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Cited by 5Hypothalamic control of reproductive hormonesSexual Differentiation and DisordersNeuroendocrine regulation and behavior

Puberty Blocker, Leuprolide, Reduces Sex Differences in Rough-and-Tumble Play and Anxiety-like Behavior in Juvenile Rats

Open access
Oliveira, Gabriela de Faria, Nguyen, Amber T, Carreras-Simons, Leykza, Niepsuj, Thomas, Gadelhak, Salma H, Johnson, Aimee K, Abdalla, Ashwakh, Lev, Eden +5 more·2024·Endocrinology
DOI 10.1210/endocr/bqae046PMC PMC11033216
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Abstract

We examined the effect of the puberty blocker, leuprolide acetate, on sex differences in juvenile rough-and-tumble play behavior and anxiety-like behavior in adolescent male and female rats. We also evaluated leuprolide treatment on gonadal and pituitary hormone levels and activity-regulated cytoskeleton-protein messenger RNA levels within the adolescent amygdala, a region important both for rough-and-tumble play and anxiety-like behavior. Our findings suggest that leuprolide treatment lowered anxiety-like behavior during adolescent development, suggesting that the maturation of gonadotropin-releasing hormone systems may be linked to increased anxiety. These data provide a potential new model to understand the emergence of increased anxiety triggered around puberty. Leuprolide also reduced masculinized levels of rough-and-tumble play behavior, lowered follicle-stimulating hormone, and produced a consistent pattern of reducing or halting sex differences of hormone levels, including testosterone, growth hormone, thyrotropin, and corticosterone levels. Therefore, leuprolide treatment not only pauses sexual development of peripheral tissues, but also reduces sex differences in hormones, brain, and behavior, allowing for better harmonization of these systems following gender-affirming hormone treatment. These data contribute to the intended use of puberty blockers in stopping sex differences from developing further with the potential benefit of lowering anxiety-like behavior.

AI snapshot

Written from the paper’s full text.

TL;DR

Daily leuprolide acetate in juvenile rats reduced anxiety-like behavior and eliminated several sex differences in rough-and-tumble play, hormones, and amygdala ARC mRNA. The authors propose this as a model for how puberty blockade may reduce sex differences and anxiety during adolescent development.

Key findings

  • Leuprolide delayed vaginal opening in females (P = .0002; t = 4.829) and reduced anxiety-like behavior in both sexes, with treated animals spending more time in the open arm of the elevated plus maze (F(1,44) = 7.103; P = .011).
  • Leuprolide eliminated sex differences in rough-and-tumble play: control males showed higher total play frequency than control females (P < .001), while leuprolide-treated males showed lower frequency than control males (P < .001) and resembled control females; leuprolide did not alter female play. Similar effects were reported for play duration and pinning.
  • Leuprolide lowered FSH (F(1,28) = 5.390; P = .028) and reduced or eliminated sex differences in testosterone, TSH, GH, and corticosterone; for example, leuprolide reduced male testosterone to female-like levels (P < .001) and increased male corticosterone to female-like levels (P = .026).
  • In the amygdala, control females had higher ARC mRNA than control males (P = .006), and leuprolide increased ARC mRNA in males to female-like levels (P = .001); ARC mRNA was negatively correlated with pinning (P = .029; R = 0.289) and testosterone (P = .040; R = 0.307), while testosterone was positively correlated with pinning (P < .001; R = 0.728).

Methods

  • Juvenile Sprague Dawley rats (female = 24, male = 24) received daily subcutaneous leuprolide acetate (25 µg/kg) or saline from postnatal day (PD) 27 to PD 39, with n = 12 per sex per treatment.
  • Rough-and-tumble play was scored from PD 27 to PD 37; anxiety-like behavior was assessed with the elevated plus maze on PD 38 and 39; tissue and serum were collected on PD 40.
  • Analyses used 2-way ANOVAs with Tukey post hoc tests, Pearson correlations, and Grubbs outlier testing; amygdala ARC mRNA was measured by qPCR, and hormones by LC-MS/MS and multiplex assay.

