Women's Sexual Function and Dysfunction. Irwin Goldstein MD

Modulation of female genital sexual arousal by sex steroid hormones

Abdulmaged M Traish, Noel N Kim

Introduction

The important role of sex steroids in modulating sexual function in women has been recognized for many years. However, the great majority of investigative efforts have focused on the regulation of female reproductive function by sex steroid hormones. Correspondingly, knowledge in this area has contributed significantly to successful clinical management of contraception, infertility, and gynecologic disorders by the use of sex steroid hormones or synthetic hormone analogs as therapeutic agents. In contrast, the physiology of peripheral genital sexual arousal and its regulation by sex steroid hormones has received limited attention. Receptors for sex steroid hormones (estrogens, progestins, and androgens) are widely expressed in the brain and genital tissues, suggesting that steroid hormones may modulate sexual function at both central and peripheral levels. It should be noted that most of the studies with sex steroid hormones have focused on the effects of androgens on female sexual desire, and little attention was paid to female sexual arousal response.1,2

With regard to sex steroid hormones, critical evaluation of the literature points to the fact that sexual function studies in women are almost entirely clinical in nature and that, in some instances, the data remain controversial. There is a great deal of skepticism regarding the therapeutic value of sex steroid hormones in treating female sexual dysfunction, especially peripheral genital arousal dysfunction in women. Unfortunately, an accurate appraisal of the usefulness of sex steroid hormones in the management of female sexual dysfunction remains elusive due to the paucity of objective clinical criteria and the limited preclinical and clinical studies investigating the underlying pathophysiology. Employment of objective criteria of biologic effectiveness, together with clinical observations, are needed in order to have a critical and effective appraisal of the role of sex steroid hormones in modulating sexual function in women. Thus, caution must be exercised when interpreting clinical studies, especially when objective criteria are not available and since most of the work in the literature is anecdotal. Furthermore, preclinical studies are needed to develop reliable measures for evaluating objective physiologic parameters to be tested in response to sex steroid treatments. This requires development of experimental models and validating the physiologic responses to be measured. In this chapter we discuss the critical role of steroid hormones in regulating female genital arousal. There is a vast literature on sex steroid hormones and human physiology, and this chapter is not intended to cover such literature, but to focus on sex steroid and peripheral sexual arousal function and dysfunction. To this end, we present historical highlights in investigation of the relationship between sex steroids and sexual function in women, review the biochemistry of steroid metabolism, and summarize recent research efforts.

Historical perspective

It has been known for quite some time that while estrogens have little or no effect on sexual interest, they are important and necessary for the maintenance of structural and functional integrity of genital organs. The action of androgens in sexual interest was noted as an incidental finding in studies on the effects of androgens on menopause.3 Loeser reported that all female patients who were treated with testosterone had experienced an enlargement of the clitoris and increased sexual drive.4 The author suggested that androgens play a key role in enhancing libido in women. This observation was further confirmed by several investigators.5-9 To this end, it was thought that the effects of androgens on increasing libido were universal observations. It was further suggested that a combination of estrogens and androgens exerted optimal effects on libido and sexual response. The increased libido and sexual response were attributed in part to changes in the external genitalia (increased sensitivity to engorgement and hypertrophy of the clitoris and the vulva) and increased sensation secondary to sexual stimulation.

It should be noted that stimulation of sex drive in women by estrogen is not commonly encountered in clinical experience in spite of the fact that sexual receptivity in the female of lower animals is controlled by estrogens. However, administration of androgens in females intensifies sexual interest.10 As early as 1938, Shorr et al. observed that libido and sexual responses were greater when testosterone was administered together with estrogen rather than administering estrogen alone.3 Along with reported heightened sexual desire, easier attainment of orgasm and heightened satisfaction during intercourse were also observed. It was not clear whether the effects of androgens were at the central nervous system or at the genital level or both.

In 1943, Salmon and Geist suggested that androgens have threefold action in women: (a) increased susceptibility to psychosexual stimulation, (b) increased sensitivity of the external genitalia, and (c) greater intensity of sexual gratification.The authors suggested that endogenous androgens in the normal mature woman may act as the physiologic sensitizer of both the psychic and somatic components of the sexual mechanism. The authors further suggested that dryness of the vagina occurring during androgen treatment may lead to dyspareunia and might be ameliorated by estrogen administration.8

At present, with the exception of local vaginal administration of estrogen, there is no established and accepted scientific rationale for pharmacologic management of women with sexual dysfunction by sex steroid hormones or hormone analogs. In fact, doubts have been cast on the potential usefulness of sex steroid therapy in light of the data from the hormone replacement study that was conducted in the USA as part of the Women’s Health Initiative (WHI). The trials with estrogen therapy alone did not show increased risk of breast cancer and decreased risk of colon cancer and osteoporosis coupled with decreased fracture risk, and no change in heart disease was noted as opposed to estrogen and progesterone trials in postmenopausal women. These trials with estrogen plus progesterone and estrogen alone therapy were prematurely terminated when the data indicated increased risk of stroke and general lack of benefit.

Nevertheless, a number of studies suggest that steroid hormones are important in modulating the physiology of genital organs and sexual response in women. Bachmann et al.11 reported that estrogen deficiency associated with the postmenopausal state resulted in: (1) loss of collagen and adipose tissue in the vulva; (2) attenuated maturation of vaginal epithelial cells; (3) thinning and loss of elasticity of the vaginal wall with loss of premenopausal ridges; (4) bleeding and ulceration of the vaginal epithelium, even with minor trauma; (5) delayed onset of lubrication with sexual stimulation; and (6) increased vaginal pH leading to heightened vulnerability to urogenital pathogens and flora. In a series of studies, Sarrel and co-workers12-15 reported that women with plasma estradiol levels less than 50pg/ml had significantly more complaints of vaginal dryness and increased frequency and intensity of dyspareunia and burning than women with estradiol values greater than 50 pg/ml. Several investigators have shown that treatment with estradiol increases vaginal blood flow and lubrication, improves epithelial maturation indices, normalizes vaginal pH, and prevents vaginal atrophy.15-17

