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REVIEW Biochemical Factors Modulating Female Genital Sexual Arousal Physiology

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Abdulmaged M. Traish, PhD,* Ella Botchevar, MA, and Noel N. Kim, PhD
*Boston University School of MedicineDepartments of Biochemistry and of Urology, Boston, MA, USA; Boston University School of MedicineDepartment of Urology, Boston, MA, USA; Institute for Sexual Medicine, San Diego, CA, USA DOI: 10.1111/j.1743-6109.2010.01903.x

ABSTRACT

Introduction. Female genital sexual arousal responses are complex neurophysiological processes consisting of central and peripheral components that occur following sexual stimulation. The peripheral responses in sexual arousal include genital vasocongestion, engorgement and lubrication resulting from a surge of vaginal and clitoral blood ow. These hemodynamic events are mediated by a host of neurotransmitters and vasoactive agents. Aim. To discuss the role of various biochemical factors modulating female genital sexual arousal responses. Methods. A comprehensive literature review was conducted using the PubMed database and citations were selected, based on topical relevance, and examined for study methodology and major ndings. Main Outcome Measures. Data from peer-reviewed publications. Results. Adrenergic as well as non-adrenergic non-cholinergic neurotransmitters play an important role in regulating genital physiological responses by mediating vascular and non-vascular smooth muscle contractility. Vasoactive peptides and neuropeptides also modulate genital sexual responses by regulating vascular and non-vascular smooth muscle cells and epithelial function. The endocrine milieu, particularly sex steroid hormones, is critical in the maintenance of tissue structure and function. Reduced levels of estrogens and androgen are associated with dramatic alterations in genital tissue structure, including the nerve network, as well as the response to physiological modulators. Furthermore, estrogen and androgen deciency is associated with reduced expression of sex steroid receptors and most importantly with attenuated genital blood ow and lubrication in response to pelvic nerve stimulation. Conclusions. This article provides an integrated framework describing the physiological and molecular basis of various pathophysiological conditions associated with female genital sexual arousal dysfunction. Traish AM, Botchevar E, and Kim NN. Biochemical factors modulating female genital sexual arousal physiology. J Sex Med 2010;7:29252946. Key Words. Vagina; Clitoris; Sex Steroid Hormones; Sexual Arousal; Blood Flow; Sexual Dysfunction

Introduction

exual function in women is often described in relation to the response phases of desire, arousal, orgasm, and resolution. Genital sexual arousal is a neuro-vascular process under both central and peripheral regulation, and is profoundly inuenced by cognitive and social factors. This review focuses on peripheral mechanisms within the genital tissues that regulate an important part of the female genital sexual arousal response.
2010 International Society for Sexual Medicine

The genital arousal response in women is manifested by engorgement and swelling of genital tissues, clitoral engorgement, increased compliance of the vaginal wall, and production of lubricating mucus and uid transudate from the cervix, periurethral glands, and vagina [1]. These physiological events are dependent upon the structural integrity of the genital tissues and the function of neural, endocrine, and vascular systems that regulate and coordinate the genital arousal response (Figure 1). Genital vasocongestion and vaginal
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Traish et al.

Figure 1 Interaction between cellular constituents of the vagina and the physiological processes mediated by them. (Adapted from Traish AM et al. Drug Discovery Today: Disease Mechanisms 2004;1:9197).

lubrication result from increased blood ow to the clitoris, vagina, and labia. These hemodynamic processes are regulated by the tone of the vascular smooth muscle of the erectile tissue and blood vessels within the genital tissues. Vaginal compliance is largely determined by the tone of the nonvascular smooth muscle within the vaginal muscularis and the skeletal muscle that supports the vagina. While there is limited understanding of the regulatory mechanisms modulating genital tissue smooth muscle tone and how these mechanisms are altered by disease states, increasing research efforts within the past decade have extended earlier studies and helped elucidate some of the basic physiology of genital arousal in women (Figure 2).
Modulation of Female Genital Arousal Response by Neurotransmitters

A number of studies have demonstrated the presence of adrenergic, cholinergic, and nonadrenergic non-cholinergic (NANC) neurotransmitters in vaginal and clitoral tissue from various
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species (Table 1) [214]. Immunohistochemical studies in vaginal and clitoral tissues demonstrated the presence of nerve bers containing tyrosine hydroxylase (a marker for cathecholamines that has been used to identify sympathetic nerve bers) [15], choline acetyltransferase (a marker for cholinergic nerves), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP) and substance P [3,911,16]. Despite the presence of cholinergic nerve bers, no functional evidence has been reported for acetylcholine as a direct regulator of genital blood ow [17]. Interestingly, extra-neuronal choline acetyltransferase and acetylcholine have been detected in rat and human epithelial cells, including the epithelium of the vagina, and have been postulated to regulate cell growth [18]. Thus far, the most compelling evidence indicates that the adrenergic, VIP and nitric oxide (NO) signaling systems play important roles in regulating genital blood ow. These systems are described in Table 1 and discussed in the following subsections. In contrast, few studies have investi-

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Figure 2 Biochemical pathways regulating vaginal smooth muscle contractility. Mechanisms that regulate vascular and non-vascular smooth muscle contractility in the vagina are outlined. The importance of each pathway in the 2 different types of smooth muscle has not been fully elucidated. Other regulatory pathways such as the prostanoid and endothelin systems have not been extensively investigated and are not depicted. Potential targets for therapeutic intervention are highlighted within the shaded boxes. Inhibitory regulation is represented by a dashed arrow. Purinoceptor-mediated mucin secretion occurs only in epithelial cells (see inset box). Abbreviations: AR = adrenoceptor; 5-AMP = 5-adenosine monophosphate; 5-GMP = 5-guanosine monophosphate; cAMP = adenosine 3,5-cyclic monophosphate; cGMP = guanosine 3,5-cyclic monophosphate; Ca 2+ = calcium; DAG = diacylglycerol; IP3 = inositol trisphosphate; L-Arg = L-arginine; NO = nitric oxide; NOS = nitric oxide synthase; PDE = phosphodiesterase; PR = purinoceptor; PK-G = protein kinase G; VIP = vasoactive intestinal polypeptide; VIP-R = VIP receptor. (Used with permission, Traish AM et al. copyright, Drug Discovery Today: Disease Mechanisms 2004; 1: 9197).

gated the role of peptide neurotransmitters in regulating genital arousal.

Regulation of Vaginal and Clitoral Smooth Muscle Contractility by Adrenergic Neurotransmitters In the basal, non-stimulated state, norepinephrine from sympathetic nerve terminals causes vascular and non-vascular smooth muscle contraction in the genital organs, maintaining low genital blood ow and a high degree of tone to the vaginal wall. The adrenergic system is also thought to be important in terminating the engorged state after genital arousal has ceased. Stimulation of the hypogastric nerve results in marked reduction in clitoral and vaginal blood ow (Traish AM et al. unpublished work). Expression of a1- and a2-adrenergic receptors has been demonstrated in the vagina by radioligand binding studies and organ bath preparations of vaginal and clitoral tissue and in vivo animal studies have provided evidence for the function of these receptors [5,19]. In organ bath studies, exogenous norepinephrine causes dose-dependent contraction in tissue strips of clitoral corpus cavernosum

and vagina [5,19,20]. Furthermore, in isolated strips of rat vaginal tissue, contraction to norepinephrine has been shown to be attenuated by the a-adrenergic receptor antagonists prazosin and phentolamine [21]. In anesthetized rabbits, intravaginal injection of the a-adrenergic receptor antagonist phentolamine mesylate (500 mg/kg) dissolved in 10% (v/v) N,N-dimethylformamide in sterile, deionized water in a total volume of 0.1 mL, caused a marked increase in the amplitude and duration of the engorgement response with and without pelvic nerve stimulation [5]. In clinical studies, phentolamine mesylate has been suggested to improve vaginal lubrication in postmenopausal women with female sexual arousal disorder [22]. In addition, the a-2 adrenergic receptor agonist clonidine impaired both vaginal engorgement (photoplethysmography) and subjective sexual response (self report) when administered to healthy volunteers [23]. Elevated systemic blood pressure and positive chronotropic/ inotropic effects on the heart, secondary to moderate sympathetic activation, would facilitate
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Table 1
Class Neuropeptides

