Name: Establishment of Rat Models Mimicking Gender-affirming Hormone Therapies (Video) | JoVE Journal
Uploaded: 2025-01-10T13:50:00.000+00:00
Duration: 6 min 24 s
Description: Learn to establish rat models mimicking gender-affirming hormone therapies using testosterone and estrogen with anti-androgen. This method evaluates long-term effects and environmental chemical interactions in transgender health research.

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<li>Kinnear, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+model+to+investigate+the+impact+of+testosterone+therapy+on+reproduction+in+transgender+men%5BTitle%5D)+AND+%22Human+Reprod%22%5BJournal%5D)">A mouse model to investigate the impact of testosterone therapy on reproduction in transgender men.</a> Human Reprod. 34 (10), 2009-2017 (2019).</li>
<li>Gómez, Á, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+adult+male+rat+feminization+treatments+on+brain+morphology+and+metabolomic+profile%5BTitle%5D)+AND+%22Hormones+Behav%22%5BJournal%5D)">Effects of adult male rat feminization treatments on brain morphology and metabolomic profile.</a> Hormones Behav. 125, 104839(2020).</li>
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<li>Coleman, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standards+of+care+for+the+health+of+transgender+and+gender+diverse+people%2C+version+8%5BTitle%5D)+AND+%22Int+J+Transgender+Health%22%5BJournal%5D)">Standards of care for the health of transgender and gender diverse people, version 8.</a> Int J Transgender Health. 23 (sup 1), S1-S259 (2022).</li>
<li>Sudhakar, D., Huang, Z., Zietkowski, M., Powell, N., Fisher, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Feminizing+gender%E2%80%90affirming+hormone+therapy+for+the+transgender+and+gender+diverse+population%3A+An+overview+of+treatment+modality%2C+monitoring%2C+and+risks%5BTitle%5D)+AND+%22Neurourol+Urodynamics%22%5BJournal%5D)">Feminizing gender‐affirming hormone therapy for the transgender and gender diverse population: An overview of treatment modality, monitoring, and risks.</a> Neurourol Urodynamics. 42 (5), 903-920 (2023).</li>
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<li>Tassinari, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((short-term+exposure+to+titanium+dioxide+nanoparticles+in+Sprague-Dawley+rat%3A+focus+on+reproductive+and+endocrine+systems+and+spleen%5BTitle%5D)+AND+%22Nanotoxicology%22%5BJournal%5D)">short-term exposure to titanium dioxide nanoparticles in Sprague-Dawley rat: focus on reproductive and endocrine systems and spleen.</a> Nanotoxicology. 8 (6), 654-662 (2014).</li>
<li>Fortune, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+early+stages+of+follicular+development%3A+activation+of+primordial+follicles+and+growth+of+preantral+follicles%5BTitle%5D)+AND+%22Animal+Reprod+Sci%22%5BJournal%5D)">The early stages of follicular development: activation of primordial follicles and growth of preantral follicles.</a> Animal Reprod Sci. 78 (3-4), 135-163 (2003).</li>
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<li>Rasinger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low+dose+exposure+to+HBCD%2C+CB-153+or+TCDD+induces+histopathological+and+hormonal+effects+and+changes+in+brain+protein+and+gene+expression+in+juvenile+female+BALB%2Fc+mice%5BTitle%5D)+AND+%22Reprod+Toxicol%22%5BJournal%5D)">Low dose exposure to HBCD, CB-153 or TCDD induces histopathological and hormonal effects and changes in brain protein and gene expression in juvenile female BALB/c mice.</a> Reprod Toxicol. 80, 105-116 (2018).</li>
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<li>Falvo, R. E., Kaltenbach, C. C., Pancoe, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determination+of+testosterone+concentration+in+the+plasma+of+normal+and+androgen-sterilized+female+rats%2C+using+a+competitive+protein+binding+technique%5BTitle%5D)+AND+%22Neuroendocrinology%22%5BJournal%5D)">Determination of testosterone concentration in the plasma of normal and androgen-sterilized female rats, using a competitive protein binding technique.</a> Neuroendocrinology. 10 (4), 229-234 (1972).</li>
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<li>Raja, N. S., Rubin, E. S., Moravek, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Review+of+animal+models+investigating+the+reproductive+effects+of+gender-affirming+hormone+therapy%5BTitle%5D)+AND+%22J+Clin+Med%22%5BJournal%5D)">A Review of animal models investigating the reproductive effects of gender-affirming hormone therapy.</a> J Clin Med. 13 (4), 1183(2024).</li>
</ol>

The authors declare that the research was conducted without any commercial or financial relationships that could potentially create a conflict of interest.

The implementation of rodent models mimicking GAHT is crucial to studying the potential specific susceptibility and vulnerability of TG people and the long-term outcomes of the therapies, usually lasting all their lives.
Given the scarce number of similar studies in the literature, the critical point of this experiment is the selection of the doses to set the models; such doses should be sufficiently low to be compatible with long-term administration in animals without causing adverse effects,...