Androgen insufficiency, in women who are treated with adequate doses of estrogens, is also associated with sexual dysfunction.13,1^22 When supraphysiologic doses are used, androgen replacement in women with sexual dysfunction is associated with changes in the external genitalia, including increased sensitivity, engorgement and hypertrophy of the clitoris, and vulvar hyperemia.6,7,9 It has been reported that women with higher levels of testosterone exhibit significantly greater increases in vaginal blood flow in response to erotic stimuli than those with lower levels of testosterone.23,24 Moreover, exogenous administration of androgens has significantly increased subjective ratings of sexual arousal in postmenopausal women.25-27 In a limited study (nine patients), Tuiten et al.27 showed that arousal response in young women given testosterone undecanoate orally and allowed to watch erotic video resulted in marked arousal response, albeit delayed by several hours after treatment. In oophorectomized women treated with testosterone, those who had a higher ratio of testosterone to sex hormone-binding globulin had greater sexual arousal response.19 In a recent review, Sherwin suggested that combined estrogen and testosterone therapy enhances sexual desire, interest, and sometimes the frequency of intercourse.1 In addition, the overall quality of life was improved in naturally and surgically menopausal women on this combined treatment. Even more compellingly, administration of transdermal testosterone improved sexual function and psychologic well-being in women who had undergone oophorectomy and hysterectomy.28,29 Treatment of women with adrenal insufficiency with dehydroepiandrosterone also improved overall well-being and sexual function.30,31 In separate studies, androgen replacement therapy with dehydroepiandros- terone in pre- and post-menopausal women with sexual dysfunction and androgen insufficiency significantly decreased sexual distress, significantly increased sexual function in the domains of desire, arousal, lubrication, satisfaction, and orgasm, and normalized androgen blood levels to values within the physiologic range.32

Thus, there is growing evidence that an imbalance in the sex steroid hormonal milieu may be responsible for some of the sexual complaints in women. It is well recognized that sex steroid hormones play an important role in reproductive function and sexual desire. However, their role in modulating genital arousal is poorly understood. Insufficiency in sex steroid hormones may not only alter sexual desire at the level of the central nervous system, but also modify peripheral genital tissues, resulting in decreased blood flow and lubrication, altered mucification and smooth muscle contractility, and adverse effects on clitoral and vaginal arousal. It is our view that basic and clinical research in the next decade will shed light not only on the mechanisms of hormonal regulation of female sexual arousal but also on therapeutic values of androgens and estrogens in the management of female sexual arousal dysfunction.

Biosynthesis and metabolism of sex steroid hormones in women

Biosynthesis of androgens in the ovaries and adrenals

Androgen hormones are a class of C-19 steroids, produced by the gonads and the adrenals.33-35 Steroids with androgenic activity include testosterone, 5a-dihydrotestosterone (5a-DHT), A4-androstenedione, A5-androstenediol, 5a-androstane-3P, 17b-diol, dehydroepiandrosterone (DHEA), and 3a-hydroxy- androsterone (Fig. 5.5.1). Approximately 25% of androgen biosynthesis takes place in the ovaries, 25% is produced by the adrenal gland, and the remaining 50% is produced in the periphery.33,34,36 In women, essentially all of the androgens detected in the urine are of adrenal origin.10 In Addison’s disease, the output of urine androgens in the female approaches zero.

In the ovaries, cholesterol is metabolized to pregnenolone, which serves as the precursor for the synthesis of sex steroids.

Biosynthesis of testosterone from pregnenolone proceeds via participation of several key enzymes in two interrelated pathways, namely, the A5 and A4 pathways (Fig. 5.5.2). In the A5 pathway, hydroxylation of pregnenolone by 17a-hydroxylase and subsequent cleavage of the C-17,20 side chain by the C- 17,20-lyase produces dehydroepiandrosterone. The latter is converted to A5-androstenediol via 17P-hydroxysteroid dehydrogenase (17P-HSD). This derivative is converted into testosterone by the enzyme complex, 3P-hydroxy-A5-steroid dehydrogenase (3P-HSD), A4,5-isomerase. In the A4 pathway, pregnenolone is converted first into progesterone by 3P-HSD, A4,5-isomerase. Progesterone is then hydroxylated at the C17 position by the 17a-hydroxylase and becomes the substrate for the C-17,20-lyase, which converts 17a-hydroxyprogesterone to A4-androstenedione. This last product is metabolized to testosterone by the action of the 17P-hydroxysteroid dehydrogenase.

In the adrenal gland, cholesterol is metabolized to pregnenolone, which serves as the precursor for the synthesis of glucocorticoids and androgens. Similar to the ovarian synthetic pathway, pregnenolone is converted to dehydroepiandrosterone by the actions of 17a-hydroxylase and C-17,20-lyase (Fig. 5.5.2). At this stage, dehydroepiandrosterone is converted to A4-androstenedione via the enzyme complex, 3P-hydroxy- steroid dehydrogenase, A4,5-isomerase, and this product is then metabolized to testosterone by the action of the 17P-hydroxy- steroid dehydrogenase.

Biosynthesis of estrogens in the ovaries

Figure 5.5.1. Structures of steroid hormones with androgenic activity.

Figure 5.5.2. Biosynthesis of progesterone and androgenic steroid hormones. DHEA, dehydroepiandrosterone; ЭР-HSD, 3P-hydroxy-A5-steroid dyhydrogenase; 17P-HSD, 17P-hydroxy-A5-steroid dehydrogenase.

