Establishing the causative link between CFAP221 variants and asthenoteratozoospermia in humans.

PURPOSE: To identify the novel genetic causes of male infertility related to asthenoteratozoospermia in two unrelated Chinese families. METHODS: Whole-exome sequencing (WES) and sanger sequencing were performed on peripheral blood samples from two infertile patients' families. Papanicolaou stain, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) revealed the morphology and ultrastructure of the infertile patients' spermatozoon. Immunofluorescence staining and single-cell RNA sequencing analysis identified the expression of CFAP221 in the different stages of spermatogenesis sperm cells in mouse and human testes. Bioinformatics analysis predicted the protein interactions involving CFAP221. RESULTS: Pathogenic biallelic variants in CFAP221 were detected in two unrelated infertile men by whole-exome sequencing. Abnormalities in the morphology and ultrastructure of sperm flagella were detected in the two patients. Moreover, during the spermatogenesis, CFAP221 was primarily localized in the flagella of elongating and elongated sperm in humans and mice. Bioinformatics analysis predicted that CFAP221 interacts with flagellum development related proteins such as CFAP74, CFAP194, CFAP246, and CFAP297, and is co-expressed with these proteins in various spermatids during mouse spermatogenesis. Furthermore, intracytoplasmic sperm injection (ICSI) treatment can rescue male infertility caused by harmful variants in CFAP221. CONCLUSION: Our findings suggested that CFAP221 is a novel causative gene for male infertility and our findings would guide clinical ICSI treatment and diagnosis of male infertility.