
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

72365
10.1038/s41598-024-72365-4
Article
The vaginal microbiome of transgender men receiving gender-affirming hormonal therapy in comparison to that of cisgender women
Feil Katharina 1
Pabst Lisa 1
Reider Simon simon.reider@i-med.ac.at

3
Schuchter Stefanie 1
Ciresa-König Alexandra 2
Toth Bettina 1
1 grid.5361.1 0000 0000 8853 2677 Department of Gynecological Endocrinology and Reproductive Medicine, Medical University of Innsbruck, Innsbruck, Austria
2 grid.5361.1 0000 0000 8853 2677 Department of Obstetrics and Gynecology, Medical University of Innsbruck, Innsbruck, Austria
3 grid.5361.1 0000 0000 8853 2677 Department of Internal Medicine 2, Medical University of Innsbruck, Innsbruck, Austria
14 9 2024
14 9 2024
2024
14 2152614 5 2024
6 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The vaginal microbiome of trans men and menopausal women is suspected to be similar due to a lack of estrogen leading to the absence of lactobacilli. However, data are scarce. We performed an analysis of the vaginal microbiome of trans men (n = 25) in comparison to that of menopausal (n = 25) and premenopausal women (n = 25). The vaginal microbiome of trans men and menopausal women showed a higher alpha diversity than that of premenopausal women. Various beta diversity indices (e.g., Bray‒Curtis (Un-)Weigthed Unifrac), showed significant differences in community composition between trans men and premenopausal (p < 0.001) and menopausal women (p < 0.001). The vaginal microbiome of trans men is characterized by a loss of Lactobacillus and an increase in bacteria associated with the intestinal flora (e.g., Campylobacter, Anaerococcus, Dialister, Prevotella). The abundance of Dialister and Prevotella decreased with the length of hormonal therapy in trans men. The Nugent score, Pap smear and HPV status did not differ between the study groups. The vaginal microbiome of trans men differs from that of premenopausal women but shows similarities to that of menopausal women. The duration of hormonal therapy in trans men may have important impacts on the vaginal microbiome and thus possibly on the risk for STIs.

Keywords

Gender incongruence
Testosterone
Estradiol
Menopause
Subject terms

Endocrine system and metabolic diseases
Microbiome
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Gender incongruence describes the discrepancy between the assigned and the experienced gender. The global prevalence of transgender and gender diverse people (TGD) is currently estimated to be 0.3–4.5% of the general adult population and 2.5–8.4% of the general child and adolescent population1. We refer to TGD individuals who were assigned female at birth and identify as male as trans men (TM). For comparison, cisgender women were assigned female at birth and identify as female.

The presence of gender incongruence leads to enormous distress, and TGD people are more likely to suffer from depression and suicidal thoughts and face increased discrimination2–8. Treatment options such as gender-affirming hormone therapy (GAHT) or surgical interventions often result in significant improvements in quality of life4,9–11.

GAHT via exogenous testosterone treatment in TM leads to the induction of male puberty with clitoral enlargement, increased sexual desire and reduced vaginal bleeding or induced amenorrhea1,12. Since menstrual bleeding can cause distress in many TM, suppression of estradiol levels and thus menstruation via progestins or gonadotropin-releasing-homone analogs (GnRH-a) is often desired1.

TM may undergo genital surgical interventions such as vaginectomy or colpectomy with the construction of a neophallus (metoidioplasty or phalloplasty). However, only a small percentage (approximately 2%) of TM ultimately undergo metoidioplasty or phalloplasty, and the majority retain the vagina13.

Recommendations regarding gynecological care in TM follow those for cisgender females14, although there is a paucity of knowledge about changes in internal genitalia under testosterone. The known effects of low estrogen levels in TM and MW are vaginal dryness, irritation, pain, and bleeding during vaginal penetration, resulting in a significant health burden15–17.

The vaginal epithelium of TM shows a significant reduction in proliferation and loss of normal architecture with absence of intermediate and superficial layers under the influence of testosterone16. Maturation of superficial layers is mediated by estrogen, which increases the thickness and production of glycogen15. The metabolism of glycogen to lactic acid by lactobacilli primarily Lactobacillus spp., lowers the vaginal pH, promotes the proliferation of other lactobacilli and reduces the risk for bacterial vaginosis (BV)15. A vaginal microbiome deficient in lactobacilli is associated with impaired epithelial maturation, increased mucosal inflammation, changes in epithelial barrier integrity, and increased susceptibility to sexually transmitted infections (STIs)15.

