Gender-affirming hormone therapy (GAHT) plays a crucial role in the care of many transgender individuals who seek alignment between their gender identity and the sex assigned at birth. Trans women may undergo GAHT which usually combines the administration of estrogens and anti-androgens to suppress endogenous testosterone production and to develop feminine secondary sexual characteristics (Hembree et al., 2017; Coleman et al., 2022). Following feminizing GAHT, trans women without any surgical contraindications can opt for a sex reassignment surgery (SRS) which typically consists of a bilateral orchidectomy, as well as a gender-affirming vulvoplasty or vaginoplasty (van der Sluis et al., 2023).

While various effects of feminizing GAHT on the physical transition have been extensively studied (Schneider et al., 2017; Trans Primary Care, 2023), its impact on the testicular microenvironment remains a subject of ongoing research. Previous studies mainly focused on the impact of GAHT on spermatogenesis, yielding contrasting outcomes, ranging from maturation arrest (Lu and Steinberger, 1978; Sapino et al., 1987; Schulze, 1988; Payer et al., 2009; Leavy et al., 2017; Jindarak et al., 2018; Matoso et al., 2018; Schneider et al., 2019; Vereecke et al., 2020; de Nie et al., 2022a) to full spermatogenesis (Jiang et al., 2019; Alford et al., 2020). Besides this, varying degrees of tubular hyalinization were found after GAHT (Sapino et al., 1987; Schulze, 1988; Venizelos and Paradinas, 1988; Jindarak et al., 2018; Sinha et al., 2021; de Nie et al., 2022b). Some studies also suggested dedifferentiation of Sertoli cells (Schulze, 1988) and/or dedifferentiation or loss of Leydig cells (Rodriguez-Rigau et al., 1977; Sapino et al., 1987; Schulze, 1988; Venizelos and Paradinas, 1988; Kisman et al., 1990; Matoso et al., 2018; Cornejo et al., 2022). These discrepancies may stem from the limited sample sizes in most studies and the differences in GAHT regimens, involving diverse hormone types, dosages, modes of administration or adherence to treatment (Hembree et al., 2009). Moreover, previous findings on the testicular cells mainly relied on histological outcomes and lacked the use of specific markers to accurately identify the condition of these cells.

Therefore, this study aims to thoroughly characterize the spermatogonial stem cell niche in a large cohort of trans women subjected to a standardized GAHT regimen. This research entails (immuno)histochemistry to examine the general condition of the tissue and the maturity/functionality of the somatic cells. Comparative differential gene expression and functional analysis are performed to compare testicular tissue of trans women with that of cisgender adults, as well as peri- and prepubertal boys.

As many individuals with gender dysphoria seek medical treatment, the need to comprehend the impact of hormone therapy on their bodies, and more particularly on their fertility increases. Despite existing research on the effect of GAHT on spermatogenesis in trans women, the exact influence on the testicular environment remains inadequately explored. This study aimed to fill this knowledge gap by thoroughly examining the spermatogonial stem cell niche of 106 trans women subjected to a standardized GAHT regimen, consisting of estrogens and CPA.

Upon analyzing the testicular histology, it became evident that most trans women either showed a half-open or absent lumen, aligning with the observations in the peri- and prepubertal controls, respectively. Only 23% of the trans women displayed an open lumen, resembling the normal status in adults. Moreover, unlike the controls, a significant portion of trans women displayed mild (41%), moderate (29%), or severe (8%) tubular hyalinization. Earlier studies already brought up varying degrees of hyalinization, basal membrane thickening, and seminiferous tubule atrophy as a result of GATH (Sapino et al., 1987; Schulze, 1988; Venizelos and Paradinas, 1988; Schneider et al., 2019). More recent work on 50 trans women, treated with varying types of GAHT, demonstrated a thickening of the tubular wall in 82% of the participants (Matoso et al., 2018). Similarly, in another examination of 136 adult trans women undergoing estrogen and CPA treatment, a majority exhibited tubular hyalinization, ranging from 76% to 92%, depending on the cessation of the GAHT for 4 weeks, or its continuation before SRS, respectively (de Nie et al., 2022b). Additionally, most of these participants exhibited an absent or half-open lumen, regardless of whether the GAHT was discontinued. Conversely, two other recent studies, one involving 173 trans women undergoing mixed GAHT (Jindarak et al., 2018), and another one including 85 trans women treated with estrogen and spironolactone (Sinha et al., 2021), only reported hyalinization in 6–28% of the participants, respectively. These variations in results could be attributed to differences in study methodologies as the latter two studies focused exclusively on severe cases of hyalinization, while both our study and the one conducted by De Nie and colleagues considered a broader spectrum, encompassing milder forms of hyalinization.

