Short and long duration testosterone treatments induce reversable subfertility in female mice using a gestational model of gender-affirming hormone therapy.

study questionHow does testosterone gender-affirming hormone therapy (T-GAHT) impact breeding success in female mice? SUMMARY ANSWER: T-GAHT causes reversible subfertility in female mice and persistent changes to reproductive tract anatomy, gene expression, and hormone receptors. WHAT IS KNOWN ALREADY: Adult female mice implanted with capsules containing 10 mg of testosterone mimic many aspects of reproductive phenotypes of T-GAHT patients, who may desire future gestation while pausing T-GAHT. In mice, oocytes retrieved from T-GAHT mice had decreased IVF rates, and T cessation prior to stimulation improved these outcomes. However, the effects of T-GAHT on breeding have not been examined. STUDY DESIGN, SIZE, DURATION: Adult female CD1 mice were subcutaneously implanted with capsules containing 10 mg of testosterone or blank controls. In separate studies, capsules were removed after 6 ('short') or 12 weeks ('long' n = 15/group), then mice were paired with proven-breeder CD1 males. Breeding pair success and pup development (15-20/group) were measured for first and second litters, then terminal measurements were taken from dams and their adult offspring (10/group). PARTICIPANTS/MATERIALS, SETTING,

methodsThe reproductive success of explanted T-GAHT and control mice was investigated by pairing them with proven-breeder CD1 males. Regular observations of dams and litters enabled analysis of fertility and the development of male and female pups for two litters. Terminal measures for dams and/or adult offspring focused on endpoints tied to reproductive tract function and gestation, including reproductive hormones, vaginal cytology, sperm analysis and ovarian and uterine anatomy, histology, and gene expression. MAIN RESULTS AND THE ROLE OF CHANCE: All but one T-GAHT dams gave birth, but the time between pairing and their first birth was longer than controls after long (22.3 ± 1.3 days vs 24.5 ± 3.1) and short (23.2 ± 1.4 days vs 25.5 ± 4) treatments. Dams given long T-GAHT treatment had fewer pups in their first litters (11.9 ± 2.7 pups vs 7.8 ± 3.1) but pup number was unaltered after short treatment (11.5 ± 2.4 pups vs 11.4 ± 3.7). Further, offspring from first litters displayed accelerated puberty. Fertility differences and offspring developmental effects were absent for second gestations and litters. Despite fertility rescue, several anatomical, genetic, and histological changes persisted in T-GAHT dams after two litters. Offspring reproductive system outcomes were not significantly altered once dam fertility was restored. This study powerfully demonstrates a subfertile phenotype in T-GAHT-treated animals that is rescued over time and identifies gonadotropin and steroid hormone signaling as potential mechanisms for further investigation. LARGE SCALE DATA: No large-scale data were generated in this study. LIMITATIONS, REASONS FOR CAUTION: Significant effects of T-GAHT on dam terminal measures may be unrelated to subfertility, and similar endpoints must be examined during the subfertile period to identify and fully understand their roles in T-GAHT-dependent reproductive changes. WIDER IMPLICATIONS OF

findingsThe assumption that T-GAHT causes irreversible damage to reproduction has harmfully informed public opinion, medical practice, and government policies. The finding in T-GAHT mice that fertility and offspring outcomes are not permanently impacted are of translational relevance and opens avenues to be tested first in non-human primate models and then humans. STUDY FUNDING/COMPETING INTEREST(S): NIH R01 HD098233, NIH T32 DK071212. The authors declare no competing interests.