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• Novel variants of ZP1, ZP2 and ZP3 are associated with female infertility. • In-vitro functional studies of ZP protein variants indicate pathogenesis. • The ZP3 variant was overcome with ICSI treatment. • Targeted genetic diagnosis of ZP genes may guide fertilization methods. Which genetic variants might explain the causes of empty follicle syndrome (EFS) and abnormal zona pellucida (ZP) and affect the success of treatment with assisted reproductive technologies (ART)? Whole-exome sequencing was performed in probands with EFS and abnormal ZP. Sanger sequencing was used for variant validation. Using HEK-293T cells, the effects of ZP1 and ZP2 variants on protein expression were explored by western blotting, and the effect of the ZP1 variant on protein location was investigated via immunofluorescence. The protein structure was also analysed to investigate the pathogenicity of variants. A homozygous nonsense variant in ZP1 (c.874C>T, p.Gln292*) was detected in a patient with EFS. A novel homozygous frameshift variant in ZP2 (c.836_837delAG, p.Glu279Valfs*6) and a novel heterozygous missense variant in ZP3 (c.1159G>A, p.Val387Met) were identified in two patients with ZP morphological abnormalities, respectively. Western blotting and immunofluorescence analysis showed that the ZP1 variant results in a premature stop codon, leading to the truncated ZP1 protein. The ZP2 variant, which is situated in the N-terminus, triggers the degradation of a premature termination protein. Additionally, the patient with the ZP3 variant achieved clinical pregnancy following intracytoplasmic sperm injection treatment. These findings expand the mutational spectrum of ZP1, ZP2 and ZP3 , and provide new evidence for genetic diagnosis of female infertility. The targeted genetic diagnosis of ZP genes is recommended to choose appropriate fertilization methods and improve success rates of treatment with ART. [ABSTRACT FROM AUTHOR]

Research question: More than 100 variants have been identified in the TUBB8 gene, which account for approximately 30% of infertile women with oocyte maturation defects. But what is the correlation between the highly phenotypic diversity and genetic variability? Are there other variants in TUBB8 related to female infertility? Design: TUBB8 resequencing was performed in 80 female subjects who were experiencing infertility and were seeking treatment with assisted reproductive technologies (ART), or had ever experienced ART failure due to oocyte maturation defects. All variants were evaluated with pedigree analysis, population frequency, in-silico analysis and molecular modelling. The effects of the variants on oocytes/arrested embryos were assessed by morphological observations, immunostaining, embryo biopsies and chromosome euploidy analysis. Results: Nine missense variants and two frameshift variants from an additional 15 families were identified, including four novel variants and seven previously reported recurrent variants. These TUBB8 variants were related to highly variable phenotypes, including abnormalities in oocyte maturation or morphology, fertilization failure, embryonic development abnormalities and implantation failure. Also further clarified were the incomplete penetrance of heterozygous p.E108K, the likely benign significance of heterozygous p.A313V and the clinical effect of a novel variant of p.R380C. Conclusions: This study significantly expands the variant spectrum of the TUBB8 gene and, together with the available findings on TUBB8 variants and female infertility, will potentially facilitate the genetic counselling of infertile women in future. [ABSTRACT FROM AUTHOR]