As we reported earlier, most trans women in this cohort lacked ongoing spermatogenesis, primarily showing spermatogonia-only patterns (Vereecke et al., 2020). This previous report mentioned a positive correlation between the number of spermatogonia and the serum T, LH, and inhibin B levels, and a negative correlation with the serum AMH levels and the age at the time of surgery. In the current study, a negative correlation could be observed between the serum AMH levels and the degree of tubular hyalinization and spermatogenesis. Moreover, the regression analysis indicated that higher LH values were associated with differentiation up to secondary spermatocytes and, even more, with differentiation up to round spermatids, while an older age at the time of surgery was indicative of a spermatogonia-only pattern or the complete absence of germ cells.

Examining the PTMCs, a major component of the tubular wall, was crucial to delve deeper into the abnormalities observed in the tubular wall of trans women. The PTMCs provide structural support to the seminiferous tubules, contribute to their contractile function, and exhibit a smooth muscle phenotype characterized by intensive expression of ACTA2, distinguishing them from fibroblasts (Volkmann et al., 2011; Mayerhofer, 2013). Our results indicated that nearly half of the included trans women showed a ‘disconnected’ ACTA2 pattern. The occurrence of this phenotype might be linked to excessive deposition of extracellular matrix, causing spacing between the PTMCs and a more intensive thickening of the tubular wall. Moreover, 16% of the trans women displayed an ‘interrupted’ pattern, while 6% presented an ‘absent’ pattern, reminiscent of the patterns of peripubertal and prepubertal boys, respectively. However, by looking at the morphology of the tissue, the latter two patterns are more likely a reflection of the hyalinization within the tubular wall, causing a loss of the PTMCs rather than their rejuvenation. Noteworthy, our regression analysis revealed that as trans women are older at the time of surgery, they are more likely to have an ‘interrupted’ or an ‘absent’ pattern. Nevertheless, these disturbed patterns do not seem to be age-related, as older adult controls only exhibited a slightly thicker ACTA2 layer, without a ‘disconnected’ or ‘interrupted’ appearance. Apart from this, ACTA2 expression is stimulated by androgens (Schlatt et al., 1993) which are suppressed in trans women undergoing GAHT. This may result in a loss of ACTA2 expression, causing a shift in the PTMC phenotype as smooth muscle cells are known to do so in response to changes in the local environment (Owens et al., 2004). Adult Klinefelter syndrome participants, for example, also typically possess low T levels and exhibit an ‘interrupted’ or ‘absent’ ACTA2 pattern (Van Saen et al., 2020). Interestingly, the regression analysis pointed out that higher serum T values were indicative of ‘intact’ ACTA2 patterns, and to a somewhat lesser extent of ‘disconnected’ ACTA2 patterns.

Since it was previously suggested that the Sertoli cells of trans women might transform into immature cells (Schulze, 1988), their maturation state was of particular interest. Almost all trans women in this cohort exhibited only semi-mature Sertoli cells, expressing both the mature marker AR and the immature marker AMH, resembling the peripubertal controls. A minority of the trans women in this cohort showed mixed phenotypes, in which the presence of semi-mature tubules accounted for a significant portion of the total tubule count. Only a few trans women exclusively exhibited mature Sertoli cells, and none showed solely immature tubules. As T is essential for maintaining the differentiated phenotype and function of Sertoli cells (Gholami et al., 2023), these findings suggest that the disrupted steroid hormone levels after GAHT could cause a partial dedifferentiation of the Sertoli cells (Nistal et al., 2013). Upon comparing Sertoli cell maturity among the different stages of spermatogenesis, it became apparent that although the group without spermatogonia exhibited the highest ratio of mature/semi-mature tubules, those with only spermatogonia and partial differentiation followed an increasing trend. Moreover, the ratio of immature/semi-mature tubules decreased as participants displayed more advanced stages of germ cell differentiation. These observations could possibly be attributed to the older age of participants within the group without spermatogonia, with an age of 46.3±12.7 years old compared to 34.3±13.0 years old in the spermatogonia-only group and 27.9±8.7 years old in the group with partial differentiation. A less pronounced age difference was also noted between the only spermatogonia and partial differentiation groups. Such an age-related variation aligns with earlier findings on this cohort, indicating that suppressing spermatogenesis might be more challenging in younger individuals (Vereecke et al., 2020). The age-related differences in drug sensitivity, particularly the higher metabolization in younger people (Cherry and Morton, 1989), or the use of transdermal estrogens in older participants could contribute to the observed trend. Nevertheless, the age at SRS did not emerge as a predictor for the number of AMH⁺ tubules, whereas elevated serum T levels were contraindicative for having 100% AMH⁺ tubules. Previous research also indicated a correlation between intratesticular AMH levels and the duration of GAHT (Schneider et al., 2021). Our study did not find a correlation between serum AMH levels and GAHT duration, but there was a significant positive correlation between serum AMH and the percentage of AMH⁺ tubules.

