Mutational disruption of transcription factors binding and regulatory networks in a case of unexplained total fertilization failure.

backgroundFertilization involves fusion between sperm and metaphase II (MII) oocyte, initiating a cascade of events including oocyte activation, resumption of meiosis, formation and interdigitation of male and female pronuclei, and zygote formation. Total Fertilization Failure (TFF), characterized by the disruption of any of these processes, occurs in 1-3% of intracytoplasmic sperm injection (ICSI) cycles. The genetic basis of TFF remains largely unexplored. TFF can occur in cases where no single genetic cause is apparent, suggesting a potential polygenic contribution.

methodsA couple (34 year-old female and 35 year-old male) affected by TFF, and the mother of the affected female patient. A multi-tiered genomic, transcriptomic and functional investigation was conducted to uncover potential genetic contributors to TFF in the couple. Assisted oocyte activation (AOA) was employed upon definition of the putative genetic cause by single-oocyte RNA-seq analyses. Fertilization and embryonic development were monitored using a time-lapse incubator (EmbryoScope) to evaluate the efficiency of AOA in rescuing oocytes.

resultsRNA sequencing revealed aberrant expression of genes involved in meiosis, zona pellucida biogenesis, and calcium signaling. Through open-chromatin-mediated gene regulatory network (oGRN) analysis, we identified 45 transcription factors (TFs) linked to putative regulatory regions, and considered as key regulators including ZNF121, ZFP28, ZNF394, YY1, VEZF1, and SP2. Genetic analysis identified 17 transcription factors (TFs) with disrupted binding patterns to the proband's genome, associated with fertilization and early embryonic development. Gene Ontology (GO) enrichment analysis revealed that VEZF1, ZNF148, SP2, ZNF121, and ZFP28 were central regulators of key biological processes related to reproduction. Reactome pathway analysis further showed that TFAP2D, YY1, ZFP28, ZNF260, and ZNF121 were highly interconnected within critical signaling pathways, including calcium signaling, which is essential for oocyte activation and fertilization. AOA successfully rescued fertilization in 25% of oocytes, resulting in one embryo reaching the 8-cell stage.

conclusionsThis study integrates oocyte gene expression profiles with parental genomic data to identify transcriptional dysregulations associated with TFF. Key disruptions in gene regulatory networks and TFs binding were inferred, potentially affecting critical processes in oogenesis, fertilization, and early embryonic development. AOA by exogenous calcium administration partially rescued fertilization, highlighting its potential as a therapeutic intervention. Our genomic analyses at the single-oocyte resolution provide new insights into new potential polygenic causes of TFF.