A compound heterozygous mutation in ZP1 and two novel heterozygous cis mutations in ZP3 causes infertility in women presenting with empty follicle syndrome.

BACKGROUND: Infertility, as a major human reproductive health problem, affects approximately 17.5% of the global population. The emergence of assisted reproductive technology, particularly in vitro fertilization (IVF), has resolved the reproductive challenges of most infertile couples. In IVF, there exists a group of women who are unable to retrieve oocytes from typically developing follicles despite a positive response to ovarian stimulation, a condition clinically referred to as empty follicle syndrome (EFS). The pathogenesis of EFS is still unclear, but increasing evidence suggests that genetic factors play a very significant role. A clinical and experimental analysis of two pedigrees was performed in this study to investigate the genetic basis of EFS. METHODS: Genomic DNA was extracted from peripheral blood samples for whole-exome sequencing in EFS patients. The identified variants were validated by Sanger sequencing. Computer simulation of protein structure was used to assess the pathogenicity of the variants on the proteins. The effects of the ZP1 and ZP3 variants on protein expression were probed via western blotting, and the effects of the variants on protein localization were probed by immunofluorescence. ELISA and Co-IP were used to detect the effects of variants on protein secretion and interactions. RESULTS: In this study, we identified a compound heterozygous mutation in ZP1 (c.[2T > A]; [1429G > T]) and two novel ZP3 heterozygous cis mutations (c.[724G > T;815 A > G]) from two EFS patients, respectively. These mutations are highly conserved between different species. Through in vitro experiments, we showed that the ZP1 (p.[Met1?]; [Gly477*]) mutations result in reduced protein expression, whereas the ZP3 (p.[Asp242Tyr; Asn272Ser]) mutations lead to increased protein expression. However, neither mutation affected the subcellular localization of the ZP proteins. Bioinformatic analysis revealed that these mutations disrupt the conformation of the ZP protein, which may affect its stability and binding capability. Functional experiments showed that the ZP1 and ZP3 mutations altered the interaction between themselves and ZP2 proteins; the ZP1 mutation inhibited ZP1 protein secretion, whereas the ZP3 mutation increased the secretion of ZP3 protein, which may affect ZP assembly. CONCLUSIONS: Our study has enriched the mutational spectrum of the ZP gene by identifying mutations in the causative genes ZP1 and ZP3 associated with EFS. In vitro experiments exploring the effects of mutations on ZP protein expression and function confirmed that ZP is an important genetic cause of EFS, thus broadening our understanding of the genetics of female infertility. We emphasize the importance of genetic analysis in the diagnosis and prognosis of "genuine" EFS (GEFS) and recommend that EFS patients strive for a successful pregnancy through an oocyte donation program.