The vulva refers to the female external genitalia comprising the mons pubis, clitoris, labia majora, labia minora, and perineum (Fig. 3.1). They are supported by superficial and deep muscles of the perineum and their fasciae (Yavagal et al. 2011).

The labia minora are two small cutaneous folds 3–4 cm long, situated between the labia majora and extended from the clitoris anteriorly to the fourchette posteriorly (Putz and Pabst 2008). Anteriorly each labium is divided into two portions: the upper division passes above the glans of the clitoris to fuse with the opposite part and form the preputium clitoridis; the lower division passes under the clitoris forming the frenulum of the clitoris with its contralateral part. The labia minora are rich in sebaceous glands, connective tissue, and vascular erectile tissue with a considerable number of sensory nerve endings and receptors (Netter 2010). They are covered with keratinized skin that contains sebaceous glands but lacks sweat glands or hair follicles. The epidermis is thinner than on the labia majora, and the degree of keratinization diminishes gradually. From the inner third of the labia minora toward the vestibule, the epithelium is nonkeratinized, endodermal-originated mucosal tissue.

The vestibule of the vagina extends from the glans clitoridis to the posterior fourchette between the labia minora, up to their internal border. It contains the vaginal orifice, external urethral meatus, vestibular bulbs, and the openings of the greater vestibular glands (also known as Bartholin’s glands). The vaginal orifice is below the opening of the urethra, and it is characterized by the presence of the hymen (a circumferential hairless skin with variable shape) (Standring 2008). The urethral orifice (lower third of the urethra) is surrounded by the erectile tissue of the clitoral bulbs, partly considered the equivalent of the male urethral corpus spongiosum (O’Connel and DeLancey 2005). It has both a sexual and protective function. It gets very congested during physiologic sexual arousal, contributing to genital congestion and the formation of the so-called orgasmic platform (Masters et al. 1986). Meanwhile, it constitutes a kind of physiologic airbag, protecting the urethra from the “mechanical” trauma of repeated sexual thrusting at intercourse. When women suffer from vaginal dryness and/or inadequate genital arousal due to different etiologies, including low desire, poor foreplay and/or vestibular pain with dyspareunia, and/or hyperactive pelvic floor mechanically narrowing the vaginal entrance, the lack of this protective cuff increases the urethral and bladder vulnerability to the “mechanical” trauma of the intercourse, contributing to recurrent cystitis (r-UTIs). Sixty percent of r-UTIs are complained of 24–72 h after the intercourse and are referred to as “postcoital cystitis,” a powerful contributor of chronic bladder pain syndrome (Graziottin et al. 2014b). The vestibular bulbs (recently renamed as “bulbs of the clitoris” (O’Connel and DeLancey 2005) are two erectile organs situated laterally to the vaginal orifice directly beneath the skin of the labia minora and joined together (pars intermedia) and extended to the base of the glans. They are in contact with the greater vestibular glands posteriorly and covered by the bulbocavernosus muscles superficially. The greater vestibular glans (Bartholin’s) are two small glands situated one on either side of the vaginal orifice and through a 2 cm long duct opened between the hymen, and the labia minora (Standring 2008) extends anteroposterior from the frenulum of the clitoris to the fourchette and laterally to the labium minus on each side. The vestibule is covered by endodermal-originated mucosa. Its border is referred to as the Hart line, which is the junction between the keratinized and nonkeratinized labia majora. Numerous mucous glands, called the minor vestibular glands, open in the vestibule. It exhibits a high concentration of sensory free ends with a dense and shallow ramification.

The clitoris is an erectile structure, homologue to the male penis, formed by two corpora cavernosa and the glans, covered by the prepuce. Only a fifth (or less) is visible (glans), while the rest is hidden under the skin (O’Connel et al. 1998, 2008; O’Connel and DeLancey 2005). The corpora cavernosa are made of cavernous erectile tissue and diverge and follow the pubic rami on each side, forming the crura. It represents the hidden part of the clitoris which is covered by the ischiocavernosus muscle: it may reach 7 or more cm in length. The glans of the clitoris is the free extreme of it. It’s 4–7 mm long and covers the distal part of the corpora cavernosa from which it is not dependent (Williams and Bannister 2008). It represents the most innervated part of the clitoris, full of free nerve endings, Krause-finger corpuscles, and corpuscles of Pacini and Meissner (Yang et al. 2006). The clitoris is connected to the mons pubis and pubic symphysis by the suspensory ligament which influences the clitoridis stability during sexual intercourse. The urethra lies surrounded by this complex with the body directly anterior to it, flanked superficially by the bulbs and deeply by the crura. In anatomy texts the bulbs are referred to as the bulbs of the vestibule and appear as if they form an erectile structure of the labia minora (Standring 2008; Netter 2010). However, according to most recent studies, the bulbs relate most closely to the clitoris and urethra so that they would be renamed the bulbs of the clitoris (O’Connel and DeLancey 2005). O’Connell refers to the “clitoral complex,” composed of the distal vagina, urethra, and clitoris, as “the” location of female sexual activity (O’Connell et al. 2008). Recently, the term clitoral-urethral-vaginal complex (CUV) (Jannini and d’Amati 2006) has been proposed, to include the specialized area called the “G-spot,” encompassing the anterior vaginal wall and the embedded structures. The vagina is related anteriorly to the base of urinary bladder and urethra, so strictly that some anatomists recently wrote that “the urethra is embedded in the vaginal wall” (O’Connel and DeLancey 2005). This very close relationship contributes to the high comorbidity between the bladder, vaginal, and sexual symptoms.