Limitations

  • Paper-stated: longitudinal studies are needed to determine whether leuprolide’s reductions in anxiety-like behavior are permanent, and additional studies are needed to examine whether effects are reversible or somewhat permanent.
  • Paper-stated: several observations appeared to affect males rather than females; the authors note that longer treatment might reveal more dramatic effects in females once estradiol levels increase.
  • Paper-stated and evident: LH could not be analyzed because 27% of samples were below assay detectability; the study is a short-window rat model, so it does not directly establish long-term or human clinical outcomes.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1210/endocr/bqae046

Cites

31 of 31 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

19 of 41 cited sources checked · 5 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Female rats had significantly higher ARC mRNA levels than male rats.

    “Our findings are consistent with previous studies indicating that female rats had significantly higher ARC mRNA levels than male rats (15).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [15] Randesi, 2019, Sex differences in neuroplasticity- and stress-related gene expression and protein levels in the rat hippocampus following oxycodone conditioned place preference, Neuroscience source

      The Results (Plasticity genes and related proteins) report significantly higher Arc mRNA in saline-treated females than saline-treated males in the lateral hippocampus (p = 0.048). The claim omits those condition and region qualifiers; the Discussion notes that after oxycodone CPP, males’ Arc mRNA rose to levels equivalent to females’. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    At the developmental stage in the paradigm, there is a notable disparity in the levels of testosterone and estradiol.

    “At the developmental stage in our paradigm there is a notable disparity in the levels of testosterone and estradiol (24).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] Bell, 2018, Comparing postnatal development of gonadal hormones and associated social behaviors in rats, mice, and humans, Endocrinology source

      The Rats section reports different developmental patterns: estradiol peaks around postnatal day 15, while testosterone remains low from days 1 to 19. That supports a difference in their developmental trajectories, but the cited paper does not identify the stage used in the citing authors’ paradigm or establish a direct disparity between the two hormones’ levels at that stage. The Conclusion’s figure caption says its curves are scaled to each hormone’s own maximum, rather than to one another; page numbers were not provided in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Findingcitation

    Control male rats engaged in higher levels of rough-and-tumble play behavior compared to female rats.

    “Consistent with previous studies, we found that control male rats engaged in higher levels of rough-and-tumble play behavior compared to female rats (18).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Auger, 2009, Brain sex differences and the organisation of juvenile social play behaviour, J Neuroendocrinol source
  • Premisecitation

    Previous studies have shown that the profile of estrogen levels between males and females are similar throughout the juvenile and prepubescent periods.

    “Previous studies have shown that the profile of estrogen levels between males and females are similar throughout the juvenile and prepubescent periods (24).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] Bell, 2018, Comparing postnatal development of gonadal hormones and associated social behaviors in rats, mice, and humans, Endocrinology source

      The Rats section reports that estradiol levels remain low and equivalent in male and female rats until P39. The claim does not specify rats: the Humans section says girls have higher estradiol levels than boys from age two until puberty, and the Mice section says longitudinal levels in males are undetermined. The similarity is therefore supported for rats, not across the species covered by the source. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Findingown data

    Leuprolide lowered FSH levels in male and female rats.

    “Using a 2-way ANOVA, we found a main treatment effect was leuprolide lowering FSH levels (F [1, 28] = 5.390; P = .028) Fig 3A.”
    Quote in the paperNot a substantial claim
  • Classified as probably not a substantial claim. 21
  • May combine two claims. 31

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Females have higher levels of neurogenesis within the newborn amygdala.

    “Previous research has shown that females have higher levels of neurogenesis within the newborn amygdala (30)”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [30] Krebs-Kraft, 2010, Sex difference in cell proliferation in developing rat amygdala mediated by endocannabinoids has implications for social behavior, Proc Natl Acad Sci U S A source

      The Discussion says neonatal females had more new amygdala cells, but attributes the difference to astrocyte genesis and explicitly reports no sex difference in neurogenesis. The Abstract likewise says females had more newly generated glial cells, not neurons. (LLM, conf=0.95)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pubertal development proceeds in a typical manner on withdrawal of puberty blockers.

    “While pubertal development is paused using puberty blockers, studies indicate that pubertal development proceeds in a typical manner on withdrawal of puberty blockers.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [7] Rew, 2021, Review: puberty blockers for transgender and gender diverse youth—a critical review of the literature, Child Adolesc Ment Health source
    • cites [8] Vrouenraets, 2015, Early medical treatment of children and adolescents with gender dysphoria: an empirical ethical study, J Adolesc Health source

      The section on possible physical or psychological harmful effects discusses uncertainty about bone-density catch-up and long-term outcomes, but does not describe pubertal development after blockers are withdrawn. Searches for withdrawal, resumption of puberty, and reversibility found no passage addressing whether development then proceeds typically. (LLM, conf=0.70)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    In rats, reproductive physiology and behavior are delayed with leuprolide acetate but later restored following cessation of the puberty blockade.

