CBLL1 is hypomethylated and correlates with cortical thickness in transgender men before gender affirming hormone treatment.

Gender identity refers to the consciousness of being a man, a woman or other condition. Although it is generally congruent with the sex assigned at birth, for some people it is not. If the incongruity is distressing, it is defined as gender dysphoria (GD). Here, we measured whole-genome DNA methylation by the Illumina © Infinium Human Methylation 850k array and reported its correlation with cortical thickness (CTh) in 22 transgender men (TM) experiencing GD versus 25 cisgender men (CM) and 28 cisgender women (CW). With respect to the methylation analysis, TM vs. CW showed significant differences in 35 CpGs, while 2155 CpGs were found when TM vs. CM were compared. With respect to correlation analysis, TM showed differences in methylation of CBLL1 and DLG1 genes that correlated with global and left hemisphere CTh. Both genes were hypomethylated in TM compared to the cisgender groups. Early onset TM showed a positive correlation between CBLL1 and several cortical regions in the frontal (left caudal middle frontal), temporal (right inferior temporal, left fusiform) and parietal cortices (left supramarginal and right paracentral). This is the first study relating CBLL1 methylation with CTh in transgender persons and supports a neurodevelopmental hypothesis of gender identity.

Testosterone administration affects 1H-MRS metabolite spectra in transgender men.

BACKGROUND: Recently, a variety of studies using different neuroimaging techniques attempted to identify the existence of a brain endophenotype in people with gender dysphoria (GD). However, despite mounting neuroimaging work, brain gender differences and effects of gender-affirming hormone therapy (GAHT) at the metabolite level remain understudied. METHODS: Thirty-one transgender men (TM) before and after testosterone administration (7.7 months ± 3.5 months), relative to 30 cisgender men (CM) and 35 cisgender women (CW) underwent magnetic resonance spectroscopy (1H-MRS) at two time points. Two brain regions were assessed, i.e. the lateral parietal cortex and the amygdala/anterior hippocampus. Associated metabolites that were measured include N-acetyl aspartate (NAA), creatine (Cr), choline (Cho), glutamate and glutamine (Glx), myo-inositol (mI), glycine (Gly) and their respective ratios. RESULTS: A critical time by group interaction revealed an effect of GAHT in the lateral parietal cortex of TM. MI+Gly/Cr ratios decreased upon initiation of GAHT. In addition, NAA/Cr and Cho/Cr ratios were lower in CW when compared to CM in the lateral parietal cortex. Glx levels and Glx/Cr ratios in TM differed from those in CW in the amygdala/anterior hippocampus. Interestingly, pubertal age of onset of gender dysphoria (i.e. GD) in TM differentially affected testosterone-mediated effects on Cr concentration and NAA/Cr ratios when compared to childhood and adult GD onset in the amygdala/anterior hippocampus. CONCLUSION: This 1H-MRS study demonstrated that testosterone administration shifts mI+Gly/Cr ratios in the parietal cortex. In the amygdala/anterior hippocampus, modulation of metabolite concentrations by age of onset of GD is suggestive for a possible developmental trend.

Gender minority stress in transgender people: a major role for social network.