Synthesis of estrogen from androgens in the ovary is thought to involve both the thecal layer and the granulosa. The theca cells have a rich blood supply, and steroids synthesized in the theca can readily pass into the circulation. In contrast, the granulosa cell layer is relatively avascular, and steroids formed in these cells cross into the theca interna in order to enter the circulation. Both the theca and the granulosa express the aromatase enzyme systems for synthesis of estrogens (Fig. 5.5.3). In the theca cells, androstenedione and estradiol are derived from 17-hydroxypregnenolone (A5 pathway). In the granulosa, pregnenolone is readily converted into progesterone, suggesting that the primary mechanism of synthesis is through the A4 pathway. Estradiol is the major steroid detected in ovarian venous blood. The synthesis of estrogens from androgens is regulated by the gonadotrophic hormones luteinizing hormone (LH) and follicle- stimulating hormone (FSH). Follicle-stimulating hormone acts mainly on the granulosa cells, while luteinizing hormone acts on multiple sites, including the theca, stroma, luteum, and granulosa. The theca interna expresses luteinizing hormone receptors that regulate androgen biosynthesis, mainly androstenedione and testosterone. Androgens (D4-androstenedione and testosterone) produced by the thecal compartment diffuse into the follicular fluid, where they are converted into estrogens by the granulosa cells or released into the ovarian vein. The granulosa cells express follicle-stimulating hormone receptors, and an increase in follicle-stimulating hormone levels upregulates the number of follicle-stimulating hormone receptors due to increased granulosa cell number. Furthermore, follicle-stimulating hormone upregulates aromatase activity in the granulosa, thus increasing conversion of androgens into estrogens. Estradiol via autocrine or paracrine mechanisms increases the mitogenic activity, independent of that of follicle-stimulating hormone. Estradiol augments the activity of follicle-stimulating hormone in increasing aromatase activity and increasing the conversion of androgens to estrogens. In menopause, serum levels of follicle-stimulating hormone and luteinizing hormone rise,37 probably due to the loss of feedback mechanism. However, the increased levels of follicle- stimulating hormone and luteinizing hormone may explain the temporarily early rise in androgen levels in postmenopausal women due to the activity of these hormones on the interstitial cells. Eventually, the loss of expression of receptors for follicle- stimulating hormone and luteinizing hormone in ovaries of postmenopausal women and inactivation of steroidogenic enzymes result in loss of synthesis of androgens and estrogens in the ovaries.38 This suggests that the adrenals become the major source of androgens in the circulation in postmenopausal women.38

Figure 5.5.3. Biosynthesis of estrogens. In the ovaries, androgen precursors are primarily derived from 17-OH-pregnenolone or progesterone (see Fig. 5.5.2). Conversion of dehydroepiandrosterone (DHEA) sulfate or DHEA to androgens primarily takes place in the adrenals or other peripheral target tissues. Эв-HSD, 3P-hydroxy-A5-steroid dehydrogenase; 17P-HSD, 17P-hydroxy-A5-steroid dehydrogenase.

Peripheral conversion of androgens in target tissues

Conversion of precursor steroids, derived from adrenal or ovarian origin, into active androgens in peripheral tissues is an important pathway of androgen metabolism.35 Thus, dehydroepiandrosterone and A4-androstenedione may be converted into testosterone and 5a-DHT in target tissues.34,36 Labrie and his colleagues suggested that in postmenopausal women almost 100% of active sex hormones are derived from peripheral conversion of the steroid precursor dehydroepiandrosterone and dehydroepiandrosterone sulfate (DHEA-S) into active estrogens and androgen hormones.34,38 This concept suggests that active androgen hormones could be made on demand by the target tissues from precursors of ovarian or adrenal origin. This would also suggest that conversion of dehydroepiandrosterone and A4-androstenedione from adrenal or ovarian origin to testosterone and estradiol may take place in many tissues.

The conversion of dehydroepiandrosterone and A4- androstenedione into specific metabolites in the peripheral target tissues is catalyzed by tissue-specific, unidirectional 17P- hydroxysteroid dehydrogenases.36 A family of several enzymes have been cloned and characterized to date. These enzymes may play an important role in providing target tissues with active sex steroid hormones, via a well-controlled intracellular pathway. Thus, local conversion of dehydroepiandrosterone or dehydro- epiandrosterone sulfate into A4-androstenedione (via 3P-HSD) or A5-androstenediol (via 17P-hydroxysteroid dehydrogenase) leads to production of testosterone. Testosterone may be converted locally into 5a-dihydrotestosterone (via 5a-reductase) or into estradiol (via aromatase enzyme). A4-Androstenedione may be converted locally into estrone via the aromatase and into estradiol via 17P-hydroxysteroid dehydrogenase.36 Since different target tissues express specific and selective isoforms of 17P-hydroxysteroid dehydrogenase, it is likely that conversion of the adrenal androgen precursors into active androgen derivatives is regulated by a given tissue’s specific physiologic requirement.

Effects of plasma binding proteins on availability of bioactive androgens and estrogens

Plasma sex steroid binding proteins act as depot storage and transport sex steroid hormones from the site of synthesis to the target tissue. It is presumed that the free fraction of sex steroid hormone that enters the cells elicits the biologic response. Thus, the availability of bioactive hormone is dependent on the levels of plasma binding proteins and the affinity of these steroids for the protein. Since the free hormone also acts on the pituitary to regulate further synthesis of steroids and the free hormone is preferentially inactivated by the hepatic metabolism, plasma proteins play an important role in regulating the bioavailable steroids in the plasma. However, it remains unclear what role plasma binding proteins play in modulating the physiologic response. It is possible that changes in the levels of sex hormone-binding globulin may contribute significantly to androgen insufficiency in women. Sex hormone-binding globulin binds testosterone and estradiol with high affinity but does not bind androstenedione or estrone, neither does it bind dehydroepiandrosterone except for negligible amounts. The concentration of sex hormone-binding globulin increases in response to estrogens and increases fivefold during pregnancy. Sex hormone-binding globulin synthesis is influenced not only by estrogens but also by thyroid hormones.

Modulation of female genital arousal by sex steroid hormones

Estrogens are important in regulating the development, growth, and maintenance of many organs and tissues in women. In addition to their critical role in reproductive function, estrogens are important as metabolic regulators and modulate the development and growth of the mammary gland, genital tissue, bone, and skin. The role of estrogens in sexual behavior in rodents is well studied and well recognized.39,40 However, their role in human sexual behavior and sexual function is not well understood. The decline in circulating estrogen levels associated with menopause is thought to be responsible for many of the sexual complaints seen in postmenopausal women.41,42 Preclinical and clinical studies suggest that estrogens modulate genital hemodynamics and are critical for maintaining structural and functional integrity of vaginal tissues.11,15,43,44 Estrogen deprivation may lead to decreased pelvic blood flow, resulting in diminished vaginal lubrication, clitoral fibrosis, thinning of the vaginal wall, and decreased vaginal submucosal vasculature.42 In addition, estrogen deficiency leads to involution and atrophy of the genital organs, adversely affecting cervical, endocervical, and glandular mucin production. In contrast, estrogen replacement in postmenopausal women increases pelvic blood flow, reestablishing vaginal integrity and lubrication.42

Androgens modulate the function of many organs and tissues in women, including the pituitary, bone, adipose tissue, kidney, skeletal muscle, blood, ovaries, uterus, vagina, oviduct, clitoris, and mammary gland, and they regulate secondary sex characteristics.10 Androgens are not only essential for the development of reproductive function in women and hormonal homeostasis, but also represent the immediate precursors for the biosynthesis of estrogens. Androgens affect sexual desire, bone density, adipose tissue distribution, mood, energy, and wellbeing. Consequently, an imbalance in androgen biosynthesis or metabolism in women may have undesirable effects on general health and on sexual and reproductive functions.45 Although clinical studies have indicated that androgens modulate sexual arousal responses,6,18-21,2^32,46 no investigations have addressed the mechanisms by which androgens facilitate such responses.