Traish et al.
Biochemical and physiological modulators of female genital sexual arousal response
Biochemical modulator Vasoactive intestinal polypeptide (VIP) Source Vipergic nerves innervating clitoral and vaginal tissues. Proposed physiological function Mediates nonadrenergic noncholinergic (NANC) relaxation of clitoral and vaginal vascular and non-vascular smooth muscle. PHM is a VIP precursor; mediates vasodilation and relaxation of the non-vascular smooth muscle. Mediates nonadrenergic noncholinergic (NANC) relaxation response of clitoral and vaginal vascular and non-vascular smooth muscle. Mediates tone in specic vascular beds. Mediates sensory function.

Peptide histidine methionine (PHM) Pituitary adenylate cyclase activating polypeptide (PACAP) Calcitonin gene-related peptide (CGRP) Substance P

Co-localizes with VIP nerve endings innervating clitoral and vaginal tissues. Nerve endings of clitoral and vaginal nerve ber network.

Neuropeptide Y (NPY) C-anking peptide of NPY (CPON) Neurotransmitters Norepinephrine (NE)

Sensory C-bers innervating clitoral and vaginal smooth muscle. Sensory C-bers innervating clitoral and vaginal vascular and non-vascular smooth muscle. Nerve endings innervating vaginal smooth muscle; localized near blood vessels. Sympathetic/parasympathetic nerve bers innervating sub-epithelial blood vessels. Sympathetic adrenergic nerve bers innervating vaginal and clitoral tissue.

Vasoconstrictor activity and attenuation of blood ow. Mediates vasodilation in specic vascular beds; involved in sensory pathways. Mediates vascular and non-vascular smooth muscle contractility in the genital organs, attenuates blood ow and maintains a high degree of tone to the vaginal wall. Mediates NANC neurogenic relaxation response of vaginal and clitoral cavernosal smooth muscle; regulates vaginal blood ow and vaginal wall contractility. No physiological function has been dened to date. Maintain genital tissue structural integrity and facilitate pelvic nerve-stimulated genital blood ow; modulate neural and endothelial NOS. Maintain genital tissue structural integrity; maintains adrenergic nerve bers; enhances VIP-induced relaxation of vaginal smooth muscle; modulate neural and endothelial NOS; facilitates pelvic nerve-stimulated genital blood ow.

Nitric oxide (NO)

NANC nerve bers innervating vaginal and clitoral tissue; endothelial cells.

Acetylcholine (Ach) Sex steroid hormones Estradiol

Cholinergic nerves innervating vaginal and clitoral tissue. Ovaries, peripheral tissues, adipose tissue and breasts.

Testosterone

Ovaries and adrenals.

genital engorgement by increasing the pressure gradient between the larger resistance arteries and the local genital circulation. Thus, genital (clitoris and vagina) tissue expresses functional a1- and a2-adrenergic receptors, which modulate the tone of the vascular and non-vascular smooth muscle in the clitoris and vagina, suggesting that the adrenergic pathway is an important regulator of genital hemodynamics.

Regulation of Vaginal and Clitoral Smooth Muscle Contractility by NO Recent studies in animal models have suggested that the NO/cGMP pathway is an important signaling mechanism that modulates clitoral and vaginal blood ow [16,20,24,25]. Immunostaining with antibodies against nNOS in the cavernosal
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body of the clitoris revealed a rich network of nerve bundles and terminal branches, suggesting that the NO/cGMP pathway contributes to tissue vasocongestion and physiological arousal during sexual stimulation [26,27]. In addition to immunohistochemical demonstration of neuronal and endothelial NOS, contractility experiments in isolated clitoral cavernosal tissue strips indicated that non-adrenergic, non-cholinergic relaxation to electrical stimulation was potentiated by the phosphodiesterase (PDE) type 5 inhibitor sildenal and sensitive to tetrodotoxin, the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME), and the guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) [16, 20,24,25]. In human vaginal tissue, immunoreactivity for PDE type 5 was localized in the endothe-

Biochemical Factors Modulating Female Sexual Arousal Physiology lium and smooth muscle of blood vessels [28]. In vivo studies in rat, rabbit, and dog have also provided further support for this mechanism in the clitoris and vagina. Using inhibitors of key enzymes in the NO/cGMP pathway, changes in genital tissue engorgement (using near-infrared spectroscopy) and vaginal blood ow (using laser Doppler owmetry) were investigated. Administration of L-NAME or ODQ markedly attenuated genital engorgement and vaginal blood ow in response to pelvic nerve stimulation, whereas administration of sildenal enhanced genital engorgement and vaginal blood ow [2931]. Topical administration of a novel NO donor, linear polyethylenimineNO/nucleophile adducts (DS1), enhanced vaginal blood ow in anesthetized rats [32]. In addition, vaginal and clitoral smooth muscle cells in culture have been shown to express PDE type 5 [33,34]. Moreover, in rat tissue, clitoral relaxation induced by neurovascular derived NOS occurs via activation of protein kinase G (PKG)-dependent calcium-activated potassium channels (BKCa) [35]. Clinical studies have shown that in healthy women, sildenal signicantly improved arousal, orgasm, and enjoyment when compared with placebo [36,37]. However, other clinical studies have shown no improvement in womens sexual function [38,39]. Thus, while the efcacy of PDE type 5 inhibitors in treating female sexual dysfunction remains unresolved, data from laboratory studies suggest that the NO/cGMP pathway is an important regulator of vaginal and clitoral hemodynamics [26,27,29,33,34,4043]. The role of NO in mediating relaxation of the non-vascular smooth muscle in the vagina remains unclear. Giraldi et al. using organ bath studies with strips of rat vagina have shown that L-NAME completely inhibits the relaxation response caused by electrical stimulation [3]. However, L-NAME only partially reduced neurogenic relaxation in studies using rabbit vaginal tissue strips [40]. The relaxation response could not be inhibited by protease inhibitors, potassium channel blockers, or purinergic receptor antagonists. Whether these differences are due to variability between different species remains to be resolved [20]. A novel regulator of the NO pathway that may have particular relevance in genital tissues is the enzyme arginase, a metalloenzyme that catalyzes the hydrolysis of L-arginine to form L-ornithine and urea. Arginase appears to play a role in regulating L-arginine bioavailability and can indirectly regulate NO synthase activity. The advent of 2(S)amino-6-boronohexanoic acid (ABH), a transition

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state analog inhibitor of arginase, permitted further characterization of the arginase enzyme. In anesthetized rabbits, intravenous administration of the arginase inhibitor ABH (46 mg/kg dissolved in 40% propylene glycol) followed by pelvic nerve stimulation caused a 2.1-fold increase in genital tissue oxyhemoglobin and a 34% increase in vaginal blood ow, suggesting that arginase has the ability to regulate genital blood ow by modulating NOS [44].

