To date, the effects of long-term testosterone (T) administration on the human vagina are not completely understood. Thus, the aim of this study was to investigate the effects of long-term T treatment on vaginal tissue histology, estrogen receptor alpha (ERα) and beta (ERβ) expression and proliferation in female to male transsexual subjects (FtM). We compared vaginal samples from FtM subjects with those of premenopausal women (PrM) and postmenopausal women (M) not receiving any hormonal treatment for at least 2 years. Vaginal tissue samples from 16 FtM subjects treated with T (intramuscular injections of 100 mg Testoviron Depot/7–10 days for at least 1 year), undergoing sex reassignment surgery, and 16 PrM and 16 M subjects undergoing a vaginal hysterectomy for prolapse, were collected. For each sample, morphology, glycogen content, proliferation (ki-67), ERα and ERβ expression were evaluated. Vaginal samples from FtM showed a loss of normal architecture of the epithelium, intermediate and superficial layers were completely lost, and glycogen content was depleted. T administration resulted in a strong proliferation reduction when compared with both M and PrM subjects. Stromal and epithelial ERα as well as ERβ were significantly decreased in FtM when compared with PrM subjects. In conclusion, our data suggests that systemic T administration at supraphysiological dosage, determines profound changes in histomorphology and reduces ERs expression and proliferation of vaginal epithelium.
In recent years, testosterone (T) has gained attention as a potential treatment for women with hypoactive sexual desire disorder.1, 2, 3, 4, 5, 6, 7 Although still controversial, especially with regard to its long-term effects, T treatment both administered alone or in combination with classic hormone replacement therapy, has been shown to provide a small, but significant improvement in different aspects of sexual function, such as sexual interest, genital sensation, orgasmic response and sexual satisfaction.8
Genital sexual arousal results from the synergy of both central and peripheral mechanisms. In this regard, the vagina is the key organ of sexual response, but to date, limited studies are available concerning the effects of long-term T administration on the human vaginal structure.9 Numerous animal studies have investigated the effects of T alone or in combination with other sex steroid hormones on the vagina, but in some cases results are still controversial.10 In ovariectomized rats, T administration alone does not influence vaginal epithelium growth11 and has no effect on the muscularis, but when administered with estradiol (E2), it reduces E2-induced proliferation.11 Similar antagonistic effects of T on estrogens (E) have been suggested in other systems such as bone, and to a certain extent, in the endometrium.12 All together, this animal data seems to indicate that sex steroids have different and perhaps complementary roles in the modulation of the various systems that regulate the physiological function of the human vagina.13 Therefore, the collection of information on the effects of T on vaginal tissue is important, not only for a better and more comprehensive understanding of the physiology of the vagina, but also for the tuning of hormonal replacement treatment in different conditions.
For this purpose, in this study, we evaluated the effects of high dose T administration on the structure of the human vaginal epithelium of young women, undergoing T treatment for a disorder of gender identity.14, 15, 16, 17 We compared the vaginal tissue structure of these women with that of premenopausal (PrM) and menopausal women.
Materials and methods
Forty-eight women were included in our study, of which 16 were PrM, 16 were postmenopausal (M) and 16 were female with gender identity disorder (female to male, FtM).
PrM women had regular menstrual cycles (28–36 days) during the last 2 years, and were subjected to hysterectomy for vaginal prolapse. M women were also selected among those undergoing hysterectomy for vaginal prolapse. The diagnosis of menopause was carried out according to the definition of the American College of Obstetricians and Gynecologist.18 All M patients had developed permanent amenorrhea for at least 2 years.
FtM subjects received hormonal treatment (100 mg Testoviron depot i.m. every 7–10 days) for at least 1 year before undergoing hysterectomy and bilateral annessiectomy for sex reassignment. At study inclusion, all FtM subjects were currently taking T even if they were at different interval times from their last injection.
Inclusion criteria for PrM and M women were: absence of major medical illnesses including oncological problems assessed by medical history, and routine laboratory tests, no regular medication intake in the prior 6 months and no hormonal treatments for at least 1 year. None of the women in any of the groups received any drugs known to affect hormone metabolism.