Recent work has suggested that progesterone is an important signaling molecule in peripheral nerves, where it promotes myelin sheath formation by activating expression of specific hormone-sensitive genes.47 However, the role of progesterone on peripheral vaginal arousal is poorly understood. In the following section, we provide a brief discussion of the experimental data published to date on sex steroid hormones in modulating physiologic parameters of genital arousal as well as recent observations from our laboratory and others. Specifically, we will discuss the role of steroid hormones in modulating tissue structure, blood flow, lubrication and mucification, neurotransmitter biosynthesis and function, smooth muscle contractility, and expression of sex steroid receptors in genital tissue.

Effects of androgens and estrogens on vaginal tissue structure

Estrogens are crucial in maintaining tissue structure. In rat studies, it has been noted that ovariectomy caused considerable changes in vaginal epithelium and, to a limited extent, the muscularis and lamina propria. Estradiol treatment restored the changes in tissue structure as determined by histologic assays (M. A. Pessina et al., unpublished observations). The thickness of the vaginal epithelium was dramatically reduced by ovariectomy and was restored by estrogen administration (Fig. 5.5.4). Similar observations were noted when estradiol was coadministered with testosterone or progesterone. However, administration of testosterone alone or progesterone alone did not normalize the vaginal epithelium to that observed in intact control rats.

In intact animals, the muscularis layer is comprised of circular and longitudinal smooth muscle fibers. The muscle fibers are most notable in the upper layer of the vagina. Ovariectomy reduced the volume of the muscularis layer (M. A. Pessina et al., unpublished observations), and it was noted that there was considerable deposition of connective tissue between muscle bundles. Estradiol treatment of ovariectomized animals resulted in increased cross-sectional area of the muscularis, but compared with control animals, individual muscle fibers appeared enlarged, and there was less connective tissue between the muscle bundles. Testosterone treatment resulted in restoration of the muscularis fiber bundles, but not to the same extent as that noted for estradiol. Clearly, the effects of estradiol alone or testosterone alone on the fine structure of the muscularis need to be examined in more detail. Treatment of ovariectomized rats with progesterone did not have any significant effects on the muscularis layer. When ovariectomized rats were treated with estradiol plus testosterone or estradiol plus progesterone, the muscularis fiber bundle was restored with an appearance similar to that of control intact animals (Pessina et al., unpublished observation).

Examination of blood vessel density in the lamina propria confirmed that there is a rich vascular network of blood vessels. Interestingly, we (Pessina et al., unpublished data) observed no differences in vaginal tissues from intact and ovariectomized animals at 4 weeks. It is possible that the short period after ovariectomy does not produce sufficient changes in the lamina propria and that longer periods of time may be needed to observe such changes.

Figure 5.5.4. Effect of sex steroid hormones on vaginal epithelium. Vaginal tissue was obtained from intact control rats (C) or ovariectomized rats infused with vehicle (V) or physiologic concentrations of estradiol (E) or testosterone (T). Fixed tissue sections were stained with Gill's hematoxylin and photographed at x200 magnification to highlight the epithelial layers.

Modulation of genital blood flow by estrogens and androgens

In studies with animal models, hormone depletion by ovariectomy resulted in a significant reduction in vaginal blood flow after pelvic nerve stimulation when compared to controls.43,48,49

Estradiol or estradiol plus testosterone treatment of ovari- ectomized animals increased pelvic nerve-stimulated genital blood flow when compared to controls. Treatment of ovari- ectomized animals with testosterone alone did not result in increased genital blood flow in the rabbit,50 but was effective in increasing blood flow in the rat (Stankovic et al., unpublished observations). These observations suggest that estrogens and androgens regulate the vascular components of genital tissues.

Evidence suggests that sex steroid hormones may modulate blood flow by regulating the activities of vasoactive intestinal polypeptide (VIP) and neural and endothelial nitric oxide synthase (NOS) in the vagina.51-58 In ovariectomized rabbits, treatment with sex steroids had no effect on vasoactive intestinal polypeptide content. However, the binding affinity of vasoactive intestinal polypeptide to tissues of the genital tract was greatest in ovariectomized rabbits treated with estrogen and progesterone.55 Furthermore, administration of vasoactive intestinal polypeptide to postmenopausal women receiving no hormone replacement failed to increase vaginal blood flow, whereas those receiving hormone replacement exhibited increases in blood flow that were comparable to premenopausal women.56 In both rats and rabbits, total nitric oxide synthase in the vagina has been observed to be downregulated by estrogen and upregulated by progesterone.51-54 When specific isoforms of nitric oxide synthase are considered, estrogen has been reported to upregulate endothelial nitric oxide synthase in the rat vagina when assessed by immunohistochemistry.59 Using Western blot analyses, we have also corroborated these findings in ovari- ectomized rats replaced with estradiol (Traish et al., unpublished observations). However, other studies in rabbits have shown that ovariectomy increases both endothelial and neural nitric oxide synthase in the clitoris and vagina.57 Using an ovariectomized rabbit model, our studies indicate that neural nitric oxide synthase protein and enzyme activity in the vagina is downregulated by estrogen but upregulated by androgens.58

While the consistency and significance of these data remain unresolved, there appears to be differential regulation of nitric oxide synthase isoforms by various sex steroids, and this regulation may be cell type and species specific. In addition, while the regulation of phosphodiesterase (PDE) type 5 in female genital tissues by sex steroid hormones has not been examined, the sensitivity of the nitric oxide pathway to sex steroids may provide a partial explanation for the inconsistent results of several clinical studies with sildenafil in women in which both positive and negative results were obtained.60-64 It is possible that the endocrine status (sex steroid hormone insufficiency) of the patient may play an important role in determining whether phosphodiesterase type 5 inhibitors will be effective in facilitating the genital arousal response.

