We will conduct a prospective, controlled clinical trial of ovarian and menstrual cyclicity in TGN initiating TRT (Fig. 8). Enrollment will include 20 TGN and 20 cisgender female (CF) control subjects who report female gender identity congruent with female sex assignment at birth. All subjects will be age \>18 y, with female sex assignment at birth, regular menstrual cycles, and body mass index 18-35 kg/m2. Subjects with history of prior T use, cancer, chemotherapy, or radiation of the brain, abdomen, or pelvis, current use of hormonal medications (including, but not limited to, metformin, insulin, progestins, or estrogenic medications), current endocrinopathy (including, but not limited to, PCOS, androgen secreting tumor, diabetes, or pituitary, thyroid, or adrenal disease), and renal, hepatic, cardiac, or hematologic disease will be excluded. TGN subjects will undergo baseline endocrine and menstrual cycle evaluation, followed by intramuscular (i.m.) administration of testosterone cypionate (TC) 50 mg (standard dose) every 7 d for 32 wks (Fig. 8). After 24 wks, an aromatase inhibitor, letrozole (LET, 2.5 mg/d oral), will be co-administered with TC for 8 wks to block estrogen synthesis and examine whether T's effects are independent of E2 signaling. TGN will collect first morning-voided urine daily at home for assessment of hormone levels during one complete menstrual cycle before TRT begins and continuing during TRT. Untreated CF controls will collect urine daily during one menstrual cycle. Urinary concentrations of LH, FSH, estrone (E1) conjugates, and pregnanediol glucuronide (PdG) will be measured via ELISA. Studies of steroidogenesis during the menopausal transition have demonstrated accuracy of monitoring urinary hormone metabolite levels to reconstruct ovarian cyclicity and ovulatory patterns170-173; we can use this methodology to track progression of any declining HPG axis output over time. All subjects will complete a daily uterine bleeding log using REDCap® and undergo weekly measurement of serum FSH, LH, E2, and P4 (ELISA), and T (LC-MS/MS) for confirmation of hormone dynamics demonstrated by urine hormone metabolite studies. TGN will also undergo weekly transvaginal ultrasound (US) using a 4- to 9-MHz probe to obtain three-dimensional (3D) pelvic imaging following initiation of TRT for confirmation of corpus luteum (CL) formation and regression analysis of hormone secretion patterns, endometrial thickness, antral follicle count (AFC), and volume of the uterus and ovaries. Changes in follicle dynamics will be studied with 3D US determination of the mean diameter of each antral in 1-mm increments from 2 to 9 mm, as in our prior reports174,175.

The primary endpoint will be Evidence of Luteal Activity (ELA), as defined by a 3-fold increase in urinary PdG level over baseline170,176. Basal urinary PdG levels, normalized to urinary creatinine excretion, will be determined based upon the minimum daily PdG level detected per cycle or 4-wk interval, as previously described170,176. Secondary endpoints will include mean serum FSH and LH levels, peak urinary LH concentration, and cumulative LH surge count. Follicular phase function will be defined per cycle (or per 4-wk interval in amenorrheic subjects) by creatinine-adjusted urinary E1 AUC. Five-day moving averages of creatinine-adjusted urinary FSH and LH levels will be calculated, with LH surge defined as a 3 standard deviation increase in the 5-d moving LH average, as previously described170,176. The proportion of subjects demonstrating an LH surge will be compared during successive 4-wk intervals.

We have already demonstrated successful recruitment and retention of TGN and CF participants in longitudinal studies involving daily urine collection and serial pelvic US (e.g., our study in Fig. 2 and others)174,175. TGN subjects will be serially evaluated for adverse effects of TRT per The Endocrine Society recommendations63. Details regarding safety monitoring are in the Human Subjects Section. We calculate that 15 subjects/group will have \>95% power to detect a 30% decrease in the proportion of subjects with ELA at baseline compared to the final 4-week TC study interval (wks 21-24). Although we anticipate a larger, more clinically meaningful decrease, we have utilized a conservative target to maximize the study's power. Although we have had no dropouts in our current TGN pilot study, we factored a 20% dropout rate into our enrollment target of 20 subjects per group to ensure achieving sufficient power.