The individual&#39;s psychological perception of being male, female, neither, both, or somewhere in between1 is called gender identity. It can match the biological sex (cisgender) or can be different (transgender - TG). A TG man is an individual born as a female but who identifies himself as a man. A TG woman is born as a male but identifies herself as a woman2. It is estimated that, at present, there are 25 million TG people worldwide3, most of them suffering from gender dysphoria, a psychological condition characterized by incongruence between their gender and the one they were assigned at birth4, which can result in social discrimination and difficulties at&#160;work and in family, often leading to depression, anxiety, and stress5. For such reasons, TG people often go through gender-affirming hormone therapy (GAHT) and/or gender-affirming surgery. GAHT for TG men is characterized by the administration of testosterone (T) (Table 1), and for TG women, estrogens (E2) plus antiandrogens (Table 2)6.
GAHT usually lasts for the entire life of the individual, acting continuously on the endocrine system7,8. So, it is important to analyze the GAHT&#39;s impact on the health of TG people and its potential long-lasting effects. Moreover, TG people are exposed, as the general population, to chemical contaminants -- in particular, the endocrine disrupters (ED) -- that&#160;target the endocrine system as the GAHT, resulting in overstimulation9.
ED are&#160;a group of chemicals affecting the organisms and/or their progeny by altering different hormonal and metabolic processes, such as the secretion, activation, synthesis, release, and binding of natural hormones. Since ED&#160;are widespread in the environment, food and products of everyday use (e.g., plastic bottles and containers, liners of metal food cans, detergents, flame retardants, food, toys, cosmetics and pesticides, etc.) general population is continuously exposed during the whole life10. In addition, even low-dose exposure to EDs can lead to tissue and organ damage, and a common phenomenon associated with ED exposure is the occurrence of sex-specific effects both in laboratory animals and in humans11. As an example, exposure to the food contact material,&#160;bisphenol A (BPA), is linked to health risks such as endometriosis and polycystic ovary syndrome in women, as well as reduced fertility, due to its estrogenic effects12. In men, BPA can lower&#160;levels and diminish sperm quality13. Additionally, pesticides are associated with a higher risk of breast cancer in women and significant fertility issues in men13. So far, no specific tools are available to study the toxicological effects of environmental contaminants, including ED, in TG people9.
The present study aims to describe methods and parameters selected for the development of two TG animal models: a demasculinizing-feminizing model (dMF) and a defeminizing-masculinizing model (dFM). In particular, the selection of suitable dose levels, time, and way of administration of the hormones on the basis of the current human therapies are assessed14. Moreover, tissue and functional biomarkers that define the models uniquely are identified and characterized. In addition, the efficacy and tolerability of therapy in animals as well as the selection of the most appropriate markers for use in long-term studies, are described in detail together with the techniques used for such purposes.
In order to characterize the models, the following endpoints are analyzed: testosterone (T) serum level (the best biomarker to evaluate the success of GAHT in both models9,15); estradiol (E2) serum level (both models); thyroid stimulating hormone (TSH) and thyroxine (T4) (dMF model); sperm count (dMF model); clitoral dimension (dFM model); histopathological analysis of reproductive organs, liver and thyroid (for both models). In addition, gene expression of the following sex-specific liver cytochrome P450 isoforms (CYP450s, for both models) are also analyzed16,17: CYP2C11 (specifically expressed in the male liver), CYP3A18 (expressed 25 times more in male liver than in female), CYP2C12 (specifically expressed in the female liver), and CYP2C6 (predominantly expressed in female liver, but present at lower levels, also in the male).