Modulation of Vaginal and Clitoral Function by Neuropeptides The distributions of neuropeptides in human clitoris was investigated by Hauser-Kronberger et al. using immunohistochemistry and demonstrated a complex network of nerve bers containing VIP, PHM, NPY, C-anking peptide of NPY (CPON), CGRP, and substance P [11]. Immunohistochemical studies have also localized VIP-containing nerves to the vaginal wall. Several reports have suggested that VIP is involved in the regulation of clitoral and vaginal vascular and non-vascular smooth muscle tone [4549]. Exogenous VIP has been shown to cause a concentration-dependent increase in vaginal blood ow and lubrication [47,50], and in vitro organ bath studies showed that VIP facilitates vaginal and clitoral smooth muscle relaxation [19]. However, there has been no conclusive experimental evidence for the functional involvement of endogenous VIP because of the lack of specic and selective antagonists for VIP receptors. PHM is a VIP precursor and is co-localized in nerve bers with VIP in the female reproductive tract. In the vagina, PHM is present in higher concentrations than VIP and can cause relaxation of the nonvascular smooth muscle by acting through the same receptors as VIP [13,40]. PHM has also been reported to increase vaginal blood ow in women [49]. Physiological studies in organ baths have demonstrated that exogenous VIP induced relaxation of pre-contracted vaginal non-vascular smooth muscle [19,40,51,52]. VIP caused a dosedependent relaxation in vaginal strips from intact and ovariectomized animals [19]. VIP-induced relaxations were augmented in tissue strips from ovariectomized animals treated with testosterone (T) and were attenuated in tissue from animals treated with estradiol (E2), suggesting modulation of VIP action by steroid hormones. Aughton et al. investigated the relaxation response induced in rabbit vaginal strips by VIP and pituitary adenylate
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2930 cyclase activating polypeptide (PACAP). The authors demonstrated relaxation in a dose dependent fashion for VIP and PACAP, and these responses were attenuated in the presence of a VIP antagonist (VIP 628) and a PACAP antagonist (PACAP 638), respectively, suggesting specic physiological response to these peptides [51]. Elevated levels of cAMP are associated with VIPinduced relaxation, further implicating unique pathways in genital smooth muscle contractility and demonstrating the complexity of this signaling mechanism [40]. NPY has been detected at relatively high concentrations in human, rabbit, and rat vagina [3,9,5355]. Because of its primary localization within nerve bers near blood vessels, it has been postulated that NPY regulates blood ow in the vagina. In studies with isolated tissue in organ baths, NPY constricted small arteries from human cervix and had an additive effect on that of norepinephrine [53]. In rabbits, NPY decreased myometrial blood ow [55]. While NPY does not alter the contractility of the vaginal muscularis [51], these studies suggest that NPY has vasoconstrictor activity in the uterus and cervix, and may have a similar function in the vagina. While substance P and CGRP are known to be involved primarily in sensory pathways, these peptide neurotransmitters have been shown to mediate vasodilation in specic vascular beds of the brain and ovaries [56,57]. Their roles in modulating the sexual arousal response remain to be elucidated. Thus, the exact mechanisms by which neurotransmitters modulate genital arousal responses are subjects of continuing investigation.
Modulation of Female Genital Arousal Response by Sex Steroid Hormones

Traish et al. pathway contributes to tissue vasocongestion and physiological arousal response during sexual stimulation [26,27,60,61]. The role of NO/cGMP in the clitoris may be different from that of the vagina. Similar to that in the vagina, the clitoris expresses ERa [60] and estrogens have an important role in the clitoral physiological response. Similar to studies in the vagina, clitoral blood ow is thought to be a valid and sensitive tool to measure female genital response [62]. Also, treatment of women with female sexual arousal disorder with tibolone signicantly increased clitoral peak systolic and end diastolic velocity with improvement of sexual function, as measured by McCoy Female Sexuality Questionnaire (MFSQ), after 6 months of treatment [63]. These data suggest that these different genital organs, while exhibiting different anatomical structure, share signaling pathways and hormonal responses that regulate their physiological process, thus contributing and augmenting the genital sexual arousal response. There is growing evidence that sex steroids (androgens and estrogens) regulate the structure and function of genital organs and modify the sexual arousal response. Sex steroid hormones (e.g., E2, T, 5a dihydrotestosterone (5a-DHT) progesterone) regulate distinct cellular components of the vagina (Figure 3). Each of these cellular interactions inuences specic physiological events such as growth and function of neurons, blood vessels, smooth muscle, and epithelial cells. Androgens and estrogens regulate: (i) synthesis, secretion, and reuptake of neurotransmitters; (ii) vascular and nonvascular smooth muscle contractility; (iii) mucication, keratinization, and permeability of the epithelium; and (iv) production of autocrine and paracrine growth factors and vasoactive and trophic substances. Further, androgens and estrogens may regulate the synthesis and deposition of the connective tissue matrix within the vagina. In women, E2 is important in regulating the development, growth, and maintenance of many organs and tissues. In addition to their critical role in reproductive function, estrogens are important metabolic regulators and modulate development, growth, and function of the mammary glands, genital tissue, bone, and skin. The role of estrogens in sexual behavior in rodents is well recognized [64,65]. However, their role in human sexual behavior and sexual function is not well understood. The decline in circulating E2 levels associated with menopause is thought to be responsible

The role of the clitoris in genital sexual arousal is well accepted; however, to what extent the clitoris and vagina contribute to this process is debatable. Sexual stimulation of the clitoris has been shown to increase cerebral blood ow in the left secondary and right dorsal primary somatosensory cortex, providing the rst account of neocortical processing of sexual clitoral information [58]. Similar to studies in the vagina, clitoral tissue from human and animals showed that the clitoris is innervated with pudendal and hypogastric nerves [59]. Further, immunostaining with antibodies against nNOS in the cavernosal body of the clitoris revealed a rich network of nerve bundles and terminal branches suggesting that the NO/cGMP
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Figure 3 Proposed mechanisms of sex steroid hormone action in regulating vaginal function. Binding of steroid hormones to specic receptors (intracellular or membrane bound) leads to genomic and non-genomic activation of cellular processes. (With permission, Traish AM et al. copyright, Drug Discovery Today: Disease Mechanisms 2004; 1: 9197).

for many of the sexual complaints in postmenopausal women [66,67]. Pre-clinical and clinical studies suggest that E2 modulates genital hemodynamics and is critical for maintaining the structure and function of vaginal and clitoral tissues [43,6871]. Estrogen deprivation leads to decreased pelvic blood ow, resulting in diminished vaginal lubrication, clitoral brosis, thinning of the vaginal wall, and decreased vaginal submucosal vasculature [66]. In addition, estrogen deciency 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 ow, re-establishing vaginal integrity and lubrication.

Androgens (T and 5 a dihydrotestosterone) 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 regulate secondary sex characteristics [72]. It should be emphasized that 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. T affects sexual desire, bone density, adipose tissue distribution, mood, energy, and well being. Consequently, an imbalance in androgen biosynthesis or metabolism in women may have undesirable effects on general health and on sexual and reproductive functions
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2932 [73]. Although clinical studies have indicated that androgens modulate sexual arousal responses, no investigations have addressed the mechanisms by which androgens facilitate such responses [7484]. In the following section, we provide a brief discussion of the experimental data published to date on sex steroid hormones (estrogens and androgens) in modulating physiological parameters of genital arousal, as well as recent observations from our laboratory and others.

