The PCDfert project aimed to increase knowledge and understanding of motile ciliopathies, male fertility and biological processes related to sperm tail formation. This was achieved through training and creation of collaboration opportunities for the research fellow. Results have been communicated to researchers, clinicians and the general public through seminar, conference and workshop presentations. For sperm sample collection and characterization of the male fertility in PCD patients, we updated the ethics for sperm sample collections, organized site approvals for patient recruitment (primarily from the Royal Brompton Hospital RBH, London and Seacroft Hospital, Leeds) and for subsequent sample collection and sperm quality analysis (Hammersmith Hospital, London). We were able to start sperm collection and recruited >25 adult male PCD patients for the study. Unfortunately, the Covid-19 pandemic stopped sperm collection, but the preliminary results show different sperm phenotypes depending on the mutated gene. This has revealed a stratification of motile ciliopathy patients into a range of more or less severely affected male germ cells according to genotype and factors still remaining to be elucidated. These results underline the importance of identification of the real prevalence of male infertility in PCD as well as the exact causes of sperm defects. We applied for additional funding (BBSRC) to continue sperm collection and analysis in 2021 – this significant funding was successfully awarded (01/21). The developed pipeline and knowledge can be utilized in a recently established national PCD adult clinical service for the UK, where centers will be able to inform patients of their fertility status. A review article about known effects of PCD mutations on male fertility has been published in collaboration with Dr Amelia Shoemark and Prof Michael Loebinger at RBH. Several additional papers are in preparation for publications.
Novel PCD gene variant identification was performed for a Finnish PCD patient cohort. Gene panel and whole exome sequencing was utilized for candidate variant detection. Loss-of-function variants in PCD candidate genes CFAP300 and HYDIN were identified as causative mutations explained 50% of studied Finnish cases. We have developed a Sanger sequencing based method for diagnosis of PCD caused by these variants within the Finnish population.
For functional studies of transition zone formation specific antibodies were tested in human sperm samples and during mouse spermiogenesis. For the first time we have tested and developed methods for localization of proteins within the spermatid transition zone and investigated the effect of inhibited IFT on transition zone formation. Super resolution microscopy imaging was used for protein localization in wild type and Tctex1d2 (component of retrograde IFT dynein complex) mutant mouse model to establish the transition zone structure during spermiogenesis. These results show novel data with differential localization of transition zone proteins in the sperm head tail connecting piece. The importance of functional IFT in transition zone formation was revealed, in addition to the requirement for specialized protein transport to the sperm tail. Furthermore, a new collaboration was established with Newcastle University, to investigate the specific role of transition fiber protein CEP164 in spermiogenesis.
During the fellowship the Researcher has been able to train in ciliopathy related subjects and methods, improved knowledge and skills in microscopy and high speed video imaging and gained knowledge to conduct research in collaboration with clinicians and hospital settings. The expertise of the Researcher has enabled development of systematic studies of male fertility in PCD patients and experiments to resolve the similarities and differences in cilia/sperm tail development. The Researcher has been able to develop an extensive international collaboration network in the field of cilia research, especially in wider Europe through engagement in the pan-European BEAT-PCD network (previous COST Action and current European Respiratory Society Clinical Research Collaboration).
