The Impacts of Extended-Release Leuprolide Acetate on Juvenile Rodent Reproductive Hormones and Organ Development: A Focus on Puberty Blocker Effects

Rithika R. Nurani^1;2, Thomas M. Niepsuj^1;2, Gabriela de Faria Oliveira¹, Aimee K. Johnson¹, Amber T. Nguyen¹, Kristin M .Ebert³, Anthony P. Auger¹, Joan S. Jorgensen²

1. Department of Psychology, University of Wisconsin – Madison

2. Department of Comparative Biosciences, School of Veterinary Medicine, University of Wisconsin – Madison

3. Division of Pediatric Urology, School of Medicine and Public Health, University of Wisconsin – Madison

Abstract Text:

Leuprolide Acetate, a GnRH agonist (GnRHa), is widely used to suppress gonadal hormones and delay secondary sex characteristic maturation in cases of precocious puberty or gender dysphoria. This study investigates the effects of a 28-day extended-release Leuprolide Acetate (LA) depot suspension administered to juvenile rodents before puberty onset. We hypothesized that LA treatment would block puberty, alter reproductive hormonal feedback circuits, and reduce gonad and endocrine-sensitive organ sizes. Forty-eight rats (24 male, 24 female), weight-matched (~50g), were administered LA (0.6mg) or vehicle control once, via subcutaneous injection, at postnatal day (P) 23, prior to puberty onset (n=12 per group). Body weight and vaginal opening was assessed on P23, 34, and 44. Rats were euthanized on P44 after 21 days of drug exposure. Gonads were harvested, weighed, and processed for histology. Serum was collected to measure anterior pituitary and steroid hormone levels. Sex hormones from testes and ovaries were also normalized to organ weight. Puberty indicators: At P23, vaginal opening was absent in all females. By P34, it was present in all control females and 25% of LD females, with no additional openings by P44, indicating blocked puberty onset in 75% of treated females. Histology of preputial anatomy is underway to assess puberty onset in males. Anterior pituitary hormone profiles: FSH was significantly decreased and almost undetectable in LA treated females and males. Prolactin levels decreased in LA females to levels seen in males, with no differences between groups. Surprisingly, LH levels remained unchanged in males but significantly increased in LA treated females compared to controls. Other pituitary hormones, including GH and TSH were unaffected by LA. Gonad morphology: After normalization to body size, LA animals had significantly smaller gonads compared to control (~3 times smaller ovaries and ~1.5 times smaller testes). Sex hormone profiles: In LA treated females, serum progesterone was significantly lower and 17-OH progesterone was below the detectable limit compared to controls. Estrone and estradiol levels were too low to detect in both groups. Testosterone was not different between control and treated males. Normalization of serum steroid values to ovary or testis weight did not alter reported outcomes of serum alone.  This study underscores the impact of long-term GnRH agonist (LA) on pubertal maturation and hormone signaling across the hypothalamic-pituitary-gonad axis. Serum levels of P4, 17-OH, LH, and FSH indicate a disruption in typical ovarian steroid production. Further histological analysis quantifying stages of follicle maturation stages are underway to assess impacts on folliculogenesis. Serum T levels indicated testis output was unaffected, suggesting that Leydig cell function was robust despite a significantly smaller organ size. Testicular histology is under investigation to assess impacts on spermatogenesis. The GnRHa treatment reduced FSH as expected, but the increase of LH in females, and no effect in males was unexpected. This finding may indicate discrete time frames related to HPG axis and peripubertal specific sex difference development in GnRH influence on gonadotrope activity. While GnRHa treatment is typically administered after puberty begins in relevant patients, this study initiated treatment before puberty to determine the effect on juvenile development omitting typical pubertal changes. With the use of puberty blockers in gender-affirming care, further research should evaluate reversibility and long-term reproductive health implications. Funding source: Wisconsin-Partnership-Program.