Traish et al. gen is required to down-regulate its own receptor in the vagina. It may be that in the vagina, downregulation of ERa by estrogen is a mechanism that may be important to impart a refractory phase to attenuate the effect of E2 surges during the estrus cycle. Ovariectomized animals treated with a physiological dose of T had increased expression of ERa levels in the vagina (Figure 4). Immunohistochemical data supported these ndings [96]. These data suggest that T does not counteract the upregulation of ERa in ovariectomized rats. Further, these data suggest that there is minimal conversion of exogenous T to E2 in the rat vagina. Whether conversion of T to E2 is also minimal in vaginal tissue in women remains to be conrmed. In contrast, radioligand binding studies showed that AR levels decreased in rat vaginal tissue from ovariectomized animals and increased with estrogen or androgen treatment [95]. Pelletier et al. also showed that in mice, AR mRNA expression is down regulated by ovariectomy and up-regulated by E2 administration [97]. These studies investigated the early temporal relationship of AR expression 312 hours after E2 injection.

Effects of Steroid Hormones on Estrogen and Androgen Receptor Expression in the Vagina Several studies have shown the presence of estrogen receptors (ER), progesterone receptors (PR), and androgen receptors (AR) in the vagina by biochemical and immunochemical assays [8592]. While the effects of steroid hormones on the regulation of ER and PR in reproductive organs have been extensively investigated, there are limited studies on the regulation of expression of ER, PR, and AR in the vagina [93]. Sex steroid receptors are regulated differentially in different target tissues by sex steroid hormones. It has recently been reported that expression of the ERb isoform is diminished or lost in the vagina of postmenopausal women, suggesting that the loss of physiological response may be mediated by this receptor isoform [92]. Because hormone replacement therapy is used to treat various symptoms in post-menopausal women, it will be important to determine how androgens and estrogens regulate the expression of ER, PR, and AR in vaginal tissue. Moreover, these studies will be invaluable in correlating the changes in receptor expression with changes in neurotransmitter function and modulation of the physiological parameters of vaginal arousal. The regulation of ER and AR expression in the rat vagina under various endocrine manipulations has been examined [94,95]. Because intact female rats undergo an estrous cycle, a variation in ER concentration is expected due to the changing levels of E2 in the serum. After 4 weeks, ovariectomized animals exhibited increased levels of ERa in total vaginal extract, whereas E2 replacement (at physiological concentrations) down-regulated vaginal ERa [9496]. These changes in ERa were conrmed by radioligand binding and Western blot analyses [9496]. Interestingly, treatment of ovariectomized rats with a sub-physiological dose of E2 did not decrease ERa levels when compared to ovariectomized rats receiving no hormone [94,96]. This suggests that a critical level of estroJ Sex Med 2010;7:29252946

Effects of Androgens and Estrogens on Vaginal Tissue Structure Estrogens are crucial in maintaining genital tissue structure. Both clitoral and vaginal tissues are considered target organs for androgens and estrogens (Table 1). The clitoral corpus cavernosum smooth muscle requires androgens and estrogens for growth and maintenance of function. Androgen and estrogen-induced deciency results in genital atrophy. In animal studies, we and others have noted that ovariectomy caused considerable changes in vaginal epithelium and, to a limited extent, the muscularis and lamina propria (Figure 5). E2 treatment restored the changes in tissue structure as determined by histological assays [98]. The thickness of the vaginal epithelium was dramatically reduced by ovariectomy and was restored by estrogen administration. Similar observations were noted when E2 was co-administered with T or progesterone. However, administration of T alone or progesterone alone did not normalize the vaginal epithelium to that observed in intact control animals. In intact animals, the muscularis layer is comprised of circular and longitudinal smooth muscle bers. Ovariectomy reduced the volume of the muscularis layer, and there was considerable deposition of connective tissue between muscle bundles

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Figure 4 Effects of ovariectomy and androgen treatment on ERa and AR expression in vaginal tissue. In vaginal tissue extracts from intact control or ovariectomized (Ovx) rats, saturationbinding assays for estrogen and androgen receptors were carried out using [3H]estradiol and [3H]R1881, respectively, and specic binding data were subjected to Scatchard analyses (upper panels). Western blot analyses (lower panels) were also conducted on vaginal tissue extracts from intact (C) and ovariectomized rats infused with vehicle (V) or testosterone at doses of 55 mg/day (T3) or 5.5 mg/day (T1). Continuous infusion of vehicle or hormone was started 2 weeks after ovariectomy and was maintained for 2 weeks. (Adapted from reference 95, with permission).

based on Massons trichrome staining [98]. E2 treatment of ovariectomized animals resulted in increased cross-sectional area of the muscularis, but the appearance of these bers was different from that of control animals. The differences in the morphological appearance are represented by enlarged individual muscle bers compared with those in tissue from control animals, and there was less connective tissue between the bundles in the treated animals. T treatment resulted in restoration of the muscularis ber bundles but not to the same extent as that noted for E2. Clearly the effects of E2 alone or T alone on the ne structure of the muscularis need to be examined in more detail. When ovariectomized animals were treated with E2 and T or E2 and progesterone, the muscularis ber bundle was restored with an appearance similar to that of control intact animals [98].

Effects of Sex Steroid Hormones on Vaginal Nerve Network It has been known for almost 40 years that adrenergic nerves of the female genital tract are sensitive

to changes in the hormonal milieu [99,100]. In animal studies, estrogen treatment increased the norepinephrine content of adrenergic nerves. In addition, administration of a mixture of E2 and progesterone to ovariectomized pigs caused an increase in vaginal norepinephrine content [101]. In contrast to the effects of estrogen on adrenergic neurotransmitter content, a recent report indicates that the density of innervation (assessed by the pan-neuronal marker PGP9.5) increased in the rat vagina subsequent to ovariectomy and was reduced by estrogen administration [102]. 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 post-menopausal women. However, in other studies using rats, there were no signicant changes in vaginal nerve distribution or density (PGP 9.5, TH, nNOS) after ovariectomy or with E2 or progesterone replacement. In
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Figure 5 Effects of ovariectomy and estrogen or androgen treatment on vaginal tissue structure. Female rats were left intact (Control) or ovariectomized and infused with vehicle (V) or estradiol (E) at the indicated doses (mg/day). Hormone treatment was started 2 weeks after ovariectomy and was maintained for 2 weeks. Vaginal tissue was xed, sectioned and subjected to Massons trichrome staining (upper panels). Panels 13 represent xed and paraffin-embedded vaginal tissue sections subjected to immunostaining procedures using anti-PGP 9.5 primary antibody and counterstained with Gills hematoxylin. In vaginal tissue from intact rats (panel 1), nerve bers extending from the lamina propria into the epithelial layer were occasionally observed (see arrow in panel 1; magnication, X-600). PGP-positive bers were commonly observed surrounding blood vessels (arrows) (panel 2) as depicted in tissue sections from intact control (panel 2) and testosterone infused (11 mg/day for 14 days), ovariectomized animals (panel 3) (magnication, X-400) (Adapted from reference 98, with permission).

contrast, T signicantly increased the density of adrenergic nerve bers and this effect was attenuated when E2 was co-administered with T [98]. These observations suggest differential regulation of vaginal innervation by androgens and estrogens and underscore the need for additional detailed assessments of the effects of various hormones, alone or in combination, on vaginal nerve ber density and distribution.
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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. Ovariectomy reduces smooth muscle relaxation to electric eld stimulation and to exogenous VIP in organ bath studies [19]. Estrogen treatment of ovariectomized

Biochemical Factors Modulating Female Sexual Arousal Physiology animals failed to improve the relaxation response to either electrical stimulation or VIP. In contrast, androgen treatment enhanced relaxation to electrical stimulation and restored VIP-induced relaxation, suggesting that androgens may modulate neurotransmitter function. These observations suggest that androgens facilitate vaginal smooth muscle relaxation while estrogens attenuate this response [19]. In addition, tissue from T-infused ovariectomized animals developed signicantly greater contractile force to norepinephrine. These observations suggest that T may be an important regulator of vaginal non-vascular smooth muscle contractility. However, it should be stressed that these studies were performed with supraphysiological levels of hormones. Recently, Labrie et al. [103,104] have presented data showing that dehydroepiandrosterone (DHEA) given intra-vaginally improved not only arousal but also had similar benecial effects on orgasm. This study suggests that improvement in vaginal arousal and sensation also improves orgasmic function. However, it remains to be seen if this is a direct effect on vaginal function or on other genial organs. Future studies using plasma concentrations that approximate the physiological range are required to corroborate these ndings.