Recently, Winston Mc Pherson et al. showed that the vaginal microbiome of TM was less likely to contain Lactobacillus and more likely to be enriched with > 30 other species in comparison to a cisgender control group18. Since the majority of the individuals in the control group were using hormonal contraceptives and a significant portion of the individuals in the TM group were using vaginal estrogens, the study showed a distinct bias.

Vaginal health in TM is largely unknown, as are the possible effects of testosterone therapy on the vaginal microbiome.

The objectives of this study were to map and characterize the vaginal microbial composition of TM with testosterone treatment. Furthermore, a comparison was made with physiological hormonal situations in cisgender women during the reproductive phase and in menopause.

Methods

Study design

This was a cross-sectional controlled study conducted at the Medical University of Innsbruck (MUI), Department of Gynecological Endocrinology and Reproductive Medicine with prospective enrolment between 01/2022 and 02/2023.

The aim of this study was to analyze the vaginal microbiome of TM undergoing GAHT and compare it to the one of cisgender women. Because the vaginal microbiome of cisgender women changes throughout life due to hormonal changes two control groups of cisgender women—premenopausal and menopausal—were established.

In total, 75 participants were enrolled in this study, see Fig. 1. Of these, 25 were self-identifying TM assigned a female sex at birth and were recruited during routine follow-ups. The remaining 50 participants were cisgender women and were recruited during the consultation unit at the Department of Gynecological Endocrinology and Reproductive Medicine, MUI, as well as during consultations at the Department of Gynecology and Obstetrics, MUI. The cisgender women were, according to the inclusion criteria, divided into one premenopausal control group consisting of 25 premenopausal women and one menopausal control group consisting of 25 menopausal women.Fig. 1 Flowchart showing study population.

All TM were diagnosed with gender dysphoria according to the DSM-V criteria, were aged between 18 and 32 years and had been prescribed GAHT ± GnRH-a for at least 10 months. TM were excluded if they underwent hysterectomy or had contraindications for GAHT.

Premenopausal women aged 18–45 years with regular menstrual cycles (27–35 days) were included. Cycle disorders such as amenorrhea, oligomenorrhea or polymenorrhea were defined as exclusion criteria.

Menopausal women were included if they were aged > 45 years with amenorrhea for at least 1 year.

For all controls, the exclusion criteria included hysterectomy, current pregnancy, current hormone replacement therapy (HRT) and current medication that might influence the composition of the vaginal microbiome, such as hormonal contraception or vaginal estrogen.

Demographic data included age at start, duration and form of GAHT, time since last menstrual bleeding, length of menstrual cycle, medication, sexual activity, sex of sexual partners, contraception, HPV-status and last PAP smear result.

In all TM and controls, after exclusion criteria were ruled out, two vaginal swabs were taken at the same time during one of the above-described consultations by a medical professional. For one TM the two vaginal swabs were taken before hysterectomy (gender-affirming surgery) in the operating room instead of during the consultation. The cottons swabs were then used for further analyzation. One of them was directly sent to the Institute of Hygiene and Medical Microbiology of the Medical University Innsbruck, for Nugent scoring. The other cotton swab was stored at – 80 °C and then send on for sequencing and microbiome analysis at IMGM Laboratories in Munich.

Current testosterone and estradiol levels were analyzed in TM as well as current estradiol levels in the premenopausal controls (Fig. 2A). Estradiol levels of menopausal women were not measured as they are, if inclusion criteria are met, expected to be under detection limit (which is below 25 ng/l at the Institute for Medical and Chemical Laboratory Diagnostics, MUI) or in the menopausal reference interval (25–138 ng/l). Fig. 2 Measured serum estradiol concentrations. (A) Serum estradiol concentrations in trans men (n = 25) and premenopausal women (n = 17). Cisgender men (11–43 ng/l), premenopausal (31–533 ng/l) and menopausal serum estradiol reference ranges (25–138 ng/l). For menopausal women serum estradiol concentrations below 25 ng/l may occur but are not detected by the Central Insitute for Medical and Chemical Laboratory Diagnostics Innsbruck. Significant difference in serum estradiol concentration comparing TM with the premenopausal controls (p < 0.001). (B) Serum estradiol concentrations in trans men undergoing GAHT without (n = 15) and with (n = 10) supplementary gonadotropin-releasing-hormone analogs medication (p = 0.014).