Modulation of vaginal lubrication by estrogens and androgens

Vaginal lubrication is an indicator of tissue health and increases significantly during genital sexual arousal, facilitating sexual intercourse. Production of vaginal fluid transudate and glycoproteins (such as mucin) contributes to the overall lubrication process. In estrogen-deprived animals, production of vaginal transudate, as measured by fluid weight absorbed by cotton swabs, was markedly decreased compared with controls and was restored by estrogen treatment.50 However, treatment with testosterone alone did not improve vaginal transudate production.48

Vaginal mucin production, assessed by tissue sialic acid content, has been reported to be stimulated by low doses of estrogen and reduced by high doses of estrogen.65-67 We have observed a significant decrease in vaginal sialic acid concentration in ovariectomized, vehicle-treated animals compared with the intact control group. Further decreases in vaginal sialic acid concentration were noted in the animals treated with high doses of estradiol, whereas testosterone treatment restored sialic acid to that of the vehicle treated group (Traish et al., unpublished observations). With regard to testosterone, these data are consistent with the findings of Kennedy and Armstrong, who have shown that androgens increase vaginal mucification in the rat.68,69 Treatment of ovariectomized rats with topical dehydro- epiandrosterone resulted in complete reversal of vaginal atrophy and stimulated proliferation and mucification of the vaginal epithelium.70

While vaginal fluid transudate production is dependent on genital blood flow and development of pressure within the vascular bed of the lamina propria, it remains unclear whether the synthesis and secretion of glycoproteins are modulated by acute hemodynamic events. Genital atrophy, in conjunction with diminished genital blood flow, secondary to estrogen deprivation, may bring about structural and functional changes in the genital tissues that negatively affect lubrication. Thus, while some effects of steroid hormones on vaginal lubrication have been noted, their exact roles and the mechanisms by which they act remain incompletely characterized.

Effects of androgens and estrogens on vaginal smooth muscle contractility

Genital sexual arousal induces changes in the tissue properties of the vaginal canal that are in part regulated by the tone of the smooth muscle within the muscularis layer. We have demonstrated that ovariectomy reduces smooth muscle relaxation to electric field stimulation and to exogenous vasoactive intestinal polypeptide in organ bath studies.71 Estrogen treatment of ovariectomized animals reduced the relaxation response. In contrast, androgen treatment facilitated vasoactive intestinal polypeptide-induced relaxation, suggesting that androgens may modulate neurotransmitter function. These observations suggest that androgens facilitate vaginal smooth muscle relaxation while estrogens attenuate this response. However, it should be stressed that these studies were performed with supraphysiologic levels of hormones. Future studies using plasma concentrations that approximate the physiologic range are required to corroborate these findings.

Effects of sex steroid hormones on vaginal neurotransmitters and innervation

Aside from the effects of sex steroids on nitric oxide production and vasoactive intestinal polypeptide receptors (see previous section on genital blood flow), it has been known for almost 40 years that adrenergic nerves of the female genital tract are sensitive to changes in the hormonal milieu.72,73 In animal studies, estrogen treatment increased the norepinephrine content of adrenergic nerves. In addition, administration of a mixture of estradiol and progesterone to ovariectomized pigs caused an increase in vaginal norepinephrine content.74 In contrast to the effects of estrogen on adrenergic neurotransmitter content, a recent report indicates that the density of innervation (assessed by the panneuronal marker PGP 9.5) increased in the rat vagina subsequent to ovariectomy and was reduced by estrogen administration.75 These changes were attributed to true axonal proliferation, rather than altered tissue volume, and consisted of adrenergic, cholinergic, and calcitonin gene-related peptide-containing nerves, which can mediate vasoconstriction and nociception. It was suggested that similar changes may explain the sensitivity and hyperalgesia of the vagina in postmenopausal women.

In our own studies using rats, we found no significant changes in vaginal nerve distribution or density after ovariectomy or with estradiol or progesterone replacement. In contrast, testosterone significantly increased the density of adrenergic nerve fibers, and this effect was attenuated when estradiol was coadministered with testosterone (Pessina et al., unpublished observations). These observations suggest differential regulation of vaginal innervation by sex steroid hormones. Clearly, additional studies undertaking detailed analyses of vaginal nerve fiber density and distribution are necessary to assess the effects of various hormones, alone or in combination, on vaginal histology and function.

Effects of steroid hormones on estrogen and androgen receptor expression in the vagina

Several studies have shown the presence of steroid receptors in the vagina by biochemical and immunochemical assays.76-83

While the effects of steroid hormones on the regulation of estrogen and progesterone receptors in reproductive organs have been extensively investigated,84 there are limited studies on the regulation of expression of sex steroid hormone receptors in the vagina. Steroid receptors are regulated differentially in different target tissues by sex steroid hormones. It has recently been reported that expression of the beta isoform of the estrogen receptor (ER) is diminished or lost in the vagina of postmenopausal women, suggesting that the loss of physiologic response may be mediated by this receptor isoform.83 Since hormone replacement therapy is used to treat various symptoms in postmenopausal women, it will be important to determine how sex steroids regulate the expression of vaginal steroid hormone receptors. Moreover, these studies will be invaluable in correlating the changes in receptor expression with changes in neurotransmitter function and modulation of the physiologic parameters of vaginal arousal (vaginal blood flow, lubrication, mucification, and smooth muscle contractility).

We have investigated the regulation of expression of estrogen and androgen receptors in the rat vagina under various endocrine manipulations.49 Since intact animals undergo an estrous cycle, a variation in estrogen receptor concentration is expected because of the changing levels of estradiol in the serum. After 4 weeks, ovariectomized animals exhibited increased levels of estrogen receptor in the total vaginal extract, whereas estradiol replacement (at physiologic concentrations) downregulated vaginal estrogen receptor. These changes in estrogen receptor were confirmed by radioligand binding and Western blot analyses. Ovariectomized animals treated with a physiologic dose of testosterone exhibited no change in estrogen receptor levels. Immunohistochemical data (unpublished observations) supported these biochemical data. For these reasons, we believe that estrogen receptor is negatively regulated by estradiol in the vagina, but unaffected by testosterone. Interestingly, treatment of ovariectomized rats with a subphysiologic dose of estradiol did not decrease estrogen receptor levels when compared with ovariectomized rats receiving no hormone.49 This suggests that a critical level of estrogen is required to downregulate its own receptor in the vagina. We suggest that downregulation of estrogen receptor by estrogen in the vagina is a mechanism that may be important to impart a refractory phase to attenuate the effect of estradiol surges during the estrus cycle.