For each woman in the three study groups, biopsies from the proximal–anterior, proximal–posterior, distal–anterior and distal–posterior vaginal wall were taken. These biopsies provide representative samples of the two portions of the vagina, the proximal of Mullerian origin and the distal derived from the urogenital tract.
The Human Subject Committee of S. Orsola-Malpighi Hospital approved this study, and specimens were obtained with written informed consent.
For each patient, four tissue biopsies from proximal and distal, anterior and posterior vagina were collected. Tissue specimens were fixed in 10% phosphate buffered formalin for histological and immunohistochemical analysis. Samples of vaginal tissue from each patient underwent standard haematoxylin–eosin staining for morphological analysis.
Periodic acid schiff staining
Periodic acid schiff staining of each vaginal biopsy was performed to assess glycogen stores. Briefly, the formalin fixed and paraffin-embedded specimens were cut into slices of 4 μm thickness and mounted on glass slides. Thin sections were deparaffinized, rehydrated in distilled water,and then pretreated for 5 min with 1% periodic acid (FlukaChemie, Buchs, Switzerland). After washing in double distillate water, the sections were left to react with Schiff’s reagent (Sigma-Aldrich, St Louis, MO, USA) for 15 min at room temperature. Nuclei were counterstained for 2 min with Mayer’s haematoxylin solution (Sigma-Aldrich, Milan, Italy).
Formalin fixed, paraffin-embedded vaginal tissue samples were cut into 4 μm sections. After deparaffinization and inhibition of the endogenous peroxidase activity, slides were subjected to antigen retrieval in citrate buffer pH 6, and were then incubated with 10% normal goat serum for 45 min. Sections were stained overnight at 4 °C with mouse monoclonal anti-Ki-67 antibody (1:50; Dako Cytomation, Glostrup, Denmark) and mouse monoclonal anti-estrogen receptor alpha (ERα) and anti-ER beta (ERβ) antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Primary antibodies were revealed using goat anti-mouse (ki-67 and ERα) or goat anti-rabbit (ERβ) immunoglobulins conjugated to peroxidase (EnVision, Dako Cytomation, Denmark) and 3,3′-diaminobenzidine (Sigma-Aldrich, USA) as chromogen. In the negative controls, immunostaining was performed without the primary antibody.
Section evaluations for Ki-67, ERα and ERβ were performed at a magnification of × 20 using an image cytometer consisting of a single 2/3′′ CCD (charge-coupled device ) color camera (JVC Professional Europe, London, UK) mounted on a microscope (DMLB microscope, Leica Microsystems, Wetzlar, Germany) equipped with a motorized scanning table (Märzhäuser, Wetzlar, Germany) controlled by Cytometrica software (C&V, Bologna, Italy). For Ki-67, immunostaining results were expressed as the number of positive cells per linear mm of vaginal mucosa as previously described,19 while for ERα an Allred score19 was used to rate the immunostaining in each sample. The proportion of immunoreactive cells in the vaginal mucosa and stroma was assessed using the following scale: 1=no/few positive cells, 2=5–10% of positive cells, 3=30% of positive cells, 4=50% of positive cells and 5=more than 50% of positive cells, while the stain intensity was graded using the following scale 1=weak, 2=moderate and 3=strong. The Allred score results from the addition of the percentage score and the intensity score.20 The total score for each sample resulted from the addition of the Allred score from four different random fields.
