Impaired in vitro fertilization outcomes following testosterone treatment improve with washout in a mouse model of gender-affirming hormone treatment

Abstract

Background: The impact of gender-affirming testosterone on fertility is poorly understood, with ovarian histopathologic studies showing variable results, some with a detrimental effect on reproductive capacity and uncertain reversibility. Assisted reproductive outcome data are restricted to small case series that lack the ability to inform clinical practice guidelines and limit fertility preservation counseling for transgender and nonbinary individuals.

Objective: This study aimed to determine the impact of current testosterone and testosterone washout on in vitro fertilization outcomes in a mouse model for gender-affirming hormone treatment. We hypothesized that current or previous testosterone treatment would not affect in vitro fertilization outcomes.

Study design: C57BL/6N female mice (n=120) were assigned to 4 treatment groups: (1) current control, (2) current testosterone, (3) control washout, and (4) testosterone washout. Testosterone implants remained in situ for 6 or 12 weeks, representing the short- and long-term treatment arms, respectively. Current treatment groups underwent ovarian stimulation with implants in place, and washout treatment groups were explanted and had ovarian stimulation after 2 weeks. Oocytes were collected, fertilized, and cultured in vitro, with one arm continuing to the blastocyst stage and the other having transfer of cleavage-stage embryos. Statistical analysis was performed using GraphPad Prism, version 9.0 and R statistical software, version 4.1.2, with statistical significance defined by P<.05.

Results: Current long-term testosterone treatment impaired in vitro fertilization outcomes, with fewer mature oocytes retrieved (13.7±5.1 [standard deviation] vs 28.6±7.8 [standard deviation]; P<.0001) leading to fewer cleavage-stage embryos (12.1±5.1 vs 26.5±8.2; P<.0001) and blastocysts (10.0±3.2 vs 25.0±6.5; P<.0001). There was recovery of in vitro fertilization outcomes following washout in the short-term treatment cohort, with incomplete reversibility in the long-term cohort. Testosterone did not negatively affect maturity, fertilization, or blastulation rates.

Conclusion: In a mouse model of gender-affirming hormone treatment, testosterone negatively affected oocyte yield without affecting oocyte quality. Our findings suggest that testosterone reversibility is duration-dependent. These results demonstrate the feasibility of in vitro fertilization without testosterone discontinuation while supporting a washout period for optimization of mature oocyte yield.

Keywords: assisted reproductive technology; fertility; fertility preservation; transgender medicine.

FIGURE 1. Experimental design and model profile

(A1) Control or T silastic tubing implants placed subcutaneously for 6 or 12 weeks representing short and long-term treatment cohorts. For immediate groups, implants remained in place at time of IVF while in cessation groups implants were removed with a 14 day washout period before IVF. (A2) Ovarian stimulation protocol utilized for all mice. (B) Embryo culture design with (B1) arm cultured to blastocyst stage and (B2) cultured to 2-cell embryo stage with subsequent transfer into pseudopregnant mice. (C1) Cyclicity of one representative control and (C2) one representative T-treated mouse. (D) Percent cyclicity for mice after implant placement in control and T cohorts. (E) Weekly T levels obtained prior to implant placement then biweekly throughout study duration for all treatment cohorts. hCG, human chorionic gonadotropin; IVF, in vitro fertilization; T, testosterone.

FIGURE 2. Hormones for long-term treatment cohorts

A, Terminal estradiol, B, progesterone, and C, AMH for immediate and washout cohorts. Bars are mean±SD. Significance was determined by P<.05. AMH, anti-Müllerian hormone; SD, standard deviation.

FIGURE 3. In vitro fertilization and embryo culture outcomes

Ovarian stimulation outcomes for long-term treatment cohort including (A) total oocytes and (B) mature oocytes for both immediate treatment and washout cohorts. (C) Maturity and (D) fertilization rates for immediate and washout cohorts. Culture outcomes including (E) 2-cell embryos, (F) blastocysts, (G) blastulation rates, and (H) live birth rates for long-term treatment and washout cohorts. ns, not significant.

FIGURE 4. Assessment of reversibility following testosterone washout in long-term cohort

A, Dot-plot of IVF outcomes in long-term cohort with mean and confidence interval represented. B, Difference in means for IVF outcomes between T and T washout cohorts. Significance was determined by P<.05. IVF, in vitro fertilization; T, testosterone.

FIGURE 5. Hormones for short-term treatment cohorts

Terminal (A) estradiol, (B) progesterone, and (C) AMH for immediate and washout cohorts. (D) Testosterone levels obtained before implant placement and then biweekly for study duration of short-term treatment cohorts. Bars are mean±SD. Significance was determined by P<.05. AMH, anti-Müllerian hormone; ns, not significant; SD, standard deviation.

FIGURE 6. In vitro fertilization and embryo transfer outcomes following short-term treatment

Ovarian stimulation results for immediate and washout cohorts including (A) total oocytes, (B) mature oocytes, (C) fertilization rate, (D) 2-cell embryos, and (E) live birth rate.