Estradiol increases cortical and trabecular bone accrual and bone strength in an adolescent male-to-female mouse model of gender-affirming hormone therapy

Abstract

The effects of gender-affirming hormone therapy on the skeletal integrity and fracture risk in transitioning adolescent trans girls are unknown. To address this knowledge gap, we developed a mouse model to simulate male-to-female transition in human adolescents in whom puberty is first arrested by using gonadotrophin-releasing hormone analogs with subsequent estradiol treatment. Puberty was suppressed by orchidectomy in male mice at 5 weeks of age. At 3 weeks post-surgery, male-to-female mice were treated with a high dose of estradiol (~0.85 mg) by intraperitoneal silastic implantation for 12 weeks. Controls included intact and orchidectomized males at 3 weeks post-surgery, vehicle-treated intact males, intact females and orchidectomized males at 12 weeks post-treatment. Compared to male controls, orchidectomized males exhibited decreased peak bone mass accrual and a decreased maximal force the bone could withstand prior to fracture. Estradiol treatment in orchidectomized male-to-female mice compared to mice in all control groups was associated with an increased cortical thickness in the mid-diaphysis, while the periosteal circumference increased to a level that was intermediate between intact male and female controls, resulting in increased maximal force and stiffness. In trabecular bone, estradiol treatment increased newly formed trabeculae arising from the growth plate as well as mineralizing surface/bone surface and bone formation rate, consistent with the anabolic action of estradiol on osteoblast proliferation. These data support the concept that skeletal integrity can be preserved and that long-term fractures may be prevented in trans girls treated with GnRHa and a sufficiently high dose of GAHT. Further study is needed to identify an optimal dose of estradiol that protects the bone without adverse side effects.

Fig. 1

Serum estradiol concentrations are elevated in Orx male-to-female mice administered estradiol by silastic implantation. Serum (a) estradiol (pg/mL) and (b) testosterone (ng/mL) in intact female, intact male, and male Orx controls and in male Orx mice administered estradiol (+ estradiol). The text below each experimental group indicates the number of samples below the limit of detection (LOD) of the LC/MS assay. c Seminal vesicle mass (mg) in male controls, Orx males and male Orx mice administered estradiol (+ estradiol) at 3 weeks post-surgery (8 weeks of age) for 12 weeks (20 weeks of age), n = 10/group. Serum hormone concentrations are plotted on a logarithmic scale. Shown are individual values, medians, interquartile ranges, and minimum and maximum values. *P < 0.05, **P < 0.01, ***P < 0.000 5, ****P < 0.000 1

Fig. 2

Pubertal suppression decreases peak cortical bone accrual, which is increased by subsequent estradiol treatment in Orx male-to-female mice. a Representative μCT images showing the femoral cross-section averaged over the imaging depth of 0.68 mm at the mid-diaphysis. Scale on left indicates cortical thickness. b Periosteal circumference (mm), (c) cortical thickness (mm), (d) medullary volume (mm³), and (e) polar moment of inertia (mm⁴) in male controls and Orx males 3 weeks post-surgery (8 weeks of age) and in female controls, male controls, Orx males and Orx males administered estradiol (male Orx + estradiol) at 3 weeks post-surgery (8 weeks of age) for 12 weeks (20 weeks of age) (n = 10/group). *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.000 1. Shown are individual values, medians, interquartile ranges, and minimum and maximum values

Fig. 3

Estradiol treatment in Orx male-to-female mice increases whole-bone strength. a Mean force (N) vs. displacement (mm) for each group with error bars representing SEM from which the following parameters (b–d) were derived: (b) stiffness (N/mm), (c) maximum force (N), (d) postyield displacement (mm) in female controls (n = 5), male controls (n = 10), Orx males (n = 10) and Orx males administered estradiol ( + estradiol) (n = 5) at 3 weeks post-surgery (8 weeks of age) for 12 weeks (20 weeks of age). *P < 0.05, **P < 0.01, ***P < 0.000 5, ****P < 0.000 1. Shown are individual values, medians, interquartile ranges, and minimum and maximum values

Fig. 4

Pubertal suppression decreases peak trabecular bone accrual, which is markedly increased with subsequent estradiol treatment in Orx male-to-female mice. Representative 3D reconstructed μCT images showing (a) the distal femur and (b) trabecular bone in the distal femoral metaphysis. Scale on left indicates trabecular thickness. c Trabecular bone volume as a % of bone volume to tissue volume ((BV/TV)/%), (d) trabecular thickness (Tb.Th/mm), (e) trabecular number (Tb.N/mm⁻¹) and (f) trabecular separation (Tb.Sp/mm) in male controls (n = 10) and Orx males (n = 10) 3 weeks post-surgery (8 weeks of age) and in female controls (n = 10), male controls (n = 10), Orx males (n = 9) and Orx males administered estradiol (male Orx + estradiol) (n = 10) at 3 weeks post-surgery (8 weeks of age) for 12 weeks (20 weeks of age). **P < 0.005, ***P < 0.000 5, ****P < 0.000 1. Shown are individual values, medians, interquartile ranges, and minimum and maximum values

Fig. 5

Estradiol treatment in Orx male-to-female mice increases trabecular bone accrual by stimulating bone formation. a Osteoclast number/bone perimeter [(N.Oc./B.Pm)/(No./mm⁻¹)], (b) osteoclast surface/bone surface ((Oc.S/BS)/%), (c) mineralizing surface/bone surface ((MS/BS)/%), (d) mineral apposition rate (MAR/(μm/d)), (e) bone formation rate (BFR/(μm²/μm/d)) in the distal femoral metaphysis of male controls (n = 4 for (a, b), n = 7 for (c–e)) and Orx males (n = 4 for (a, b), n = 6 for (c–e)) 3 weeks post-surgery (8 weeks of age) and in female controls (n = 7–8), male controls (n = 9), Orx males (n = 7) and Orx males administered estradiol (male Orx + estradiol) (n = 5–6) at 3 weeks post-surgery (8 weeks of age) for 12 weeks (20 weeks of age). *P < 0.05, **P < 0.005, ***P < 0.000 5. Shown are individual values, medians, interquartile ranges, and minimum and maximum values

Fig. 6

Estradiol treatment in Orx male-to-female mice increases trabecular bone arising from the growth plate. a Bone area as a percentage of total area ((BA/TA)/%), (b) trabecular thickness (Tb.Th/μm), (c) trabecular number (Tb.N/mm⁻¹) and (d) trabecular separation (Tb.Sp/μm) in the metaphyseal primary spongiosa of male controls and (e) trabecular and cortical bone volume as a percentage of total metaphyseal volume measured at low-, mid- and high-density thresholds in male controls (n = 10) and Orx males (n = 10) 3 weeks post-surgery (8 weeks of age) and in female controls (n = 10), male controls (n = 10), Orx males (n = 9) and Orx males administered estradiol (male Orx + estradiol) (n = 10) at 3 weeks post-surgery (8 weeks of age) for 12 weeks (20 weeks of age). For (a–d), the inset depicts the region of interest for primary spongiosa measurements extending 265 μm from the growth plate. *P < 0.01, **P < 0.005, ***P < 0.001, ****P < 0.000 1. For (e), the colors used for P values match the density level of bone analyzed; data are the mean ± standard error; *P < 0.05, **P < 0.005, ***P < 0.000 5, ****P < 0.000 1. Shown are individual values, medians, interquartile ranges, and minimum and maximum values