Androgen production within the human vagina

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Insight on the intracrinology of menopause: androgen production within the human vagina
Ilaria Cellai, Vincenza Di Stasi, Paolo Comeglio, Elisa Maseroli, Tommaso Todisco, Chiara Corno, Sandra Filippi, Sarah Cipriani, Flavia Sorbi, Massimiliano Fambrini, Felice Petraglia, Irene Scavello, Giulia Rastrelli, Gabriele Acciai, Fabio Villanelli, Giovanna Danza, Erica Sarchielli, Giulia Guarnieri, Annamaria Morelli, Mario Maggi, Linda Vignozzi


Abstract

In this study, we investigated steroidogenic gene mRNA expression in human vaginas and verified the ability of human vagina smooth muscle cells (hvSMCs) to synthesize androgens from upstream precursor dehydroepiandrosterone (DHEA). As a readout for androgen receptor (AR) activation, we evaluated the mRNA expression of various androgen-dependent markers.

hvSMCs were isolated from the vagina tissues of women undergoing surgery for benign gynecological diseases. In these cells, we evaluated the mRNA expression of several steroidogenic enzymes and sex steroid receptors using real-time RT-PCR. Androgen production was quantified with liquid chromatography tandem-mass spectrometry (LCMS/MS).

In vaginal tissues, AR mRNA was significantly less expressed than estrogen receptor α (ERα), whereas in hvSMCs, its mRNA expression was higher than progestin and both estrogen receptors. In hvSMCs and in vaginal tissue, when compared to ovaries, the mRNA expression of pro-androgenic steroidogenic enzymes (HSD3β1/β2, HSD17β3/β5), along with 5α-reductase isoforms and sulfotransferase, resulted as being more abundant. In addition, enzymes involved in androgen inactivation were less expressed than in the ovaries. The LCMS/MS analysis revealed that, in hvSMCs, short term DHEA supplementation increased Δ4- androstenedione levels in spent medium, while increasing testosterone (T) and dihydrotestosterone (DHT) secretion after a longer incubation. Finally, androgenic signaling activation was evaluated through AR-dependent marker mRNA expression, after DHEA and T stimulation.

*This study confirmed that the human vagina is an androgen-target organ with the ability to synthesize androgens, thus providing support for the use of androgens for local symptoms of the genitourinary syndrome in menopause.




Introduction

Sex steroids are hormones derived from cholesterol with fundamental roles in the development and functioning of female sexual organs, such as the vagina (1-5). Two main classes of sex steroids, namely androgens, and estrogens, are secreted (the latter not in a significant amount) by the outer cortex of adrenal glands and, in a sexually dimorphic manner, also by the gonads, showing dramatic fluctuations throughout the different stages of life (6).

The human vagina is extremely sensitive to changes in the hormonal milieu throughout a person’s lifespan. Among sex steroids, estradiol has been historically considered the most important hormone for vaginal health. Therefore, a wide spectrum of genital and sexual symptoms (including dryness, burning, irritation, lack of lubrication, discomfort or pain, urgency, dysuria, and recurrent urinary tract infections), encompassed by the term “Genitourinary syndrome of menopause” (GSM), (7), has been closely tied to the drop of estrogen level after menopause. More recently, the role of androgens in the development and treatment of GSM has been receiving increased attention (8). In fact, the fall in ovarian estrogen production at menopause is accompanied by a progressive, and aging-dependent, decline in androgens (9). Nevertheless, to protect peripheral tissues from circulating hormonal imbalances, small amounts of estrogens and androgens are also produced in situ from inactive precursors by cell-specific sets of enzymes. This specific local production and activity of hormones, which differs from the classical processes, is defined as “intracrinology” (10). A peculiar intracellular enzymatic equipment - by which dehydroepiandrosterone (DHEA), an almost biologically inactive androgen, is locally converted into testosterone (T) and 17β-estradiol - has been evoked to protect several peripheral tissues, including the vagina (11) from hormonal deficiency. Indeed, adrenal DHEA, which is considered the only source of sex steroids in menopausal women, starts to decline at about the age of 30 years, resulting, on average, in a 60% decrease after menopause. In peripheral tissues, a parallel 60% decrease in androgen biosynthesis from DHEA has therefore been hypothesized (11,12). Consistent with this view, a novel approach aimed at treating GSM symptoms using intravaginal DHEA has been postulated.