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Modulation of Genital Blood Flow by Estrogens and Androgens In studies with animal models, hormone depletion by ovariectomy resulted in signicantly reduced vaginal blood ow responses following pelvic nerve stimulation when compared to controls [29,70,94]. E2 alone, T alone, or E2 plus T treatment of ovariectomized animals increased pelvic nerve-stimulated genital blood ow when compared to controls [94,95]. Similar to that in the vagina, the clitoris expresses ERa [26] and estrogens have an important role in the clitoral physiological response. Also, similar to studies in the vagina, clitoral blood ow is thought to be a valid and sensitive tool to measure female genital response [63]. Treatment of women with female sexual arousal disorder with tibolone, a synthetic steroid that gives rise to several biologically active metabolites with estrogenic, androgenic and progestogenic activity, signicantly increased clitoral peak systolic and end diastolic velocity with improvement of sexual function, as measured by the MSFQ, after 6 months of treatment [63]. These data suggest that the vagina and the clitoris, while exhibiting different anatomical structure,

share signaling pathways and hormonal responses that regulate their function with regard to genital sexual arousal. Evidence suggests that sex steroid hormones may modulate blood ow by regulating the activities of VIP and neural and endothelial NOS in the vagina [43,105111]. In ovariectomized rabbits, treatment with sex steroids had no effect on VIP content. However, the binding afnity of VIP to tissues of the genital tract was greatest in ovariectomized rabbits treated with estrogen and progesterone [109]. Furthermore, administration of VIP to post-menopausal women receiving no hormone replacement failed to increase vaginal blood ow, whereas those receiving hormone replacement exhibited increases in blood ow that were comparable to pre-menopausal women [108]. In both rats and rabbits, NOS isoforms in the vagina are differentially regulated by estrogen [105108]. Estrogens up-regulate endothelial NOS in the rat vagina when assessed by immunohistochemistry and Western blot analyses [112,113]. Using Western blot analyses, we have also corroborated these ndings in ovariectomized rats treated with E2 (unpublished data). Interestingly, however, other studies in rabbits have shown that ovariectomy increases both endothelial and neural NOS in the clitoris and vagina, and that neural NOS protein and enzyme activity in the vagina is downregulated by estrogen [43,111]. While the discrepancies and signicance of these data remain unresolved, there appears to be differential regulation of NOS isoforms by various sex steroids, and this regulation may be cell type and species specic. In addition, while the regulation of PDE type 5 in female genital tissues by sex steroid hormones has not been fully examined, the sensitivity of the NO pathway to sex steroids may provide a partial explanation for the inconsistent results of clinical studies with PDE5 inhibitors in women in which both positive and negative results were obtained [3638,114,115]. It is possible that the endocrine status (sex steroid hormone insufciency) of the patient may play an important role in determining whether PDE 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 signicantly during genital sexual arousal, facilitating sexual intercourse. Production
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2936 of vaginal uid transudate and glycoproteins (e.g., mucin) contributes to the overall lubrication process. Vaginal uid is derived from several sources including plasma transudate and cervical secretion. Further, its chemical composition is represented by a complex mixture of ions (Na+, K+, Ca 2+ and Cl-), sialoglycoproteins, albumin, small organic acids including amino acids, lactate, and urea [116123]. The vaginal uid ion concentration differs from that found in plasma. For instance, K+ ions are 600% higher in vaginal uid compared to plasma and Na+ and Cl- are 45% less than that found in plasma [116119]. In estrogen-deprived animals, production of vaginal transudate, as measured by uid weight absorbed by cotton swabs, was markedly decreased compared to controls and was restored by estrogen treatment [124]. However, treatment with T alone did not improve vaginal transudate production [29]. The transport of vaginal uid transudate is also regulated by aquaporins (AQP), water channel proteins that can reversibly and dynamically translocate between the cytoplasm and plasma membrane. Positive immunoreactivity to AQP-1, 2, and 3 has been demonstrated in rat vagina [125]. AQP-1 was mainly associated with blood vessels, whereas AQP-2 and AQP-3 were mainly associated with the epithelial cytoplasm and plasma membrane, respectively. The translocation of AQP-1 and AQP-2 to the plasma membrane was induced by pelvic nerve stimulation [125]. Further, AQP-2 expression was decreased in vaginal tissue of ovariectomized rats, while E2 administration restored AQP-2 to levels found in intact control animals [126]. 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 [127129]. Consistent with previously mentioned observations, we have also noted a signicant decrease in vaginal sialic acid concentration in ovariectomized, vehicle-treated animals compared to the intact control group. Further decreases in vaginal sialic acid concentration were noted in the animals treated with high doses of E2, whereas T treatment restored sialic acid to that of the vehicle treated group (unpublished work). With regard to T, these data are consistent with the ndings of Kennedy and Armstrong, who have shown that androgens increase vaginal mucication in the rat [120,121]. Treatment of ovariectomized rats with topical DHEA resulted in complete reversal of vaginal atrophy
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Traish et al. and stimulated proliferation and mucication of the vaginal epithelium [130]. While vaginal uid transudate production is thought to be dependent on genital blood ow and development of pressure within the vascular bed of the lamina propria [47,48,131135], it remains unclear if lubrication is mediated mainly by blood ow. Thus far, no biochemical mechanisms have been demonstrated to account for a direct link between lubrication and blood ow. In fact, agonists of P2Y2 receptors applied directly to the vaginal wall of ovariectomized animals increased vaginal lubrication in the absence of pelvic nerve stimulation of blood ow, suggesting an independent mechanism [136]. Thus, other potential mechanisms may come into play and contribute to vaginal lubrication, independent of blood ow. The synthesis and secretion of glycoproteins which also contribute to the lubrication process is modulated by estrogens and androgens [120,121]. Genital atrophy, in conjunction with diminished genital blood ow, secondary to estrogen deprivation, may bring about structural and functional changes in the genital tissues that negatively affect synthesis of sialo-glycoproteins and attenuate lubrication. Thus, while some effects of estrogens and androgens on vaginal lubrication have been noted, their exact roles and the mechanisms by which they modulate this physiological process remain incompletely characterized.
Purinergic Receptors and Vaginal Moisture

Purinergic receptor ligands have been studied for their effects on the physiology of various systems throughout the body [137]. P2Y2 receptor agonists, in particular, are currently being studied in human clinical trials for their pro-secretory effect on the ocular surface for the treatment of dry eye disease and for their ability to increase mucociliary clearance of upper and lower airways (e.g., for the treatment of chronic bronchitis, cystic brosis, and sinusitis) [138]. Gorodeski et al. rst reported P2Y2 receptor responses in human endocervical cells in vitro, and have recently presented evidence of P2Y2 receptor gene expression in these cells [139,140]. Studies of cervical mucus quantity and composition in response to uctuating hormone levels in women have shown that certain mucin subtypes are upregulated during ovulation, presumably to facilitate sperm penetrance [141]. Stimulation of the P2Y2 receptor activates phospholipase C, which results in increased inositol trisphosphate levels and release