The study was approved by the ethics committee of the MUI (Decision Identifier 1055/2017). All participants provided written and informed consent at enrollment. All methods, procedures and experimental measurements were performed in accordance with the relevant guidelines and regulations. Study numbers were used in place of participant names; no patient identifiers were retained.

Measurement of serum hormone concentrations

The blood samples were analyzed at the Institute for Medical and Chemical Laboratory Diagnostics, MUI. The serum hormone concentration of estradiol was analyzed using Cobas8000 Estradiol III (third generation assay), which uses a competitive test principle with two monoclonal antibodies that are specifically directed against 17ß-estradiol. The testosterone concentration was determined using HPLC–MS/MS measurement platforms, which are carried out using selected reaction monitoring scans in positive mode.

Genomic DNA isolation and 16S rRNA sequencing

DNA isolation was conducted using the NukEx Pure RNA/DNA Kit (Gerbion, Germany) according to IMGM SOP AA-0290, based on the manufacturer’s instructions. PBS samples were used as negative controls to identify potential contaminants for every isolation batch. The DNA was eluted in 50 μl of elution buffer and quantified using a highly sensitive fluorescent dye-based QubitTM dsDNA HS Assay Kit (Thermo Fisher Scientific, Massachusetts).

The V3-V4 region of the bacterial 16S rRNA gene was amplified according to established protocols using the 341 F/805 R primer pair (5ʹ-CCTACGGGRSGCAGCAG-3ʹ and 5ʹ-GACTACHVGGGTATCTAATCC-3ʹ).

Sequencing was performed on an Illumina MiSeq device with V2 chemistry, resulting in paired-end reads 250 bp in length (IMGM, Munich). The sequencing results were delivered as .fastq files, and quality control was performed with fastqc. Demultiplexing and processing of sequences were performed in R (19, version 4.3.1) and RStudio (version 2023.06.2+561, Posit Software, US) using the dada2 package according to published workflows20). After removal of the primers, the reads were trimmed to 245 base pairs for both the forward and reverse reads, and the percentage of merged reads was 94.9% (76.3–99.8%). A sequence table was generated, and chimeric reads were removed within dada220. Taxonomic assignments were obtained from the Silva reference database (v13821).

The generated 16S sequence data are available from the European Nucleotide Archive (accession number PRJEB71387).

Statistics and bioinformatics

Metagenomic analyses, including calculations of α-diversity and β-diversity, were performed using the phyloseq R package22, and differential abundance was assessed using DESeq223. Differences in α-diversity between the study groups were compared using ANOVA with post hoc Tukey tests, and β-diversity was assessed by permutational ANOVA (PERMANOVA) on Bray‒Curtis dissimilarity matrices of samples24. For analysis of differential abundance of taxa according to the duration of menopause, a generalized linear model implemented in the R package Maaslin2 was used25.

A detailed description of the bioinformatic analysis environment and computational steps of this analysis, as well as all relevant scripts and resources, is available from GitHub: https://github.com/reider-si/MBVagTrans

Results

Study cohort

The vaginal microbiome composition was analyzed in 25 TM and 50 cisgender women (n = 25 premenopausal; n = 25 menopausal; Table 1). The mean age was 23 ± 4.24 years in the TM group, 33.28 ± 4.60 years in the premenopausal group and 64.32 ± 7.43 years in the MW group. Table 1 Characteristics of transgender men and cisgender women.

