Biology of Female Sexual Function
Ricardo Munarriz, MD, Noel N. Kim, PhD, Irwin Goldstein, MD and Abdul M. Traish, PhD
Excerpt from North American Clinics
Female sexual dysfunction is defined as disorders of sexual desire, arousal, orgasm and/or sexual pain, which results in significant personal distress and may have an impact on the quality of life and interpersonal relationships. Although each specific condition can be separately defined in medical terms, clinically there is significant overlap in afflicted patients. The limited available data on female anatomy, physiology, biochemistry and molecular biology of the female sexual response makes this field particularly challenging to clinicians, psychologists and basic science researchers alike.
The sexual response cycle consists of desire, arousal, orgasm and resolution (both physiologic and psychologic). Desire is the mental state created by external and internal stimuli that induces a need or want to partake in sexual activity. Desire may be said to consist of: 1) biologic roots, which in part are based on hormones such as androgen and estrogen, 2) motivational roots, which are in part based on intimacy, pleasure and relationship issues and 3) cognitive issues such as risk and wish. Arousal is the state with specific feelings and physiologic changes usually associated with sexual activity involving the genitals. Arousal may be said to consist of: 1) central mechanisms including activation of thoughts, dreams and fantasies, 2) non-genital peripheral mechanisms such as salivation, sweating, cutaneous vasodilation and nipple erection and 3) genital mechanisms such as clitoral, labial and vaginal engorgement. Orgasm is the altered state of consciousness associated with primarily genital sensory input. Orgasm consists of multiple sensory afferent information from trigger points such as clitoris, labia, vagina, periurethral glans, etc., which pass centrally to supraspinal structures likely involving the thalamic septum. Following sufficient sensory stimulation, central neurotransmitter discharge during orgasm results in repeated 1-second motor contractions of the pelvic floor (3 – 8/orgasm) followed in 2 – 4 seconds by repeated uterine and vaginal smooth muscle contraction. Pleasurable sensory information is also carried to the cortical pleasure sites.
Epidemiology of Female Sexual Dysfunction
Well-designed, random-sample, community-based epidemiologic investigations of women with sexual dysfunction are limited. Current data reveals that up to 76% of women have some type of sexual dysfuntion. U.S. population census data suggest that approximately 10 million American women ages 50-74 self-report complaints of diminished vaginal lubrication, pain and discomfort with intercourse, decreased arousal, and difficulty achieving orgasm. Recently, Laumann and Rosen found that sexual dysfunction is more prevalent in women (43%) than in men (31%) and is associated with various psychodemographic characteristics such as age, education, and poor physical and emotional health. More importantly, female sexual dysfunction is associated with negative sexual relationship experiences.
Anatomy and physiology of genital sexual arousal
There is a paucity of data concerning the anatomy, physiology, pathophysiology of sexual function in women. The female external genitalia consist of various structures. The vagina is a midline cylindrical organ that connects the uterus with the external genitalia. The vaginal wall consists of three layers: a) an inner mucous type stratified squamous cell epithelium supported by a thick lamina propia, that undergoes hormone-related cyclical changes, b) the muscularis composed of outer longitudinal smooth muscle fibers and inner circular fibers, and c) an outer fibrous layer, rich in collagen and elastin, which provides structural support to the vagina. The vulva, bounded by the symphysis pubis, the anal sphincter and the ischial tuberosities, consists of labial formations, the interlabial space, and erectile tissue. The labial formations are two paired cutaneous structures: a) the labia majora are fatty folds covered by hair-bearing skin that fuses anteriorly with the mons veneris, or anterior prominence of the symphysis pubis, and posteriorly with the perineal body or posterior commissure b) The labia minora are smaller folds covered by non-hearing skin laterally and by vaginal mucosa medially, that fuses anteriorly to form the prepuce of the clitoris, and posteriorly in the fossa navicularis. The interlabial space is composed of the vestibule, the urinary meatus, and vaginal opening and is bounded by the space medial to the labia minora, the fossa navicularis and the clitoris. The clitoris is a 7-13 cm Y shaped organ comprised of glans, body, and crura. The body of the clitoris is surrounded by tunica albuginea and consists of two paired corpora cavernosa composed of trabecular smooth muscle and lacunar sinusoids. Finally, the vestibular bulb consists of paired structures located beneath the skin of the labia minora and represents the homologue of the corpus spongiosum in the male.
