Dyspareunia, or pain experienced by women during penetrative sexual activities, affects the psychological and sexual health of more than one in five Canadian women [1], yet its pathophysiology is poorly understood [2-4] and evidence for management approaches is limited.[5] It is thought that pelvic floor muscle (PFM) dysfunction is implicated in many forms of dyspareunia, while the nature and aetiology of this involvement remain largely unknown. The goal of this study is to understand if and how PFM dysfunction contributes to the pain experienced by women with provoked vestibulodynia (PVD), the most common cause of dyspareunia. This goal will be achieved through implementing an innovative and comprehensive approach to measuring the neuromuscular function of the PFMs. Understanding the pathophysiology of PVD is essential to the development of effective interventions to improve the health and quality of life of the many Canadian women who suffer from dyspareunia.

Despite its high prevalence, little is known about the aetiology of dyspareunia. Dyspareunia is often sub-classified by the location (generalized vs a specific region) and by the stimulus that induces pain (provoked vs spontaneous). As such, PVD is characterized by severe sharp and/or burning pain felt at the entrance to the vagina (i.e. the vulvar vestibule) when pressure is applied to this area (i.e. provoked). [2] PVD is considered to be the most common cause of dyspareunia in pre-menopausal women, with a prevalence of about 12% of women, [1] while it remains underreported due to stigma embarrassment and shame. [6] Among women who seek intervention for PVD, involuntary PFM activation is thought to play a significant role in the onset and/or maintenance of pain. [7-15] Indeed, PVD is sometimes accompanied by intense, involuntary contraction of the PFMs [3], termed vaginismus (VAG). Yet it is not known whether PVD and VAG are distinct entities, or whether VAG exists along some continuum of PVD severity; [8,16] a matter of much debate in the literature.[17,19]

There is evidence to suggest that PFM dysfunction is a feature of PVD even in the absence of VAG.[7-15,20] When assessed through intravaginal palpation, women with PVD (without VAG) present with higher PFM tone (i.e., greater resistance to a passive manual stretch of the PFMs), difficulty relaxing their PFMs following a contraction, and lower PFM strength when compared to asymptomatic women.[7-9] Yet strength and tone graded by palpation are subjective, and studies were performed without assessor blinding. Some of these palpation findings have been corroborated using electromyography (EMG), including higher than normal tonic PFM activation,[14,21,22] poor ability to achieve a PFM contraction [10], impaired ability to relax the PFMs after activation [4], and/or poor ability to sustain a consistent activation level on voluntary PFM contraction,[10,15,23] yet the latter findings have been refuted by others.[9,20,24-26] While this more objective evidence exists, studies on PFM involvement in PVD using EMG have been small (n<12), have carried high risk of bias, [27] and authors have often failed to report whether women with PVD had concurrent VAG, perpetuating the lack of clarity around diagnosis.

Indeed, it is often difficult to determine on clinical examination whether or not VAG is present in women with PVD [3] and the new Diagnostic and Statistical Manual of Mental Disorders (DSM-5) omits any distinction between PVD and VAG, referring to both as genito pelvic pain penetration disorder (GPPPD17,21). Yet PFM involvement in PVD that is accompanied by VAG (PVD+VAG) may be distinct from PFM dysfunction in PVD with no concurrent VAG. [4,27-31] While trying to understand the nature of PFM involvement in PVD, it remains prudent to evaluate the PFMs while distinguishing between those with PVD+VAG and those with PVD alone.[19] This study will contribute to our knowledge of the nature of PFM involvement in PVD, and will inform on differences in PFM involvement between those with PVD alone and those with PVD+VAG.

Objectives and hypotheses: The overall goal of this project is to determine whether alterations in tonic, voluntary, reflex or behavioural responses of the superficial and/or the deep layer of the (PFMs) are implicated in PVD and/or PVD+VAG. There are four main aims with associated hypotheses:

Aim 1: To measure and compare pressure pain threshold (PPT), Temporal Summation (TS) at the vulvar vestibule, pain reported during the tampon test, and sexual function.

Hypothesis 1 (a): Compared to pain-free controls, women with PVD and PVD+VAG will demonstrate (a) greater self-reported pain during the tampon test. (b) lower PPT, (c) higher TS, and (d) lower sexual function.

Aim 2: To measure corticomotor excitability of projections to the PFMs through motor evoked potentials (MEPs) and cortically mediated silent periods (cSPs).

Hypothesis 2(a) Compared to pain-free controls, women with PVD and PVD+VAG will have larger MEPs and shorter cSPs in the bulbocavernosus, pubovisceral, and external anal sphincter muscles in response to transcranial magnetic stimulation (TMS) of the cortical area corresponding to the PFMs.

Hypothesis 2(b) Differences will be evident between women with PVD and PVD+VAG.

Aim 3: To measure the amplitude and timing of EMG responses to pressure applied to the vulvar vestibule and the posterior thigh Hypothesis 3 (a): Compared to pain-free controls, women with PVD and PVD+VAG will have higher incidences of anticipatory responses (ie. responses recorded before pressure is applied) recorded from the PFMs (bulbocavernosus, pubovisceral, external anal sphincter) and remote (uppper trapezius, hip adductor) muscles when standardized pressure stimuli are applied to the posterior vaginal fourchette regardless of the intensity of the pressure (low vs moderate).