Biochemical Factors Modulating Female Sexual Arousal Physiology of calcium from the endoplasmic reticulum. This stimulates cellular functions such as increased mucin secretion and chloride efux, resulting in increased surface liquid and hydration of secretions. In a recent study, the expression of P2Y2 receptors in vaginal and cervical tissues and the effects of two novel P2Y2 receptor agonists INS45973 and INS365 on vaginal moisture were determined [136]. Compared to control, vaginal moisture was signicantly diminished in ovariectomized animals treated with vehicle. Topical application of INS365 (8.1%) and INS45973 (0.9%) increased vaginal moisture in ovariectomized animals to levels that were comparable to or signicantly higher than control animals, respectively. In situ hybridization studies indicated that P2Y2 receptor mRNA was localized to endocervical and cervical glands, epithelium, and stratied squamous epithelium of the vagina. Thus, stimulation of P2Y2 receptors in the cervix and vagina enhance vaginal moisture in the estrogen-deprived state. P2Y2 receptor agonists may provide a potential nonhormonal alternative for treating vaginal dryness in post-menopausal women.
Pathophysiology of Female Genital Arousal Disorder

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Female sexual dysfunction consists of multiple disorders classied into the diagnostic categories of desire, arousal, orgasm, and pain. Each of these categories involves both psychological and physiological aspects and requires subjective as well as objective assessments. However, this discussion will be limited to peripheral physiological mechanisms that directly affect the genital organs and can be objectively assessed. In its most general terms, arousal disorder is dened as the persistent or recurrent inability to attain or maintain sufcient sexual excitement that causes personal distress [142]. Women diagnosed with sexual arousal disorder may have sexual complaints of diminished vaginal lubrication, increased time for arousal, diminished vaginal and clitoral sensation, and difculty with orgasm. These conditions may exist, in part, due to disruptions in the normal vascular, neural, and/or endocrine/paracrine regulatory mechanisms with concomitant changes in genital tissue structure or cellular organization. Genital sexual arousal is associated with changes in the hemodynamics of the clitoris and vagina and is controlled by numerous and overlapping parasympathetic and sympathetic neural

pathways. It has been demonstrated that centrally administered apomorphine alters vaginal blood ow through central dopamine receptors [143]. Further, stimulation of the paraventricular nucleus resulted in changes in vaginal arousal as measured by the ratio of the oscillation frequencies. Previous articles have provided detailed discussion of the physiology and neurophysiology of central regulation of genital arousal [2,144146]. Further, the development of animal models for assessing vaginal sexual arousal and the advent of new centrally acting pharmacotherapeutic agents should facilitate the investigation of the role of the central nervous system in regulating genital sexual arousal and the potential development of new pharmacological agents for the treatment of female sexual arousal dysfunctions. Interestingly, in some women, changes in genital responsiveness with arousal (e.g., vasocongestion) may appear normal, yet these women may suffer from female sexual arousal disorder (FSAD). Recently, Laan et al. [146] showed no signicant differences in mean and maximum vaginal pulse amplitude (VPA) and in latency of VPA response in women with and without FSAD. These observations suggest that impaired genital responsiveness may not be a valid diagnostic criterion. Further, it has been recently [144,145] demonstrated that slow oscillations in vaginal blood ow are correlated with subjective physiological arousal and display diminished responsiveness in women with FSAD. Similar ndings were reported in the animal model [144]. The authors suggested that slow oscillations in vaginal blood ow are entirely independent of vaginal vasocongestion since women with FSAD demonstrated a normal vasocongestion response to visual sexual stimulation. There is an increased awareness for the role of physiologic factors mediating female sexual arousal and a new appreciation for the role of organic pathophysiologic conditions resulting in female sexual arousal disorders. However, the cellular and molecular mechanisms responsible for disruptions in normal genital arousal remain unknown. Based upon current knowledge of physiology of female sexual function, we shall address both known and postulated peripheral mechanisms within the genital tissues that can contribute to female genital arousal dysfunction.

Vascular Insufciency Vascular insufciency states have been associated with disorders and diseases of the heart, brain, eye,
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2938 and bladder, as well as the penis (erectile dysfunction) [147150]. The association between vascular insufciency and sexual arousal disorders has been reviewed by Goldstein and Berman [151], and it may impair blood ow as shown in experimental animal models. However, its impact on impairment of genital sexual arousal in women remains to be established. There is limited understanding of local regulatory mechanisms modulating clitoral, vaginal, and labial blood ow and how these mechanisms are altered by disease states. A developing hypothesis is that following sexual stimulation, diminished arterial inow is a major factor contributing to impaired genital arousal, as manifested by inadequate genital engorgement. While animal models of disease that exhibit genital arousal dysfunction are generally lacking, several published studies provide insight into some potential patho-physiological mechanisms [4,20,24,30 32,42,71,94,95,103,104,125,136,143,144]. In the female rabbit model, Park et al. induced atherosclerosis of the ilio-hypogastric-pudendal arterial bed by injuring the intima with a balloon catheter and maintaining the rabbits on a high cholesterol diet for 16 weeks [152]. When compared to control animals, rabbits with atherosclerotic lesions had signicantly diminished vaginal and clitoral blood ow following pelvic nerve stimulation, as well as reduced development of pressure in vaginal and clitoral tissues [152]. Upon histological examination, clitoral and vaginal tissues from atherosclerotic animals exhibited diffuse brosis. In a separate study, clitoral corpus cavernosum tissue from atherosclerotic animals was found to have signicantly decreased smooth muscle content with a concomitant increase in connective tissue [153]. While specic pathophysiological processes have yet to be explicitly demonstrated in vaginal or clitoral tissues, it seems likely that the development of atherosclerotic plaques within the blood vessels feeding the genital tissues and the progression of disease would be similar to what has been described in the heart and coronary vessels (cf [154]. for review). Atherosclerotic blood vessels that have developed signicant stenosis may not be able to maintain sufcient perfusion such that female genital tissues are exposed to chronic ischemia and hypoxia. In penile cavernosal tissue, pro-brotic mechanisms related to decreased perfusion and oxygenation have been postulated [155]. These mechanisms are based on the concept that tissue oxygen tension regulates the local production of vasoactive factors, growth factors, and
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Traish et al. cytokines that can modulate extracellular matrix metabolism. An important contributor to vascular disease is a dysfunctional endothelium. A healthy endothelium serves to provide an anti-thrombotic, antiinammatory, and anti-atherogenic surface while also regulating vascular tone and permeability. Diseased or damaged endothelium may be a major contributor to vascular insufciency of female genital tissues. Endothelium-dependent relaxation of blood vessels has been shown to be compromised in animal models of atherosclerosis, hypertension, diabetes, aging, smoking, and renal failure [156159]. While it remains unclear whether endothelial dysfunction is a cause or a consequence of any of these disease states, its existence as a common pathological state warrants consideration and further investigation. Given the vascular nature of genital tissue and the importance of blood ow during the genital arousal response, perturbations in endothelial function are likely to be important mechanisms mediating genital arousal dysfunction.

