ABSTRACT

The main cause of vulvar pain and dyspareunia is provoked vulvodynia (PV), also known as vestibulodynia, and formerly termed “vulvar vestibulitis”. It affects 8-10% of women of all ages. Although the etiology remains an enigma, peripheral neurologic mechanism has recently been identified as a factor associated with vulvodynia. The aim of the present review is to present and discuss neuroproliferation as a main cause of PV.

Studies comparing Immunohistochemical staining of specimens from women with PV to controls, and animal models of PV, have been analysed for evidence of inflammation and neuroproliferation.

The density of nerve fibres in the vestibular stroma of women with PV was 10 times greater than that in the vestibular stroma of non-affected women controls. Moreover, the fibres penetrated the basal membrane and continued vertically, reaching close to the surface of the epithelial surface. An increase in the number of stromal mast cells has also been demonstrated in women with PV. Heparanase, discharged from mast cells, degrades the connective tissue and epithelial basement membrane, allowing the proliferating nerve fibres to penetrate the degraded epithelial basement membrane into the epithelium. This intraepithelial hyperinnervation results in local hyperesthesia characteristic of PV. In a mouse model a significant increase has been depicted in the density of nerve fibres in 40% of the mice repeatedly infected with fungal antigen. An increase in mast cells number has been depicted in a mouse model as well.

A peripheral neurologic mechanism leading to neuroproliferation has been recognised in women with PV and animal models. Future research should consider this pathogenesis.

INTRODUCTION

Provoked vulvodynia (PV), also called “vestibulodynia” and formerly termed “vulvar vestibulitis”, is the primary cause of vulvar pain and dyspareunia.¹ It affects 8-10% of women of all ages and significantly impairs their quality of life. Its most recognizable symptom is dyspareunia, and it is characterized by extreme symmetric sensitivity at the vestibule, close to the hymenal base. This concealed location of sensitivity has made diagnosis difficult and has led to myths regarding the development of PV, involving hurtful statements like “it is all in your head.” Recent research has proven that the pain arises from local, organic, and vestibular sensitivity. Approximately 50% women with PV experience sensitivity only in the posterior half of the vestibule.² The others have both anterior and posterior allodynia.

The data provided in Table 1 indicate that idiopathic vulvar pain and vulvodynia have been recognized since the first century AD. However, between 1928 and 1976, this syndrome was not reported in the medical literature, possibly due to ignorance owing to failure in curing vulvar pain and vulvodynia. Various treatments have been attempted for PV, but so far, the effective treatment is vestibulectomy, a surgical removal of the vestibule.

Although progress in the diagnosis and treatment of PV has been made,^1,2 the etiology remains unknown. Nevertheless, a breakthrough in understanding the pathogenesis of vulvodynia was achieved with the introduction of a list of evidence-based factors associated with vulvodynia in the 2015 Consensus Terminology and Classification of Persistent Vulvar Pain and Vulvodynia (Table 2).³ Peripheral neurologic mechanism is a remarkable factor associated with vulvodynia;^1,3 however, the trigger for this mechanism remains unknown. One hypothesis is that laxity of the uterosacral ligaments causes loss of support to the Frankenhauser’s plexus, initiating the development of the pain of PV.⁴ The present review discusses this peripheral neurologic mechanism as a cause of PV.

Peripheral neurologic findings in women with provoked vulvodynia

As shown in Table 3, several studies have reported on hyperinnervation and nerve sprouting in the stroma of the vestibule of women with PV.^5-15 The proliferating nerve fibres were reportedly nociceptors.¹⁶ Further, increased vanilloid receptor VR1 (TRPV1) density caused an increase in allodynia.¹⁷ To obtain accurate and objective quantitative data regarding hyperinnervation, we applied computerized histomorphometry to images of immunohistochemically stained sections from women with PV and controls; when the immunohistochemical S100 stain was used, the density of nerve fibres in the vestibular stroma of women with PV was 10 times greater than that in the vestibular stroma of controls.⁸ Moreover, these nerve fibres showed collateral sprouting that resembled nerve-ending trauma, which develops in women with chronic inflammation or major trauma such as amputation.

We have also proposed an objective histopathological criterion for establishing PV diagnosis: the total calculated area of nerve fibres (per the same microscopic field) is 10 times higher in women with PV than in controls.⁸ Although tissue analysis is not currently required for PV diagnosis, a morphological diagnosis may be useful for research purposes.