The vestibule and the vulva “receive” secretions from the vagina. Their health is modulated (also) by the vaginal microbiota, i.e., the microorganisms living in a specific organ or tissue. Lactobacilli are the microorganism typical of the fertile age. Lactobacilli, Gram-positive bacilli, the so-called Doderlein’s bacilli, represent the most prevalent microorganisms (up to 90 %) in the normal vaginal ecosystem, and they present two mechanisms to interfere with pathogens: (1) adherence to the mucus, forming a barrier which prevents colonization by pathogens; (2) production of antimicrobial compounds such as lactic acid, hydrogen peroxide, and bacteriocin-like substances (Boris and Barbés 2000). The vaginal acid system is facilitated by Lactobacilli, that metabolizes glycogen into lactic acid lowering vaginal pH to a normal value of 4.2. In physiologic conditions, lactobacilli include 90 % of the vaginal ecosystem during the fertile age, the remaining 10 % being composed by different commensal germs. The ecosystem is important for limiting the growth of pathogenic bacteria (Stumpf et al. 2013). The concept of “pathogenic biofilms” (Graziottin and Zanello 2015) is currently referring to structured germ communities living inside a self-produced polysaccharidic network adhering to the vaginal mucosa and/or to inert medical devices. They can contribute to recurrent vaginitis and cystitis with their associated sexual comorbidities, such as introital dyspareunia and postcoital cystitis.

“There is no considerable muscle in the body whose form and function are more difficult to understand than those of the levator ani, and about which such nebulous impressions prevail” (Dickinson 1889). Despite a century of medical progress since Dickinson offered this observation, the details of levator ani muscle anatomy remain poorly understood (Lawson 1974; Bustami 1988–1989; Kearney et al. 2004).

The pelvic floor consists of different muscles’ layers: the pelvic diaphragm, the urogenital diaphragm, the superficial trigonal muscles and the lateral muscles (Standring 2008). The pelvic floor is important for the support of the pelvic organs, to assist fecal and urinary continence and to improve pelvic-spinal stability; furthermore, it plays a key role for sexual pleasure. The pelvic diaphragm is formed by the levator ani and the coccygeus muscles (Kearney et al. 2004).

The coccygeus muscle forms a triangular structure attached to the spine of the ischium and to the lateral surface of the coccyx and S5. This muscle does not contribute to active movement of the pelvic floor; in fact the effective contractile support structure is represented by the levator ani muscle. The components of the levator ani muscle are the puborectal, iliococcygeal, and pubovisceral (pubococcygeus) muscles, further subdivided into pubovaginal, puboperineal, and puboanal. This terminology was accepted in 1998 by the Federative Committee on Anatomical Terminology (International Anatomical Nomenclature Committee 1983).

The iliococcygeus originates from the tendinous arch of levator ani and forms a diaphragm between the anus and the coccyx. The puborectalis originates from the pubic bone forming a ring around the rectum. The pubococcygeus with its three branches originates from the pubic bone and insert into the perineal body, the vaginal wall, and into the tissue between the internal and external anal sphincter. In the axial plane, the puborectal muscle can be seen lateral to the pubovisceral muscle and decussating dorsal to the rectum. The course of the puboperineal muscle near the perineal body is visualized in the axial plane. The coronal view is perpendicular to the fiber direction of the puborectal and pubovisceral muscles and shows them as “clusters” of muscle on either side of the vagina. The sagittal plane consistently demonstrates the puborectal muscle passing dorsal to the rectum to form a sling that can consistently be seen as a “bump.” This plane is also parallel to the pubovisceral muscle fiber direction and shows the puboperineal muscle (Margulies et al. 2006).