	TM (n = 25)	Premenopausal controls (n = 25)	P (trans: premenopausal)	Menopausal Controls (n = 25)	P (trans: menopausal)	
Age (years)a	23 ± 4.24	33.28 ± 4.60	 < 0.001	64.32 ± 7.43	 < 0.001	
Sexual activityb	
 Yes	13 (52)	25 (100)	 < 0.001	9 (36)	0.248	
 No	10 (40)	0 (0)	 < 0.001	15 (60)	0.248	
Last sexual intercourseb	
 Last week	7 (28)	18 (72)	0.358	6 (24)	1.000	
 Last month	3 (12)	3 (12)	0.358	3 (12)	1.000	
Vaginal intercourseb,d	
 Yes	11 (44)	25 (100)	 < 0.001	25 (100)	 < 0.001	
 No	10 (40)	0 (0)	 < 0.001	0 (0)	 < 0.001	
Sex of sexual partner(s)b	
 Male	7 (28)	24 (96)	 < 0.001	25 (100)	 < 0.001	
 Female	7 (28)	1 (4)	 < 0.001	0 (0)	 < 0.001	
 Both	3 (12)	0 (0)	 < 0.001	0 (0)	 < 0.001	
Condomsb	
 Yes	6 (24)	3 (12)	0.270	NA	NA	
 No	16 (64)	22 (88)	0.270	25 (100)	0.007	
Medicationb,c	
 Yes	18 (72)	15 (60)	0.551	21 (84)	0.496	
 No	7 (28)	10 (40)	0.551	4 (16)	0.496	
Data presented as.

aMean ± SD.

bFrequency (percent).

cOther than GAHT.

dPenetrative vaginal intercourse in the past.

NA not available.

Statistical analysis by Mann–Whitney-U test. Significant p-values (P < 0.05) are marked in bold.

The mean duration of GAHT in TM was 33.56 ± 28.40 months. In addition to testosterone therapy, 10 TM also received GnRH-a, but no TM received progestin. In total, 21 TM applied testosterone undecanoate intramuscular, four testosterone transdermal and one TM both, transdermal and intramuscular testosterone. The average duration since menopause in the MW was 14.48 ± 8.77 years. On average vaginal swabs of premenopausal women were taken on the 12.24 ± 6.17 cycle day.

Medications for all groups included levothyroxine (n = 20), antidepressants (n = 15), antihypertensives (n = 7), cholesterol-lowering medications (n = 5), and other medications. Women in the premenopausal and MW groups were not taking any hormonal medications. Overall, 13 TM, nine MW and all premenopausal women reported sexual activity (defined as sexual intercourse over the last 12 months). Sexual intercourse in this context may include receptive vaginal penetration, oral intercourse, anal intercourse, or manual stimulation. Of the sexually active study participants, 53.8% of the TM, 72% of the premenopausal women and 66.7% of the MW had the last sexual intercourse in the week prior to sample collection. Regarding sex of their sexual partners, 38.5% of the TM, 96% of the premenopausal women and all MW had sexual intercourse only with male partners. 46.2% of the TM and 4% of the premenopausal women had sexual intercourse only with female partners and 15.4% of the TM with both. Only six TM and three premenopausal women used condoms. PAP smear results were available for 22 TM, 20 premenopausal women and 24 MW. The remaining results were not known. The PAP smear results of all TM and premenopausal women were normal (PAP II equals NILM). Three MW had an abnormal PAP smear (PAP IIID equals LSIL). The HPV status was available for 21 TM, 2 premenopausal women and 11 MW. Four TM and four 4 MW tested positive for HPV. Among these, high risk types of HPV were found in all TM (HPV 53, 59, 82) and three women (HPV 73).

Serum hormone concentrations

The serum testosterone concentration in TM (5.23 ± 2.41 µg/l) was within the adult male cisgender reference (3.10—8.30 µg/l) for 19 TM, slightly below that of the adult male cisgender reference for 4 TM and above that for 2 TM..

The serum estradiol concentration of TM (40.72 ± 19.85 ng/l) was within the adult cisgender male reference interval (11–43 ng/l) for the majority of TM (n = 20) and above the cisgender male reference interval for a subset (n = 5).

Compared to the serum estradiol reference intervals for cisgender females, all TM were within the menopausal reference range (25–138 ng/l) and 18 TM within the premenopausal reference intervals (follicular phase: 31–90 ng/l, mid-cycle: 60–533 ng/l, luteal phase: 60–232 ng/l). The highest serum estradiol measured in the TM group was 98 ng/l. Estradiol levels of 17 premenopausal women (174.88 ± 113.05 ng/l) were within the premenopausal reference intervals. Estradiol levels of eight premenopausal women were missing, as they did not consent to a blood sample being taken. Menopausal estradiol levels were not measured. There was a significant difference in serum estradiol concentration comparing the TM with the premenopausal controls (p < 0.001; Fig. 2A).