In another study, to further characterize PV hyperinnervation, we stained the tissue excised during vestibulectomy of women with severe PV using protein gene product (PGP) 9.5.¹⁰ PGP 9.5 detects the thinnest unmyelinated sensory C fibres (<1 mm thick). We demonstrated that in women with PV, the innervation not only occurred in the stroma (Figure 1a) but also penetrated the basal membrane and continued vertically for >50% of the distance to the epithelial surface (Figure 1b), almost reaching the epithelial surface. Very thin intraepithelial nerve fibres ending within the basal layers of the epithelium were also detected. Parallel sections incubated with only secondary antiserum were not stained. Control tissues showed no intraepithelial innervation. As such, in addition to the increased density of nociceptors, the nociceptors were located much closer to the mucosal surface. This combination can explain the severe allodynia experienced by women with PV.

Women with primary PV (identified at their first attempt at intercourse or physical contact) reportedly have a higher degree of neuroproliferation than those with secondary PV.¹¹ In such cases, the hyperinnervation may be congenital. In another study involving 10 women with PV, sensitive areas contained increased numbers of mechanoreceptive nociceptor axons relative to nontender foci and to that in 7 controls.¹⁴ The sensitive areas also contained increased T-cell, macrophage, and B-cell numbers. Local renin-angiotensin system protein levels were increased because more T-cells and B-cells expressed angiotensinogen. In another study, the hyperinnervation was more pronounced in foci with marked B-cell but not T-cell infiltration.¹⁵

Inflammation and provoked vulvodynia

Acute inflammation may lead to chronic sensitization that persists even after the inflammation resolves.^18,20 However, results regarding cytokine, prostaglandin E2 levels, T-cell, B-cell, natural killer cell, and macrophage numbers have been inconsistent in women with PV.²¹ Studies on proinflammatory vaginal or plasma cytokine profiles in vulvodynia are ongoing.^22-24

Some women with vulvodynia have reported recurrent yeast infections;²⁵ this, together with the reduced systemic number of natural killer cells in women with vulvodynia compared with that in controls,²⁶ led to the hypothesis that deficiency in natural killer cell numbers is correlated with recurrent Candida infections in these women. Vulnerability to Candida infection associated with the reduced capability to stop the resulting inflammation has been described; however, a causal relationship between Candida infection and vulvodynia development cannot be determined via these observations.

Mast cells and hyperinnervation

Findings concerning hyperinnervation in women with PV prompted a search for agents that could enhance nerve growth. One of the potential agents may be mast cells. Mast cells discharge various mediators from their granules, such as histamine, tryptase, nerve growth factor (NGF), and bradykinin.^27,28 Some of these directly cause pain, whereas the others cause pain by stimulating peripheral neurons. An increase in the number of mucosal mast cells has been demonstrated in women with PV,^29-31 and in an animal model of vulvodynia.³² We assessed women with severe PV undergoing vestibulectomy and compared them with those of a similar age undergoing vestibular biopsy due to other conditions.³³ Mast cell detection was conducted not only using Giemsa stain (Figure 2) but also immunostaining for c-KIT (Figure 3) and mast cell tryptase.^8,10,33 c-KIT is a membrane proto-oncogene encoding a type 3 tyrosine kinase protein receptor. In contrast, tryptase is a specific protein present in granules secreted from mast cells. Positive tryptase staining indicates that the mast cells were activated and degranulated. Using both c-KIT and tryptase staining, we observed a significant number of mast cells in the superficial dermis, mostly activated, in women with PV compared to that in controls. An increase in the number of stromal mast cells has also been demonstrated in animal models of PV.^29-31 In another study, although the number of mast cells was not increased, they were activated in sensitive vestibular areas, as indicated via chymase discharge.¹⁴ Chymase is one of three peptidases capable of converting angiotensin I to angiotensin II, which is a promoter of axon sprouting and regeneration.³⁴

While some studies have not demonstrated an increase in mast cell numbers in women with PV, these studies were limited because they only used Giemsa staining or c-KIT staining but not a marker of mast cell activation, such as tryptase.³⁵ Another limitation was that these studies sampled only one fixed area of the vestibule, regardless of whether that focus was sensitive. In another study that compared tender and non-tender foci, mast cell numbers were significantly increased in the tender foci.¹¹

Heparanase (HPSE) is another enzyme secreted by mast cell granules.³⁶ We immunostained surgical specimens from women undergoing vestibulectomy and from controls for HPSE and demonstrated the presence of this enzyme in the subepithelial cells of the vestibule in women with PV (Figure 4).^10,33 HPSE is a mammalian endoglycosidase (endo-alpha-D-glucuronidase) that can cleave heparan sulfate, a proteoglycan that stabilizes the connective tissues,³⁶ thereby releasing heparin-binding growth factors, enzymes, and plasma proteins that further weaken the extracellular matrix. Conceivably, HPSE degrades the connective tissue and epithelial basement membrane, allowing proliferating nerve fibres to penetrate the degraded epithelial basement membrane to the epithelial surface. This intraepithelial hyperinnervation results in local hyperesthesia characteristic of PV.