The urogenital diaphragm consists of the deep transverse perineal muscle with the superior and inferior fascia. The perineal membrane is composed of two regions, one dorsal and one ventral. The dorsal portion consists of bilateral transverse fibrous sheets that attach the lateral wall of the vagina and perineal body to the ischiopubic ramus. This portion is devoid of striated muscle. The ventral portion is part of a solid 3-dimensional tissue mass in which several structures are embedded. It is intimately associated with the compressor urethrae and the urethrovaginal sphincter muscle of the distal urethra, with the urethra and its surrounding connective tissue. In this region the perineal membrane is continuous with the insertion of the arcus tendineus fascia pelvis. The levator ani muscles are connected with the cranial surface of the perineal membrane. The vestibular bulb and clitoral crus are fused with the membrane’s caudal surface (Stein and DeLancey 2008). The superficial trigonal muscle is composed of the bulbocavernosus, ischiocavernosus, and the superficial transverse perineal muscles in the anterior triangle and the anal sphincter in the posterior triangle. The superficial transverse perineal originates from the ischial tuberosity and insert on the perineal body (Stein and DeLancey 2008). The ischiocavernosus muscle extends from the ischial tuberosity to the clitoral crura inserting on to the body of the clitoris (this muscle compresses the crura of the clitoris and retard the return of blood through the veins contributing to maintain the erection). The bulbocavernosus muscle occupies each lateral side of vagina between the perineal body and the clitoris body (it diminishes the orifice of the vagina and with its anterior fibers contributes to the erection of the clitoris) (Standring 2008). Female longitudinal anal muscles or conjoint longitudinal coats (CLCs) are attached to the subcutaneous tissue along the vaginal vestibule on the anterior side of the external anal sphincter. Lateral to the CLCs, the external anal sphincter also extends anteriorly toward the vaginal side walls. The anterior part of the CLCs originates from the perimysium of the levator ani muscle. In terms of topographical anatomy, the female anterior CLCs are likely to correspond to the lateral extension of the perineal body (a bulky subcutaneous smooth muscle mass present in adult women), supporting the vaginal vestibule by transmission of force from the levator ani (Kinugasa et al. 2013).

In addition to muscles, the pelvic organs are supported by connective tissue organized in different layers of fasciae and ligaments. Magnetic resonance studies offer new insights to the traditional anatomic readings (Tunn et al. 2001, 2003).

The endopelvic fascia covers the pelvic organs and connects them to the lateral pelvic wall. It’s made up of a combination of elastin, collagen, mucopolysaccharides, and adipose and neurovascular tissue. The fascia covering the levator ani muscle continues with the endopelvic fascia above, perineal fascia below, and obturator fascia laterally (Yavagal et al. 2011). The levator ani muscles and their superior and inferior fascia combined together form the so-called pelvic diaphragm (Ashton-Miller and DeLancey 2007).

The broad ligaments connect the uterus to the lateral pelvic walls on both side, and on its upper end it encases the fallopian tubes, round ligaments, utero-ovarian ligaments, and the ovaries (Standring 2008).

The round ligaments extend from the lateral side of the uterine body and passing through the inguinal canal insert into the labia majora (Standring 2008).

The uterosacral ligaments support the cervix and the upper part of the vagina by their attachment to the sacrum, having also an important role of contention function in sexual intercourse.

The cardinal ligaments, or Mackenrodt’s ligaments, extend from the cervix to the posterolateral pelvic wall (Ramanah et al. 2012).

The fascia of the obturator internus covers the pelvic surface of the muscle; it arches beneath the obturator vessels and nerve, completing the obturator canal, and at the front of the pelvis is attached to the back of the superior ramus of the pubis. Below it’s attached to the falciform process of the sacrotuberous ligament and to the pubic arch. Thickening in the obturator fascia is called the arcus tendinous fascia pelvis, extended from the pubis anteriorly to the ischial spine (Ziouziou et al. 2013). Alcock’s canal syndrome or pudendal nerve entrapment (Labat et al. 2008) is a condition caused by the compression of the pudendal nerve in the canal, resulting in a neuralgia in the area of distribution of the pudendal nerve (vulva, vagina, clitoris) (Oelhafen et al. 2013).