There is a significant difference comparing the serum estradiol levels of TM on testosterone with (35.60 ± 22.20 ng/l) and without (44.13 ± 18.09 ng/l) supplementary GnRH-a medication (p = 0.014; Fig. 2B).

Nugent score

In total, 72% of TM, 72% of MW and 88% of premenopausal women had a Nugent score of 0–3. 28% of TM, 20% of MW, and 8% of premenopausal women had a Nugent score of 4–6. 8% of the MW and 4% of the premenopausal woman had a Nugent score of 7–10, indicating BV (Fig. 3).Fig. 3 Nugent scores within the study population. Nugent score of 0–3 (negative) no evidence of bacterial vaginosis; Nugent score of 4–6 (intermediate); Nugent score of 7–10 (positive) presence of BV.

Microbiome: diversity analysis

Alpha diversity

Alpha diversity was markedly different between TM and control groups (Fig. 4A). However, in TM, it was more similar to that in MW.Fig. 4 Analysis of microbial diversity in the vaginal microbiome. (A) Alpha- diversity analyzed by intraindividual change of Observed Richness, Shannon- and Inverse Simpson indices comparing trans men, post- and premenopausal women (ANOVA with post- hoc Tukey HSD). (B) Changes in alpha-diversity values in TM regarding supplementary GnRH-a medication (student’s t-test). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Furthermore, a significant effect of the length of GAHT on the Shannon index in TM was observed. The calculations revealed that the alpha diversity decreased with increasing length of GAHT in TM (p = 0.047, Pearson correlation).

As there was a significant difference in serum estradiol concentrations in TM with and without supplementary GnRH-a medication we further analyzed and compared the alpha diversity values for these sub-groups. However no significant differences could be found (Fig. 4B).

Beta diversity

The main hypothesis postulated that the overall community composition differed significantly between the TM group and the control groups. All beta diversity indices (Bray–Curtis, Weighted Unifrac, Unweighted Unifrac; Fig. 5) showed significant differences in community composition between TM and the premenopausal group (p < 0.001; Permutational multivariate analysis of variance [PERMANOVA]) as well as between TM and the MW group (p < 0.001; PERMANOVA).Fig. 5 Beta diversity analysis using multidimensional scaling (MDS) of (A) Bray–Curtis, (B) Weighted Unifrac and (C) Unweighted Unifrac distances. (R2 and p values from PERMANOVA analysis).

Microbiome: taxonomic composition and differential abundance

Different taxonomic profiles of TM and control groups

The taxonomic distribution on the phylum level was dominated by Firmicutes in all study groups (these include lactobacilli, Fig. 6A). There was also an increase in the abundance of Bacteroidota in the TM group compared to the control groups.Fig. 6 (A) Taxonomic distribution compared between trans men, post- and premenopausal women at the phylum level. Heatmap of relative abundance for individual samples. (B) Differentially abundant microbial taxa (amplicon sequence variant, ASV) between study groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (Negative binomial model, pairwise comparisons of the trans men group against the menopausal and premenopausal groups).

The microbiome of the TM group was characterized by a loss of Lactobacillus and an increase in bacteria more commonly associated with the intestinal flora (e.g., Campylobacter, Anaerococcus, Dialister, Prevotella) compared to the premenopausal group (Fig. 6B; Table 2 ). Lactobacillus was the most abundant taxon in the premenopausal group. Streptococcus, Prevotella, Lactobacillus and Alloscardovia were the most abundant taxa in the MW group. Prevotella, Streptococcus, Dialister, Fenollaria, Anaerococcus and Peptoniphilus were the most abundant taxa in the TM group. Comparing TM to MW revealed an even greater reduction in Lactobacillus in TM. Overall, the taxonomic composition of the microbiome in TM was strikingly similar to that in MW (Supplementary Fig. 1). Table 2 Differential abundance of taxa at genus level. Values are relative abundances (%).