There is limited understanding of the precise location of autonomic neurovascular structures related to the uterus, cervix, and vagina. Uterine nerves arise from the inferior hypogastric plexus formed by the union of hypogastric nerves (sympathetic T10-L1) and the splanchnic fibers (parasympathetic S2-S4). This plexus has three portions: Vesical plexus, the rectal plexus, and the uterovaginal plexus (Frankenhauser’s ganglion), which lies at the base of the broad ligament, dorsal to the uterine vessels, and lateral to the uterosacral and cardinal ligament. This plexus provides innervation via the cardinal ligament and uterosacral ligaments to the cervix, upper vagina, urethra, vestibular bulbs and clitoris. At the cervix, sympathetic and parasympathetic nerves form the paracervical ganglia. The larger one is called the uterine cervical ganglion. It is at this level that injury to the autonomic fibers of the vagina, labia, cervix may occur during hysterectomy. The pudendal nerve (S2-S4) reaches the perineum through Alcock’s canal and provides sensory and motor innervation to the external genitalia.
Large gaps exist in our knowledge of how the central nervous system controls female sexual function. Limited data suggest that descending supraspinal modulation of female genital reflexes emanates from: 1) brainstem structures such as the nucleus paragigantocellularis (inhibitory via serotonin), locus ceruleus (norepinephrine, nocturnal engorgement during REM sleep) and midbrain periaqueductal gray, 2) hypothalamic structures such as the medial pre-optic area, ventromedial nucleus and paraventricular nucleus and 3) forebrain structure such as the amygdala. Multiple factors interact at the supraspinal levels to influence the excitability of spinal sexual reflexes such as: 1) gonadal hormones, 2) genital sensory information via the mylenated spinothalamic pathway and the unmyelinated spinoreticular pathway and 3) input from higher cortical centers of cognition.
The sexual arousal responses of the multiple genital and non-genital peripheral anatomic structures are largely the product of spinal cord reflex mechanisms. The spinal segments are under descending excitatory and inhibitory control from multiple supraspinal sites. The afferent reflex arm is primarily via the pudendal nerve. The efferent reflex arm consists of coordinated somatic and autonomic activity. One spinal sexual reflex is the bulbocavernosus reflex involving sacral cord segments S 2,3 and 4 in which pudendal nerve stimulation results in pelvic floor muscle contraction. Another spinal sexual reflex involves vaginal and clitoral cavernosal autonomic nerve stimulation resulting in clitoral, labial and vaginal engorgement.
In the basal state, clitoral corporal and vaginal smooth muscles are under contractile tone. Following sexual stimulation, neurogenic and endothelial release of nitric oxide (NO) plays an important role in clitoral cavernosal artery and helicine arteriolar smooth muscle relaxation. This leads to a rise in clitoral cavernosal artery inflow, an increase in clitoral intracavernosal pressure, and clitoral engorgement. The result is extrusion of the glans clitoris and enhanced sensitivity.
In the basal state, the vaginal epithelium reabsorbs sodium from the submucosal capillary plasma transudate. Following sexual stimulation, a number of neurotransmitters including NO and vasoactive intestinal peptide (VIP) are released modulating vaginal vascular and nonvascular smooth muscle relaxation. Dramatic increase in capillary inflow in the submucosa overwhelms Na-reabsorption leading to 3-5 ml of vaginal transudate, enhancing lubrication essential for pleasurable coitus. Vaginal smooth-muscle relaxation results in increased vaginal length and luminal diameter, especially in the distal two-thirds of the vagina (Fig. 1). Vasoactive intestinal polypeptide is a non-adrenergic non-cholinergic neurotransmitter that plays a role in enhancing vaginal blood flow, lubrication and secretions.
