Objective: To establish if the cessation of testosterone (T) therapy reverses T-induced acyclicity in a transgender mouse model that allows for well-defined T cessation timing. Design: Experimental laboratory study using a mouse model. Setting: University-based basic science research laboratory. Animals: A total of 10 C57BL/6NHsd female mice were used in this study. Intervention(s): Postpubertal C57BL/6NHsd female mice were subcutaneously implanted with T enanthate (n ¼ 5 mice) or placebo (n ¼ 5 mice) pellets. Pellets were surgically removed after 6 weeks to ensure T cessation, after which the mice were followed for four estrous cycles after the resumption of cyclicity. Main Outcome Measure(s): Primary outcomes included daily vaginal cytology and weekly T levels before, during, and after T enanthate or placebo pellet implantation and removal. Secondary outcomes included ovarian follicle distribution and corpora lutea numbers, body metrics, and terminal diestrus hormone levels. Result(s): T-treated mice (100%) resumed cycling within one week of T pellet removal after six weeks of T therapy. T levels were significantly elevated during T therapy and decreased to control levels after surgical pellet removal. No detectable differences were observed in the follicle count, corpora lutea formation, diestrus hormone levels, or body metrics after four estrous cycles, with the exception of persistent increased clitoral area between T-treated mice and controls. One T-treated mouse was sacrificed early due to vaginal prolapse and not included in subsequent analyses. Conclusion(s):Our results demonstrated a close temporal relationship between estrous cycle return and T levels dropping to control levels following T pellet removal. The return of regular cyclic ovulatory function is also supported by the formation of corpora lutea and the lack of detectable differences in key reproductive parameters as compared to controls four cycles after T cessation. These results may be relevant to understanding the reversibility of T-induced amenorrhea and possible anovulation in transgender men interested in pausing T to pursue pregnancy or oocyte donation. Results may be limited by the duration of T treatment, lack of functional testing, and physiological differences between mice and humans. (Fertil Steril Sci Ò 2021;2:116-23. Ó2021 by American Society for Reproductive Medicine.

STUDY QUESTION Can mice serve as a translational model to examine the reproductive consequences of pubertal suppression with GnRH agonist (GnRHa) followed by testosterone (T) administration, a typical therapy in peripubertal transmasculine youth? SUMMARY ANSWER An implanted depot with 3.6 mg of GnRHa followed by T enanthate at 0.45 mg weekly can be used in peripubertal female mice for investigating the impact of gender-affirming hormone therapy in transmasculine youth. WHAT IS KNOWN ALREADY There is limited knowledge available in transgender medicine to provide evidence-based fertility care, with the current guidelines being based on the assumption of fertility loss. We recently successfully developed a mouse model to investigate the reproductive consequences of T therapy given to transgender men. On the other hand, to our knowledge, there is no mouse model to assess the reproductive outcomes in peripubertal transmasculine youth. STUDY DESIGN, SIZE, DURATION A total of 80 C57BL/6N female mice were used in this study, with n = 7 mice in each experimental group. PARTICIPANTS/MATERIALS, SETTING, METHODS We first assessed the effectiveness of GnRHa in arresting pubertal development in the female mice. In this experiment, 26-day-old female mice were subcutaneously implanted with a GnRHa (3.6 mg) depot. Controls underwent a sham surgery. Animals were euthanized at 3, 9, 21 and 28 days after the day of surgery. In the second experiment, we induced a transmasculine youth mouse model. C57BL/6N female mice were subcutaneously implanted with a 3.6 mg GnRHa depot on postnatal day 26 for 21 days and this was followed by weekly injections of 0.45 mg T enanthate for 6 weeks. The control for the GnRH treatment was sham surgery and the control for T treatment was sesame oil vehicle injections. Animals were sacrificed 0.5 weeks after the last injection. The data collected included the day of the vaginal opening and first estrus, daily vaginal cytology, weekly and terminal reproductive hormones levels, body/organ weights, ovarian follicular distribution and corpora lutea (CL) counts. MAIN RESULTS AND THE ROLE OF CHANCE GnRHa implanted animals remained in persistent diestrus and had reduced levels of FSH (P = 0.0013), LH (P = 0.0082) and estradiol (P = 0.0155), decreased uterine (P < 0.0001) and ovarian weights (P = 0.0002), and a lack of CL at 21 days after GnRHa implantation. T-only and GnRHa+T-treated animals were acyclic throughout the treatment period, had sustained elevated levels of T, suppressed LH levels (P < 0.0001), and an absence of CL compared to controls (P < 0.0001). Paired ovarian weights were reduced in the T-only and GnRHa+T groups compared with the control and GnRHa-only groups. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Although it is an appropriate tool to provide relevant findings, precaution is needed to extrapolate mouse model results to mirror human reproductive physiology. WIDER IMPLICATIONS OF THE FINDINGS To our knowledge, this study describes the first mouse model mimicking gender-affirming hormone therapy in peripubertal transmasculine youth. This model provides a tool for researchers studying the effects of GnRHa-T therapy on other aspects of reproduction, other organ systems and transgenerational effects. The model is supported by GnRHa suppressing puberty and maintaining acyclicity during T treatment, lower LH levels and absence of CL. The results also suggest GnRHa+T therapy in peripubertal female mice does not affect ovarian reserve, since the number of primordial follicles was not affected by treatment. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the Michigan Institute for Clinical and Health Research grants KL2 TR 002241 and UL1 TR 002240 (C.D.C.); National Institutes of Health grants F30-HD100163 and T32-HD079342 (H.M.K.); University of Michigan Office of Research funding U058227 (A.S.); American Society for Reproductive Medicine/Society for Reproductive Endocrinology and Infertility grant (M.B.M.); and National Institutes of Health R01-HD098233 (M.B.M.). The University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core Facility was supported by the Eunice Kennedy Shriver NICHD/NIH grants P50-HD028934 and R24-HD102061. The authors declare that they have no competing interests.