Imbalances in Endocrine Milieu There is growing evidence that alterations in the sex steroid hormonal milieu may, in part, contribute to sexual dysfunction (see previous section on sex steroid hormones). At present, there is no clear evidence-based rationale for pharmacologic management of women with sexual dysfunction by sex hormones or hormone analogues. Bachmann et al. reported that estrogen deciency associated with the post-menopausal state results in loss of collagen and adipose tissue in the vulva, attenuated maturation of vaginal epithelial cells, thinning and loss of elasticity of the vaginal wall with loss of pre-menopausal ridges, bleeding and ulceration of the vaginal epithelium after minor trauma, delayed onset of lubrication with sexual stimulation, and an increase in vaginal pH leading to heightened vulnerability to urogenital pathogens and ora [68]. Sarrel reported that women with plasma E2 levels less than 50 pg/mL had signicantly more complaints of vaginal dryness, frequency and intensity of dyspareunia and burning compared to women with E2 values greater than 50 pg/mL [160163]. Several investigators have shown that treatment with E2 increases vaginal blood ow and lubrication, improves epithelial maturation indices, normalizes vaginal pH, and prevents vaginal atrophy [160,163,164]. Recently, Labrie et al. [103,104] have presented data showing that DHEA given intra-vaginally improved not only arousal but also

Biochemical Factors Modulating Female Sexual Arousal Physiology had benecial effects on orgasm. This study suggests that improvement in vaginal arousal and sensation also improves orgasmic function. However, it remains to be seen if this is a direct effect on vaginal function or on other genital organs. A more detailed discussion on androgen insufciency in women has been reviewed recently and is not to be addressed here [165]. Nevertheless, it is clear that steroid hormones have a dramatic impact on genital arousal and further laboratory and clinical research is needed to gain a better understanding of the physiology of steroid hormones in sexual function and dysfunction.

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Diabetes Diabetes causes multiple medical complications, affecting vascular, neural, and endocrine mechanisms. However, the effects of diabetes on sexual function in women have received limited attention in basic and clinical research [166170]. The most noted common sexual dysfunction in women with diabetes is decreased genital arousal with inadequate lubrication [166,171,172]. Enzlin reported that women with diabetes had higher incidence of sexual dysfunction (27% vs. 15%; P = 0.04) compared to controls, but a signicant difference was found only for decreased lubrication [173]. Further, depression is the major predictor of sexual dysfunction in women with type 1 diabetes [173,174]. Women with diabetes exhibit decreased sensation at both genital and extra-genital sites [175]. In addition, diabetic women may have decreased physiological arousal responses to erotic stimuli when compared to non-diabetic women [176]. In laboratory studies, vaginal tissue from streptozotocin-induced diabetic rats exhibited attenuated contractile responses to exogenous norepinephrine as well as diminished relaxation to exogenous NO or calcitonin gene-related peptide [21]. Further, both neurogenic contraction and relaxation responses elicited by electrical stimulation were inhibited in vaginal tissue from diabetic animals when compared to control responses. However, the mechanisms responsible for the observed changes remain unclear [21,177]. Vaginal tissues from diabetic animals (models of both type I and type II diabetes) were observed to have decreased epithelial thickness, decreased muscularis volume and overall wall thickness, and atrophic submucosal vasculature and increased brosis [71,177179]. Also, the cross-sectional area of connective tissue and immunostaining for TGF-b1 was signicantly higher in vaginal tissue from dia-

betic animals. Diabetes reduced ERa and AR expression, reduced eNOS and nNOS expression and altered connective tissue histo-architecture in the vagina [177179]. Studies in female rabbits showed that the diabetic state inhibits both baseline and nerve-stimulated clitoral blood ow and causes diffuse clitoral cavernosal brosis [180]. The vaginal blood ow response to pelvic nerve stimulation is also signicantly attenuated in diabetic rats when compared to responses in control animals [169,177]. Interestingly, type II diabetic female mice have decreased plasma E2 and increased plasma T levels that are associated with atrophic and brotic changes in the vagina, while administration of exogenous E2 to diabetic mice restores many of the histological (Figure 6) and biochemical alterations [178,179]. A more thorough discussion of steroid hormones and diabetes in female sexual dysfunction has been recently reviewed elsewhere [169,170]. Thus, the diabetic condition may cause a state of vascular insufciency and hormonal imbalance that leads to adverse changes in tissue structure. However, as the clinical ndings suggest, there are most certainly other aspects that need to be explored. In general, diabetic complications are thought to arise from increased oxidative stress, which is associated with an array of hyperglycemia-induced alterations. These changes include protein kinase C activation, formation of advanced glycation end-products (AGEs), and increased activity of the polyol and hexoseamine pathways [181]. While the consequences of these individual pathways in various tissues have been extensively investigated, recent work suggests that enhanced superoxide generation within the mitochondria is the underlying mechanism that directly or indirectly stimulates these metabolic pathways. Nevertheless, the mechanisms by which diabetes modulates female genital arousal responses are not well understood and require further investigation.

Antidepressant Medications Antidepressants have long been associated with adverse effects on sexual function [182184]. Angulo et al. have performed laboratory studies to investigate the effects of various antidepressants on vaginal and clitoral blood ow [185]. In female rabbits, acute administration of venlafaxine (5 mg/ kg) and duloxetine (1 mg/kg), mixed inhibitors of serotonin and norepinephrine reuptake, signicantly inhibited the increase in genital blood ow that is normally observed after pelvic nerve stimuJ Sex Med 2010;7:29252946

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Traish et al.

Figure 6 Effects of type 2 diabetes and estradiol supplementation on vaginal tissue structure. At 8 weeks of age, animals in the estradiol treated diabetic group were implanted subcutaneously with pellets containing 17b-estradiol (0.83 micrograms/ day) through a small incision on the nape of the neck (E2). The control (C) and untreated diabetic (Db) groups of animals were implanted with pellets devoid of estradiol. Massons trichrome staining of vaginal tissue sections from control and diabetic rats with or without exogenous estradiol treatment (bar = 50 mm; E = epithelium; LP = lamina propria; M = muscularis). Lower Panels: Histomorphometric data for the epithelium and muscularis are shown in the graphs (Adaped from reference 179, with permission).

lation. The specic serotonin reuptake inhibitor paroxetine (5 mg/kg) also inhibited the genital blood ow response after pelvic nerve stimulation. Interestingly, serotonin itself or the highly selective inhibitor of serotonin reuptake escitalopram did not appreciably affect the genital blood ow response. Thus, it seems unlikely that the inhibitory effect on genital blood ow caused by venlafaxine, duloxetine, and paroxetine is due to increased serotonin levels. In the same study, administration of L-arginine completely blocked the inhibitory effect of paroxetine on genital blood ow while the alpha-adrenergic antagonist phentolamine prevented the inhibitory effect of venlafaxine. The inhibitory effect of duloxetine was partially attenuated by either L-arginine or phentolamine and completely blocked by the combination of L-arginine and phentolamine. These data suggest that some serotonin reuptake inhibitors can also inhibit the production of NO in genital tissues and thereby attenuate genital blood
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ow, while others may increase the availability of norepinephrine in the genital vascular bed and cause vasoconstriction. While this initial study provides some insight, further elucidation of the mechanisms responsible for the inhibitory effects of antidepressants on sexual function is required. In addition to the roles of the NO, serotonin, and alpha-adrenergic signaling pathways, it also remains important to distinguish between the central and peripheral effects of various antidepressant drugs.
Summary

The physiology of genital arousal is highly dependent on the structural and functional integrity of the tissue, involving complex neurovascular processes modulated by numerous local neurotransmitters, vasoactive agents, sex steroid hormones, and growth factors (Table 1). The vascular nature of genital tissue lends itself to many parallel com-