Experimental models for investigation of female sexual genital arousal
I Results from in vivo animal studies:
The absence of established animal models to investigate female sexual genital arousal has hampered progress in this field. Recently, Park et al., investigated vaginal and clitoral hemodynamics in female New Zealand White rabbits in response to pelvic nerve stimulation (PNS) in order to mimic genital arousal in response to sexual stimulation. This elegant study showed that pelvic nerve-stimulation caused an increase in vaginal blood flow, vaginal wall pressure, vaginal length, clitoral intracavernosal pressure and clitoral blood flow and a decrease in vaginal luminal pressure. This study represents an approach to study genital arousal in an animal model and paved the way for the investigation of genital arousal in a laboratory setting. Using a rat model, Vachon et al., confirmed genital hemodynamic changes reported by Park et al., in the rabbit model. More recently, Giuliano et al., further demonstrated that PNS induced an increase in vaginal wall tension and a decrease in vaginal vascular resistance in the rat model. In addition, this study showed that atropine did not significantly affect vaginal blood flow response to pelvic nerve stimulation despite the fact that cholinergic fibers innervate vascular smooth muscle in the rat vagina, suggesting that acetylcholine may not be the primary neurotransmitter responsible for the increase in vaginal engorgement during sexual arousal. These studies documented that genital arousal is a neurovascular event characterized by increase in genital blood flow and smooth muscle relaxation. These hemodynamic changes are mediated by neurotransmitters and vasoactive agents and modulated by the hormonal milieu. Park et al., investigated the effects of estrogen deprivation and replacement on genital hemodynamics. They reported that ovariectomy significantly reduced vaginal and clitoral blood flow in response to pelvic nerve stimulation. We also investigated the effects of ovariectomy and estrogen and androgen treatment on genital blood flow using a novel, non-invasive laser oximetry technique. In contrast to the observations made by Park et al. we found that ovariectomy did not significantly alter genital blood flow in the rabbit model. The discrepancy may be attributed to differences in methodologies. In our studies, we determined genital blood flow two-weeks post ovariectomy, while Park et al. performed their studies six weeks after ovariectomy. The longer period of estrogen deprivation may have produced tissue structural changes that altered the engorgement response. Since the female rabbit remains in continuous diestrus until mounted, serum estrogen levels are normally low (32-38 pg/ml), and ovariectomy does not produce a dramatic decrease in estrogen levels (22-25 pg/ml). As a consequence, genital hemodynamic changes before and after ovariectomy may be minimal. In addition, laser oximetry was used in our studies to assess changes in genital blood flow, whereas Park et al., used laser Doppler-flowmetry. Further studies using other animal models that undergo menstrual cycling (e.g. rat) are necessary to investigate this discrepancy.
Park et al., also reported that estrogen replacement normalized genital hemodynamics to control levels. In our studies, treatment of ovariectomized animals with estradiol significantly increased pelvic nerve-stimulated genital blood flow above control levels (Fig.2). Interestingly, treatment with testosterone did not restore blood flow to that observed in control animals. Park et al., also noted marked thinning of the vaginal epithelial layers, decreased vaginal submucosal microvasculature, and diffuse clitoral cavernosal fibrosis in ovariectomized animals. In addition, the percentage of clitoral cavernosal smooth muscle was significantly decreased in ovariectomized animals. These studies suggest that estrogens modulate genital hemodynamics and are critical for maintaining tissue structural integrity.
Vaginal lubrication, an estrogen-dependent physiological process, is one of the indicators of genital arousal and tissue integrity. Min et al., showed that vaginal lubrication in ovariectomized animals under basal conditions and after pelvic nerve stimulation was reduced and normalized with estrogen treatment (Fig 3 and 4). In contrast, androgen treatment of ovariectomized animals with testosterone alone or in combination with estradiol did not restore vaginal lubrication to that observed in control animals. Finally, it was noted that ovariectomy caused vaginal atrophy and reduced vaginal epithelial cell maturation, which was normalized by estrogen but not androgen treatment.