This proposed mechanism raised the hypothesis that blocking HPSE activity might halt PV development. Such blockade may be achieved by an anti-HPSE agent such as unfractionated or low-molecular-weight heparin; heparin is reportedly a powerful inhibitor of the activity of HPSE on antithrombin-binding oligosaccharides.³⁷ We therefore conducted a randomized controlled trial of an anti-HPSE agent (enoxaparin) for the treatment of PV.³³ This treatment led to a greater reduction of clinical and subjective pain scores in women treated with enoxaparin than in a placebo-controlled group. Pain reduction was correlated with a diminishing number of intraepithelial-free nerve fibres in the enoxaparin group but not in the placebo group.³³ The anti-HPSE therapy may have worked by strengthening the stroma, thereby inhibiting neuronal sprouting and proliferation.

Genetic predisposition to neuroproliferation and provoked vulvodynia

Consequent to our studies associating mast cell increase and activation, HPSE excretion, and hyperinnervation with vulvodynia, we examined a possible genetic predisposition of women with PV to these phenomena.³⁸ We examined several single nucleotide polymorphisms (SNPs) in TRPV1, NGF, and HPSE genes in 65 women with moderate or severe primary PV, comparing them to 126 healthy, ethnically matched controls. Genotyping analyses revealed a novel, statistically significant and elevated prevalence of SNPs in TRPV1 and in the promoter region of NGF in women with PV than in controls. The amino acid substitution resulting from SNPs in TRPV1 modifies the functional properties of the channel and induces increased TRPV1 protein expression due to an increased copy number.³⁹ Women with PV exhibit lower thresholds for heat pain as well as increased expression of the receptor.^17,40

Our finding of a polymorphism in NGF is further supported by another study that demonstrated increased blood levels of NGF in response to Candida antigen stimulation in women with vulvodynia than in controls.⁴¹ In another study, an increase in NGF levels was detected in vestibular tissues from women with PV, but only in areas with abundant B-cells.¹⁵ NGF plays a role in the development of peripheral nerves by promoting neuronal growth and survival.^42,43 NGF produces pain directly by acting on the nerves and indirectly by affecting mast cells.⁴⁴ NGF also increases TRPV1 expression.⁴⁵ Additionally, hyperalgesia was shown to be extensively mediated by NGF-positive regulation of the TRPV1-promoter system via Sp1 and Sp4 transcription factors.^34,46

Animal models of provoked vulvodynia

Animal models of PV have been developed to monitor the progression of PV. Hyperinnervation has been elicited in the vestibule of mice and rodents in which vulvodynia was induced via various topical vestibular stimuli. Hyperinnervation persisted even after the initial local inflammatory reaction subsided. In an established mouse model,⁴⁷ a significant increase in the density of nerve fibres, immunostained by PGP 9.5, developed in 40% of the mice repeatedly infected with Candida albicans strain SC5314 or by zymosan (a mixture of fungal antigens). There was a 300-400% increase in the number of nerve fibres in the mice that developed hyperinnervation. Furthermore, an elevation in the density of peptidergic fibres, as revealed by calcitonin gene-related peptide (CGRP) levels was depicted in the allodynic group. The hyperinnervation was observed throughout the lamina propria, with some thin nerves penetrating the lamina propria beneath the epithelium. An increase in sympathetic innervation, as revealed by the vesicular monoamine transporter 2 levels, was shown in allodynic mice. No inflammation was noted in the tissues. The finding of hyperinnervation and allodynia in only 40% of the treated mice was attributed to the variable genetic predisposition of the mice to vulvodynia.

