L26/P-399 A selective JAK1/JAK2 inhibitor, ruxolitinib, increases follicle retention, including primordial follicle, after exposure to cyclophosphamide in xenografted human ovarian tissue

Abstract

Study question Can systemic Ruxolitinib (Ruxo) treatment increase the retention of primordial follicles (PrFs) after cyclophosphamide (Cp) exposure in a human ovarian tissue xenograft model? Summary answer Administration of Ruxo before Cp exposure increases follicle viability and retention of PrFs in xenografted human ovarian tissue. What is known already Gonadotoxicity, manifesting as a reduction in PrFs, is a well-known side effect of Cp treatment. Current fertility preservation efforts focus primarily on banking oocytes, embryos, or ovarian tissue, as effective strategies to safeguard ovarian reserve in situ remain elusive. We have previously linked inflammatory signaling within the mouse ovary to Cp exposure and shown that pretreatment with Ruxo before Cp administration increased retention of PrFs. Building on these results, we utilized this treatment protocol to test the fertoprotective effect of Ruxo in human ovarian cortical fragments from four different organ donors xenografted into immunocompromised mice. Study design, size, duration We thawed four fragments (∼24x4x1 mm) of human ovarian tissue, each from an organ donor aged 21-28 years. Each strip was divided into six equal pieces, and tissue was evenly distributed among the experimental groups. Mice underwent ovariectomy and co-transplantation of ovarian tissue with endothelial cells (n = 24 grafts bilaterally in 12 NSG mice). Two weeks transplant, mice harboring xenografts representing an equal distribution of donors were treated for 15 days before graft recovery. Participants/materials, setting, methods Mice were divided into three groups for daily intraperitoneal injections. Control (DMSO 14%, daily); Cp (60 mg/kg once a week, with daily DMSO 14%); and Ruxo (100 mg/kg daily) -Cp (60 mg/kg once a week). Two weeks following the first Cp injection, grafts were harvested, fixed in 4% paraformaldehyde, and underwent histologic evaluation with H&E staining and light microscopy imaging. Serum from xenograft-bearing mice was tested for estradiol, progesterone, and AMH levels. Main results and the role of chance In a histological analysis, follicle counts showed a significantly higher (p = 0.03) ratio of PrFs to growing (primary + secondary) follicles in Ruxo-Cp (0.18) versus Cp (0.00) groups. To account for interpatient variability, we calculated the percentage of viable follicles derived from each condition relative to the entire count from each patient tissue fragment; this calculation was performed for all four donors for each follicle type. When comparing PrF percentage, the Ruxo-Cp group exhibited a significantly higher (p = 0.03) median retention rate (14.29%) compared to Cp (0.00%). The retention rate in the Ruxo-Cp group was not significantly different from the CTRL group. Comparing the percentage of total (PrF + growing) follicles, the Ruxo-Cp group (16.35%) had a higher (p = 0.007) median percentage than the Cp group (4.17%). The CTRL group median percentage (16.00%) was also significantly higher (p = 0.047) than the Cp group median percentage (p = 0.047). Serum levels of estradiol and AMH were undetectable in all mice. Progesterone serum median levels did not differ significantly between groups: CTRL 2.15 ng/mL, Cp 1.79 ng/mL, and Ruxo-Cp 2.31 ng/mL. Limitations, reasons for caution These results demonstrate a benefit to PrF retention and follicle viability. As indicated by lack of circulating estradiol in xenografted mice, however, these experiments were limited to assessment of follicle viability/retention at early growth phases. Further experiments will be needed to measure activity/output over longer intervals. Wider implications of the findings Ruxolitinib, an FDA-approved medication, shows promise in protecting ovarian reserve after Cp exposure in mice and human ovarian xenografts. Further investigation in these and other preclinical models is warranted to determine whether application of Ruxolitinib should be tested in clinical trials of prophylactic treatments for in-situ fertility preservation. Trial registration number No