Biochemical Factors Modulating Female Sexual Arousal Physiology parisons from the already established eld of cardiovascular biology. However, it is also well known that different vascular beds can yield diverse responses to the same disease state. Thus, there are most likely mechanisms that are unique to the genital tissues and their vasculature. For example, D5-androstenediol, a steroid hormone possessing both androgenic and estrogenic activity, binds to a unique nuclear receptor that may be preferentially expressed in the vagina [186,187]. In addition, the alpha-adrenergic and purinergic signaling systems, the neurotransmitter VIP, and the enzyme arginase have all been shown to regulate the genital arousal response. In the clitoris and the vagina, norepinephrine binds to a1 and a2-adrenergic receptors and results in a cascade of signaling that produces smooth muscle contraction. Stimulation of the hypogastric nerve results in attenuation of blood ow to the genital tissues. In contrast, NO activates guanylyl cyclase in clitoral and vaginal tissues, increasing cGMP and resulting in relaxation of vascular and non-vascular smooth muscle. This has been well established in organ bath studies [20,30]. Similarly, VIP has been shown to stimulate G-protein coupled receptors that activate adenylyl cyclase to produce cAMP, thus modulating vascular and non-vascular smooth muscle contractility. The advent of validated female animal models and assessment techniques will facilitate future research endeavors. Thus, there is much to learn about the normal physiological mechanisms of genital arousal. It should be noted that to date, there is no FDA-approved, pharmacological agent for treating female sexual arousal dysfunction. However, a host of chemical agents have been used off-label without discerning the mechanism of action, safety or adverse events. In light of the discussion provided above, several potential biochemical modulators may serve as targets for development of new strategies and pharmacotherapeutic agents for treatment of female genital sexual arousal disorder. These include inhibitors of enzymes that cleave neuropeptides (such as VIP). Thus, these inhibitors may prolong the half lives of such vasodilator peptides and increase their action in genital tissue and facilitating genital arousal. Similarly, utilization of alpha blockers that antagonize a1 and a2-adrenergic receptors will attenuate the response to adrenergic stimuli and contribute to increased blood ow and genital arousal. Moreover, the knowledge gained from the role of sex steroid hormones on maintaining genital tissue structural and func-

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tional integrity could be used to improve the genital sexual arousal response. Research using diabetic animals suggested that estrogen supplementation improved tissue innervation and NOS expression [178,179]. This approach has been recently shown to be effective in improving the genital arousal response in women [103,104]. It is believed that the precursor DHEA, which was administered intravaginally, is potentially converted to T and E2 within the vaginal tissue, resulting in restoration of tissue integrity and physiological function. This may contribute to improving the genital arousal response without the systemic effects of these steroids. Finally, nonhormonal agents, such as P2Y2 receptor agonists, may be developed to improve vaginal lubrication [136]. Additional understanding of the cellular and molecular mechanisms of normal physiology, as well as pathogenesis, will help to identify new potential points of intervention and more specic targets for the treatment of female genital sexual arousal dysfunction.

Acknowledgments

This work was supported by the Department of Biochemistry and Department of Urology, Boston University School of Medicine. Corresponding Author: Abdulmaged Traish, PhD, Laboratory for Sexual Medicine Research, Boston University, 700 Albany Street, W-607, Boston, Massachusetts 02118, USA. Tel: 617-638-4578; Fax: 617 638 5412; E-mail: atraish@bu.edu Conict of Interest: None.

Statement of Authorship

Category 1
(a) Conception and Design Abdulmaged M. Traish; Noel N. Kim (b) Acquisition of Data Abdulmaged M. Traish; Noel N. Kim (c) Analysis and Interpretation of Data Abdulmaged M. Traish; Noel N. Kim

Category 2
(a) Drafting the Article Abdulmaged M. Traish; Ella Botchevar; Noel N. Kim (b) Revising It for Intellectual Content Abdulmaged M. Traish; Noel N. Kim; Ella Botchevar J Sex Med 2010;7:29252946

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18 Klapproth H, Reinheimer T, Metzen J, Mnch M, Bittinger F, Kirkpatrick CJ, Hhle KD, Schemann M, Rack K, Wessler I. Non-neuronal acetylcholine, a signaling molecule synthesized by surface cells of rat and man. Naunyn Schmiedebergs Arch Pharmacol 1997;355:51523. 19 Kim NN, Min K, Pessina MA, Munarriz R, Goldstein I, Traish AM. Effects of ovariectomy and steroid hormones on vaginal smooth muscle contractility. Int J Impot Res 2004;16:4350. 20 Cellek S, Moncada S. Nitrergic neurotransmission mediates the non-adrenergic non-cholinergic responses in the clitoral corpus cavernosum of the rabbit. Br J Pharmacol 1998;125:16279. 21 Giraldi A, Persson K, Werkstrm V, Alm P, Wagner G, Andersson KE. Effects of diabetes on neurotransmission in rat vaginal smooth muscle. Int J Impot Res 2001;13:5866. 22 Rosen RC, Phillips NA, Gendrano NC 3rd, Ferguson DM. Oral phentolamine and female sexual arousal disorder: A pilot study. J Sex Marital Ther 1999;25:13744. 23 Meston CM, Gorzalka BB, Wright JM. Inhibition of subjective and physiological sexual arousal in women by clonidine. Psychosom Med 1997;59:399407. 24 Park JK, Kim JU, Lee SO, Hwang PH, Yi HK, Kim YG, Cho KW. Nitric oxide-cyclic GMP signaling pathway in the regulation of rabbit clitoral cavernosum tone. Exp Biol Med 2002;227:102230. 25 Vemulapalli S, Kurowski S. Sildenal relaxes rabbit clitoral corpus cavernosum. Life Sci 2000;67:239. 26 Martin-Alguacil N, Pfaff DW, Shelley DN, Schober JM. Clitoral sexual arousal: An immunocytochemical and innervation study of the clitoris. BJU Int 2008;101:140713. 27 Martin-Alguacil N, Schober J, Kow LM, Pfaff D. Oestrogen receptor expression and neuronal nitric oxide synthase in the clitoris and preputial gland structures of mice. BJU Int 2008;102:171923. 28 DAmati G, di Gioia CR, Bologna M, Giordano D, Giorgi M, Dolci S, Jannini EA. Type 5 phosphodiesterase expression in the human vagina. Urology 2002;60:1915. 29 Min K, Munarriz R, Kim NN, Goldstein I, Traish A. Effects of ovariectomy and estrogen and androgen treatment on sildenal-mediated changes in female genital blood ow and vaginal lubrication in the animal model. Am J Obstet Gynecol 2002;187:13706. 30 Kim SW, Jeong SJ, Munarriz R, Kim NN, Goldstein I, Traish AM. Role of the nitric oxide-cyclic GMP pathway in regulation of vaginal blood ow. Int J Impot Res 2003;15:35561. 31 Angulo J, Cuevas P, Cuevas B, Bischoff E, Saenz de Tejada I. Vardenal enhances clitoral and vaginal blood ow responses to pelvic nerve stimulation in female dogs. Int J Impot Res 2003;15:13741. 32 Pacher P, Mabley JG, Liaudet L, Evgenov OV, Southan GJ, Abdelkarim GE, Szabo C, Salzman AL. Topical administration of a novel nitric oxide donor, linear polyethyleniminenitric oxide/nucleophile adduct (DS1), selectively increases vaginal blood ow in anesthetized rats. Int J Impot Res 2003;15:4614. 33 Traish A, Moreland RB, Huang YH, Kim NN, Berman J, Goldstein I. Development of human and rabbit vaginal smooth muscle cell cultures: Effects of vasoactive agents on intracellular levels of cyclic nucleotides. Mol Cell Biol Res Commun 1999;2:1317. 34 Park K, Moreland RB, Goldstein I, Atala A, Traish A. Sildenal inhibits phosphodiesterase type 5 in human clitoral corpus cavernosum smooth muscle. Biochem Biophys Res Commun 1998;249:6127. 35 Gragasin FS, Michelakis ED, Hogan A, Moudgil R, Hashimoto K, Wu X, Bonnet S, Haromy A, Archer SL. The

Category 3
(a) Final Approval of the Completed Article Abdulmaged M. Traish; Noel N. Kim; Ella Botchevar

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