In preliminary radioligand binding studies, we have observed androgen receptor levels in rat vaginal tissue to increase after ovariectomy and be down-regulated with estrogen treatment but unaltered by testosterone (unpublished observations). Similarly, progesterone receptor levels in rat vaginal tissue increased after ovariectomy and were down-regulated with estrogen treatment but unaltered by testosterone. Immunohistochemical and Western blot analyses yielded inconsistent trends with regard to androgens and with progesterone receptors, suggesting that detection limits of the antibody may be compromised by stability of the androgen receptor and progesterone receptor due to loss of a specific epitope on the protein in ovariectomized animals. The inconsistency between ligand binding studies and immunochemical assays may be attributed to partial proteolysis of the receptor in the absence of ligand. Pelletier et al. have shown that androgen receptor mRNA expression in mice is downregulated by ovariectomy and upregulated by estradiol administration.85 These studies investigated the early temporal relationship of androgen receptor expression 3-12 h after estradiol injection.

Summary and conclusions

Sex steroid hormones are critical in maintaining the structural and functional integrity of genital tissues and therefore are critical for genital arousal (genital blood flow, lubrication, muci- fication, and sensation). While the effect of androgens on sexual desire is well established, the role of sex steroid hormones in genital sexual arousal is not well understood and remains a subject of debate and controversy. Better understanding of the role of sex steroid hormones in modulating female sexual function requires investigation of the biochemical, cellular, and physiologic mechanisms by which sex steroid hormones modulate sexual function in general and genital sexual arousal in particular in experimental models. Future investigative efforts will benefit from the establishment of a host of experimental models and the advancement in biochemical and molecular biologic approaches for preclinical research. With the emerging consensus on female sexual dysfunction and sex steroid insufficiency, it is anticipated that the coming years will bring new advancement toward better understanding and management of female sexual dysfunction with sex steroid hormones.

References

1. Sherwin BB. Randomized clinical trials of combined estrogen- androgen preparations: effects on sexual functioning. Fertil Steril 2002; 77 (Suppl 4): S49-S54.

2. Burger HG, Hailes J, Menelaus M et al. The management of persistent menopausal symptoms with oestradiol-testosterone implants: clinical, lipid and hormonal results. Maturitas 1984; 6: 351-8.

3. Shorr E, Papanicolaou GN, Stimmel BF. Neutralization of ovarian follicular hormone in women by simultaneous administratin of male sex hormone. Proc Soc Exp Biol Med 1938; 38: 759-62.

4. Loeser A. Subcutaneous implantation of female and male hormone in tablet from in women. BMJ 1940; 1: 479-82.

5. Greenblatt RB, Wilcox EA. Hormonal therapy of fibromyomas of the uterus. South Surg 1941; 10: 339-46.

6. Greenblatt RB, Mortara F, Torpin R. Sexual libido in the female. Am J Obstet Gynecol 1942; 44: 658-63.

7. Salmon U. Rationale for androgen therapy in gynecology. J Clin Endocrinol 1941; 1: 162-79.

8. Salmon U, Geist SH. Effects of androgens upon libido in women. JCmEndocringl 1943; 3: 235-8.

9. Carter AC, Cohen EJ, Shorr E. The use of androgens in women. Vitam Horm 1947; 5: 317-91.

10. Dorfman RI, Shipley RA. Androgens: Biochemistry, Physiology and Clinical Significance. New York: Wiley, 1956: 152-217.

11. Bachmann GA, Ebert GA, Burd ID. Vulvovaginal complaints. In Lobo RA, ed. Treatment of the Postmenopausal Woman: Basic and Clinical Aspects. Philadelphia: Lippincott Williams & Wilkins 1999: 195-201.

12. Sarrel PM. Sexuality in the middle years. Obstet Gynecol Clin North Am 1987; 14: 49-62.

13. Sarrel PM. Ovarian hormones and vaginal blood flow: using laser Doppler velocimetry to measure effects in a clinical trial of postmenopausal women. Int J Impot Res 1998; 10 (Suppl 2): S91-3.

14. Sarrel PM. Effects of hormone replacement therapy on sexual psychophysiology and behavior in postmenopause. J Womens Health Gend Based Med 2000; 9 (Suppl 1): S25-S32.

15. Sarrel PM, Wiita B. Vasodilator effects of estrogen are not diminished by androgen in postmenopausal women. Fertil Steril 1997; 68: 1125-7.

16. Semmens JP, Wagner G. Estrogen deprivation and vaginal function in postmenopausal women. JAMA 1982; 248: 445-8.

17. Utian WH, Shoupe D, Bachmann G et al. Relief of vasomotor symptoms and vaginal atrophy with lower doses of conjugated equine estrogens and medroxyprogesterone acetate. Fertil Steril 2001; 75: 1065-79.

18. Sherwin BB, Gelfand MM. The role of androgen in the maintenance of sexual functioning in oophorectomized women. Psychosom Med 1987; 49: 397-409.

19. Sherwin BB, Gelfand MM, Brender W. Androgen enhances sexual motivation in females: a prospective, crossover study of sex steroid administration in the surgical menopause. Psychosom Med 1985; 47: 339-51.

20. Davis SR, McCloud P, Strauss BJ et al. Testosterone enhances estradiol’s effects on postmenopausal bone density and sexuality. Matuîitâs 1995; 21: 227-36.

21. Davis SR, Burger HG. The rationale for physiological testosterone replacement in women. Baillieres Clin Endocrinol Metab 1998; 12: 391-405.

22. Bachmann G, Bancroft J, Braunstein G et al. Female androgen insufficiency: the Princeton consensus statement on definition, classification, and assessment. Fertil Steril 2002; 77: 660-5.

23. Schreiner-Engel P, Schiavi RC, Smith H et al. Sexual arousability and the menstrual cycle. Psychosom Med 1981; 43: 199-214.