The arterial supply of the vulva is derived from the external and internal pudendal arteries. The internal pudendal artery is a branch of the anterior division of the internal iliac artery, and the veins drain into the internal iliac vein (Netter 2010). The inferior rectal artery supplies the anal canal; the perineal artery supplies the superficial perineal muscles; and the posterior labial branch gives artery to the bulbs of the vestibule and dorsal and deep arteries of the clitoris. The superficial and deep external pudendal arteries are branches of the femoral artery, and they supply the labia majora with branches of the pudendal artery (Beech and Adams 2009). The internal pudendal arteries are the key resistance vessels controlling the peripheral circulatory component of sexual responses in both male and females. Structurally, the pudendal artery has a smaller lumen diameter and wall thickness and much lower wall-to-lumen ratio compared to that of the male. The lumen of this artery also tapers as it travels distally and becomes the clitoral artery. Based on its smaller wall thickness, as expected, the female pudendal artery does not contract to the same magnitudes attained by the male pudendal. However, the sensitivity to adrenergic-mediated contraction is not different between male and women (Hannan et al. 2012).

Many of the differences between the male and female pudendal arteries can be explained by the hemodynamic demands of their genital organs. The volume of blood and inflow pressures required to fill the penis are much greater than the demands of the female genitalia, when sexual purposes are considered. Furthermore, various clinical studies have confirmed the difference in the volume of blood as well as the pressures achieved by the genital organs during orgasm in both sexes. In fact, the volume of blood required to fill the clitoral tissue during a sexual response is one tenth than that required to fill the penis (10 ml vs 100 ml) (Maravilla and Yang 2008). Furthermore, the intracavernosal pressure within the penis reaches suprasystolic values during orgasm/ejaculation, whereas the vaginal, clitoral, and labial pressures only increase approximately 30–40 mm/Hg at peak sexual response (Kandeel et al. 2001; Sommer et al. 2001). Thus, the male pudendal artery needs to be able to withstand greater inflow of blood at higher pressures, and these requirements are reflected in the increased wall-to-lumen ratio of the pudendal artery. The pudendal artery in the female rat is very similar anatomically to that of women. In women, the origin of the internal pudendal artery is also located on the internal iliac artery but appears to arise much further down after the obturator, vesicular, and inferior gluteal branches (Beech and Adams 2009). In both species, the internal pudendal artery gives off branches supplying the labia and distal vaginal wall and terminates as the common clitoral artery with branches forming the clitoral cavernous and dorsal clitoral arteries (Fătu et al. 2006; O’Connel and DeLancey 2005). There is also evidence in both men and women of accessory pudendal arteries which arise off the inferior vesical, obturator, and external pudendal arteries and supply the genital tissues.

The internal pudendal artery has markedly heightened susceptibility to vascular damage compared to other vessels in the body. Evidence suggests that the female may also be susceptible to vascular pathologies contributing to sexual dysfunction. Indeed, vaginal/clitoral engorgement is a central nervous system-driven event leading to increases in blood flow to the genital organs: an event that precedes arousal (Traish et al. 2010). This increased blood flow to the vagina, clitoris, and labia is responsible for the vasocongestion, engorgement, and lubrication in the sexual arousal response.

The pudendal nerve arises from the sacral plexus; it is formed by the second, third, and fourth sacral nerve roots. It passes between the piriformis and coccygeus muscles and leaves the pelvis through the lower part of the greater sciatic foramen. It then crosses the spine of the ischium being situated between the sacrospinous and the sacrotuberous ligament (Robert et al. 1998) and reenters the pelvis through the lesser sciatic foramen. It goes along the lateral wall of the ischiorectal fossa with the internal pudendal vessels (the pudendal artery lies on its medial side), contained in a duplication of the obturator fascia called Alcock’s canal (Schraffordt et al. 2004) and divides at the level of the perineum in three terminal branches: the dorsal nerve of the clitoris, the perineal nerve, and the inferior rectal nerve, providing the sensory branches to the skin of the perineal area, labia majora, and clitoris (Tagliafico et al. 2014; Mahakkanukrauh et al. 2005). It also innervates the external anal sphincter (inferior rectal nerve) and deep muscles of the urogenital triangle (perineal nerve). The perineal nerve is situated below the internal pudendal artery and divides into a posterior labial branch and a muscular branch. The dorsal nerve of the clitoris is the deepest division of the pudendal nerve. Considering the relatively small size of the clitoris, even inclusive of the crura and bulbs, in comparison to the penis, the size of the dorsal nerve of the clitoris is proportional to its extraordinary sensory capacity, albeit it is small in absolute terms. The dorsal nerve supplies the clitoris. The pudendal nerve is the most important human nerve in terms of pleasure perception. At the same time, it is also critical in sexual pain disorders, namely, introital dyspareunia and vaginismus.