    “For example, recent studies in rats indicate that while reproductive physiology and behavior are delayed with the usage of leuprolide acetate, these are later restored following cessation of the puberty blockade (7, 8).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [7] Rew, 2021, Review: puberty blockers for transgender and gender diverse youth—a critical review of the literature, Child Adolesc Ment Health source
    • cites [8] Vrouenraets, 2015, Early medical treatment of children and adolescents with gender dysphoria: an empirical ethical study, J Adolesc Health source

      The section on possible physical or psychological harmful effects discusses uncertainty about puberty suppression and concerns about fertility, but does not report a rat study. Searches for leuprolide acetate, rats, reproductive behavior, and recovery after stopping treatment found no passage addressing the claimed finding. The indexed passages provide no page numbers. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Those receiving gender-affirming health care exhibit lower levels of anxiety, depression, and suicidal ideation.

    “Recent studies have shown that those receiving gender-affirming health care exhibit lower levels of anxiety, depression, and suicidal ideation (13, 14)”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [13] Costa, 2015, Psychological support, puberty suppression, and psychosocial functioning in adolescents with gender dysphoria, J Sex Med source

      The CGAS at Follow-Up section reports improved psychosocial functioning among adolescents receiving psychological support and, for some, puberty suppression. The Discussion says the study assessed only a measure of psychosocial well-being; it does not establish lower levels of anxiety, depression, or suicidal ideation specifically. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [14] de Vries, 2012, Clinical management of gender dysphoria in children and adolescents: the dutch approach, J Homosex source

  • May combine two claims.

    Premisecitation

    The ARC protein is an inducible transcription factor associated with increased synaptic plasticity and is expressed at higher levels in females compared to males.

    “The ARC protein is an inducible transcription factor associated with increased synaptic plasticity and has been reported to be expressed at higher levels in females compared to males (15).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Randesi, 2019, Sex differences in neuroplasticity- and stress-related gene expression and protein levels in the rat hippocampus following oxycodone conditioned place preference, Neuroscience source

      The Discussion describes Arc as an immediate early gene involved in synaptic plasticity, not as a transcription factor. The Results report higher ARC protein immunoreactivity in females than males in specific rat hippocampal regions and experimental conditions, but do not establish that females generally have higher ARC levels. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Puberty is associated with increased vulnerability to anxiety, depression, and suicidal ideation.

    “In addition to the physiological changes associated with puberty, this developmental period is also associated with dramatic shifts in mental health risk, such as increased vulnerability to anxiety, depression, and suicidal ideation (9-11)”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [9] Pfeifer, 2021, Puberty initiates cascading relationships between neurodevelopmental, social, and internalizing processes across adolescence, Biol Psychiatry source

      The Abstract and “Growing Gender Disparities in Internalizing Disorders During Adolescence” describe increased adolescent vulnerability to depression and anxiety in the context of pubertal development. The review discusses non-suicidal self-injury and mentions suicide risk in relation to romantic relationships, but it does not establish increased vulnerability to suicidal ideation specifically during puberty. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [10] Johnson, 2020, Invalidation experiences among non-binary adolescents, J Sex Res source
    • cites [11] Tordoff, 2022, Mental health outcomes in transgender and nonbinary youths receiving gender-affirming care, JAMA Netw Open source

      The Discussion (page unavailable) says transgender and nonbinary adolescents are at increased risk of depression, anxiety, and suicidality, and the Results (page unavailable) report high rates of these outcomes in the study cohort. The paper does not establish that puberty itself increases vulnerability, or that the finding applies to youths generally. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    TNB adolescents receiving gender-affirming hormone therapy had reduced anxiety and depression symptoms.

    “studies examining the effects of gender-affirming health care found that TNB adolescents receiving gender-affirming hormone therapy had reduced anxiety and depression symptoms (11).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [11] Tordoff, 2022, Mental health outcomes in transgender and nonbinary youths receiving gender-affirming care, JAMA Netw Open source

      The Results (page unavailable) associate receipt of puberty blockers or gender-affirming hormones with lower odds of moderate to severe depression, but report no association with moderate to severe anxiety. The study analyzed the two interventions together, so it does not establish the stated outcomes for hormone therapy alone. (LLM, conf=0.91)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Previous findings have reported that peak GH levels at PD 30 and 90 are higher in males compared with females.