The significant role of Leydig cells in male masculinization prompts intriguing questions about their status in trans women undergoing GAHT. Earlier work already mentioned a reduction in their number (Rodriguez-Rigau et al., 1977; Sapino et al., 1987; Venizelos and Paradinas, 1988; Kisman et al., 1990; Payer et al., 2009; Sinha et al., 2021; Cornejo et al., 2022), their regression to an immature phenotype (Matoso et al., 2018), or their dedifferentiation into fibroblast-like cells (Schulze, 1988). Something that immediately stood out in the present analysis was the fact that, contrary to adult controls, trans women exhibited very small Leydig cell clusters, reminiscent of what was observed in peripubertal controls. Interestingly, estrogens promote Leydig cell engulfment by macrophages by stimulating Leydig cells to produce growth arrest-specific 6, which mediates phagocytosis of apoptotic cells by bridging cells with surface-exposed phosphatidylserine to macrophage receptors (Yu et al., 2014). INSL3 was used to further assess the Leydig cells, as it is exclusively produced by Leydig cells and is only indirectly regulated by the hypothalamic-pituitary-gonadal axis, making it a reliable marker to assess the Leydig cell population size and Leydig cell maturity (Ivell et al., 2013; Anand-Ivell et al., 2022). Nevertheless, it should be considered that excessive E2 concentrations might suppress INSL3 expression (Laguë and Tremblay, 2009; Sansone et al., 2019). Within this cohort, the functional maturity of the Leydig cells varied widely, ranging from 2% to 97%. Only in 47% of the trans women, the functional maturity of the Leydig cells exceeded 70%, which was similar to the adult controls. Comparing scores across the different stages of spermatogenesis revealed significantly fewer functionally mature cells in the groups with no or only spermatogonia compared to those with partial differentiation. This was expected since adequate T levels, produced by Leydig cells, are essential for supporting spermatogenesis (Smith and Walker, 2014). The regression analysis indicated that age-related differences between the groups could also account for this effect. This could again be attributed to variations in GAHT sensitivity related to age but also to the tendency of Leydig cells to lose functionality with increasing age (Anand-Ivell et al., 2022). However, even the oldest control (77 years of age) still had 72% functionally mature Leydig cells. Additionally, this analysis implied that participants with lower FSH levels tended to have fewer functionally mature Leydig cells, aligning with findings by Sapino and colleagues who observed a decrease in Leydig cell numbers correlated with low serum gonadotropin levels (Sapino et al., 1987). In this study, functional Leydig cells were identified with the marker CYP11a1 (Lottrup et al., 2014). However, as shown by Guo and colleagues, and reflected in the prepubertal controls, CYP11a1 only gradually appears in Leydig cells around puberty (Guo et al., 2020), meaning that truly immature Leydig cells could not be identified using this approach. Only the use of an immature Leydig cell marker (delta-like homolog 1) could confirm their presence. Unfortunately, our attempts to optimize the immunofluorescence protocol for this marker were unsuccessful. Due to the negative feedback of estrogens on steroidogenic enzymes, it was considered whether Leydig cells could lose their CYP11a1 signal. While a few cells were CYP11a1^-/INSL3⁺, most Leydig cells seemed unaffected, suggesting that CYP11a1 expression is persistent to maintain basal T levels (Table 1).

Regarding the differential gene expression analysis, this study showed clear segregation between prepubertal and adult control tissue based on spermatogenesis-related genes in P3. Due to the lack of spermatogenesis, transgender testicular tissue clustered with prepubertal rather than adult samples. In fact, it has been shown that the spermatogonia present in transgender tissue express transcripts of early undifferentiated spermatogonia, similar to the early undifferentiated spermatogonia in cisgender tissue of all ages (Guo et al., 2020). In addition, also in other expression patterns (P1 and P2), transgender tissue was closer to prepubertal and peripubertal (Peripubertal 1) tissues than to adult tissue. P1 is a pattern that includes biological processes related to tissue morphology and development, and cell location establishment. This is in line with the histological findings regarding the spermatogonial stem cell niche, as well as the findings in the literature (Guo et al., 2020) pointing to a rejuvenation of transgender tissue, particularly Sertoli cells. On the other hand, P4 reveals that transgender testicular tissue is deprived of - or at least less rich in - mitotic and meiotic cells in comparison to controls. Also, the observation that seminiferous tubules in transgender tissue shrink (Leavy et al., 2017), which may be simply due to germ cell loss, but that they also become hyalinized ghost tubules, indicates that Sertoli cells are not proliferating, but rather depleting. Moreover, we show that transgender Sertoli cells exhibit a phenotype similar to that of Sertoli cells in Sertoli cell-only tubule disorders where immaturity markers and AR are concomitantly expressed (Sharpe et al., 2003). Taken together, these findings indicate that Sertoli cells in trans women have only partially dedifferentiated.