Genus	trans men	premenopausal controls	menopausal controls	
Actinomyces	0.66 ± 1.39	0.00 ± 0.00	0.57 ± 1.87	
Alloscardovia	1.43 ± 5.09	0.01 ± 0.04	3.93 ± 14.08	
Anaerococcus	4.06 ± 3.34	0.09 ± 0.29	3.07 ± 5.57	
Atopobium	0.92 ± 1.18	2.31 ± 6.57	4.52 ± 10.42	
Bifidobacterium	0.02 ± 0.10	0.44 ± 2.19	4.06 ± 14.92	
Campylobacter	1.48 ± 2.89	0.06 ± 0.19	0.93 ± 1.84	
Corynebacterium	0.01 ± 0.06	0.02 ± 0.09	0.83 ± 2.82	
DNF00809	0.15 ± 0.42	0.07 ± 0.21	0.00 ± 0.00	
Dialister	6.10 ± 6.33	0.15 ± 0.45	1.12 ± 2.12	
Escherichia-Shigella	0.06 ± 0.18	0.54 ± 2.04	4.44 ± 14.45	
Ezakiella	0.87 ± 1.24	0.01 ± 0.02	0.68 ± 1.40	
Fenollaria	2.25 ± 2.52	0.00 ± 0.01	0.55 ± 1.78	
Finegoldia	2.29 ± 4.78	0.35 ± 0.74	3.96 ± 6.13	
Fusobacterium	0.90 ± 2.46	0.00 ± 0.01	0.50 ± 1.78	
Gardnerella	2.36 ± 5.52	3.15 ± 7.76	0.39 ± 1.39	
Hat002	0.01 ± 0.04	0.34 ± 0.58	0.08 ± 0.20	
Howardella	0.46 ± 1.10	0.01 ± 0.03	2.63 ± 10.10	
Lactobacillus	6.00 ± 8.97	71.66 ± 35.72	24.28 ± 34.48	
Moryella	0.49 ± 1.08	0.00 ± 0.00	0.02 ± 0.11	
Parvimonas	0.76 ± 1.41	0.00 ± 0.00	0.56 ± 1.87	
Peptoniphilus	2.72 ± 2.43	0.11 ± 0.22	1.74 ± 2.74	
Peptostreptococcus	0.52 ± 0.8	0.02 ± 0.09	0.32 ± 0.67	
Porphyromonas	5.65 ± 10.79	0.03 ± 0.10	1.29 ± 3.40	
Prevotella	25.17 ± 17.55	1.31 ± 4.29	6.76 ± 10.29	
Sneathia	0.01 ± 0.03	0.64 ± 2.40	0.14 ± 0.70	
Streptococcus	7.41 ± 10.23	0.21 ± 0.79	12.37 ± 24.57	
Sutterella	0.19 ± 0.30	0.00 ± 0.01	0.01 ± 0.02	
Ureaplasma	0.02 ± 0.0	0.57 ± 1.74	0.07 ± 0.29	

Factors influencing vaginal microbiome composition

Next, taxa abundance by sexual activity was analyzed. The top 20 most abundant taxa regarding sexual activity in TM and MW are shown in Fig. 7. In MW, sexual activity led to a difference in distribution which was not the case in TM. Sexually active MW seem to have a numerically lower abundance of Lactobacillus and a greater abundance of Prevotella and Gardnerella than non-sexually active MW (Fig. 7A). Interestingly, there seemed to be no difference in the abundance of Lactobacillus and Prevotella as well as any other taxa depending on sexual activity in TM (Fig. 7B). Taking the sex of the partner into account did not provide any further insight.Fig. 7 Taxonomic distribution at the genus level. (A) Abundance of bacteria in the vaginal microbiome of menopausal women with regard to sexual activity. (B) Abundance of bacteria in the vaginal microbiome of trans men with regard to sexual activity (all differences statistically not significant; negative binomial model).

At the genus level, a correlation between the length of GAHT in TM and the presence of certain bacteria was detected. The abundances of Dialister (− 1.21, p = 0.004) and Prevotella (− 0.64, p = 0.030) decreased with the length of GAHT in TM (Fig. 8).Fig. 8 Taxonomic distribution at the genus level. Abundance of bacteria in the vaginal microbiome of trans men with length of testosterone therapy (generalized linear model).

Discussion

The aim of this study was to characterize the vaginal microbiome in TM undergoing GAHT. In addition, we compared the microbiome of TM to that of women with low and normal estradiol levels to gain insight into possible endocrine effects. Significant differences were identified across the groups, underscoring the complex interplay between hormonal status, microbiome diversity, and taxonomic composition in these populations.