BACKGROUND: Gender minority individuals, on average, experience higher rates of mental health problems. Mounting work suggests that gender minority stress (GMS) contributes to mental health outcomes in transgender/gender-nonconforming individuals. AIM: We assessed whether GMS decreased in transgender people after initiating gender-affirming hormone therapy (GAHT), and we identified social predictors and hormonal associations for GMS at 2 time points. METHODS: GMS was surveyed through self-report questionnaires tapping into proximal and distal stressors and coping constructs following the minority stress framework. Eighty-five transgender persons wishing to undertake hormonal interventions were assessed prospectively at start of GAHT and after 7.7 ± 3.5 months (mean ± SD). Sixty-five cisgender persons served as a control group. OUTCOMES: (1) Proximal stressors were surveyed by the Beck Depression Inventory II, State-Trait Anxiety Inventory, Scale for Suicide Ideation, Suicidal Thoughts/Attempts, Stigma Consciousness Questionnaire, and Perceived Stress Scale; (2) distal stressors by the Everyday Discrimination Scale; and (3) coping constructs by the Resilience Scale, social network, social standing, and Marlowe Crowne Social Desirability Scale. RESULTS: Transgender people experienced higher rates of proximal stressors (Beck Depression Inventory II, State-Trait Anxiety Inventory, Scale for Suicide Ideation, Suicidal Thoughts/Attempts, Perceived Stress Scale) and had lower protective factors (social standing) prior to and during GAHT than cisgender people. Social network and resilience were lower in transgender people relative to cisgender peers only at baseline. Prospectively, decreasing trait anxiety was observed in transgender people. Social factors were adequate predictors of multiple GMS constructs. Specifically, a major role for social network emerged. As for hormonal associations, only serum estradiol levels in transgender women with GAHT were negatively associated with trait anxiety and suicidal thoughts/attempts but positively with resilience and social desirability. CLINICAL IMPLICATIONS: Stimulating a social environment supportive of diverse identities, particularly by investing in social networks as a resource for resilience, is likely to alleviate GMS. STRENGTHS AND LIMITATIONS: Longer duration of interventions with sex steroid treatment, with continued resilience-enhancing strategies, is needed to observe further alleviation of GMS in transgender persons. Also, objective and subjective GMS identification with heteronormative attitudes and beliefs should be surveyed for good measure when assessing GMS. CONCLUSION: Transgender people experienced more GMS throughout study visits than cisgender people did. With a relatively short period of GAHT, some significant changes in and predictors for experienced GMS emerged.

Changes in Serum Testosterone and Adrenal Androgen Levels in Transgender Women With and Without Gonadectomy.

BACKGROUND: Initiating feminizing gender-affirming hormone therapy (GAHT) in transgender women causes a steep decline in serum testosterone. It is unknown if testosterone concentrations change further and whether adrenal androgen levels change during feminizing GAHT and after gonadectomy. This limits clinical decision making in transgender women with symptoms attributed to GAHT or gonadectomy. METHODS: Transgender women (n = 275) initiating estradiol and cyproterone acetate (CPA) were included at baseline, and had follow-up visits after 3 months, 12 months, and 2 to 4 years. During follow-up, 49.5% of transgender women underwent a gonadectomy. Total testosterone (TT), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), and androstenedione (A4) were measured using liquid chromatography tandem mass spectrometry. RESULTS: After 3 months of GAHT, mean TT, calculated free testosterone (cFT), and A4 decreased by 18.4 nmol/L (95% CI, -19.4 to -17.4, P < 0.001 [ie, -97.1%]), 383 pmol/L (95% CI, -405 to -362, P < 0.001 [ie, -98.3%]), and 1.2 nmol/L (95% CI, -1.4 to -1.0, P < 0.001 [ie, -36.5%]), respectively, and remained stable thereafter. DHEA and DHEAS decreased by 7.4 nmol/L (95% CI, -9.7 to -5.1 [ie, -28.0%]) and 1.8 µmol/L (95% CI, -2.2 to -1.4 [ie, -20.1%]), respectively, after 1 year and did not change thereafter. After gonadectomy, CPA therapy is stopped, which induced no further change in TT, cFT, DHEA, DHEAS, and A4 compared with those who did not undergo gonadectomy. CONCLUSIONS: Our findings confirm that after an initial drop, testosterone levels in transgender women remain stable. Adrenal androgens decrease in the first year of CPA and estrogen supplementation and remain unchanged after gonadectomy. Androgens did not change after gonadectomy and cessation of CPA. Correlates with clinical symptoms remain to be elucidated.

Thrombophilia and hormonal therapy in transgender persons: A literature review and case series.