Pattern P5 was the most specific for transgender tissue. This pattern, although including a variety of biological processes, points to inflammation and fibrosis, in line with what is observed histologically. Noteworthy, Leydig and PTMCs in the testes of trans women display altered transcriptomes not related to rejuvenation (Guo et al., 2020), which may point to potential malignant alterations due to GAHT and to the important role of T in keeping these cells healthy after maturation. Although a recent report shows no increased risk of testicular cancer in GAHT patients (de Nie et al., 2022b), several other reports point to a tight correlation between elevated levels of estrogen and germ or Leydig cell cancers (Huseby, 1980; Bouskine et al., 2008; Chandhoke et al., 2018; Fénichel and Chevalier, 2019). In another recent report, routine pathology examination following orchidectomy was advised even though malignant findings were rare (Bonapace-Potvin et al., 2022). Also interesting to note, the GO term ‘response to estrogen’ (eight genes, adjusted p-value<1.55e-2) is part of P5, showing that alterations at the testis level result from a combination of estrogen and anti-androgen therapy, and not solely from T suppression.

A common shortcoming in testis biology and male fertility research, testis toxicology, and endocrine disruption testing is the lack of human testicular tissue, particularly from prepubertal and peripubertal boys. Our findings suggest that transgender testicular tissue which expresses AMH, AR, CYP11a1, INSL3, and ACTA2 contains spermatogonia, and exhibits minimal tubular hyalinization, may serve as a valuable alternative for this. Moreover, a recent study showed that testicular cells from GAHT-treated trans women can preserve their function, and that spermatogenesis can be successfully restored (de Nie et al., 2023). The great advantage of using trans women’s testicular tissue is that it is abundant since it is treated as medical waste material. This advantage has particular interest for the testing of compounds, whether it be drugs or environmental toxicants, as this application always requires large amounts of samples. In fact, the use of digested trans women’s testicular tissue for the testing of compounds in vitro has been described in the literature (Mincheva et al., 2020).

While this study provides valuable insights, it is essential to recognize its limitations. The first limitation is that the hormone levels were measured during the last visit before SRS rather than on the day of surgery, and not considering the time of last administration and blood draw, explaining suboptimal E2 serum levels in many participants. Nevertheless, this approach offers insight into the hormonal status throughout treatment, rather than 2 weeks post-GAHT cessation. It is however important to mention that, despite the recommended follow-up interval of 6–12 months (Hembree et al., 2017), hormone measurements for four participants dated back more than a year before the surgery. Moreover, for eight participants, only hormone measurements from less than 1 year after the initiation of treatment were available. Another limitation of this study is the lack of information regarding the testicular function of the participants before the start of the hormonal treatment. It is possible that certain transgender-specific factors such as tucking, wearing tight underwear, and having a low masturbation frequency might have negatively impacted their testicular health (de Nie et al., 2020). Nevertheless, all trans women in this study showed normal serum T values before initiating GAHT. Finally, it was challenging to draw strong conclusions from the regression analysis within the current cohort, and the available predictors are deemed inadequate for making predictions at an individual participant level. On the other hand, this study features a large cohort of 106 trans women who were subjected to a consistent hormone regimen involving both estrogens and a fixed dose of CPA, ensuring robust statistical analyses and reliable outcomes. Since in ENIGI the practice has changed over the recent years toward reduction in prescribed daily CPA dosage (Kuijpers et al., 2021) or abandoning CPA altogether in favor of GnRH analogues as anti-androgen treatment, a re-evaluation in this more recent cohort is advisable.

In summary, our findings show that trans women undergoing GAHT with E2 and CPA exhibit a partially rejuvenated spermatogonial stem cell niche. In the tubular wall, PTMCs continue to express ACTA2 although in a disturbed pattern, likely due to fibrotic processes. Additionally, there is evidence of a regressed seminiferous epithelium and a collapsing lumen. Sertoli cells have partially dedifferentiated (expression of both AMH and AR) and resemble those of peripubertal boys. Leydig cells also show a distribution similar to that of peripubertal tissue and a decreased INSL3 expression. The transcriptomic profiles of transgender and control testicular tissue reveal ongoing inflammation but also confirm the microscopy findings that point to a loss of mature characteristics - a partial rejuvenation - of the spermatogonial stem cell niche in transgender testicular tissue. Hence, testicular tissue from trans women holds the potential to be used as a surrogate for (pre)pubertal tissue in in vitro applications.