Vaginal tissue biopsies were homogenized in 1% Triton X-100 lysis buffer supplemented with a cocktail of protease inhibitors (NaCl 0.1 mol l−1, Tris-HCl 0.01 mol l−1 pH 7.6, EDTA 0.001 mol l−1, 1% Triton X-100, leupeptin 10 mg ml−1, pepstatin 1 mg ml−1, aprotinin 2 mg ml−1, antipain 2 mg ml−1, phenylmethylsulfonyl fluoride100 ng ml−1, dithiothreitol 1 mmol l−1 and Na3VO4 1 mmol l−1). Total protein concentration in each sample was determined by the Lowry method. Afterwards, 50 μg of total protein for each sample was electrophoresed on 8% SDS polyacrylamide gels (8% polyacrylamide, 10% SDS) under denaturing conditions (5% β-mercaptoethanol) and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% non-fat dry milk in 0.1% phosphate-buffered saline-Tween, and incubated overnight at 4 °C with mouse monoclonal anti-ERα antibody (1:500; Santa Cruz Biotechnology, USA). The membranes were then washed three times with 0.1% phosphate-buffered saline-Tween, and incubated with anti-mouse horseradish peroxidase secondary antibody. After thorough washing, the signal was detected by incubating the membranes with peroxide and luminol enhancer solution (ECL-Advance, Amersham Biosciences, Munchen, Germany), and then exposing them to a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA). Densitometry was performed using NIH ImageJ software (http://rsbweb.nih.gov/ij/).
All assays were performed in our hospital routine diagnostic laboratory using established commercial assays, as previously described.17
Statistical analysis was performed using SPSS 11.0 software (SSPS Inc Chicago, IL, USA). Ordinal variables were analyzed by the Kruskal-Wallis test with Dunn’s post hoc. One-way analysis of variance with post hoc Bonferroni test was used to analyze continuous variables normally distributed. A P-value< 0.05 was considered to be statistically significant.
Demographic, anthropological and hormonal data of the subjects included in the study is shown in Table 1. As previously reported in literature,14 in our FtM subjects, high dose T administration also did not induce any clinically relevant adverse effects.
Morphological analysis of vaginal biopsies
This group showed the typical histological features observed in vaginal epithelium. A parabasal layer composed of two to four rows of polygonal cells, covered specifically the basal cell layer, composed of a single row of columnar-like cells. The superficial zone consisted of normally maturating keratinocytes. Intracytoplasmic glycogen was present.
Vaginal epithelium showed features of atrophy in all patients. Specifically, the basal layer was covered by up to 6–8 layers of parabasal cells, and only in three cases did focal intracytoplasmic glycogen persist. Marked hyperkeratosis with hypergranulosis were present in three cases, mainly in samples taken from the posterior vaginal wall.
Premenopausal women (FtM)
Who received high doses of T: vaginal epithelium was considerably thinner than is usually seen. It showed a basal cell layer, composed of a single layer of columnar-like cells, oriented perpendicularly to the basal lamina. There were up to 10 parabasal layers, all composed of polygonal cells, with scant eosinophilic cytoplasm, oriented perpendicularly to the basal lamina. No intermediate or superficial layers and no intracytoplasmic glycogen were seen. Submucosal vessels were markedly dilated and filled with erythrocytes.
Mean epithelium thickness in the three groups of women is reported in Figure 1 panel A. Epithelium was significantly thinner in the FtM groups when compared with the M and PrM groups.
Mild inflammatory infiltration, mainly composed of mature lymphocytes, was additionally seen in all three patient groups.
Periodic acid schiff staining
Although the periodic acid Schiff staining allows only a qualitative evaluation, the glycogen store seems to be diminished in M when compared with PrM. T administration is associated with a complete loss of glycogen store in the vaginal mucosa, which may be related to a modification of normal vaginal epithelium (Figure 2).
Ki-67-positive immunostaining was observed in the nuclei of epithelial cells of the vaginal mucosa of all experimental groups (Figure 1 panels c–e). As expected, the positive nuclei per mm of vaginal mucosa rate was higher in PrM, and although not statistically significant, it decreased in M. Women who underwent T treatment showed only few positive nuclei in the vaginal mucosa (Figure 1 panel b). No statistically significant differences were detected when proximal and distal parts of the vagina were compared (see Supplementary Material).