Background: Venous thromboembolism (VTE) is a rare side effect of hormonal therapy in transgender persons. Prothrombotic genetic variants can increase this risk. For this reason, previous VTE and/or genetic thrombophilia may be considered by some as contraindications to hormonal treatment. Aim: To formulate directions for clinical practice about the indications for thrombophilia screening and when to consider combination therapy of therapeutic anticoagulation and hormonal treatment as a safe alternative to withholding hormonal treatment. Methods: We conducted a literature search and describe a case series. All adult patients with gender dysphoria and a known prothrombotic genetic variant or history of VTE were invited by letter to participate in this study. Results: In our center, thrombophilia screening before start of hormonal treatment was restricted to those with a personal or family history of VTE. Sixteen individuals with a history of VTE and/or an underlying prothrombogenic condition were described. The time of follow up varied from 4 months to 20 years. Seven trans women had a positive thrombophilia screening (2 Factor V Leiden (FVL), 1 FVL + anticardiolipin antibodies, 1 FVL + high Factor VIII coagulant activity, 1 protein C deficiency, 1 prothrombin mutation, 1 positive lupus anticoagulant). Three trans women experienced an unprovoked VTE after start of hormonal therapy of which one lead to a positive thrombophilia screening. One VTE event in a trans woman was assumed to be provoked by surgery. Five trans men were identified with a prothrombogenic mutation (3 FVL, 1 protein C deficiency, 1 prothrombin mutation). One trans man, with a negative thrombophilia screen, experienced multiple provoked VTE events before start of hormonal therapy. Conclusion: Based on our literature review and case series we offer guidance when confronted with patients with previous VTE and/or genetic thrombophilia requesting hormonal interventions.

Gender-affirming hormonal treatment changes neural processing of emotions in trans men: An fMRI study.

BACKGROUND: Some transgender people desire a transition through gender-affirming hormone treatment (GAHT). To date, it is unknown how GAHT changes emotion perception in transgender people. METHODS: Thirty transgender men (TM), 30 cisgender men (CM), and 35 cisgender women (CW) underwent 3 Tesla functional magnetic resonance imaging (fMRI) while passively viewing emotional faces (happy, angry, surprised faces) at two timepoints (T0 and T1). At T0 all participants were hormone-naïve, while TM immediately commenced testosterone supplementation at T0. The second scanning session (T1) occurred after 6-10 months of GAHT in TM. All 3 groups completed both T0 and T1 RESULTS: GAHT in TM shifted the neural profile whilst processing emotions from a sex-assigned at birth pattern at T0 (similar to CW) to a consistent with gender identity pattern at T1 (similar to CM). Overall, the brain patterns stayed the same for the cis people at T0 and T1. CONCLUSIONS: These findings document the impact of hormone treatment, and testosterone supplementation specifically, on emotion perception in TM.

The ENIGI (European Network for the Investigation of Gender Incongruence) Study: Overview of Acquired Endocrine Knowledge and Future Perspectives.

Literature on the efficacy and safety of gender-affirming hormonal treatment (GAHT) in transgender people is limited. For this reason, in 2010 the European Network for the Investigation of Gender Incongruence (ENIGI) study was born. The aim of this review is to summarize evidence emerging from this prospective multicentric study and to identify future perspectives. GAHT was effective in inducing desired body changes in both trans AMAB and AFAB people (assigned male and female at birth, respectively). Evidence from the ENIGI study confirmed the overall safety of GAHT in the short/mid-term. In trans AMAB people, an increase in prolactin levels was demonstrated, whereas the most common side effects in trans AFAB people were acne development, erythrocytosis, and unfavorable changes in lipid profile. The main future perspectives should include the evaluation of the efficacy and safety of non-standardized hormonal treatment in non-binary trans people. Furthermore, long-term safety data on mortality rates, oncological risk, and cardiovascular, cerebrovascular and thromboembolic events are lacking. With this aim, we decided to extend the observation of the ENIGI study to 10 years in order to study all these aspects in depth and to answer these questions.

Osteoporosis and Bone Health in Transgender Individuals.

This review discusses the changes in bone mass, structure, and metabolism that occur upon gender-affirming hormonal treatment (GAHT) in transgender adults and adolescents, as well as their clinical relevance. In general, available evidence shows that GAHT in transgender adults is not associated with major bone loss. In transgender adolescents, pubertal suppression with gonadotropin-releasing hormone agonist monotherapy impairs bone development, but at least partial recovery is observed after GAHT initiation. Nevertheless, a research gap remains concerning fracture risk and determinants of bone strength other than bone mineral density. Attention for bone health is warranted especially in adult as well as adolescent trans women, given the relatively high prevalence of low bone mass both before the start of treatment and after long-term GAHT in this population. Strategies to optimize bone health include monitoring of treatment compliance and ensuring adequate exposure to administered sex steroids, in addition to general bone health measures such as adequate physical activity, adequate vitamin D and calcium intake, and a healthy lifestyle. When risk factors for osteoporosis exist the threshold to perform DXA should be low, and treatment decisions should be based on the same guidelines as the general population.