This analysis revealed that the microbiome of TM is characterized by an increase in alpha diversity, a loss of lactobacilli and an increase in bacteria more commonly associated with the intestinal flora. Alpha diversity declined with the duration of GAHT. Furthermore, the abundance of Dialister and Prevotella decreased similarly with increasing GAHT length in TM. Neither sexual activity nor the sex of the partner seemed to influence the top 20 taxa of the TM.

To our knowledge, this is the first study to compare the vaginal microbiomes of TM, MW and premenopausal women. Although it has often been hypothesized that there are similarities between the microbiomes of TM and MW, this study demonstrated that the vaginal microbial diversity of TM was more similar to that of MW, despite the younger age and premenopausal status of the TM group..

A previous study evaluated the effect of testosterone on the vaginal microbiome18. In contrast to our study, sample collection was not performed by a medical professional; instead, vaginal swabs were self-collected. A significantly greater Shannon diversity index was detected in TM (n = 25) than in cisgender women (n = 8)18. However, most women (75%) in the control group used hormonal contraceptives or intrauterine devices, and 16% of the TM applied vaginal estrogen, potentially leading to biased study results. Hormonal contraceptives could have an impact on the vaginal microbiome, possibly even on diversity26,27. To reduce this potential bias, the exclusion criteria in the present study included hormonal contraceptives and vaginal estrogen. Furthermore, changes in the vaginal microbiome throughout the menstrual cycle have been reported, with increased alpha diversity during menstruation26,28. A recently published study by our group showed that uterine microbiota plasticity varies during the menstrual cycle29. Uterine flushing in the follicular, ovulatory and luteal phases revealed high alpha diversity in the follicular phase of healthy women in comparison to women with recurrent pregnancy losses or recurrent implantation failures29. Furthermore, an increase in the abundance of Firmicutes throughout the menstrual cycle of healthy women with a simultaneous decrease in alpha diversity has been shown29. To standardize the procedure and minimize potential bias, the sampling of premenopausal controls was performed in the mid-cycle phase in our study.

As expected, the premenopausal group showed higher serum levels of estradiol overall. Testosterone often suppresses follicular development and thus leads to lower estrogen levels. Estradiol levels in TM were low in most but not all TM. This further demonstrates that high testosterone levels are not necessarily converted to estrogen by aromatase in TM. However, testosterone therapy alone does not always result in sufficient suppression of ovulation and estrogen production in general. If cessation of menses in TM is not achieved with testosterone therapy alone, GnRH-a can be used to induce amenorrhea. GnRH-a causes endogenous estrogen production to cease and mimic the menopausal hormonal environment. As shown in Fig. 2B, estradiol levels in TM with GnRH-a treatment are significantly lower than without GnRH-a. Estradiol levels in the group of menopausal women were not measured but has been shown to be below 25 ng/l in most cases30, which is the detection limit in the test used by the Institute for Medical and Chemical Laboratory Diagnostics, MUI. Decreased estrogen levels during menopause lead to a decrease in vaginal glycogen31. Glycogen is metabolized by Lactobacillus spp. to lactic acid, maintaining an acidic environment (pH 3.8 to 4.5), which further promotes the growth of Lactobacillus spp. and the displacement of other potentially pathogenic bacteria31–33. As estrogen levels decrease in MW, Lactobacillus abundance decreases and thus the pH increases31,34. A similar mechanism appears to affect the vaginal microbiome of TM. Alpha diversity increases in TM and MW compared to that in premenopausal women, mainly due to the loss of Lactobacillus dominance. Compared to MW, the loss of lactobacilli in TM is even more pronounced. This could be due to significantly higher testosterone levels. In individuals with polycystic ovary syndrome (PCOS), testosterone levels are elevated compared to physiological female levels. However, the levels are not in the normal male range as in TM. Increased alpha diversity in the vaginal microbiome has been observed in women with PCOS compared to healthy women35,36. In addition, several studies have shown an increase in Gardnerella and Prevotella abundance in women with PCOS35,37. However, as neither hormone levels nor cycle day were reported at the time of sampling, the significance of these findings remains unclear.

