Administration of modified rna encoding anti-mullerian hormone (modrna-amh) before cyclophosphamide bridges protein loss until resumption of secretion by a critical mass of growing follicles

Cyclophosphamide (Cp), an alkylating agent, is highly gonadotoxic, but the mechanisms driving loss of primordial follicles (PrFs) that make up the ovarian reserve remain unclear. We have shown that pretreatment of ovaries with ModRNA-AMH in advance of Cp increases PrF retention in wild-type mice. Here, we set out to define the influence of Cp across all stages of follicle development. We used 8–9-week-old C57BL/6J female mice. Each experimental arm had three mice. Mice were injected directly into the ovary with buffer or ModRNA-AMH (25 mcg), followed by intraperitoneal injection 12 hours later of saline or Cp (150 mg/kg). Ovaries were harvested 12 hours after saline/Cp injection (24 hours after buffer/ModRNA-AMH injection). Right ovaries were flash-frozen and analyzed by bulk RNA sequencing. Left ovaries were sectioned, and follicles were counted using H&E staining and light microscopy. At differential follicle counts, we found the least number of PrFs after Cp treatment (buffer/Cp: 25±11) compared with controls: buffer/saline (92±17, p=0.004), ModRNA-AMH/saline (71±28, p=0.06), and retention of PrFs with ModRNA-AMH/Cp (52±6, p=0.02), aligning with our previous results. Interestingly, while Cp significantly reduced the number of primary follicles (buffer/Cp: 19±6) compared with controls (buffer/saline: 38±5, p=0.01; ModRNA-AMH/saline: 39±3, p=0.005), the ModRNA-AMH/Cp treatment did not result in significant retention of primary follicles: 32±14. Similar results were found for secondary follicles (buffer/Cp:28±17) compared with buffer/saline (69±19, p=0.05), ModRNA-AMH/saline (54±24, p=NS), and ModRNA-AMH/Cp (39±6, p=NS); and antral follicles (buffer/Cp:12±8) compared with buffer/saline (32±6, p=0.02), ModRNA-AMH/saline (31±9, p=0.05), and ModRNA-AMH/Cp (16±7, p=NS). A comparison of ovaries exposed to Cp versus control revealed transcriptional upregulation of genes related to follicular growth and steroidogenesis and downregulation of transcripts implicated in cellular/follicle growth arrest. Importantly, pretreatment with modRNA-AMH induced restoration of gene expression levels observed in control ovaries for many transcripts. The data suggest that by 12 hours after Cp treatment, a critical mass of follicles containing granulosa cells (GCs) that naturally produce AMH is lost due to Cp toxicity. Supplementing with ModRNA-AMH attenuates both depletion of PrFs and transcriptomic changes in the ovary, mitigating damage.