    “While GH is released in pulses and varies depending on time of day, previous findings have reported that peak levels at PD 30 and 90 are higher in males compared with females (30).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [30] Krebs-Kraft, 2010, Sex difference in cell proliferation in developing rat amygdala mediated by endocannabinoids has implications for social behavior, Proc Natl Acad Sci U S A source

      The Results discuss sex differences in amygdala cell proliferation and endocannabinoid levels, not growth hormone. Searches for GH, growth hormone, pulsatile secretion, and peak levels at postnatal days 30 and 90 found no passage addressing the cited claim in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Males have greater attrition of newborn cells within the amygdala around puberty.

    “and recent data suggest that males have greater attrition of newborn cells within the amygdala around puberty (32).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [32] Mohr, 2022, Sex differences in proliferation and attrition of pubertally born cells in the rat posterior dorsal medial amygdala, Dev Cogn Neurosci source

  • May combine two claims.

    Premisecitation

    Testosterone levels in males are higher than females at birth and during adolescence.

    “Testosterone levels in males are higher than females at birth and during adolescence (24), which is replicated in the observed levels of testosterone in our control rodents.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] Bell, 2018, Comparing postnatal development of gonadal hormones and associated social behaviors in rats, mice, and humans, Endocrinology source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Previous research suggests that GnRHa reduced TSH levels within 2 weeks in humans but did not alter 3,5,3′-triiodothyronine or thyroxine levels.

    “Indeed, previous research suggests that GnRHa reduced TSH levels within 2 weeks in humans but did not alter 3,5,3′-triiodothyronine or thyroxine levels (27).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [27] Yilmaz, 2020, Is gonadotropin-releasing hormone agonist usage really leading to thyroid dysfunction?, Interv Med Appl Sci source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Human adolescent development is associated with increased anxiety and depression.

    “It is known that human adolescent development is associated with increased anxiety and depression (23).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [23] Reardon, 2009, A critical review of the empirical literature on the relation between anxiety and puberty, Clin Psychol Rev source

  • May combine two claims.

    Premisecitation

    Androgen exposure during the early neonatal time period is responsible for later juvenile rough-and-tumble play behavior, and juvenile hormone levels are not involved in its regulation.

    “Therefore, it is believed that androgen exposure during the early neonatal time period is responsible for later juvenile rough-and-tumble play behavior, and that juvenile hormone levels are not involved in its regulation (18, 25).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [18] Auger, 2009, Brain sex differences and the organisation of juvenile social play behaviour, J Neuroendocrinol source

      The 'Hormones and play development' section (page unavailable) says that perinatal testosterone exposure helps organize later juvenile social play, including through androgen receptors. It does not establish that hormone levels during the juvenile period are uninvolved in regulating play. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [25] Meaney, 1986, Testosterone implants into the amygdala during the neonatal period masculinize the social play of juvenile female rats, Brain Res source

  • May combine two claims.

    Premisecitation

    Testosterone is significantly higher in males than females on PD 40; whereas estradiol has just started to be released.

    “Testosterone is significantly higher in males than females on PD 40; whereas estradiol has just started to be released (34).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [34] Walker, 2012, Molecular profiling of postnatal development of the hypothalamus in female and male rats1, Biol Reprod source

      The Results report significantly higher testosterone in males at the measured ages except P30, but the study did not measure PD40 specifically. The Results and Discussion contradict the estradiol claim: estradiol was already present, peaked at P15 in both sexes, and decreased as the rats matured. Page numbers are not available in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Leuprolide-treated males exhibited lower levels of rough-and-tumble play duration compared with control males and engaged in play levels similar to control females.

    “Furthermore, leuprolide-treated males exhibit lower levels of play duration compared with control males (P < .001) and engaged in play levels that were similar to control females.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Leuprolide-treated males exhibited lower levels of rough-and-tumble behavior frequency compared to control males, resembling control females.

    “That is, leuprolide-treated males exhibited lower levels of rough-and-tumble behavior compared to control males (P < .001), resembling that of control females.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Leuprolide increased ARC mRNA levels in males to female levels and thereby eliminated the sex difference in ARC mRNA levels.

