IntroductionThe 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.MethodsA 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.ResultsThe 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 LimitationsIt 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.ConclusionThe 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.

Background: Brain sexual differentiation is a process that results from the effects of sex steroids on the developing brain. Evidence shows that epigenetics plays a main role in the formation of enduring brain sex differences and that the estrogen receptor a (ESR1) is one of the implicated genes. Aim: To analyze whether the methylation of region III (RIII) of the ESR1 promoter is involved in the biological basis of gender dysphoria. Methods: We carried out a prospective study of the CpG methylation profile of RIII (À1,188 to À790 bp) of the ESR1 promoter using bisulfite genomic sequencing in a cisgender population (10 men and 10 women) and in a transgender population (10 trans men and 10 trans women), before and after 6 months of gender-affirming hormone treatment. Cisgender and transgender populations were matched by geographical origin, age, and sex. DNAs were treated with bisulfite, amplified, cloned, and sequenced. At least 10 clones per individual from independent polymerase chain reactions were sequenced. The analysis of 671 bisulfite sequences was carried out with the QUMA (QUantification tool for Methylation Analysis) program. Outcomes: The main outcome of this study was RIII analysis using bisulfite genomic sequencing. Results: We found sex differences in RIII methylation profiles in cisgender and transgender populations. Cismen showed a higher methylation degree than ciswomen at CpG sites 297, 306, 509, and at the total fragment (P .003, P .026, P .001, P .006). Transmen showed a lower methylation level than trans women at sites 306, 372, and at the total fragment (P .0001, P .018, P .0107). Before the hormone treatment, transmen showed the lowest methylation level with respect to cisgender and transgender populations, whereas transwomen reached an intermediate methylation level between both the cisgender groups. After the hormone treatment, transmen showed a statistically significant methylation increase, whereas transwomen showed a non-significant methylation decrease. After the hormone treatment, the RIII methylation differences between transmen and transwomen disappeared, and both transgender groups reached an intermediate methylation level between both the cisgender groups. Clinical Implications: Clinical implications in the hormonal treatment of trans people. Strengths & Limitations: Increasing the number of regions analyzed in the ESR1 promoter and increasing the number of tissues analyzed would provide a better understanding of the variation in the methylation pattern. Conclusions: Our data showed sex differences in RIII methylation patterns in cisgender and transgender populations before the hormone treatment. Furthermore, before the hormone treatment, transwomen and