Bertin and colleagues first demonstrated that the intracrinological enzymatic machinery able to produce androgens was present in the vagina of rodents and even in the cynomolgus monkey (Macaca fascicularis), the closest animal model to the human one (13). However, data on intracrinology in the human vagina are still lacking. Hence, addressing this hypothesis appears to be of particular relevance, not only to better understand the physiology of the female genitourinary tract but also from a clinical perspective, to clarify the mechanisms of action of intravaginal DHEA.

The aim of the study was to investigate whether the human vagina possesses the proper enzymatic machinery to convert weak androgen precursors, such as DHEA, into active androgens, as already described in other species. We recently demonstrated that androgens are potent anti-inflammatory hormones in human vaginal smooth muscle cells (hvSMCs) (14), thus raising the urgent need to investigate the ability of these cells to produce their own androgens. We, therefore, aimed to examine if isolated hvSMCs maintained the ability to convert DHEA into Δ4-androstenedione and T. Moreover, we wanted to investigate the capability of DHEA, through its conversion into active androgens, to induce androgen-dependent signaling by AR stimulation on these cells. All steroid measurements were performed using an isotopic dilution liquid chromatography-tandem mass spectrometry method (LC-MS/MS), the gold standard method for steroid quantification.





Discussion

The present study shows, for the first time, that human vaginal tissue harvested from postmenopausal women possesses the machinery to convert an extremely weak androgenic precursor, DHEA, into the active androgens T and DHT. This evidence definitely substantiates the construct of intracrinology in the human vagina and highlights the androgen-producing ability of this tissue. In fact, we here have evidence of a high abundance of mRNA levels of steroidogenic enzymes downstream to HSD3β, actively involved in androgen production in the human vagina, as compared to the ovary - the most important steroidogenic organ of the female urogenital tract.




*In the present study, we also confirm that the human vagina is one of the female-specific target sites for androgens (8,25). Estrogen receptors (ERs) are expressed on the whole female genital tract, especially in the vaginal epithelium (VE), stroma, and smooth muscle cells (SMCs). Similarly, androgen receptor (AR) is diffusely located in the female genito-urinary tract, with a predominance in those areas derived from the urogenital sinus, namely: the bladder, urethra, clitoris, and distal vagina (26,27). AR is located on all vaginal compartments, including mucose, sub-mucose, stroma, the smooth muscle layer, and the endothelium, thus hypothetically mediating the direct effect of androgens on genitourinary and vaginal tissues (8).


*Finally, our data confirm that the human vagina is an androgen synthesis and target organ, supporting a more substantiated use of local androgens in the GSM.
 

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Taken together, these data are highly relevant from several points of view:

The results of this study improve our knowledge of vagina physiology by providing evidence for the presence of steroidogenic machinery in the human vagina, thus supporting, for the first time, the construct of intracrinology in this organ (5,38)


They also reinforce the view that the vagina is an androgen-dependent organ, and appears to be able to locally produce these hormones, mainly thanks to a high level of steroidogenic enzymes involved in androgen production

Finally, the observations here reported suggesting the potential mechanism and the resulting action of intravaginally administered DHEA, which has recently appeared on the market for the treatment of GSM (8,39,40) and opened new perspectives for the treatment of this common and bothersome condition
 