ER immunohistochemistry confirmed the expected ERα expression with different degrees in both the epithelium and stroma across the three patient groups, with a typical nuclear pattern (Figure 3 panels a–c). ERα immunostaining was reduced in the vaginal mucosa and stroma of M patients when compared with PrM, achieving statistical significance only in the mucosa (P<0.001) (Figure 4 panels a and b). T administration resulted in a blunted ERα expression in both the mucosa (P<0.05) and stroma (P<0.001) when compared with PrM (Figure 4 panels a and b). No statistically significant differences were detected when proximal and distal parts of the vagina were compared (see Supplementary Material). Western blot analysis of ERα showed a reduced protein expression both in M and FtM as compared with PrM, achieving the statistical significance only in FtM subjects (Figure 5).
ERβ immunohistochemistry revealed the presence of this receptor in all vaginal samples from the three groups (Figure 3 panels d–f). It was expressed mainly in the nuclei of epithelial cells near the parabasal layer and in the nuclei of subepithelial stromal cells. ERβ expression was significantly reduced in the vaginal stroma (P<0.05), but not in the mucosa of M women versus PrM, while T treatment downregulated ERβ expression both in mucosal (P<0.05) and stromal layers (P<0.01) of FtM subjects as compared with PrM subjects (Figure 4 panel c and d). No statistically significant differences were detected when proximal and distal parts of the vagina were compared (see Supplementary Material).
The purpose of this study was to evaluate the effects of high dose T administration on vaginal tissue structure, and proliferation of young women compared with untreated PrM and M women. We found that T administration induced a thinner vaginal epithelium characterized by glycogen absence, reduced proliferation and ERs expression. When compared with PrM and M women, T administration induced a thinner epithelium, which lost the intermediate and superficial layers, and in which only the basal and parabasal layers persisted. Glycogen content was completely depleted in FtM women. Overall, the histological characteristics of vaginal epithelium from FtM subjects resembled those of an epithelium with lack of maturation. On the other hand, no differences were detected in the stromal tissue between the three groups.
In our study, the complete depletion of glycogen content in FtM women occurred despite the persistence of circulating E2. In fact, as previously reported, the administration of T blocks ovarian cyclic function, but circulating E levels remain detectable, compatible with the early follicular phase of the cycle.12 These E2 levels probably come from residual ovarian activity or alternatively from the peripheral aromatization of the exogenous T. Glycogen production, normally closely related to circulating E. It is normally absent in the vaginal wall of prepubertal girls with low E levels, it is present in large amounts in the vaginal epithelium of fertile PrM women, and is again greatly reduced, but not absent, in menopausal women. These findings were also confirmed by our results. Therefore, the complete depletion of glycogen observed in our FtM population is probably due to a direct effect of T that occurred and overcame the effects of circulating E levels. In the vagina, glycogen is normally metabolized by the resident bacterial flora, which then produces lactic acid responsible for the protective acidification of the vaginal environment.21 Loss of glycogen content therefore may result in an increase of vaginal pH allowing fungal or bacterial growth. In our FtM subjects, we did not record a higher vaginal infection rate, nor has this problem ever been reported in literature. However, these subjects generally do not engage in penetrative intercourse, and therefore may be protected against infection.
