Presence of Ovarian Stromal Aberrations after Cessation of Testosterone Therapy in a Transgender Mouse Model

Some transmasculine individuals may be interested in pausing gender-affirming testosterone (T) therapy and carrying a pregnancy. The ovarian impact of taking and pausing T is not completely understood. The objective of this study was to utilize a mouse model mimicking transmasculine T therapy to characterize the ovarian dynamics following T cessation. We injected postpubertal 9–10-week-old female C57BL/6N mice once weekly with 0.9 mg of T enanthate or a vehicle control for six weeks. All T-treated mice stopped cycling and demonstrated persistent diestrus within one week of starting T therapy, while control mice cycled regularly. After 6 weeks of T therapy, one group of T-treated mice and age-matched vehicle-treated diestrus controls were sacrificed. Another group of T-treated mice were maintained after stopping T therapy and sacrificed in diestrus four cycles after the resumption of cyclicity along with age-matched vehicle-treated controls. Ovarian histological analysis revealed stromal changes with clusters of large round cells in the post T group as compared to both age-matched controls and mice at six weeks on T. These clusters exhibited periodic acid-Schiff staining, which has been previously reported in multinucleated macrophages in aging mouse ovaries. Notably, many of these cells also demonstrated positive staining for macrophage markers CD68 and CD11b. Ovarian RNA sequencing found upregulation of immune pathways post T as compared to age-matched controls and ovaries at six weeks on T. Although functional significance remains unknown, further attention to the ovarian stroma may be relevant for transmasculine people interested in pausing T to carry a pregnancy. 16 ovarian samples analyzed. 4 biological replicates per group. Groups: On T, Control for On T, Post T, Control for Post T. C57BL/6NHsd female mice (Envigo) either started testosterone (weekly subcutaneous injection of 0.9 mg testosterone enanthate) at 9-10 weeks old or received control sesame oil only injections. At six weeks on T, one cohort was sacrificed ("On T") and compared to parallel age-matched controls ("Control for On T" or "Control OT"). In a separate cohort, testosterone was stopped after 6 weeks and allowed to washout. These mice were followed until resumption of estrous cyclicity and sacrificed after 4 estrous cycles (“Post T”), with parallel age-matched controls (“Control for Post T” or “Control PT”). All mice were sacrificed in diestrus and ovaries were collected for further analysis.