The lumbar plexus is formed by the loops of communication between the anterior division of the first three and the greater part of the fourth lumbar nerves; it is situated in the posterior part of the psoas major, in front of the transverse processes of the lumbar vertebrae. It divides into many branches, giving origin to the ileoinguinal nerve and genitofemoral nerve which are important for pelvic innervation. The ileoinguinal nerve arises from the first lumbar nerve giving branches to the obliquus internus muscle and to the skin covering the mons pubis and labia majora. The genitofemoral nerve arises from the first and second lumbar nerves, and it divides into the external spermatic nerve (it accompanies the round ligament of the uterus and it gets lost upon it) and into the lumboinguinal nerve (it supplies the skin of the anterior surface of the upper part of the thigh) (Standring 2008).

Vulvar pain has several common etiologies, but a very common cause of chronic vulvar symptoms is vulvodynia. Most often, the discomfort of vulvodynia is characterized as perceptions of burning, rawness, or irritation, and dyspareunia is nearly always present as well.

The International Society for the Study of Vulvovaginal Disease (ISSVD), the International Society for the Study of Women’s Sexual Health (ISSWSH), and the International Pelvic Pain Society (IPPS) recently defined a revision of persistent vulvar pain (Bornstein et al. 2016) (Table 3.1).

Two main elements characterize this new classification:

Recent evidence from human studies has significantly expanded the understanding of pain perception and has demonstrated that a complex series of spinal, midbrain, and cortical structures are involved in pain perception.

Pain transmission from the periphery to the higher brain centers via the spinal cord is not a simple, passive process involving exclusive pathways (Fig. 3.2). The relationship between a stimulus causing pain and the way it is perceived by an individual is dramatically affected by circuitry within the spinal cord and the brain. The sensation of pain is modulated as it is transmitted upward from the periphery to the cortex. It is modulated at a segmental level and by descending control from higher centers, with the main neurotransmitters involved being serotonin, norepinephrine (noradrenaline), and the endogenous opioids.

Pain can be categorized as nociceptive, inflammatory, and pathological/neuropathic.

Nociceptive pain is produced by repetitive, prolonged, and/or excessive stimulation of pain receptors that results in receptor or nerve damage. It could originate from nociceptors located in the skin, musculoskeletal tissue, external or internal organs, or anatomical structures. Stabbing and burning characterize vulvar pain. Nociceptive pain reflects our capacity to detect the presence of potentially damaging stimuli; it is an essential early warning mechanism (Haanpää et al. 2011). It quickly indicates damaging factor(s) the body should withdraw from and/or avoid, while the resulting inflammatory process is finalized to repair and heal-

This sensation is mediated in the periphery by high threshold primary sensory neurons, the nociceptors, which transmit information via nociceptive pathways in the spinal cord to the brain. Following peripheral tissue injury or inflammation, reversible adaptive changes in the sensory nervous system lead to the generation of pain hypersensitivity, a protective mechanism that ensures proper healing of damaged tissue. The noxious stimulus is transmitted by nociceptors to the dorsal root ganglion of the spinal cord. Thinly myelinated A-δ nociceptors transmit immediate sharp pain, whereas unmyelinated C-fibers transmit delayed and longer-lasting pain.

By contrast, chronic pain, which is persistently inflammatory until it becomes neuropathic, is the results of aberrant functioning of peripheral or central nervous systems that have been pathologically modified (Watson and Sandroni 2016).

Inflammatory pain also has an adaptive function to protect healing tissues. Tissue damage triggers reversible changes of hypersensitivity in the sensory system, which last as long as inflammation persists. The mast cell is the director of the inflammatory orchestra, the leading protagonist of the tissue response to high variety of damaging factors (Fig. 3.3). Mast cells, degranulated mast cells, and mast cells close to pain fibers are significantly increased in the vestibular tissue (Bohm-Starke et al. 1999; Bornstein et al. 2004; Graziottin 2009; Graziottin et al. 2013, 2014a; Graziottin and Gambini 2016).

Inflammatory pain:

Acute vulvar pain of whatever cause is typically first nociceptive and rapidly turns into inflammatory. When the peripheral vestibular/genital tissue’s inflammation persists, a progressive cytokines’ flooding of the central nervous system takes play. Cytokines, interleukin 1 beta, tumor necrosis factor alpha, and other inflammatory molecules cross the brain barrier, hyperactive the glial cells, and particularly the microglia.

The microglia’s role shifts progressively from neuroplastic to neurotoxic, with neuroinflammation leading to sickness behavior, with changes in mood, energy, sleep patterns, cognition, and memory. Sickness behavior is initially protective, aimed at sparing energy to optimize the healing process. When the inflammatory pain becomes chronic, sickness behavior becomes maladaptive with more persistent behavioral changes (Xanthos et al. 2011; Graziottin et al. 2013, 2014a).

Critical changes consequent to central neuroinflammation include: