P-506 Oocyte/zygote/embryo maturation arrest: a functional and clinical study expanding the phenotype of NOBOX variants

Abstract

Abstract Study question Are pathogenic NOBOX variants causal for oocyte/zygote/embryo maturation arrest (OZEMA) and what is their molecular mechanism? Summary answer Expression analysis of NOBOX target genes indicates that the examined variants do not influence early folliculogenesis, suggesting a potential involvement in later developmental stages. What is known already Primary ovarian insufficiency (POI) affects around 1% of women under 40 with pathogenic variants in the transcription factor NOBOX being causal in 5-7% of cases. Historically, NOBOX has been linked to premature oocyte depletion. Functional studies in female Nobox knockout (-/-) mice reveal a substantial disruption in germ cell cyst breakdown. This disruption impairs the formation of primordial follicles and the subsequent transition from primordial to primary stages, leading to the early loss of all oocytes. Study design, size, duration Three OZEMA families are presented with two distinct NOBOX variants. A functional study is conducted to explore alterations in mRNA expression associated with the investigated NOBOX variants. Participants/materials, setting, methods Three infertile women with OZEMA underwent multiple in vitro fertilization cycles. Subsequent, exome analysis targeting fertility-related genes revealed the presence of distinct NOBOX variants. To assess their impact on NOBOX target gene expression, we generated mutant NOBOX expressing plasmids via site-directed mutagenesis and transfected them into HEK293T cells. The expression of known NOBOX target genes was monitored by RT-qPCR. Main results and the role of chance We examined three families affected by OZEMA, in which the affected women were heterozygous for NOBOX variants. In one family, we found a heterozygous paternally inherited NM_001080413.3(NOBOX): c.1797_1798delCT, p.(Arg600Aspfs*6) variant in a female with oocyte maturation arrest. In two other families, a heterozygous probably pathogenic paternally inherited NM_001080413.3(NOBOX): c.1849C>T, p.(His617Tyr) variant was detected in females experiencing embryo maturation arrest. Analysis of NOBOX target genes in HEK293T cells revealed that both variants had no impact on the mRNA expression of MOS and OCT4, both of which are implicated in early folliculogenisis. Since both MOS and OCT4 are implicated in earlier folliculogenesis stages, this supports that NOBOX might also be implicated in later stages. Contrastingly, a known pathogenic NOBOX variant, used as a positive control, showed decreased MOS and OCT4 expression. Limitations, reasons for caution Verification of our findings is necessary, given that this is the first report of NOBOX variants in individuals with OZEMA. The precise molecular mechanism underlying OZEMA remains undisclosed at this stage. These results warrant the need of genome-wide mRNASeq to confirm and expand our findings. Wider implications of the findings Since NOBOX pathogenic variants are typically linked to POI, our findings suggest a broader clinical impact, where NOBOX plays a role in oocyte maturation and early embryo development. Hence, the investigation of NOBOX variants is advisable for individuals diagnosed with OZEMA. Trial registration number Not applicable