The collection and use of all tissues were approved by the Committee for Medical Ethics of the Universitair Ziekenhuis (UZ) Brussel - Vrije Universiteit Brussel (VUB) (EC nos. 2016/V9, 2017/061, and 2022/161) and UZ Gent (EC nos. 2009/622, 2014/1175, and 2014/1175-AM01). All patients or their parents gave written informed consent to donate testicular tissue to research.

The datasets supporting the findings of this study are stored under restricted access in the VUB Institutional Data Repository, under the accession numbers VUB/BITE/1/000003 and VUB/GRAD/1/000001, due to participant privacy concerns. The first dataset contains the clinical data and histological characterisation of the patients, while the latter includes the RNA-seq raw and preprocessed data. Access to the data will be considered on a case-by-case basis and must be requested by contacting Prof. Ellen Goossens (ellen.goossens@vub.be), who will review the request, including the intended purpose of the research and any potential commercial use. A data use agreement must be completed and signed in accordance with the VUB legal department's guidelines before any data can be shared or released. Metadata for the datasets can be accessed through the VUB Research Portal at: https://researchportal.vub.be/en/datasets/characterization-of-testicular-tissue-in-trans-women and https://researchportal.vub.be/en/datasets/partial-rejuvenation-of-the-spermatogonial-stem-cell-niche-after-.

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Author details

1. Emily Delgouffe

   Biology of the Testis (BITE) Laboratory, Genetics, Reproduction and Development (GRAD) Research Group, Vrije Universiteit Brussel, Brussels, Belgium

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing, She conducted the staining procedures, performed the imaging and analysis, and processed the data

   For correspondence

   emily.delgouffe@vub.be

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5611-2173

2. Samuel Madureira Silva

   Biology of the Testis (BITE) Laboratory, Genetics, Reproduction and Development (GRAD) Research Group, Vrije Universiteit Brussel, Brussels, Belgium

   Contribution

   Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing – original draft, Writing – review and editing, He gathered the tissue samples for gene expression analysis, performed the RNA extraction, and the final gene expression analysis, and drafted the part on gene expression analysis

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0833-123X

3. Frédéric Chalmel

   Inserm, EHESP, Institut de Recherche en Santé, Environnement et Travail (IRSET), Université de Rennes, Rennes, France

   Contribution

   Resources, Formal analysis, Validation, Investigation, Methodology, He performed the differential gene expression and functional analysis

   Competing interests

   No competing interests declared

4. Wilfried Cools

   Core facility, Support for Quantitative and Qualitative Research (SQUARE), Vrije Universiteit Brussel, Brussels, Belgium

   Contribution

   Data curation, He conducted the statistical analysis

   Competing interests

   No competing interests declared

5. Camille Raets

   Core facility, Support for Quantitative and Qualitative Research (SQUARE), Vrije Universiteit Brussel, Brussels, Belgium

   Contribution

   Data curation, She conducted the statistical analysis

   Competing interests

   No competing interests declared

6. Kelly Tilleman

   Department for Reproductive Medicine, Ghent University Hospital, Ghent, Belgium

   Contribution

   Project administration, Writing – review and editing, She managed the recruitment and consent of the trans women for the gene expression analysis

   Competing interests

   No competing interests declared

7. Guy T'Sjoen

   Department of Endocrinology and Center for Sexology and Gender, Ghent University Hospital, Ghent, Belgium

   Contribution

   Resources, Project administration, Writing – review and editing, He managed the recruitment of the trans women and their clinical data

   Competing interests

   No competing interests declared

8. Yoni Baert

   1. Biology of the Testis (BITE) Laboratory, Genetics, Reproduction and Development (GRAD) Research Group, Vrije Universiteit Brussel, Brussels, Belgium
   2. In Vitro Toxicology and Dermato-Cosmetology (IVTD), Vrije Universiteit Brussel, Brussels, Belgium

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Methodology, Writing – review and editing

   Competing interests

   No competing interests declared

9. Ellen Goossens

   Biology of the Testis (BITE) Laboratory, Genetics, Reproduction and Development (GRAD) Research Group, Vrije Universiteit Brussel, Brussels, Belgium

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Validation, Writing – review and editing

   Competing interests

   No competing interests declared

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