O-073 The DNA damage-sensing cGAS-STING pathway is critical in specific Telomere Variant Sequence synthesis in men with nonobstructive azoospermia and individuals with Differences of Sex Development

Study question We aimed to investigate the common molecular mechanisms linking high DNA damage levels and impaired telomere maintenance in human infertility. Summary answer We identified a general molecular mechanism driving genome instability through altered Telomere Variant Sequence, imbalance in telomerase complex components, Androgen Receptor dysfunction, and disrupted autophagy. What is known already We previously reported increased DNA damage in the blood and gonads of men with nonobstructive azoospermia (NOA) and individual with Differences of Sex Development (DSD), which were unexpectedly associated with increased telomere length (TL). Additionally, autophagy inhibition led to TL elongation and DNA damage rescue in blood leukocytes and lymphoblastoid cell lines, linking this process to the regulatory network of the Androgen Receptor (AR) and the cytoplasmic DNA-sensing cGAS-STING signaling. Recent studies have identified Telomere Variant Sequences (TVS) that disrupt telomerase assembly and telomere maintenance, highlighting the need for investigation into their role in genome instability and infertility-related pathological phenotypes. Study design, size, duration We analyzed TVS presence in blood leukocytes of infertile individuals (n = 36). We applied GAR1-siRNA in the presence of inhibitors of AR- Enzalutamide (ENZ) and autophagy-Bafilomycin A1 (BafA1) to estimate dependance of TVS from telomerase complex protein GAR1 in DSD derived lymphoblastoid cell lines (n = 4). Third, we treated blood from DSD (n = 6) with drugs rescuing DNA damage and activating (G10) and inhibiting STING (H151) to establish interaction between cGAS-STING regulation of telomerase GAR1 and TVS synthesis. Participants/materials, setting, methods Cell lines and blood leukocytes of NOA study groups (abnormal/normal embryo development after ICSI; Spermatogonia Arrest (SGA), Sertoli Cells Only (SCO) syndrome), and DSD study groups (Swyer, Complete Androgen Insensitivity Syndromes (CAIS), Germ Cell Tumors (DSD-GCT) were analyzed with PCR for normal Telomere Sequence (TS: TTAGGG) and TVS (TVS1: TGAGGG; TVS2: GTAGGG; TVS3: TTGGGG; TVS4: TTTGGG; TVS5: TTCGGG), TL with quantitative real-time PCR. Proteins were analyzed with immunoblotting. Unpaired t test was applied for statistics. Main results and the role of chance This study demonstrates that infertile Individuals experiencing genotoxic stress, such as men with NOA and individuals with DSD, exhibit Telomere Variant Sequences (TVS) in elongated telomeres. This was linked to an increased presence of the telomerase complex protein GAR1 (Swyer: p = 0,0022; Turner, p = 0,0401; abnormal ICSI group, p = 0,0270, SGA, p = 0,0176). Despite phenotypic variability, the consistent presence of TVS3 in the blood of all infertile study groups, but its absence in controls, was notable. We observed that inhibiting both autophagy and AR signaling was associated with GAR1 upregulation in DSD cell lines (ENZ (100 nM)/BafA1 (100 mM), p = 0,0286), Telomere Length (TL) extension (ENZ, p = 0,0085), but increased DNA damage marked by gH2AX on double-strand brakes (ENZ, p = 0,0286). Using GAR1-siRNA, we confirmed its essential role in inducing TVS3 under genotoxic stress caused by AR inhibition, while autophagy block with BafA1 rescued this effect. Similarly, fresh blood samples from individuals with DSD upregulated GAR1 (p = 0,0048) and sustained STING protein levels, whereas controls downregulated both (GAR1: p = 0,0079; STING: p = 0,0159) upon BafA1 exposure. G10 (60 µM) and H151 (5 µM) treatments stimulated GAR1 (p = 0,0286) and TVS3 synthesis exclusively in DSD cells, while only G10 promoted gH2AX (p = 0,0095) and cGAS (p = 0,0043) levels in primary leukocytes from patients. Limitations, reasons for caution While the observed phenotypes in blood cell hold significant diagnostic value for infertility, further research is needed to determine whether similar molecular mechanisms influence the specification of somatic gonadal and germ cells in humans. Wider implications of the findings These findings uncover a shared molecular mechanism underlying diverse infertility phenotypes associated with genotoxic stress, providing insights for novel diagnostic and therapeutic strategies. Expanding this knowledge will help design targeted approaches to mitigate DNA damage levels in abnormal gonadal and germ cells, potentially benefiting fertility potential. Trial registration number No