Novel variants in YTHDC2 cause non-obstructive azoospermia by disrupting the mitotic-to-meiotic transition in humans and mice

STUDY QUESTION: Do variants in YTH N6-methyladenosine RNA binding protein C2 (YTHDC2) cause male infertility in humans, and what is the underlying pathogenic mechanism? SUMMARY ANSWER: Biallelic pathogenic missense variants in YTHDC2 disrupt the mitotic-to-meiotic transition, causing meiotic arrest and non-obstructive azoospermia (NOA) or severe oligozoospermia in humans. WHAT IS KNOWN ALREADY: YTHDC2 is a male germ cell-specifically expressed RNA helicase essential for meiotic progression. In mice, loss of Ythdc2 leads to meiotic arrest at the early prophase. However, clinical evidence linking YTHDC2 variants to human male infertility and the underlying mechanisms involved remains to be established. STUDY DESIGN, SIZE, DURATION: This study utilized a large cohort comprising 56 consanguineous families and 89 sporadic infertile men diagnosed with NOA or severe oligozoospermia. Through extensive genetic screening, we specifically identified five infertile men from three unrelated families who harbored candidate pathogenic variants in the YTHDC2 gene. The overall study design encompassed genetic screening followed by in vivo functional validation using a knock-in mouse model. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole-exome sequencing (WES) and bioinformatic analyses were performed on the patient cohort to screen for candidate pathogenic variants. Human meiotic defects were characterized via histological analyses and immunofluorescence staining of testicular sections. To validate the pathogenicity of the identified variant, a knock-in mouse model harboring the equivalent variant found in patients was generated by CRISPR/Cas9 technology, and analyzed for spermatogenesis and meiosis using spermatocyte spreading and immunofluorescence staining, quantitative real-time PCR, and western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: Two homozygous missense variants in YTHDC2 were identified in four NOA patients from two unrelated consanguineous families (MT1: c.3491A>T, p. E1164V; MT2: c.2639G>A, p. R880H), and compound-heterozygous missense variants were identified in a sporadic patient with severe oligozoospermia (MT3: c.1145A>G, p. D382G; MT4: c.292A>G, p. R98G). The MT1 variant (p.E1164V) is not located in any annotated domains, the other three variants reside within known functional domains of YTHDC2 protein. The knock-in mouse model carrying the MT1 variant recapitulated the patient's phenotype, with both exhibiting meiotic prophase arrest during spermatogenesis. Mechanistically, significantly decreased levels of MEIOC and RBM46, two YTHDC2-interacting proteins required for meiotic transcriptome reprogramming, were observed in the patient's testes. Concurrently, mitotic cell cycle regulators such as CCNA2, CCND1, and WEE1 were aberrantly upregulated in the patient's testicular cells expressing meiosis markers, indicating a failure to silence the mitotic program upon meiotic entry. LIMITATIONS, REASONS FOR CAUTION: This study is limited by the small sample size of patients with pathogenic YTHDC2 variants. While the MT1 variant was functionally validated in vivo, the specific pathogenic mechanisms of the other variants identified require further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide direct clinical evidence establishing the pathogenicity of YTHDC2 variants in human NOA. The study reveals a conserved role for YTHDC2 in safeguarding the mitotic-to-meiotic transition by suppressing mitotic gene expression while maintaining the meiotic program. These findings expand the genetic spectrum of male infertility and suggest YTHDC2 screening as a promising approach for the genetic diagnosis of male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Developmental Program of China (2022YFA0806303 to H.Z., and 2024YFC2706801 to H.J.); the National Natural Science Foundation of China (32470898 to H.Z., W2412028 and 32330032 to Q.S., 32470915 to B.S.); the State Key Laboratory of Reproductive Medicine and Offspring Health, Nanjing Medical University (SKLRM-K202405); and the Open Research Project of Fuyang Normal University (FYKFKT24023). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.