In another mouse vulvodynia model, allodynia was achieved through topical application of the proinflammatory hapten oxazolone.⁴⁸ In oxazolone-sensitized female ND4 Swiss mice, a single labial oxazolone challenge provoked vulvar mechanical hyperalgesia for up to 24 h. This was accompanied by neutrophil influx, increased inflammatory cytokine gene expression, and increased density of cutaneous CGRP and PGP 9.5-immunostained nerve fibres. Moreover, a 3-day consecutive oxazolone challenge caused vulvar hyperalgesia that was persistent for up to 5 days, even after the initial local inflammatory reaction subsided.

In another study performed by the same group,⁴⁹ oxazolone-challenged mice developed significant tactile hypersensitivity that persisted for >3 weeks, even after exposure to vestibular allergen was discontinued. The hypersensitivity only disappeared 6 weeks after the exposure. In the allergen-challenged mice, >3-fold increase in cutaneous vestibular CGRP nerve intensity was detected compared with that in vehicle-challenged and untreated controls. A 10-fold increase was noted in RNA expression for transcripts encoding NGF and the nerve/mast cell synapse marker cell adhesion molecule 1. This indicates that the local environment supported neuronal growth and maintenance. These sites were characterized by increases in mast cell numbers as well as regulatory CD4, CD25, FoxP3, and T cell infiltration. Local depletion of mast cells by intralabial administration of the mast cell degranulation compound 48/80 led to reduction in both nerve density and tactile sensitivity.

In a placebo-controlled study,⁵⁰ an increased number of CD68-immunoreactive cells was demonstrated in the distal vagina of C57BL/6 mice after microinjection of complete Freund’s adjuvant (CFA) versus saline. Inflammation and innervation density were assessed at 7 and 28 days following the single administration or 14 days following the repeated administration of CFA or saline. Increased mast cell numbers were demonstrated. Hyperinnervation was evident in CFA mice via staining with PGP 9.5, CGRP, substance P, vasoactive intestinal peptide, and neuropeptide Y.

DISCUSSION

The 2015 Consensus Terminology and Classification of Persistent Vulvar Pain and Vulvodynia regards the peripheral neurologic mechanism as one of several factors associated with vulvodynia.³ The data reviewed in this study support the hypothesis that neuropathy or hyperinnervation is the actual cause of allodynia in women with PV. Several groups, including ours, have successfully shown that neuroproliferation developed only in women with PV and not in healthy women. Subsequently, removal of the hyper-innervated tissue may explain the successful outcome of the surgical excision of the vestibule-vestibulectomy, in women with PV. Non-invasive treatment approaches, aiming at preventing or reducing the effect of hyperinnervation may be developed in the future.

In animal models, hyperinnervation persisted for many days, even after the topical application of an anti-irritant and the initial local inflammatory reaction subsided. In terms of the etiopathogenesis of neuroproliferation, several researchers have demonstrated a correlation between hyperinnervation and the increased presence of mast cells in local tissues and their activation in women with PV. Consequently, we hypothesized that mast cell activation triggers the increase in the numbers of nerve fibres, with granule discharge of various mediators such as NGF, histamine, bradykinin, tryptase, prostaglandins, serotonin, and leukotriene. These mediators can sensitize C fibres and induce their proliferation, causing pain. NGF may be secreted by both mast cells and neurons and can induce neuronal growth and reduce the subsequent neuronal firing threshold, causing increased pain perception.²⁹ The SNPs in NGF and TRPV1 demonstrated by us may be the reason for the uncontrolled production of NGF, leading to excessive neuroproliferation in some women with PV. This is a vicious cycle as NGF also attracts mast cells and stimulates its own synthesis via NGF receptors on the mast cell membranes and nerves alike. Nerve sprouting and penetration of the newly formed nerve fibres into the epithelium is then made possible by HPSE.

Alternatively, another potential etiological mechanism for the increased mast cell numbers in women with PV is neurogenic inflammation. In this regard, stimulated nociceptors cause neurons to discharge neuropeptides that draw and stimulate local mast cells. Indeed, electron microscopic studies have shown that mast cells are closely related to sensory nerve fibres containing neuropeptides that induce mast cell degranulation. A similar mechanism has been reported in interstitial cystitis/bladder pain syndrome, a condition that may occur simultaneously with PV.

In addition, pelvic organ prolapse has been associated with vulvodynia.⁵¹ Further studies may explore the association between pelvic organ prolapse, laxity of the uterosacral ligaments and PV, and possibly the peripheral neurologic mechanism of PV.

CONCLUSION

In conclusion, vestibular neuroproliferation has been repeatedly depicted to be a substantial associated factor of PV.