Abstract
Study question
Do whole-exome sequencing (WES) data from infertile women provide valuable information for the discovery of genes/pathways involved in extreme IVF phenotypes, i.e. oocyte/embryo developmental arrest?
Summary answer
The development of a specific bioinformatic WES pipeline revealed known and new candidate genes/pathways for isolated oocyte/embryo developmental failure,providing the foundation to scale up research.
What is known already
The use of IVF has made it possible to identify extreme and isolated infertility phenotypes such as recurrent low oocytes maturity (LMR), recurrent low fertilization rate (LFR), or preimplantation developmental arrest (PDA) that would remain concealed in natural conception attempts. Recent applications of WES in families with such extreme adverse IVF phenotypes have led to the discovery of new genes and pathways affecting unique functions of gametes and exclusive mechanisms necessary for early embryo development. Here, we apply a tailored bioinformatic approach to WES from women displaying extreme IVF phenotypes to discover new causative genes/pathways involved in unexplained infertility.
Study design, size, duration
Twenty-two infertile consanguineous women(December 2018-September 2020) suffering from long-term unexplained infertility. Eight cases were classified as PDA (<20% normally developed embryos in > 2 IVF cycles), 8 as LMR (<20% mature oocytes in > 2 IVF cycles), 4 as LFR (<20% of normally fertilized oocytes in > 2 IVF cycles). Two women with recurrent IVF failure (>10 IVF cycles) were also included. A control set of 1660 WES from oocyte donors was used to control for false-positive discoveries.
Participants/materials, setting, methods
WES at 30X was performed on enrolled women’s gDNA using Illumina short-reads technology. Following annotation, variants were filtered to prioritize putative detrimental variants in genes relevant for oocyte/embryonic development using a previously developed and validated pipeline that minimizes false-positive discoveries. Runs of homozygosity (ROH) within each sample were identified using Refined IBD software. Individual-level single-cell RNAseq (scRNAseq) dataset from 18 human oocytes was used to verify the expression of the identified target genes.
Main results and the role of chance
The variant prioritization pipeline employed identified 1,160 unique variants in 1,017 genes (average per sample 59.9 sd 8.5). 453 variants were private to this study compared to the 1000 Genomes and gnomAD databases, 3% affecting splicing and/or the gene product length. Significant 5-fold enrichment of 41 genes involved in DNA-damage and repair pathways commonly associated with ovarian function/oocyte quality was observed (p < 0.001). TP53/AKT pathway also showed significant 5-fold enrichment for 45 genes (p < 0.001). This finding is consistent with the known relationship between infertility and cell-cycle/cancer genes. Overall, 66.4% (675/1,017; 95%CI:63.4-69.3) of the targeted genes were expressed in MII human oocytes. Two women (9%) were homozygous carriers of missense pathogenic variants in known candidate genes previously associated to oocyte/embryo developmental arrest (TRIP13, chr5_901344_C/T, CADD percentile 0.999; PADI6, chr1_17394384_C/G, CADD percentile 0.999). Remarkably, four additional women were carriers of high-impact variants in JAKMIP1, a member of a recently characterized family of proteins involved in various cellular processes, including cytoskeleton rearrangement, cell polarization, and intracellular transport. High-impact JAKMP1 variants were never observed in the oocyte donor control dataset. JAKMIP1 mRNA was detected in each individual biological replicate of scRNAseq analysis of MII oocytes with a mean of 6 transcripts per million.
Limitations, reasons for caution
Functional analysis is ongoing to validate the newly identified genes, data need to be verified in different ethnicities. Nevertheless, this study demonstrates the establishment of a specific and scalable analytical framework that can be employed for the identification of genetic causes in unexplained infertility cases characterized by defective developmental patterns.
Wider implications of the findings
Scaling up this investigative approach would provide an effective strategy for discovering new genes/pathways in what is considered idiopathic infertility, further defining precision reproductive medicine interventions. Importantly, this study revealed lesions in genetic patterns involved in chronic diseases providing a molecular footprint of the well-established link between infertility and comorbidities.
Trial registration number
none
Reliability of Whole-Exome Sequencing for Assessing Intratumor Genetic Heterogeneity