A significant change in the Shannon index was correlated with the duration of GAHT in TM, as alpha diversity decreased with the length of GAHT. This suggest that prolonged testosterone therapy and subsequent amenorrhea may drive a shift towards less diverse microbial environment, similar to that observed in MW. Although the alpha diversity decreased, Lactobacillus was not dominant38. Among the TM in our study, the decrease in alpha diversity was more likely an effect of a lower abundance of Dialister and Prevotella with increasing GAHT length. Dialister and Prevotella are microorganisms associated with BV. Interestingly, none of the TM had a Nugent score of 7—10, therefore no case of BV was diagnosed in this group. However, significantly more TM and MW than premenopausal women had an intermediate flora with a Nugent score of 4–6. Both groups have low estrogen levels in common, as do prepubertal girls. A comparison of girls with vulvovaginitis and healthy prepubertal girls showed not only a Lactobacillus deficiency but also a high bacterial diversity39, comparable to the results in the TM and MW groups. Interestingly, sexual activity appeared to influence the composition of the vaginal microbiome composition in MW but not in TM. This may be overshadowed by the effects of GAHT, suggesting that additional factors other than sexual activity may play a role in shaping the vaginal microbiome in this group.

Our data show that the vaginal microbiome of TM has the lowest abundance of Lactobacillus. The shift of a Lactobacillus-dominant microbiome to a more diverse vaginal microbiome leads not only to BV40, but also to a greater risk of HIV41 and other STIs, such as herpes simplex virus, human papilloma virus (HPV), N. gonorrhoeae or Chlamydia trachomatis42–44. The lower abundance of lactobacilli and the thinning of the epithelium increase the susceptibility of the vagina to infection. Thurman et al. could show, that susceptibility to HIV is greater in MW with decreased lactobacilli than in premenopausal women45. Vaginal therapy with estradiol was able to restore mucosal integrity and modulate susceptibility to HIV45. There are no data on the impact of testosterone on vaginal susceptibility to HIV and STIs in TM46,47. TM, especially those who have sex with men, seem to be at greater risk for STIs46,47. HPV was measured as an indicator of STIs in the study population. There was no increase in the incidence of HPV infection in TM, although all of the HPV-infected TM were infected with high-risk HPV types. A previous study also demonstrated a 19% rate of high-risk HPV types in TM48. The same study cohort showed a reduction in lactobacilli in 89% of TM and a greater percentage of high-grade squamous intraepithelial lesions compared to pre- and menopausal women48. This could not be verified in our study, probably due to the small sample size (n = 25).

This study has some limitations that should be noted. The sample size was small, therefore no conclusion on a correlation between hormone levels and the vaginal microbiome could be drawn, and further analyses on covariates such as partner sex were not sound. Furthermore, in contrast to the other study groups, the TM group consisted of a considerable number of people who were either not sexually active or had partners with a vagina. The representativeness of the selected groups may be further biased by the different age groups and the single-center setting, where recruitment is independent of ethnicity. Finally, long-term data are lacking.

To support vaginal health, it is important to restore the Lactobacillus-dominated vaginal microbiome and thus potentially protect against vaginal infection and STIs, especially in sexually active individuals. Since the use of estrogens, even when applied locally, is often rejected by TM, the administration (vaginal or oral) of probiotics could be considered to prevent infections49. The use of screening programs should be encouraged. In particular, the special needs of TM for gynecological examinations should be taken into account in order to increase participation in screening.

Conclusions

The vaginal microbiome of TM differs significantly from the vaginal microbiome of premenopausal women but shows similarities to that of MW. The duration of GAHT has an important impact on the vaginal microbiome and thus possibly on the risk of infection. A deeper understanding of the composition of the vaginal microbiome is crucial for improving vaginal health.

Supplementary Information

Supplementary Figure 1.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-72365-4.

Author contributions

KF, SR and BT contributed to the conception and design of the study. LP, SS and ACK collected the data. LP and SR performed the statistical analysis. KF, LP and SR wrote the first draft of the manuscript. All the authors have read and approved the manuscript.

Data availability

The dataset supporting the conclusions of this article is available in the European Nucleotide Archive, [PRJEB71387 and https://www.ebi.ac.uk/ena/browser/view/PRJEB71387]. The datasets generated and analyzed in this study are available on GitHub: https://github.com/reider-si/MBVagTrans.

Ethics approval and consent to participate

The study was approved by the ethics committee of the MUI (Decision Identifier 1055/2017). All participants provided written and informed consent at enrollment. All procedures and experimental measurements were carried out according to the current guidelines. Study numbers were used in place of participant names; no patient identifiers were retained.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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