In summary, data derived from in vivo animal models indicates that estrogen but not androgens modulate genital blood flow, vaginal lubrication and vaginal tissue structural integrity. It should be noted that estradiol levels used in these studies were supra-physiological with potential pharmacologic effects different from those achieved physiologically. Although estrogen replacement increases vaginal lubrication and restores vaginal epithelial integrity, this therapy may not be appropriate for all patients, due to associated risk of breast and endometrial cancer. An alternative to hormonal treatment is the utilization of P2Y2 receptor agonists, which have been shown to increase mucin production and blood flow in other systems. We investigated the effects of P2Y2 receptor agonists as a feasible non-hormonal alternative for the treatment of vaginal dryness in an animal model. P2Y2 receptors are expressed in cervical and vaginal tissues, and these agonists increased vaginal lubrication under conditions of estrogen deprivation.
II. Effects of vasoactive substances on genital blood flow
Limited data are available on the effects of vasoactive substances on genital hemodynamics. Park et al., 1997 demonstrated that injection of papaverine hydrochloride and phentolamine mesylate into the vaginal spongy muscularis layer increased vaginal wall pressure and vaginal blood flow. Sildenafil, a PDE5-selective inhibitor, has been utilized in the treatment of women with sexual arousal disorders with mixed results and pre-clinical data supporting the use of this agent in the management of female sexual dysfunction remains equivocal. We have shown that sildenafil administration caused significant increase in genital blood flow and vaginal lubrication in intact and ovariectomized animals. However, this response was more pronounced in animals treated with estradiol. These data suggested that the NO-cGMP pathway is involved, at least in part, in the physiologic mechanism of female genital arousal and that sildenafil facilitates this response in an in vivo animal model.
The effects of apomorphine, a non-selective dopamine receptor agonist, on genital blood flow were investigated by Tarcan et al., who suggested that systemic administration of apomorphine improved clitoral and vaginal engorgement by increasing clitoral intracavernosal and vaginal wall arterial inflow.
In summary, data derived from in vivo animal models indicate that vasoactive agents play a role in genital arousal. Although sildenafil and apomorphine enhanced genital blood flow in the animal model, clinical use of vasoactive agents remains controversial.
Studies in organ baths:
Physiological studies of the arousal phase of the female sexual response involve, in part, an understanding of the various local regulatory mechanisms, which modulate tone in the clitoral erectile tissue and the vaginal muscularis. Immunohistochemical studies in human vaginal tissues have shown the presence of nerve fibers containing NPY, VIP, NOS, CGRP and substance P.10 Previous studies have suggested that VIP may be involved in the regulation of clitoral and vaginal smooth muscle tone but, as yet, no conclusive experimental evidence of its functional involvement has been forthcoming. There is physiological evidence supporting a role for the alpha-adrenergic system in female sexual arousal. The alpha-2 adrenergic agonist clonidine impaired both vaginal engorgement and lubrication when administered to healthy volunteers.
There is limited data on the functional activity of the inhibitory non-adrenergic non-cholinergic transmission in the clitoral corpus cavernosum. Cellek and Moncada have shown that electrical field stimulation induces NANC relaxation responses in the clitoral corpus cavernosum of the rabbit. These responses were inhibited by NG-nitro-L-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) or tetrodotoxin. In addition, the inhibitory effect of L-NAME was partially reversed by L-arginine but not by D-arginine. EFS-induced relaxations were enhanced by an inhibitor of type V cyclic GMP phosphodiesterase, zaprinast. It was concluded that nitrergic neurotransmission is responsible for the NANC relaxation responses in the clitoral corpus cavernosum of the rabbit. Furthermore, the role of phosphodiesterase type 5 inhibition in the modulation of female sexual dysfunction was investigated by Vemulapalli and Kurowski. Pretreatment of clitoral corpus cavernosum strips with sildenafil enhanced the electrical field stimulation-induced relaxations, both in magnitude and duration. Thus, the NO pathway is critical for smooth muscle relaxation in the clitoris. However, in the vagina, this pathway plays only a partial role, as demonstrated by Ziessen et al. These investigators showed that in the rat and rabbit vaginal wall, NANC relaxations were partly mediated by nitric oxide. The remaining part was neurogenic since it could be inhibited by tetrodotoxin. This non-nitrergic NANC response was not associated with any known neuropeptides or purines. Thus, the nature of the non-adrenergic, non-cholinergic neurotransmitter in the vagina remains elusive.
