STUDY QUESTION Do biallelic deleterious variants of Calreticulin 3 (CALR3) cause fertilization failure (FF), resulting in male infertility in humans? SUMMARY ANSWER Biallelic mutations in CALR3 were identified in two infertile men from unrelated families and were shown to cause FF associated with failed sperm-zona pellucida (ZP) binding. WHAT IS KNOWN ALREADY In male mice, the Calr3-knockout has been reported to cause male infertility and FF. However, the mechanism behind this remains unclear in humans. STUDY DESIGN, SIZE, DURATION Sequencing studies were conducted in a research hospital on samples from Han Chinese families with primary infertility and sperm head deformations to identify the underlying genetic causes. PARTICIPANTS/MATERIALS, SETTING,

METHODS:

Data from two infertile probands characterized by sperm head deformation were collected through in silico analysis. Sperm cells from the probands were characterized using light and electron microscopy and used to verify the pathogenicity of genetic factors through functional assays. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of FF. ICSI were administered to overcome CALR3-affected male infertility. MAIN RESULTS AND THE ROLE OF CHANCE Novel biallelic deleterious mutations in CALR3 were identified in two infertile men from unrelated families. We found one homozygous frameshift CALR3 mutation (M1 c.17_27del, p.V6Gfs*34) and one compound heterozygous CALR3 mutation (M2 c.943A>G, p.N315D; M3 c.544T>C, p.Y182H). These mutations are rare in the general population and cause acrosomal ultrastructural defects in affected sperm. Furthermore, spermatozoa from patients harbouring the CALR3 mutations were unable to bind to the sperm-ZP or they disrupted gamete fusion or prevented oocyte activation. Molecular assays have revealed that CALR3 is crucial for the maturation of the ZP binding protein in humans. Notably, the successful fertilization via SUZI and ICSI attempts for two patients, as well as the normal expression of PLCζ in the mutant sperm, suggests that ICSI is an optimal treatment for CALR3-deficient FF. LIMITATIONS, REASONS FOR CAUTION The results are based on sperm-related findings from two patients. Further studies are required to gain insight into the developmental stage and function of CALR3 in human testis. WIDER IMPLICATIONS OF THE

FINDINGS:

Our findings highlight the underlying risk of FF associated with sperm defects and provide a valuable reference for personalized genetic counselling and clinical treatment of these patients. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by the National Key R&D Program of China (2021YFC2700901), Hefei Comprehensive National Science Center Medical-Industrial Integration Medical Equipment Innovation Research Platform Project (4801001202), the National Natural Science Foundation of China (82201803, 82371621, 82271639), Foundation of the Education Department of Anhui Province (gxgwfx2022007), Key Project of Natural Science Research of Anhui Educational Committee (2023AH053287), and the Clinical Medical Research Transformation Project of Anhui Province (202204295107020037). The authors declare no competing interests. TRIAL REGISTRATION NUMBER N/A.