24. Schreiner-Engel P, Schiavi RC, White D et al. Low sexual desire in women: the role of reproductive hormones. Horm Behav 1989;

25. Hackbert L, Heiman JR. Acute dehydroepiandrosterone (DHEA) effects on sexual arousal in postmenopausal women. J Womens Health Gend Based Med 2002; 11: 155-62.

26. Tuiten A, van Honk J, Verbaten R et al. Can sublingual testosterone increase subjective and physiological measures of laboratory-induced sexual arousal? AïchGnPyikiQÜÏ 2002; 59: 465-6.

27. Tuiten A, Van Honk J, Koppeschaar H et al. Time course of effects of testosterone administration on sexual arousal in women. Arch Gen Psychiatry 2000; 57: 149-53.

28. Shifren JL, Braunstein GD, Simon JA et al. Transdermal testosterone treatment in women with impaired sexual function after oophorectomy. N Engl J Med 2000; 343: 682-8.

29. Shifren JL. The role of androgens in female sexual dysfunction. Mayo Clin Proc 2004; 79 ( Suppl): S19-S24.

30. Arlt W, Callies F, van Vlijmen JC et al. Dehydroepiandrosterone replacement in women with adrenal insufficiency. New Engl J Med 1999; 341: 1013-20.

31. Arlt W, Callies F, Allolio B. DHEA replacement in women with adrenal insufficiency - pharmacokinetics, bioconversion and clinical effects on well-being, sexuality and cognition. Endocr Res 2000; 26: 505-11.

32. Munarriz R, Talakoub L, Flaherty E et al. Androgen replacement therapy with dehydroepiandrosterone for androgen insufficiency and female sexual dysfunction: androgen and questionnaire results. J Sex Marital Ther 2002; 28 (Suppl 1): 165-73.

33. Abraham GE. Ovarian and adrenal contribution to peripheral androgens during the menstrual cycle. J Clin Endocrinol Metab 1974; 39: 340-6.

34. Labrie F, Diamond P, Cusan L et al. Effect of 12-month dehy- droepiandrosterone replacement therapy on bone, vagina, and endometrium in postmenopausal women J Clin Endocrinol Metab 1997; 82: 3498-3505.

35. Labrie F, Luu-The V, Lin S et al. Role of 17beta-hydroxysteroid dehydrogenases in sex steroid formation in peripheral intracrine tissues. Trends Endocrinol Metab 2000; 11: 421-7.

36. Luu-The V, Dufort I, Pelletier G et al. Type 5 17beta-hydroxys- teroid dehydrogenase: its role in the formation of androgens in women. MolÇdlEndoçrmgl 2001; 171: 77-82.

37. Landgren BM, Collins A, Csemiczky G et al. Menopause transition: annual changes in serum hormonal patterns over the menstrual cycle in women during a nine-year period prior to menopause. J Clin Endocrinol Metab 2004; 89: 2763-9.

38. Couzinet B, Meduri G, Lecce MG et al. The postmenopausal ovary is not a major androgen-producing gland. J Clin Endocrinol Metab 2001; 86: 5060-6.

39. Mani S. Emerging concepts in the regulation of female sexual behavior. Sca^dlP^^jol 2003; 44: 231-9.

40. Erskine MS, Lehmann ML, Cameron NM et al. Co-regulation of female sexual behavior and pregnancy induction: an exploratory synthesis. Behav Brain Res 2004; 153: 295-315.

41. Notelovitz M. Management of the changing vagina. J Clin Pract Sex 1990; Special Issue: 16-17.

42. Bachmann GA. The impact of vaginal health on sexual function. J Clin Pract Sex (Special Issue) 1990; 18-21.

43. Park K, Ahn K, Lee S et al. Decreased circulating levels of estrogen alter vaginal and clitoral blood flow and structure in the rabbit. lntUmpotRes 2001; 13: 116-24.

44. Park K, Ryu SB, Park YI et al. Diabetes mellitus induces vaginal tissue fibrosis by TGF-beta 1 expression in the rat model. J Sex Marital Ther 2001; 27: 577-87.

45. Breuer H. Androgen production in the woman. In Hammerstein J, Lachnit-Fixson U, Neumann F, Plewig G, eds. Androgenization in Women. Princeton: Excerpta Medica 1980: 21-39.

46. Sherwin BB. The impact of different doses of estrogen and progestin on mood and sexual behavior in postmenopausal women. J Clin Endocrinol Metab 1991; 72: 336-43.

47. Schumacher M, Guennoun R, Mercier G et al. Progesterone synthesis and myelin formation in peripheral nerves. Brain Res Brain Res Rev 2001; 37: 343-59.

48. Min K, Munarriz R, Kim NN et al. Effects of ovariectomy and estrogen and androgen treatment on sildenafil-mediated changes in female genital blood flow and vaginal lubrication in the animal model. 2002; 187: 1370-6.

49. Kim SW, Kim NN, Jeong Seong-Joo et al. Modulation of rat vaginal blood flow and estrogen receptors by estradiol. J Urol 2004; 172: 1538-43.

50. Min K, Munarriz R, Kim NN et al. Effects of ovariectomy and estrogen replacement on basal and pelvic nerve stimulated vaginal lubrication in an animal model. JSexMartaLThei 2003; 29 (Suppl 1): 77-84.

51. Batra S, Al-Hijji J. Characterization of nitric oxide synthase activity in rabbit uterus and vagina: downregulation by estrogen. Life Sci 1998; 62: 2093-3010.

52. Al-Hijji J, Batra S. Down regulation by estrogen of nitric oxide synthase activity in the female rabbit lower urinary tract. Urology 1999; 53: 637-41.

53. Al-Hijji J, Larsson B, Batra S. Nitric oxide synthase in the rabbit uterus and vagina: hormonal regulation and functional significance. BjolReprod 2000; 62: 1387-92.

54. Al-Hijji J, Larsson I, Batra S. Effect of ovarian steroids on nitric oxide synthase in the rat uterus, cervix and vagina. Life Sci 2001; 69: 1133-42.

55. Ottesen B, Pedersen B, Nielsen J et al. Vasoactive intestinal polypeptide (VIP) provokes vaginal lubrication in normal women. Peptides 1987; 8: 797-800.

