Examining the (patho)physiology of physiologically relevant ligands present in the male and female reproductive tracts

Abstract

Sequencing technologies like Wholes Exome Sequencing (WES) and Whole Genome Sequencing (WGS) have paved the way for the next generation of disease diagnosis, prognosis and management. However, this has yet to advance to the diagnosis of male infertility. One of the main reasons for this is the lack of understanding of the physiological regulation and function of the highly complex sperm cell. Understanding the critical roles of different proteins in maintaining sperm function is key to understanding the causes of sperm dysfunction. Currently, male infertility is clinically diagnosed by semen analysis, which results in a large number of cases being diagnosed as idiopathic (unexplained). Several known sperm molecular defects are undiagnosable by the semen analysis and result in male infertility and failed In vitro Fertilisation (IVF) and Intra Cytoplasmic Sperm Injection (ICSI) treatment. This study aimed to look at the potential of sequencing and physiological assays to diagnose idiopathic male infertility and causes of Total Fertilisation Failure (TFF) in a cohort of 50 men attending the assisted conception unit at NHS Tayside using WES to identify known causes of male infertility and then adding to the existing knowledge base by identifying novel candidate gene variants responsible for infertility. This study predicted ~30% of idiopathic male infertility cases to genetic causes and has demonstrated the power of genetics in disease diagnosis.

In this study, two novel male infertility candidate genes were proposed. This study identified a novel homozygous pathogenic beta-defensin 128 (DEFB128) variant in one patient and demonstrated the expression of this protein on the sperm cell surface. The second novel male infertility gene proposed from this study is the MROH2B gene identified in one patient with sperm that IVF treatment; however, functional validation of this protein using in vitro studies is required to understand the exact cause of physiological impairment.

Although genetic sequencing has its merits, the chances of misdiagnosis are high due to the lack of evidence linked to male infertility. Several genes are not strongly associated with TFF. This study identified these limitations and emphasised the importance of physiological assays in diagnosis. One such area of dysfunction leading to male infertility and failed IVF treatment is dysfunction in the sperm calcium channel CatSper. A number of physiological agonists can activate the CatSper channel, resulting in enhanced hyperactivated motility. Identifying means of maximising CatSper responses by different mechanisms provides therapeutic and diagnostic potential. This study identified the novel beta-defensin 119 (DEFB119) ligand that induces an intracellular calcium influx in human sperm, resulting in hyperactivated motility via a third binding site on the CatSper channel.  

Another important function of the CatSper channel is the induction of Acrosome Reaction (AR), a calcium-dependent process essential for sperm-egg binding and the initiation of fertilisation. Although CATSPER gene variants have been implicated in dysfunctional calcium signalling, AR and failed IVF treatment in humans, the involvement of other genes cannot be dismissed and remain a mystery to date. Thus, developing a functional assay to measure dysfunctional AR will predict several cases of failed IVF treatment and prevent tremendous burden on patients and the healthcare system. In this study, a novel robust and reproducible assay was developed for measuring AR in human sperm. However, this assay could not be correlated to IVF success but can be used as a screening tool and has potential in drug discovery.

In conclusion, this thesis has highlighted the significant potential of WES in diagnosis, however, functional validation is needed. This thesis also identified physiological methods to diagnose idiopathic male infertility to complement semen analysis for diagnosis and help devise treatment strategies.

Date of Award	27 Oct 2023
Original language	English
Awarding Institution	Abertay University
Sponsors	Northwood Charitable Trust
Supervisor	Sean Brown (Supervisor), Scott Cameron (Supervisor) & Douglas Lester (Supervisor)