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/6 N 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.

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The impact and reversibility of long-term gender-affirming testosterone (T) therapy on the reproductive axis of transgender men has not been well-established. Little is known about outcomes for transgender men interested in pausing T therapy to harvest oocytes or get pregnant. We previously established a translational mouse model to investigate T-induced acyclicity and ovarian perturbations. We hypothesized that the duration of T-induced acyclicity would impact the reversibility of cyclic and ovarian changes. To test this hypothesis, T-treated mice were assigned to two groups: (SHORT) 6 weeks of T therapy with immediate reversibility (1.5 mg T propionate pellet implant/removal, n = 5) and (LONG) 6 weeks of T therapy with a prolonged T washout phase (subcutaneous oil injections of T enanthate at 0.9 mg once weekly, n = 5). Control groups (placebo pellets n = 5, sesame oil vehicle injections n = 5) were run in parallel. Estrous cycles were monitored using daily vaginal cytology. Following cessation of T therapy, mice were sacrificed in diestrus after resumption of cyclicity for 4 cycles and ovarian histology examined. Data were analyzed in GraphPad Prism using Welch’s t-test or Mann-Whitney where appropriate. T therapy led to persistent diestrus within a week after T administration for all T-treated mice and none of the controls. The total duration of acyclicity was 6±1 weeks for the SHORT group, which was significantly shorter than the 11±2 weeks for the LONG group (mean ± s.d., p = 0.0079). With resumption of cyclicity, both the SHORT and LONG groups had a significantly lower percentage of days in estrus and higher percentage of days in metestrus as compared to their parallel age-matched controls. Ovarian histology for the SHORT group all showed regular corpora lutea and minimal stromal changes, however, 3/5 mice in the LONG group lacked corpora lutea and 4/5 revealed marked stromal cell hypertrophy. Similar stromal cell changes were not seen in control mice. In conclusion, the length of time of T-induced acyclicity appears to impact the development of stromal cell hypertrophy and formation of corpora lutea even after resumption of cyclicity with similar alterations to the estrous cycles. These findings may have clinical relevance for transgender men interested in fertility, based on duration of gender-affirming T therapy. Future work will aim to separate out the respective contributions of T exposure and acyclicity to the stromal phenotype.