    “Leuprolide increased ARC mRNA levels in males to female levels and thereby eliminated the sex difference in ARC mRNA levels.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    There was a trend toward leuprolide-treated females experiencing lower levels of progesterone than control females, but it did not reach statistical significance.

    “While there was a trend toward leuprolide-treated females experiencing lower levels of progesterone than control females, it did not reach statistical significance.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Leuprolide reduced juvenile rough-and-tumble play behavior in males and lowered testosterone levels.

    “In the present study, we found that leuprolide reduced juvenile rough-and-tumble play behavior in males and lowered testosterone levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Leuprolide removed sex differences in hormone levels, affecting testosterone, TSH, and corticosterone.

    “Leuprolide removed sex differences in hormone levels, affecting testosterone, TSH, and corticosterone.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Leuprolide males had higher mRNA levels of ARC than control males, eliminating the sex difference in ARC mRNA levels.

    “There was also a treatment effect within males, with leuprolide males having higher mRNA levels of ARC than control males (P = .001), eliminating the sex difference in ARC mRNA levels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Leuprolide increased male corticosterone levels to female levels, effectively eliminating the sex difference in corticosterone levels.

    “Specifically, leuprolide increased male corticosterone levels to female levels, effectively eliminating the sex difference in corticosterone levels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The sex difference in rough-and-tumble play behavior was observed both in frequency and duration of play.

    “This sex difference in rough-and-tumble play behavior was observed both in frequency and duration of play.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Leuprolide treatment eliminated the sex difference in GH levels, reducing male levels to female-typical levels.

    “and leuprolide treatment eliminated this sex difference, reducing male levels to female-typical levels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Leuprolide exposure led to increased ARC mRNA levels in male rats, eliminating the sex difference observed in control rats.

    “Leuprolide exposure led to increased ARC mRNA levels in male rats, eliminating the sex difference observed in control rats.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Several of the observations appear to affect males rather than females.

    “It is also pertinent to observe that several of our observations appear to affect males rather than females.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Many GnRH agonists are safe and FDA-approved for treating precocious puberty in adolescents and reproductive cancers in adults.

    “The use of many GnRHas has been proven to be safe and approved by the US Food and Drug Administration in the early 1990s (1) for the treatment of precocious puberty in adolescents (2), and reproductive cancers in adults (3).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [1]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [2] Tuvemo, 2006, Treatment of central precocious puberty, Expert Opin Investig Drugs source
    • cites [3] Santen, 2014, Menopausal hormone therapy and breast cancer, J Steroid Biochem Mol Biol source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    These symptoms are exacerbated in TNB adolescents.

    “which are exacerbated TNB adolescents (12).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [12] Kuper, 2019, Baseline mental health and psychosocial functioning of transgender adolescents seeking gender-affirming hormone therapy, J Dev Behav Pediatr source

  • Classified as probably not a substantial claim.

    Premisecitation

    The rhythmic secretion of GnRH from the hypothalamus acts on GnRH receptors to trigger release of FSH and LH from the pituitary.

    “The rhythmic secretion of GnRH from the hypothalamus act on GnRH receptors to trigger the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) from the pituitary (4)”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4] Marques, 2000

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    GnRH agonists desensitize GnRH receptors, suppress FSH and LH release from the anterior pituitary, lower gonadal release of estrogens and androgens, and delay development of secondary sexual characteristics.

    “GnRHas modulate this pathway, leading to desensitization of GnRH receptors. As a result, the release of FSH and LH from the anterior pituitary gland is suppressed, gonadal release of estrogens and androgens is lowered, and the development of secondary sexual characteristics is delayed (6).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6] Desforges, 1991, Gonadotropin-releasing hormone and its analogues, N Engl J Med source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    These symptoms are likely attributed to gender dysphoria, social exclusion, inadequate parental support, and discrimination experienced by TNB youth.

    “These symptoms are likely attributed to gender dysphoria, social exclusion, inadequate parental support, and discrimination experienced by TNB youth (10).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Johnson, 2020, Invalidation experiences among non-binary adolescents, J Sex Res source

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Counts and duration of rough-and-tumble play were analyzed by 2 observers unaware of group assignments.

    “Counts and duration across all observation windows were summed and analyzed by 2 observers unaware of group assignments.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All data were collected by researchers unaware of group classification.

