Correlations Between Immunohistochemical Staining and Pain-Related Assessments in Patients with Neuroproliferative Vestibulodynia

Introduction Mast cell accumulation and nerve proliferation has been hypothesized to lead to pain symptoms for patients with neuroproliferative vestibulodynia (NPV). A clinically suspected diagnosis of NPV can be confirmed by examining immunohistochemical (IHC) staining of excised vestibular specimens. Compared to controls, specimens from NPV patients have been found to contain >8 mast cells per high-power field (HPF) and increased density of nerves. Objective To assess for correlations between patient reported pain-related measures and computer-assisted histometry of excised vestibulectomy specimen IHC staining with CD117 (marker for mast cells) and PGP9.5 (marker for nerves) in a cohort of histopathologically confirmed NPV patients. Methods Patients (n=65) with lifelong or acquired NPV undergoing vestibulectomy had IHC-staining of excised vestibular tissue. Tissue sections from the 1:00-11:00 and/or 12:00 regions of the vestibule were examined and high-resolution images were captured using a microscope at 100x and 200x magnification. A minimum of 2 photomicrographs per magnification were taken for each tissue section that included epithelial basement membrane and adjacent subepithelium representative of the majority of immunostained tissue. Fractional area of positive immunostaining of all photomicrographs was determined using computer-assisted histometry by ImageJ. Mean fractional area was determined from 3 separate measurements of each photomicrograph. Correlations were made between density of IHC staining and the following assessments: pain domain of the Female Sexual Function Index (FSFI), the Short Form McGill Pain Questionnaire (SF-MPQ), and cotton-tipped swab testing. We also examined correlations with the Patient Global Impression of Improvement (PGI-I), a treatment response index and indirect assessment of pain. Data were analyzed using Spearman correlations and the Mann-Whitney test. Results The study cohort had a median age of 26 (IQR = 23-31) years and experienced symptoms for a median duration of 11 (IQR = 6-15) years. While all patients reported high levels of pain (median FSFI pain domain score = 0.0), there were no correlations between CD117- or PGP9.5-immunopositive mean fractional area and FSFI pain domain or SF-MPQ scores. There was no difference in immunopositive mean fractional area between lifelong and acquired NPV. A positive correlation between fractional area of CD117 immunostaining in the 1:00-11:00 region of the vestibule and cotton-tipped swab test pain score approached but did not reach statistical significance (r = 0.24, p = 0.063). No correlations were observed between immunostaining and PGI-I or cotton-tipped swab testing scores. Likewise, SF-MPQ domain scores had no correlation to PGI-I or cotton-tipped swab testing. Median cotton-tipped swab testing pain scores were significantly higher (p < 0.001) in patients with lifelong NPV (7.6) compared to those with acquired NPV (5.7). Conclusions Our findings suggest that the high levels of pain experienced by NPV patients over a prolonged period of time may be dissociated from the density of mast cells and nerves in vestibular tissue. We postulate that any potential threshold of mast cell accumulation and increased nerve density that results in symptoms of vestibulodynia has been exceeded in our patient cohort. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Dare Bioscience; Pfizer; Prometheus Laboratories; Sprout Pharmaceuticals.