Minority Stress and the Effects on Emotion Processing in Transgender Men and Cisgender People: A Study Combining fMRI and 1H-MRS.

BACKGROUND: Minority stress via discrimination, stigmatization, and exposure to violence can lead to development of mood and anxiety disorders and underlying neurobiochemical changes. To date, the neural and neurochemical correlates of emotion processing in transgender people (and their interaction) are unknown. METHODS: This study combined functional magnetic resonance imaging and magnetic resonance spectroscopy to uncover the effects of anxiety and perceived stress on the neural and neurochemical substrates, specifically choline, on emotion processing in transgender men. Thirty transgender men (TM), 30 cisgender men, and 35 cisgender women passively viewed angry, neutral, happy, and surprised faces in the functional magnetic resonance imaging scanner, underwent a magnetic resonance spectroscopy scan, and filled out mood- and anxiety-related questionnaires. RESULTS: As predicted, choline levels modulated the relationship between anxiety and stress symptoms and the neural response to angry and surprised (but not happy faces) in the amygdala. This was the case only for TM but not cisgender comparisons. More generally, neural responses in the left amygdala, left middle frontal gyrus, and medial frontal gyrus to emotional faces in TM resembled that of cisgender women. CONCLUSIONS: These results provide first evidence, to our knowledge, of a critical interaction between levels of analysis and that choline may influence neural processing of emotion in individuals prone to minority stress.

Epigenetics Is Implicated in the Basis of Gender Incongruence: An Epigenome-Wide Association Analysis.

INTRODUCTION: The main objective was to carry out a global DNA methylation analysis in a population with gender incongruence before gender-affirming hormone treatment (GAHT), in comparison to a cisgender population. METHODS: A global CpG (cytosine-phosphate-guanine) methylation analysis was performed on blood from 16 transgender people before GAHT vs. 16 cisgender people using the Illumina© Infinium Human Methylation 850k BeadChip, after bisulfite conversion. Changes in the DNA methylome in cisgender vs. transgender populations were analyzed with the Partek® Genomics Suite program by a 2-way ANOVA test comparing populations by group and their sex assigned at birth. RESULTS: The principal components analysis (PCA) showed that both populations (cis and trans) differ in the degree of global CpG methylation prior to GAHT. The 2-way ANOVA test showed 71,515 CpGs that passed the criterion FDR p < 0.05. Subsequently, in male assigned at birth population we found 87 CpGs that passed both criteria (FDR p < 0.05; fold change ≥ ± 2) of which 22 were located in islands. The most significant CpGs were related to genes: WDR45B, SLC6A20, NHLH1, PLEKHA5, UBALD1, SLC37A1, ARL6IP1, GRASP, and NCOA6. Regarding the female assigned at birth populations, we found 2 CpGs that passed both criteria (FDR p < 0.05; fold change ≥ ± 2), but none were located in islands. One of these CpGs, related to the MPPED2 gene, is shared by both, trans men and trans women. The enrichment analysis showed that these genes are involved in functions such as negative regulation of gene expression (GO:0010629), central nervous system development (GO:0007417), brain development (GO:0007420), ribonucleotide binding (GO:0032553), and RNA binding (GO:0003723), among others. STRENGTHS AND LIMITATIONS: It is the first time that a global CpG methylation analysis has been carried out in a population with gender incongruence before GAHT. A prospective study before/during GAHT would provide a better understanding of the influence of epigenetics in this process. CONCLUSION: The main finding of this study is that the cis and trans populations have different global CpG methylation profiles prior to GAHT. Therefore, our results suggest that epigenetics may be involved in the etiology of gender incongruence.

