Pathogenic Variants in PET Complex Encoding Genes TDRD12 and EXD1 Impair piRNA Biogenesis and Cause Male Infertility

Nonobstructive azoospermia (NOA), the most severe form of male infertility, has approximately 30% of its cases attributed to genetic factors. However, current genetic research only explains a small fraction of NOA cases.

Here, 156 infertile Chinese patients diagnosed with idiopathic male infertility were recruited. Whole exome sequencing and subsequent bioinformatic analysis were performed to find candidate pathogenic variants. Sanger sequencing was used to validate the candidate variants. Quantitative reverse-transcript PCR (RT-qPCR) was performed to detect the mRNA expression of TDRD12 and related piRNA genes. Hematoxylin and eosin staining and immunofluorescence staining were performed on testicular sections obtained from patients' biopsies. Intracytoplasmic sperm injection (ICSI) was applied for assisted fertilization. Additionally, small RNA sequencing was performed to identify the abnormalities of piRNA biogenesis.

We identified eight variants in PET (PIWI-EXD1-TDRD12) complex encoding genes TDRD12 and EXD1 from three NOA patients and one oligoasthenozoospermia patient. These variants, which were rare or absent in public human databases, were predicted to be deleterious. Notably, TDRD12 mRNA was absent in three NOA patients. Testicular histopathological analyses revealed that spermatogenesis was arrested at zygotene stage in patients carrying biallelic TDRD12 variants. Further immunofluorescence staining of L1ORF1p and small RNA sequencing revealed disruptions in piRNA biogenesis and subsequent derepression of retrotransposons. Further assisted reproduction outcomes showed that one of the individuals conceived successfully after ICSI.

Overall, this study significantly expands the mutational phenotype of PET complex-encoding genes and significantly advances our understanding of spermatogenic failure and male infertility. Our findings provide direct clinical and functional evidence that the function of the PET complex and piRNA biogenesis is highly conserved between humans and mice.