We have carried out preliminary experiments in organ bath chambers to assess clitoral and vaginal tissue responses to: a) electric field stimulation; b) alpha-adrenergic agonists; c) NO donors; and d) VIP. Electrical field stimulation resulted in a biphasic (contraction/relaxation) response in clitoral and vaginal tissue strips. Bretylium (inhibitor of NE release) abolished the contractile response induced by EFS in both tissues. Exogenously added norepinephrine caused a dose-dependent contraction in vaginal and clitoral tissues. These observations suggest that adrenergic nerves mediate the contractile response. Sodium nitroprusside and papaverine caused dose dependent relaxation of vaginal and clitoral strips pre-contracted with norepinephrine. Alpha-1 (prazosin and tamsulosin) and alpha-2 (delequamine) selective antagonists inhibited contraction of vaginal tissue strips to exogenous norepinephrine. Further studies using specific molecular probes and RNase protection assays have detected mRNA for both alpha 1A and alpha 2A adrenergic receptors in human clitoral and vaginal smooth muscle cells (Traish et al., unpublished data). Thus, vaginal and clitoral smooth muscle contraction is the result of activation of alpha-adrenergic receptors by norepinephrine released from adrenergic nerves. It remains to be determined if other vasoconstrictor agents, such as endothelin, neuropeptide Y (NPY), angiotensin or eicosanoids may play a role in regulating smooth muscle tone in these tissues.
Giraldi et al., have characterized the effect of experimental diabetes on neurotransmission in rat vagina. It was suggested that diabetes interferes with adrenergic-, cholinergic- and NANC-neurotransmitter mechanisms in the smooth muscle of the rat vagina.8 The changes in the nitrergic neurotransmission were attributed to reduction in NOS-activity, but may also be attributed to inhibition of various reactions in the L-arginine/NO/guanylate cyclase/cGMP system.
We investigated the effects of hormonal manipulations on vaginal smooth muscle contractility in response to electrical field stimulation (EFS) and vasoactive substances. Ovariectomy reduced norepinephrine-induced contractile response and treatment with estradiol or testosterone normalized the contractile response. Ovariectomy also attenuated EFS-induced relaxation response and treatment with testosterone facilitated EFS-induced smooth muscle relaxation. Moreover, VIP induced a dose-dependent relaxation response that was attenuated in tissues from ovariectomized animals or in animals treated with estradiol. In contrast, VIP-induced relaxation was facilitated in tissues from ovariectomized animals treated with testosterone. These observations suggest that testosterone and estradiol produce distinct physiological responses in vaginal smooth muscle and that androgens facilitate vaginal smooth muscle relaxation.
In summary, the data reported from several laboratories suggest that NO is a key pathway in mediating clitoral smooth muscle relaxation. However, in the vagina, NO appears to play only a partial role in mediating smooth muscle relaxation. VIP also induces vaginal smooth muscle relaxation yet its exact functional role remains to be determined. Functional alpha-adrenergic receptors are expressed in the vagina and mediate norepinephrine induced contraction. Hyperglycemia affects vaginal smooth muscle response to neurotransmission affecting multiple physiological pathways. We have observed that androgens but not estrogens at pharmacological doses enhanced smooth muscle relaxation. Further studies with hormonal manipulations at physiological doses are necessary to establish the role of hormones on vaginal smooth muscle relaxation.