Characterization of the 1H-MRS Metabolite Spectra in Transgender Men with Gender Dysphoria and Cisgender People.

Much research has been conducted on sexual differences of the human brain to determine whether and to what extent a brain gender exists. Consequently, a variety of studies using different neuroimaging techniques attempted to identify the existence of a brain phenotype in people with gender dysphoria (GD). However, to date, brain sexual differences at the metabolite level using magnetic resonance spectroscopy (1H-MRS) have not been explored in transgender people. In this study, 28 cisgender men (CM) and 34 cisgender women (CW) and 29 transgender men with GD (TMGD) underwent 1H-MRS at 3 Tesla MRI to characterize common brain metabolites. Specifically, levels of N-acetyl aspartate (NAA), choline (Cho), creatine (Cr), glutamate and glutamine (Glx), and myo-inositol + glycine (mI + Gly) were assessed in two brain regions, the amygdala-anterior hippocampus and the lateral parietal cortex. The results indicated a sex-assigned at birth pattern for Cho/Cr in the amygdala of TMGD. In the parietal cortex, a sex-assigned at birth and an intermediate pattern were found. Though assessed post-hoc, exploration of the age of onset of GD in TMGD demonstrated within-group differences in absolute NAA and relative Cho/Cr levels, suggestive for a possible developmental trend. While brain metabolite levels in TMGD resembled those of CW, some interesting findings, such as modulation of metabolite concentrations by age of onset of GD, warrant future inquiry.

Characterisation of testicular function and spermatogenesis in transgender women.

STUDY QUESTION: Does gender-affirming treatment prevent full spermatogenesis in transgender women (TW)? SUMMARY ANSWER: Adequate hormonal therapy (HT) leads to complete suppression of spermatogenesis in most TW, if serum testosterone levels within female reference ranges are obtained. WHAT IS KNOWN ALREADY: Gender-affirming treatment in transgender individuals may involve gender-affirming HT. The effects on spermatogenesis in TW remain unclear. In order to add information from a referral centre for transgender care, we wish to compare results of earlier studies with our population of TW who received a standard hormone treatment. STUDY DESIGN, SIZE, DURATION: This was a prospective cohort study part of the European Network for the Investigation of Gender Incongruence (ENIGI), conducted between 15 February 2010 and 30 September 2015. There were 162 TW were included in the ENIGI study at the Ghent University Hospital in Belgium. Participants are included in ENIGI when they first start HT, and follow-up visits occur over the next 3 years. PARTICIPANTS/MATERIALS, SETTING METHODS: The study included 97 TW who initiated HT with cyproterone acetate (CPA) plus oestrogens and proceeded with gonadectomy at the Ghent University Hospital. Testicular tissue retrieved during gonadectomy was processed and stained for four different germ cell markers by the Biology of the Testis lab at the Vrije Universiteit Brussel. Subsequent immunohistochemical staining was performed for melanoma-associated antigen A4 (MAGE-A4, marker for spermatogonia and early spermatocytes), boule homologue, RNA-binding protein (BOLL, marker for secondary spermatocytes and round spermatids), cAMP-responsive element modulator (CREM, marker for round spermatids) and acrosin (marker for acrosome visualization). Serum levels of sex steroids were measured prior to surgery. MAIN RESULTS AND THE ROLE OF CHANCE: Suppressed testosterone levels (<50 ng/dl) were found in 92% of the participants prior to surgery. The mean time between initiation of HT and surgery was 685 days. In 88% (85/97) of the sections, MAGE-A4 staining was positive. Further staining could not reveal complete spermatogenesis in any participant. LIMITATIONS, REASONS FOR CAUTION: Testicular function of the participants prior to initiation of HT was not assessed, although all participants presented with cisgender male serum testosterone values before initiation of HT. The current study only reports on people using CPA at a fixed dose and may therefore not be applicable to all TW. WIDER IMPLICATIONS OF THE FINDINGS: HT leads to complete suppression of spermatogenesis in most TW, if serum testosterone levels within female reference ranges are obtained. Serum testosterone levels are associated with the sperm maturation rate. It is important to discuss sperm preservation before the start of hormone therapy. If serum testosterone levels remain higher, spermatogenesis may still occur. STUDY FUNDING/COMPETING INTEREST(S): D.V.S. is a post-doctoral fellow of the Fonds Wetenschappelijk Onderzoek (FWO; 12M2819N). Processing of the testis specimens was funded by the Biology of The Testes (BITE) research group (Department of Reproduction, Genetics and Regenerative medicine at Vrije Universiteit Brussel (VUB)). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Vaginal bleeding and spotting in transgender men after initiation of testosterone therapy: A prospective cohort study (ENIGI).

