Fertilization and early-cleavage failure are the major causes of assisted reproductive technology failure, but genotype-guided care is underused. We conducted whole-exome sequencing with Sanger confirmation in 20 men from couples with unexplained fertilization and cleavage failure after in vitro fertilization or intracytoplasmic sperm injection (ICSI). Then, further sperm transmission electron microscopy, immunofluorescence, and, where appropriate, embryo quantitative parental contamination test (qPCT) and kinship analysis were integrated in selected cases. Six novel variants were found in four families: two each in dynein axonemal heavy chain 10 (DNAH10) and dynein axonemal heavy chain 12 (DNAH12) and one in cilia and flagella associated protein 47 (CFAP47) and phospholipase C zeta 1 (PLCZ1), respectively. DNAH10/DNAH12 variants segregated with multiple morphological abnormalities of the sperm flagella and showed “9+2” axonemal disruption and reduced dynein signals. Hemizygous CFAP47 c.8006C>T is associated with cleavage failure and altered PLCZ1 localization. Homozygous PLCZ1 c.589C>T in a consanguineous pedigree coincided with multinuclear (≥3 pronuclei) zygotes, and qPCT/kinship indicated excess paternal contribution, consistent with suspected polyspermy in multinuclear zygotes. Management-matched genotype: ICSI restored fertilization in DNAH10/DNAH12 deficiency, whereas ICSI plus assisted oocyte activation (AOA) improved cleavage and yielded ongoing pregnancies in CFAP47/PLCZ1 deficiency. These findings outline potentially actionable clinical strategies–ICSI for axonemal dynein defects and ICSI plus AOA for activation pathway defects–and support targeted genetic testing plus embryo qPCT/kinship in selected cases.

Keywords: cleavage failure; fertilization failure; male infertility; sperm; variant