A new human in vitro model of cytotypic and testosterone-producing organoids derived from testicular tissue of transgender women.

Study question: Can testicular tissue from trans women (trans tissue) be used to create human testicular organoids?

Summary answer: Testosterone-producing and cytotypic human testicular organoids with bicompartmental architecture can be successfully generated from trans tissue.

What is known already: Testicular organoids are a promising tool for studying testicular function and the effects of toxicants. Immature testicular cells are currently the most efficient at forming organoids that closely recapitulate seminiferous tubule-like architecture and functions. However, the scarcity of immature human testicular tissue limits its use in high-throughput applications. Conversely, trans tissue is abundantly available and characterized by an immature phenotype.

Study design size duration: Trans tissue-derived organoids (trans organoids) were histologically and androgenically compared to reference organoids derived from immature (prepubertal and pubertal) and adult cisgender testicular tissues. Additionally, long-term testosterone production and gonadotrophic stimulation were assessed in trans organoids. To evaluate their cytotypic and transcriptomic resemblance to reference testicular tissue stages, trans organoids were compared at the gene expression level to prepubertal, pubertal, and adult cisgender tissues, along with their tissue of origin.

Participants/materials setting methods: Testicular tissue samples from transgender women, as well as from prepubertal, pubertal, and adult cisgender donors, were used to generate testicular organoids and to compare organoid formation efficiency and testosterone production according to tissue origin. These samples also served as references for transcriptomic comparisons with organoids derived from transgender women's testicular tissue at Day 14 of culture. Testicular organoids were generated and cultured using 3D Petri Dish^® platforms. Histochemistry and immunofluorescence staining were employed to characterize cellular composition and spatial organization. Testosterone production in culture media was assessed using electrochemiluminescence immunoassays. RNA was extracted and sequenced from organoids derived from transgender women, as well as from tissue samples of all donor groups. Deconvolution and differential gene expression analyses were performed to compare the organoids with testicular tissues across all groups.

Main results and the role of chance: Trans organoids form compartmentalized, cytotypic de novo tissues similar to those from pubertal testicular tissue. Additionally, trans organoids exhibit significant testosterone production, sustain this function over extended culture periods, and respond to gonadotrophic stimulation. Deconvolved bulk RNAseq data indicate that cell population proportions within these organoids are close to those in prepubertal and pubertal testicular tissues. Gene expression clusters trans organoids alongside prepubertal and trans tissues. Functional analysis reveals that trans organoids share with prepubertal, pubertal, and trans tissues varied cellular processes. Factors such as the duration of hormone therapy, the expression of anti-Müllerian hormone-an immaturity marker-within the tubules, and the proportion of peritubular myoid cells in the donor tissue were found to predict the success of trans organoid formation.

Large scale data: The bulk RNA-seq raw and preprocessed data are stored under restricted access in the Vrije Universiteit Brussel (VUB) Institutional Data Repository (VUB/IVTD/1/000001) due to participant privacy concerns. Access to the data will be considered by contacting Prof. Yoni Baert (yoni.baert@vub.be).

Limitations reasons for caution: Hormonal data from trans women donors were not acquired in a convenient manner for this study. Deconvolution data allow only cell proportions to be compared, not absolute numbers.

Wider implications of the findings: This study highlights the potential of trans organoids as a novel and ethically sustainable human-based model for male reproductive health research, reproductive toxicology, and endocrine disruption studies. While trans tissue is a valuable replacement for immature tissue, further research should focus on optimizing organoid architecture, evaluating their utility in reprotoxicity testing, and promoting germ cell differentiation.

Study funding/competing interests: This study was conducted with financial support from the VUB Research Council (OZR4004) to S.M.S., the Scientific Research Foundation-Flanders (G026223N) and the Scientific Fund Willy Gepts to Y.B., the Strategic Research Program 89 from the VUB to E.G., and the Mireille Aerens Chair to T.V. The authors declare no conflict of interest.