Table 1. List of primers and relative fragment sizes.
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Figure 1. Characterization of hSMCs isolated from human vagina Panels A, B, C, and D show representative images of hSMCs immunostaining for specific smooth muscle markers MYH11, αSMA, and vimentin, and for the epithelial marker cytokeratin (taken as a negative control), respectively. Panel E shows a representative image of the negative control obtained omitting the primary antibodies. The quantitative analysis was performed by counting positive cells in at least 10 fields per slide of three different cell preparations obtained from three patients (scale bar=100 µm).
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Figure 2. Relative mRNA expression of sex steroid receptors in human distal vaginal tissues and cells Panels A and B show the relative mRNA expression of sex steroid receptors AR, ERα, ERβ, GPER1, and PR in human vaginal tissues derived from laparoscopic biopsies (n=10) and in smooth muscle cells (hSMCs) isolated from the corresponding vaginal samples (n=4, performed in triplicate), respectively. Data were calculated according to the 2-ΔΔCt comparative method, using ribosomal subunit 18S as the housekeeping gene for normalization. Results are expressed as arbitrary units, calculated as fold-increase vs. the lower mRNA expression value in the experiment, and are reported as mean±SEM.
* p<0.05, *** p<0.001 vs AR; °°° p<0.001 vs ERα; ## p<0.01, ### p<0.001 vs ERβ; ++ p<0.01, +++ p<0.001 vs GPER1

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Panels C and D show two representative images of nuclear immunostaining for AR receptor and the corresponding nuclear DAPI double labeling, respectively. Panel E shows a representative image of the negative control obtained omitting the primary antibodies. At least three preparations obtained from three different patients were evaluated (scale bar=100 µm).
 
Figure 3. mRNA expression of steroidogenesis pathway-related genes in human vaginal tissue The panel shows the mRNA expression of genes involved in steroidogenesis in human distal vaginal tissues derived from laparoscopic biopsies (n=29). Data were calculated according to the 2-ΔΔCt comparative method, using ribosomal subunit 18S as the housekeeping gene for normalization. Results are expressed as a percentage of ovary mRNA expression and are reported as mean±SEM.
* p<0.05, ** p<0.01, *** p<0.001 vs Ovary

Screenshot (2815).png
 
Figure 4. mRNA expression of steroidogenesis pathway-related genes in human vaginal SMCs The panel shows the mRNA expression of genes involved in steroidogenesis in human distal vaginal SMCs derived from laparoscopic biopsies. Data were calculated according to the 2- ΔΔCt comparative method, using ribosomal subunit 18S as the housekeeping gene for normalization. Results are expressed as a percentage of ovary mRNA expression and are reported as mean±SEM of three independent experiments performed in triplicate.
* p<0.05, ** p<0.01, *** p<0.001 vs Ovary

Screenshot (2816).png
 
Figure 5. Measurement in mass spectrometry of androstenedione, testosterone, and DHT concentrations by LC-MS/MS in the culture media of hvSMCs in response to stimulation with increasing doses of DHEA Panel A shows the androstenedione concentrations (expressed as nmol/L) measured in the culture media of hvSMCs after stimulation with increasing doses of DHEA (from 1 to 30 nM) at 24 and 48 h. Results are reported as mean±SEM of three independent experiments performed in triplicate.
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Panel B shows the testosterone concentrations (expressed as nmol/l) measured in the culture media of hvSMCs after stimulation with increasing doses of DHEA (from 1 to 30 nM) at 24 and 48 h. Results are reported as mean±SEM of three independent experiments performed in triplicate.
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Panel C shows the DHT concentrations (expressed as nmol/l) measured in the culture media of hvSMCs after stimulation with increasing doses of DHEA (from 1 nM to 1 µM) and physiological/supraphysiological doses of T (0.6 and 30 nM) at 48 h. Results are reported as mean±SEM of three independent experiments performed in triplicate.
°°° p<0.001 vs NT, * p<0.05, *** p<0.001 vs all lower DHEA doses (nM); ^^^ p<0.001 vs T (0.6 nM)

Screenshot (2820).png
 
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Figure 6. mRNA expression of androgen-responsive genes and PR in human vagina hSMCs The panel shows the mRNA expression of PKG1, GUCY1A2, STAMP2, and PR in human distal vaginal hSMCs derived from laparoscopic biopsies. Data were calculated according to the 2-ΔΔCt comparative method, using ribosomal subunit 18S as the housekeeping gene for normalization. Results are reported as mean±SEM of three independent experiments performed in triplicate.
° p<0.05, °°° p<0.001 vs NT; * p<0.05 all lower DHEA doses (nM); # p<0.01 vs DHEA 30 nM; @ p<0.01 vs all DHEA treatments; $ p<0.05 vs DHEA 30 nM+BICA; ^ p<0.01 vs T 0.6 nM

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