BACKGROUND: Previous studies have cross-sectionally described amenorrhea in cohorts of transgender men on intramuscular or subcutaneous testosterone injections. It remains uncertain which testosterone preparations most effectively suppress vaginal bleeding and when amenorrhea occurs after testosterone initiation. AIM: To investigate the clinical effects of various testosterone preparations on vaginal bleeding and spotting in transgender men. METHODS: This prospective cohort study was part of the European Network for the Investigation of Gender Incongruence (ENIGI). Data on the persistence and intensity of vaginal bleeding and spotting, serum sex steroid levels and body composition were prospectively and cross-sectionally assessed in 267 transgender men during a three-year follow-up period, starting at the initiation of various testosterone preparations. RESULTS: After three months of testosterone, 17.9% of transgender men reported persistent vaginal bleeding and 26.8% reported spotting. The percentages reporting vaginal bleeding and spotting decreased over the first year of testosterone (bleeding 4.7% and spotting 6.9% at 12 months, respectively), with no participants reporting vaginal bleeding or spotting after 18 months of testosterone. Factors associated with vaginal bleeding or spotting included lower serum testosterone levels and being on testosterone gel as compared to injections (e.g., esters or undecanoate preparations). If vaginal bleeding persisted, starting progestogens at three months resulted in a decrease in the intensity of vaginal bleeding and spotting. DISCUSSION: Transgender men and hormone-prescribing providers can be reassured that vaginal bleeding and spotting usually stop within three months after testosterone initiation. If not, serum testosterone levels should be measured and testosterone dose adjusted to achieve serum testosterone levels in the physiologic male range. Adding a progestin can be considered after three to six months if bleeding persists. Providers should be aware that cessation of bleeding can be more difficult to achieve in transgender men with lower serum testosterone levels or those on testosterone gel.

Lower Serum Estradiol Levels in Assigned Female at Birth Transgender People with Initiation of Testosterone Therapy: Results from the European Network for the Investigation of Gender Incongruence.

Purpose: Concerns have been raised about undesired estrogenic effects in assigned female at birth (AFAB) transgender people on testosterone therapy. How serum estradiol levels change after initiation of testosterone therapy and if these levels should be monitored remain unclear. Methods: This prospective cohort study was part of the European Network for the Investigation of Gender Incongruence. Serum levels of sex steroids were assessed in 746 AFAB transgender people during a 3-year follow-up period, starting at the initiation of hormone treatment. Results: Estradiol levels decreased from median [P25-P75] 45.6 [24.0-102.2] pg/mL to 36.5 [25.0-46.2] pg/mL over 3 years (p < 0.001); a change was already noticeable during the first 3 months (mean -17.1 pg/mL, 95% confidence interval -23.8 to -10.6, p < 0.001). Serum estradiol levels were lower in people without endogenous estradiol production from ovarian source (contraceptive users or post hystero-oophorectomy) at baseline and after 3 months, compared with people with endogenous estradiol production. Using long-acting testosterone undecanoate injections resulted in a more prominent decrease in serum estradiol values over 12 months, compared with short-acting mixed testosterone esters (p < 0.001) or testosterone gel (p = 0.001). Changes in serum estradiol were positively correlated to changes in luteinizing hormone (ρ = 0.107, p < 0.001) and negatively correlated to changes in follicle-stimulating hormone levels (ρ = -0.167, p < 0.001) and body mass index (ρ = -0.082, p < 0.001). Conclusion: Testosterone administration in AFAB transgender people resulted in decreasing serum estradiol levels. Our results suggest that testosterone therapy leads to central suppression of estradiol production, with partial restitution due to aromatization.