    “All data were collected by researchers unaware of group classification.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Lower testosterone levels correlate with increased ARC mRNA levels within the amygdala, which is associated with reduced pinning behavior.

    “Therefore, lower testosterone levels correlate with increased ARC mRNA levels within the amygdala, which is associated with reduced pinning behavior.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Leuprolide treatment not only pauses sexual development of peripheral tissues, but also reduces sex differences in hormones, brain, and behavior.

    “Therefore, leuprolide treatment not only pauses sexual development of peripheral tissues, but also reduces sex differences in hormones, brain, and behavior, allowing for better harmonization of these systems following gender-affirming hormone treatment.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Leuprolide acetate at 25 µg/kg daily in juvenile rats is a valid model for human puberty blockade and gender-affirming hormone treatment.

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationreasoning

    Leuprolide treatment allows for better harmonization of these systems following gender-affirming hormone treatment.

    “allowing for better harmonization of these systems following gender-affirming hormone treatment.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: Leuprolide acetate at 25 µg/kg daily in juvenile rats is a valid model for human puberty blockade and gender-affirming hormone treatment.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All experimental procedures were approved by the University of Wisconsin-Madison Animal Care and Use Committee in accordance with NIH guidelines.

    “All experimental procedures were approved by the University of Wisconsin–Madison Animal Care and Use Committee in accordance with US National Institutes of Health guidelines.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    For LH, several samples (27%) were below detectability of the assay, preventing analyses from being conducted.

    “For LH, several samples (27%) were below detectability of the assay, preventing analyses from being conducted.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Leuprolide treatment eliminates sex differences in TSH levels and does not necessarily push males to typical hypothyroidism levels but reduces it to female-typical levels.

    “Therefore, these data suggest that leuprolide treatment eliminates the sex differences in TSH levels and does not necessarily push males to typical hypothyroidism levels but reduces it to female-typical levels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    These data contribute to the intended use of puberty blockers in stopping sex differences from developing further with the potential benefit of lowering anxiety-like behavior.

    “These data contribute to the intended use of puberty blockers in stopping sex differences from developing further with the potential benefit of lowering anxiety-like behavior.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Leuprolide acetate at 25 µg/kg daily in juvenile rats is a valid model for human puberty blockade and gender-affirming hormone treatment.

  • May combine two claims.

    Interpretationown data

    Leuprolide exposure eliminates sex differences across several domains, underscoring its intended use in gender-affirming health care.

    “Together, our data indicate that leuprolide exposure eliminates sex differences across several domains, underscoring its intended use in gender-affirming health care.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The effects of leuprolide in adolescent rats generalize to human adolescents and TNB youth receiving gender-affirming health care.

  • May combine two claims.

    Study design and conductreasoning

    The rat model allows the study to eliminate social confounders and directly assess the effect of leuprolide treatment on anxiety.

    “Our findings in a rat model allow us to eliminate the social confounders and directly assess the effect of leuprolide treatment on anxiety.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    ARC plays an important role in plasticity and learning, and rough-and-tumble play behavior has been associated with the development and maturation of neural circuits in the amygdala.

    “ARC plays an important role in plasticity and learning, and rough-and-tumble play behavior has been associated with the development and maturation of neural circuits in the amygdala (33).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Pellis, 2007, Rough-and-tumble play and the development of the social brain, Curr Dir Psychol Sci source

  • May combine two claims.

    Interpretationown data

    Leuprolide pauses further masculinization of some systems within the amygdala and may contribute to a more feminized behavioral phenotype during this developmental period.

    “This suggests that leuprolide pauses further masculinization of some systems within the amygdala and may contribute to a more feminized behavioral phenotype during this developmental period.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    An experimenter unaware of treatment groups scored the elevated plus maze recordings.

    “An experimenter unaware of the treatment groups scored the recordings.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable). May combine two claims.

    Interpretationreasoning

    These results offer support for the use of puberty blockers to prevent further development of sex differences and alleviate anxiety-like symptoms in adolescents, particularly in a clinical setting.

    “These results offer support for the use of puberty blockers to prevent further development of sex differences and alleviate anxiety-like symptoms in adolescents, particularly in a clinical setting.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The effects of leuprolide in adolescent rats generalize to human adolescents and TNB youth receiving gender-affirming health care.
    • Rests on an assumption: Anxiety-like behavior in rats corresponds to anxiety in humans.