Hypothesis 3 (b): Compared to pain-free controls, women with PVD and PVD+VAG will have higher muscle activation amplitude responses in the PFMs (bulbocavernosus, pubovisceral, external anal sphincter) and in more remote muscles (trapezius and hip adductor muscles) after standardized pressure stimuli (both low and moderate) are applied to the posterior vaginal fourchette.

Hypothesis 3 (c): Compared to pain-free controls, women with PVD and PVD+VAG will demonstrate larger responses in the PFMs (bulbocavernosus, pubovisceral, external anal sphincter) and in more remote muscles (trapezius or hip adductors) in response to pressure that is applied to the posterior thigh.

Aim 4: To measure the amplitude of PFM activation at rest (tonic), during maximum effort voluntary contraction of the PFMs and during a straining (Valsalva) maneuver Hypothesis 4: Compared to pain-free controls, women with PVD and PVD+VAG will demonstrate (a) higher tonic PFM activation, (b) more PFM activation during straining efforts (dyssynergia) and (c) lower PFM activation on maximum voluntary contraction.

Novelty and Contributions to the advancement of knowledge: Through this study, a comprehensive picture of neuromuscular impairments in the PFMs that are associated with PVD will be generated. Through Aim 1 it is expected to find in women with PVD and PVD+VAG, greater self reported pain during tampon test, lower pressure pain threshold, higher temporal summation and lower sexual function comparing with the control group. No study has investigated corticomotor excitability of projections to the PFMs through TMS. Through Aim 2 differences in corticomotor excitability to the PFMs, evidenced through higher amplitude MEPs and shorter duration cSPs are expected to be found, between women with and without PVD as well as differences in the extent of enhanced corticomotor excitability to the PFMs between PVD and PVD+VAG. Also no study has evaluated whether differences seen in tonic or phasic responses of the PFMs between women with PVD and controls are present because of or in anticipation of pain. Studies by Van Lunsen & Ramakers [32], Reissing et al. [8] and van der Velde & Everaerd [33] suggest that the PFMs respond to generalized anxiety and in response to watching sexually threatening scenes. It is quite possible, and even probable, that anxiety or fear of pain may influence study outcomes [4,34]. Through Aim 3, the timing and magnitude of PFM activation will be evaluated to determine whether responses occur in anticipation of (i.e. before) and/or in response to pressure stimuli delivered at the vulvar vestibule. The hypotheses under Aims 2 and 3 have never before been tested and doing so will have a significant impact on our understanding of the pathophysiology of PVD. Through Aim 4, it we expect to corroborate previous findings in the literature: that women with PVD and PVD+VAG will demonstrate higher tonic PFM activation and PFM dyssynergia during straining - both being functionally related to increased corticomotor excitability.

If women with PVD+VAG exhibit higher corticomotor excitability than women with PVD alone, this finding will be a major step toward understanding PVD and PVD+VAG as separate conditions, and will set the stage for improved diagnoses and targeted treatments such as repetitive TMS35 and neurotropic medications.[36] Further, interventions that have shown some evidence for effectiveness [37] including stretching, [9,38,39] acupuncture, [40] Botox injection, [41,42] and cognitive behavioural therapy [39] may be enhanced by combining them with new interventions that focus on corticomotor inhibition. The impact of any new as well as existing interventions on corticomotor excitability can, in turn, be evaluated using the innovative yet accessible assessment approaches developed through this work.

For descriptive purposes, five on-line questionnaires will be applied to assess sexual function [43,44], pain [45] pain catastrophising [46], depression/anxiety [47,48], and central sensitization [49].

Assessment will be scheduled within the week following the start of women's last menstrual cycle to account for cyclic variations in motor and sensory thresholds [50] , and at a time that is convenient.

Inclusion Criteria:

• Premenopausal women over the age of 18
• Biologically born female
• Signs and symptoms consistent with Provoked Vestibulodynia (PVD) alone or PVD+ Vaginismus (VAG) (i.e. evidence of a vaginal muscle spasm in response to palpation) or no history of pain during sexual activities or tampon insertion to serve as a comparison group
• Not currently pregnant, or pregnant in the past six months.

Exclusion Criteria:

• Neurological condition such as stroke, multiple sclerosis, spinal cord injury, epilepsy, or history of epilepsy in the family, etc
• Metal implants (cochlear, pacemaker, etc.)
• Tendency to faint
• in Peri- or post-menopause
• express high levels of anxiety about the assessment protocol

Females with PVD alone or PVD+ VAG will be recruited at the vulvar pain clinic at The Ottawa Hospital and from local physiotherapy clinics that cater to females with pelvic floor dysfunction.

Control participants will be recruited from the local community through advertisements, word of mouth and social media.

Spreadsheets will be provided by email upon request and based on the planned use of the data.

Data will be available for 10 years after the publication of the study results.

Planned use of data for systematic review.