56. Palle C, Bredkjaer HE, Ottesen B et al. Vasoactive intestinal polypeptide and human vaginal blood flow: comparison between transvaginal and intravenous administration. J Clin Exp Pharm Physiol 1990; 17: 61-8.

57. Yoon HN, Chung WS, Park YY et al. Effects of estrogen on nitric oxide synthase and histological composition in the rabbit clitoris and vagina. Int J Impot Res 2001; 13: 205-11.

58. Traish AM, Kim NN, Huang YH et al. Sex steroid hormones differentially regulate nitric oxide synthase and arginase activities in the proximal and distal rabbit vagina. Int J Impot Res 2003; 15: 397-404.

59. Berman JR, McCarthy MM, Kyprianou N. Effect of estrogen withdrawal on nitric oxide synthase expression and apoptosis in the rat vagina. Urology 1998; 51: 650-6.

60. Basson R, McInnes R, Smith MD et al. Efficacy and safety of sildenafil citrate in women with sexual dysfunction associated with female sexual arousal disorder. J Womens Health Gend Based Med 2002; 11: 367-77.

61. Caruso S, Intelisano G, Farina M et al. The function of sildenafil on female sexual pathways: a double-blind, cross-over, placebo- controlled study. Eur J Obstet Gynecol Reprod Biol 2003; 110: 201-6.

62. Caruso S, Intelisano G, Lupo L et al. Premenopausal women affected by sexual arousal disorder treated with sildenafil: a double

192 Women's Sexual Function and Dysfunction: Study, Diagnosis and Treatment blind, cross-over, placebo-controlled study. Br J Obstet Gynaecol 2001; 108: 623-8.

63. Laan E, van Lunsen RH, Everaerd W et al. The enhancement of vaginal vasocongestion by sildenafil in healthy premenopausal women. jm womens health gend med2002;

64. Berman JR, Berman LA, Toler SM et al. Sildenafil Study Group. Safety and efficacy of sildenafil citrate for the treatment of female sexual arousal disorder: a double-blind, placebo controlled study. J Urol 2003; 170: 2333-8.

65. Carlborg L. Comparative action of various oestrogenic compounds on mouse vaginal sialic acids. II. Acta Endocrinol (Copenh) 1969; 62: 663-70.

66. Galletti F, Gardi R. Effect of ovarian hormones and synthetic progestins on vaginal sialic acid in the rat. J Endocrinol 1973; 57: 193-8.

67. Nishino Y, Neumann F. The sialic acid content in mouse female reproductive organs as a quantitative parameter for testing the estrogenic and antiestrogenic effect, antiestrogenic depot effect, and dissociated effect of estrogens on the uterus and vagina. Acta Endocrinol Suppl (Copenh) 1974; 187: 3-62.

68. Kennedy TG. Vaginal mucification in the ovariectomized rat in response to 5alpha-pregnane-3,20-dione, testosterone and 5alpha- androstan-17beta-ol-3-one: test for progestogenic activity. J Endocrinol 1974; 61: 293-300.

69. Kennedy TG, Armstrong DT. Induction of vaginal mucification in rats with testosterone and 17beta-hydroxy-5alpha-androstan-3- one. Steroids 1976; 27: 423-30.

70. Sourla A, Flamand M, Belanger A et al. Effect of dehy- droepiandrosterone on vaginal and uterine histomorphology in the rat. Jj steroid biochem mol bio 1998; 66: 137-49.

71. Kim NN, Min K, Pessina MA et al. Effects of ovariectomy and steroid hormones on vaginal smooth msucle contractility. Int J Impot Res 2004; 16: 43-50.

72. Rosengren E, Sjoberg NO. Changes in the amount of adrenergic transmitter in the female genital tract of rabbit during pregnancy. Acta Physiol Scand 1968; 72: 412-24.

73. Sjoberg NO. Increase in transmitter content of adrenergic nerves

in the reproductive tract of female rabbits after oestrogen treatment. Acta Endocrinol (Copenh) 1968; 57: 405-13.

74. Kaleczyc J. Effect of estradiol and progesterone on noradrenaline content in nerves of the oviduct, uterus and vagina in ovariec- tomized pigs. Folia Histochem Cytobiol 1994; 32: 119-26.

75. Ting AY, Blacklock AD, Smith PG. Estrogen regulates vaginal sensory and autonomic nerve density in the rat. Biol Reprod 2004;

76. MacLean AB, Nicol LA, Hodgins MB. Immunohistochemical localization of estrogen receptors in the vulva and vagina. J Reprod Med 1990; 35: 1015-16.

77. Hodgins MB, Spike RC, Mackie RM et al. An immunohistochemical study of androgen, oestrogen and progesterone receptors in the vulva and vagina. br j, obstet gynaecol 1998; 105: 216-22.

78. Chen GD, Oliver RH, Leung BS et al. Estrogen receptor alpha and beta expression in the vaginal walls and uterosacral ligaments of premenopausal and postmenopausal women. Fertil Steril 1999; 71: 1099-1102.

79. Blakeman PJ, Hilton P, Bulmer JN. Oestrogen and progesterone receptor expression in the female lower urinary tract, with reference to oestrogen status. Br J Urol Int 2000; 86: 32-38.

80. Mowa CN, Iwanaga T. Differential distribution of oestrogen receptor-alpha and -beta mRNAs in the female reproductive organ of rats as revealed by in situ hybridization. J Endocrinol 2000; 165:59-66.

81. Schwartz PE. The oestrogen receptor (ER) in vulva, vagina and ovary. Eur J Cancer 2000; 36 (Suppl 4):S31-S32.

82. Wang H, Eriksson H, Sahlin L. Estrogen receptors alpha and beta in the female reproductive tract of the rat during the estrous cycle. Biol Reprod 2000; 63:1331-1340.

83. Gebhart JB, Rickard DJ, Barrett TJ et al. Expression of estrogen receptor isoforms alpha and beta messenger RNA in vaginal tissue of premenopausal and postmenopausal women. Am J Obstet Gynecol 2001; 185:1325-1330.

84. Clark JH, Peck EJ Jr. Female sex steroids: receptors and function. Monogr Endocrinol 1979; 14:I-XII, 1-245.

85. Pelletier G, Luu-The V, Li S et al. Localization and estrogenic regulation of androgen receptor mRNA expression in the mouse uterus and vagina. JEn^o^m 2004; 180:77-85.