizing intracellular Ca2+ stores within the oocyte, therefore activating meiotic resumption and propelling early embryo development. Human sperm devoid of PLCζ protein cannot induce Ca2+ oscillations and therefore express an infertile phenotype. PLCζ protein expression has therefore been proposed as a potential marker for oocyte activation potential. A novel, medium-throughput assay was developed to determine PLCζ expression accurately and objectively. After method validation, populations of sperm from patients undergoing assisted reproduction were screened with the assay. One patient exhibited a reduced proportion of sperm cells positive for PLCζ despite having a normozoospermic sample. This individual experienced low fertilisation from IVF treatment, suggesting potential PLCζ-mediated subfertility. Overall, no relationship between PLCζ expression and fertilisation outcome was observed. Future screening should capture patients experiencing total fertilisation failure to truly appreciate the commonality of PLCζ dysregulation and determine the sensitivity of the assay. Lastly, the ability of artificial oocyte activation treatment to overcome fertilisation abnormalities resulting from artificial reproduction was investigated. The treatment add-on significantly improved fertilisation outcome, number of embryos available for transfer, and live birth rate for couples with a history of unexpected low or failed fertilisation. For patients with a known PLCζ deficiency, artificial activation returned fertilisation to normal levels, indicating the potential of a pre-treatment PLCζ assay to identify individuals at risk of fertilisation failure and modify treatment approaches. Although no sperm lacking functional CatSper or PLCζ were identified, the potential of these assays to shed light on their individual contributions to male reproductive health and diagnose sperm functional deficiencies represents an exciting prospect for application in clinical andrology.