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationreasoning

    If treatment had continued longer, it is likely more dramatic effects in females would have been observed when estradiol levels are further increasing.

    “If we had continued the treatment longer, it is likely we would have observed more dramatic effects in females when estradiol levels are further increasing.”
    Quote in the paperNot a substantial claim
47 of 47

  • The paper relies on this without support.

    Puberty is triggered, in part, by increased pulsatile release of GnRH.

    “Puberty is triggered, in part, from increased pulsatile release of GnRH.”

  • The paper relies on this without support.

    Leuprolide acetate at 25 µg/kg daily in juvenile rats is a valid model for human puberty blockade and gender-affirming hormone treatment.

    “Subjects received subcutaneous injections daily starting at 8 Am between PD 27 to PD 39, that is, a juvenile, peripubertal, individual (17).”

  • The paper relies on this without support.

    The effects of leuprolide in adolescent rats generalize to human adolescents and TNB youth receiving gender-affirming health care.

    “These findings are crucial for understanding the potential benefits of leuprolide in gender-affirming health care, as it may aid in reducing behaviors that contribute to gender dysphoria and social distress.”

  • The paper relies on this without support.

    Anxiety-like behavior in rats corresponds to anxiety in humans.

    “These results offer support for the use of puberty blockers to prevent further development of sex differences and alleviate anxiety-like symptoms in adolescents, particularly in a clinical setting.”

  • The paper relies on this without support.

    Testosterone plays a role in the organization of rough-and-tumble play behavior.

    “It has been established that testosterone plays a role in the organization of rough-and-tumble play behavior.”

  • The paper relies on this without support.

    Pinning behavior is a reliable scoring category for sex difference in rough-and-tumble play behavior.

    “we investigated the relationship between ARC, testosterone, and pinning behavior (a reliable scoring category for sex difference in rough-and-tumble play behavior).”
6 of 6

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Progesterone trend is attributed to different sexes

    Minor

    The Figure 3 caption attributes the progesterone trend to males, but the Results and Discussion attribute it to leuprolide-treated females.

1 of 1

Cites

Flagged first, then by citations

3 of the 34 references couldn’t be matched to a known paper.

Research works printed but not linked: 2

  1. Marques, 2000, Endotext, 1
  2. Breehl, 2023, Physiology, Puberty. In: StatPearls

Cited by

Sorted by citations

Related

Authors
Oliveira, G. D. F., Nguyen, A. T., Carreras-Simons, L., Niepsuj, T., Gadelhak, S. H., Johnson, A. K., Abdalla, A., Lev, E., Torres Román, S. G., Fuchs, S. N., Jorgensen, J. S., Farhat, W. A., & Auger, A. P.
Affiliations
  • University of Wisconsin–Madison (US)
  • Department of Psychology, University of Wisconsin–Madison , Madison, WI 53706 , USA
Corresponding author
Oliveira, Gabriela de Faria
ORCID
Venue
Endocrinology
Publisher
The Endocrine Society
Published
2024-03-29
Volume
165
Issue
5
ISSN
  • 0013-7227 (ISSN-L)
  • 1945-7170
DOI
10.1210/endocr/bqae046
PMC
PMC11033216
OpenAlex
W4394673654
Cited by
5
References
34
Citations by year
2026: 4 · 2025: 1
FWCI
1.63
Type
journal article
Language
en
Open access
Licence
Locations
  • Endocrinology (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
Funders
  • Wisconsin Partnership Program
  • University of Wisconsin School of Medicine and Public Health
Topics
  • Health Sciences › Medicine › Reproductive Medicine › Hypothalamic control of reproductive hormones
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
  • Social Sciences › Psychology › Social Psychology › Neuroendocrine regulation and behavior
Keywords
leuprolide, puberty blockers, gonadotropin-releasing hormone, sex differences, rough-and-tumble play, anxiety-like behavior, adolescent development, gonadal hormones, follicle-stimulating hormone, testosterone, corticosterone, growth hormone, thyroid-stimulating hormone, amygdala, young rats
MeSH terms
Puberty Inhibitors, Amygdala, Animals, Anxiety, Behavior, Animal, Corticosterone, Female, Follicle Stimulating Hormone, Luteinizing Hormone, Gonadotropin-Releasing Hormone, Male, Sex Characteristics, Sexual Maturation, Testosterone, Leuprolide, Rats, Sprague-Dawley, Rats

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-12.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-12.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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