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Transmasculine individuals are often prescribed testosterone (T) for masculinizing hormone therapy. Mouse models mimicking transmasculine T therapy require reliable long-term T administration. The objectives of this study were three-fold, namely, to compare: 1) the release dynamics of three different subcutaneous delivery systems of T enanthate administration (subcutaneous injections, commercially available pellets, and silastic implants) over a 6-week period in postpubertal C57BL/6N mice, 2) to compare the timing for T levels in plasma to return to baseline and cyclicity to resume after cessation of T between injections and pellets, 3) to utilize silastic implants to achieve sustainable increase in T levels utilizing T enanthate and crystalline T. All three modes of T administration resulted in an increase in T levels in plasma. Pharmacokinetic analyses showed a similar overall exposure to T enanthate over 6 weeks (integrated area) for, subcutaneous injection (0.45 mg two times per week and 0.90 mg one time per week), pellet (5 mg 60-day release), and silastic implant (5 mg 21 week) groups. Crystalline T had lower solubility and a decreased integrated area compared to T enanthate, even when implanted at a higher dosage, indicating different pharmacokinetic profiles based on type of T formulation when utilizing the same silastic delivery method. Surgical removal of pellets and silastic tubing led to a quick drop in T levels and resumption of estrous cyclicity, while cessation of injections required a long washout period for T levels to drop and estrous cycles to resume. Sustained elevation in T levels was achieved for at least 21 weeks with silastic implants. As all three delivery methods are able to elevate T levels in female mice for at least 6 weeks, choice of T administration method should be based on outcomes of interest and study design., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]