Regarding the proliferation of vaginal epithelium, we detected Ki-67 immunostaining in all three groups. As expected, this proliferation marker was higher in PrM women compared with M women, and was almost absent in T-treated subjects. The finding of a loss of epithelial proliferation in FtM compared with reproductive-aged women was never reported before, and indicates that pharmacological T administration could induce epithelial atrophy, despite the persisting, although lower, circulating E levels. The reduced cell maturation associated with a drastic reduction of the proliferation index may result from an antagonizing effect of T on E. This antagonizing action of T versus E is not surprising, as it has already been reported in other tissues such as the endometrium12 and breast.22
This effect was also confirmed by the reduced expression of both ERα and ERβ in FtM subjects, as shown by the immunohistochemistry and western blot analysis. Furthermore, immunohistochemical staining allowed us to discriminate from stromal and epithelial ERs expression. Both the stromal and epithelial ERα is essential for normal vaginal maturation (stratification and cornification) and maintenance.23 Experimental studies have indicated that isolated vaginal epithelium fails to stratify and cornify in vivo in response to E2 treatment,24 but when epithelium is reassociated in vivo with vaginal stroma, the growth capacity is re-established.25 Therefore, stromal ERα mediates indirectly vaginal epithelium proliferation, while the differentiative response to E, such as stratification and cornification, requires both stromal and epithelial ERα.26 Results from western blot analysis show a decreased ERα expression in FtM, in particular ERα downregulation is more pronounced in the stroma than in the epithelium, as shown by immunohistochemistry, and this is consistent with our observation of an absence of epithelial proliferation. Epithelial and stromal ERα downregulation may be related to the lack of differentiation. The further downregulation of ERα in FTM compared even with M women would suggest that this effect could be the result not only of lower E levels, but also of a direct T effect. This hypothesis would also be supported by our recent finding that long-term high dose T administration is associated with AR upregulation in FtM subjects, suggesting a direct action of T at the vaginal level through its own receptor.27 These findings are clearly different from those reported by previously published animal studies that found an increased ERα after ovariectomy, restored to normal levels only by physiological E levels.28 These differences could be explained by species specific differences in the hormonal regulation of sex steroid receptors expression. Although ERα is the predominant ER expressed at the vaginal level, ERβ also participates in the estrogenic signaling transduction. Our result confirm previous studies reporting a decrease of ERβ in the vaginal stroma but not in the mucosa.29 The unchanged ERβ expression at the mucosal level may be an attempt to partially compensate ERα downregulation, as suggested also by experimental data conducted on mutant mice lacking ERα, ERβ or both.30 No data in the literature exists on the regulation of ERβ by T. The decrease that we see in our samples may be related to the reduced circulating E levels following T administration or to a direct effect of T. Taken together, these observations indicate that ER isoforms are differently distributed and regulated by sex steroid hormones (E and T) at the vaginal level.
The morphological changes in vaginal epithelium from FtM subjects are not surprising, as they are related to the masculinization process as previously reported.9 The major findings of our study are the molecular changes underling this morphological modifications. Although the antiestrogenic effect of T was demonstrated in other tissues,12, 22 to date no data had been reported about this effect on human vagina. We found that at high dosage, T exerts a potent antiestrogenic effect also in vaginal epithelium, which results in morphological changes compatible with a lack of proliferation and tissue atrophy.
Our results did not exclude a potential beneficial effect of T on female genitalia and sexual response, if administered at lower dosage. At this purpose, physiological AR expression was just reported in different part of female genitalia,27, 31 therefore androgen may have a role in modulating several aspects of sexual response, such as clitoral engorgement and vaginal lubrification. This hypothesis is in part supported by a clinical study, in which lower dosage topical T administration show improved signs and symptoms of vaginal atrophy.32
This study has a few limitations that deserve comments: first, to evaluate the effects of T on the vaginal structure, we used a model of young women receiving high dose T administration for sex reassignment. Therefore, the effects identified in this study may not be entirely or partially found with lower doses and different administration routes of T, such as those administered in M women with hypoactive sexual desire disorder or those proposed for breast cancer patients with vaginal atrophy related to aromatase inhibitors.31
More extensive hormone levels and in particular sex hormone-binding globulin, free T and E2 levels were not available, and therefore no direct correlation between vaginal differences and these hormonal levels could be performed. In PrM and M women, total T levels are close to or below the detection limit of the commonly used assays. The accuracy and precision of the levels in this part of the curve are poor.
In summary, our results demonstrate that long-term, high dose T administration in young women has no adverse effects on vaginal epithelium, but determines substantial modifications of its histomorphology, proliferation and E receptors expression that confirm the antiestrogenic effect of T that we and others have already reported on different tissues.
About this article
Supplementary Information accompanies the paper on International Journal of Impotence Research website (http://www.nature.com/ijir)
Cervical Cancer Screening for Patients on the Female-to-Male Spectrum: a Narrative Review and Guide for Clinicians
Journal of General Internal Medicine (2015)