Establishment of Rat Models Mimicking Gender-affirming Hormone Therapies

Summary

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.

Abstract

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 been implemented through the administration of the same hormones used for human GAHT, namely, β-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.

Introduction

The individual'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 birth....

Protocol

The studies are performed following Directive 2010/63/EU, the Italian Legislative Decree n. 26 of 4 March 2014, and the OECD Principles of Good Laboratory Practice (GLP). The study protocol was approved by the Italian Ministry of Health (authorization no. 806/2021-PR). Here, 16 young sexually mature Sprague-Dawley rats of both sexes (304 ± 13 g male rats and 190 ± 7 g female rats, 8-9 weeks old) are purchased and housed in two/cage under standard laboratory conditions (22 ± 0.5 °C, 50%-60% relative hu.......

Discussion

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,.......

Materials

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