<table><tbody><tr><td>Analytical balance ABJ 320-4NM</td><td>Kern</td><td>Z741091</td><td></td></tr><tr><td>Bouin</td><td>Biooptica</td><td>05-M01008</td><td></td></tr><tr><td>Centrifuge 5415 R</td><td>Eppendorf</td><td></td><td>For eppendorf</td></tr><tr><td>Cyproterone Acetate</td><td>Sigma-Aldrich</td><td>C3412</td><td></td></tr><tr><td>D-MEM medium</td><td>Gibco</td><td></td><td></td></tr><tr><td>ExcelTaq 2X Fast Q-PCR Master Mix (SYBR, ROX), 200 RXN</td><td>Smobio&amp;nbsp;</td><td>TQ1210</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128</td><td></td></tr><tr><td>GraphPad Prism software version 5.0&amp;nbsp; for Windows</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>Hematoxylin</td><td>Biooptica</td><td>05-06002/L</td><td></td></tr><tr><td>Heosin</td><td>Biooptica</td><td>05-10007/L</td><td></td></tr><tr><td>Imaging Software</td><td>Nikon</td><td></td><td>NIS-BR</td></tr><tr><td>JMP 10 statistical software</td><td>SAS Institute&amp;nbsp;</td><td></td><td></td></tr><tr><td>Microm</td><td>Thermo Scientific</td><td>HM 325</td><td></td></tr><tr><td>Microscopy</td><td>Nikon Microphot FX</td><td></td><td></td></tr><tr><td>Mouse/Rat Testosterone ELISA</td><td>Biovendor</td><td>RTC001R</td><td>96T</td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Scientific</td><td></td><td>ND-1000</td></tr><tr><td>Paraffina</td><td>Biooptica</td><td>087910</td><td></td></tr><tr><td>Portable Balances SCOUT STX2202</td><td>OHAUS</td><td>&lt;br/&gt; 30253064</td><td></td></tr><tr><td>Primers</td><td>Life Technologies</td><td></td><td>Designed by PrimerBlast</td></tr><tr><td>Rat Estradiol ELISA</td><td>Biovendor</td><td>RTC009R</td><td>96T</td></tr><tr><td>Rat TSH(Thyroid Stimulating Hormone) ELISA Kit</td><td>ELK Biotechnology</td><td>ELK2283</td><td>96T</td></tr><tr><td>Rat TSH(Thyroid Stimulating Hormone) ELISA Kit</td><td>ELK Biotechnology</td><td>ELK2283</td><td>96T</td></tr><tr><td>SensiFASTcDNA Synthesis Kit</td><td>Bioline</td><td>BIO-65053</td><td>50 reaction</td></tr><tr><td>Sesam Oil</td><td>ACROS</td><td>AC241000010</td><td></td></tr><tr><td>Sprague Dawley rats&amp;nbsp;male and female</td><td>Envigo</td><td></td><td>8/9&amp;nbsp;weeks old</td></tr><tr><td>Standard diets</td><td>Mucedola</td><td>4RF18</td><td></td></tr><tr><td>T4(Thyroxine) ELISA Kit</td><td>ELK Biotechnology</td><td>ELK8716</td><td>96T</td></tr><tr><td>Testosterone enanthate</td><td>Sigma-Aldrich</td><td>T3006</td><td></td></tr><tr><td>Thermal Cycler LineGene 9600 Plus Bioer</td><td>Aurogene</td><td></td><td></td></tr><tr><td>Tissue processor</td><td>Shandon Excelsior ES, Thermo Scientific)</td><td></td><td></td></tr><tr><td>Total RNA purification KIT</td><td>Norgen</td><td>17200</td><td>50 column</td></tr><tr><td>Victor 3 Multilabel reader&amp;nbsp;</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>&amp;beta;-Estradiol 17-valerate</td><td>Sigma-Aldrich</td><td>E1631</td><td></td></tr></tbody></table>

establishment of rat models mimicking gender-affirming hormone therapies

Establishment of Rat Models Mimicking Gender-affirming Hormone Therapies

Transgender (TG) people are individuals whose gender identity and sex assigned at birth do not match. They often undergo gender-affirming hormone therapy (GAHT), a medical intervention that allows the acquisition of secondary sex characteristics more aligned with their individual gender identity, providing consistent results in the improvement of numerous socio-psychological variables. However, GAHT targets different body systems, and some side effects are recorded, although not yet fully identified and characterized. Therefore, TG people undergoing GAHT may be considered as a susceptible sub-group of population and specific attention should be paid in the frame of risk assessment, e.g., through the use of targeted animal models. The present work describes the procedures set to implement two rat models mimicking GAHT: the demasculinizing-feminizing model (dMF) mimicking the GAHT for TG women and the defeminizing-masculinizing model (dFM) mimicking the GAHT for TG men. The models have&#160;been implemented through the administration of the same hormones used for human GAHT, namely, &#946;-estradiol plus cyproterone acetate for dMF and testosterone for dFM, by the same routes of exposure for a 2 week period. Rats are checked daily during the treatment to evaluate health status and potentially aggressive behaviors. At sacrifice, blood and target tissues have been sampled and stored for biochemical, molecular, and histopathological analysis. Sex-specific parameters, namely, sperm count and clitoral dimensions, have also been evaluated. In addition, CYP450 isoforms, exclusively and/or preferentially expressed in male and female rat liver, are identified and characterized as novel biomarkers to verify the success of GAHT and to set the model. Thyroid involvement has also been explored as a key target in the endocrine system.

As demonstrated by Tassinari et al.14 and by Tammaro et al.33, the following results showed the success of GAHT on rats and the appropriateness of the models.
No mortality or abnormal behavior, such as aggressiveness, are recorded, and no clinical signs of toxicity or suffering (e.g., decreased activity, piloerection, and an ungroomed appearance) are observed14.
Plasma T concentrations in adult ...

The protocol describes the development of two rodent models mimicking gender-affirming hormone therapies through subcutaneous administration of testosterone or estradiol plus cyproterone acetate (used in human therapies for transgender people): setting of the doses, identification of relevant biomarkers, and evaluation of the effects.

Transgender (TG) people are individuals whose gender identity and sex assigned at birth do not match. They often undergo gender-affirming hormone therapy ...

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Establishment of Rat Models Mimicking Gender-affirming Hormone Therapies

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