madman
Super Moderator
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.
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.