Studies in cell culture:
Park et al. and Traish et al.recently sub-cultured and characterized human and rabbit vaginal and clitoral smooth muscle cells and investigated the synthesis of second messenger cyclic nucleotides in response to vasodilators and determined the activity and kinetics of phosphodiesterase (PDE) type 5.32,37 Cultured vaginal and clitoral cells exhibited growth characteristics typical of smooth muscle cells and immunostained positively with antibodies against alpha smooth muscle actin. The cells retained functional prostaglandin E, VIP and b adrenergic receptors as demonstrated by increased intracellular cAMP synthesis in response to PGE1, VIP or isoproterenol. The response to these vasoactive substances was augmented with forskolin, suggesting stabilization of G-protein activated adenylyl cyclases. Treatment with the nitric oxide donor, sodium nitroprusside, in the presence of sildenafil, a PDE type 5 inhibitor, enhanced intracellular cGMP synthesis and accumulation. Incubation of rabbit vaginal tissue with sildenafil, sodium nitroprusside and PGE1 or forskolin produced a marked increase in intracellular cGMP. These observations were similar to those obtained with cultured cells and suggest that sub-cultured cells retained functional characteristics exhibited in intact tissue. The cells retained phosphodiesterase type 5 expression as shown by specific cGMP hydrolytic activity. Sildenafil and zaprinast inhibited cGMP hydrolysis competitively and bound with high affinity (inhibition constants Ki= 7 and 250 nM, respectively). These observations suggest that cultured human and rabbit vaginal smooth muscle cells retained their metabolic functional integrity and this experimental system should prove useful in investigating the signaling pathways that modulate vaginal smooth muscle tone.
Investigation of the distribution of NOS in the rat vagina in response to ovariectomy and estrogen replacement was recently performed using immunohistochemical analyses with n-NOS and e-NOS antibodies. In intact cycling animals, e-NOS and n-NOS expression were found to be highest during proestrous and lowest during metestrous while in ovariectomized animals n-NOS and e-NOS expression declined substantially. Estrogen replacement resulted in significant increase in e-NOS and n-NOS expression, when compared with NOS in intact animals. It was suggested that estrogen plays a critical role in regulating vaginal NOS expression of the rat vagina and that NO may modulate both vaginal blood supply and vaginal smooth musculature. More recent studies have shown the opposite observation. They found that rabbit vaginal NOS activity was considerably reduced by treatment with estradiol or estradiol and progesterone. They also noted that progesterone treatment alone up-regulated vaginal NOS. NOS-containing nerves could be demonstrated in vagina by immunohistochemistry. Vaginal smooth muscle responded with relaxation after EFS, which was inhibited by NG-nitro-L-arginine. A tissue specific role for NOS in vagina was suggested based on NO-dependent response of vaginal smooth muscle, expression of relatively high NOS, which is down-regulation by estradiol and up-regulation by progesterone.
This discrepancy in NOS regulation by estrogen in these studies may be due to species differences or to methods for assessment of NOS expression and activity. We have used both immunochemical (Western blots) and enzymatic activity assays to determine regulation of vaginal NOS in the rabbit model. In this study we demonstrated that nitric oxide synthase was predominantly expressed in the proximal vagina. The reason for this tissue distribution is yet to be determined. We further observed that ovariectomy enhanced NOS activity in the proximal vagina suggesting specific regulation of NOS by sex steroid hormones. Treatment of ovariectomized animals with estrogens resulted in decreased expression and activity of NOS in vaginal tissue, consistent with the research by Al-Hijji et al. In contrast, treatment of ovariectomized animals with androgens resulted in increased NOS expression and activity. These observations suggest that NOS in vaginal tissue is regulated by androgens and estrogens in an opposite manner.
The psychosocial and relationship aspects of female sexuality have been extensively investigated. However, studies concerning the anatomy, physiology and pathophysiology of female sexual function and dysfunction are limited. The paucity of biological data may be attributed to lack of reliable experimental models and tools for the investigation of female sexual function, and to limited funding, which is critical for the development of experimental approaches.
Research efforts by a number of investigators in different laboratories are establishing experimental models needed for the investigation of the physiological mechanisms involved in the genital arousal response of sexual function. These experimental models have permitted assessment of genital hemodynamics, vaginal lubrication, regulation of genital smooth muscle contractility and signaling pathways, providing preliminary information on the role of neurotransmitters and sex steroid hormones in sexual function. Further research is needed to define the neurotransmitters responsible for vaginal smooth muscle relaxation, the role of sex steroid hormones and their receptors in modulating genital hemodynamics, smooth muscle contractility and neurotransmitter receptor expression. Finally, a global and integral understanding of the biologic aspects of female sexual function requires investigation of the vascular, neurological (central and peripheral) and structural components of this extremely complex physiological process.