Abstract
Endometriosis is a common disease characterised by extrauterine growth of endometrial-like tissue. Although the exact pathogenesis of endometriosis remains unknown, current understanding suggests that dysfunction of the endocrine and immune systems play a central role. Ectopic tissue remains responsive to hormonal fluctuations throughout the menstrual cycle, but cannot be shed during menstruation. This creates inflammation, contributing to disease symptomology and progression. Laparoscopy with histological examination of biopsied tissue is considered ‘gold standard’ for endometriosis diagnosis. The challenging landscape of healthcare, combined with long waitlists for endometriosis laparoscopy, has resulted in the eight-year diagnostic delay for endometriosis in Aotearoa New Zealand. This indicates a critical need for the development of diagnostic biomarkers to streamline or replace surgical diagnosis.
Neutrophil Extracellular Traps (NETs) are a mechanism of immunological defence where neutrophils release webs of DNA and antimicrobial peptides. NETs are essential for effective immune function, but excessive NETosis is associated with manifestation of many inflammatory diseases, and therefore may may be associated with the inflammatory disease of endometriosis.
This thesis had three aims. The first aim was to assess the feasibility of NETs as an endometriosis biomarker using a NET-specific flow cytometry panel. The second aim was to characterise NET burden throughout the menstrual cycle. The final aim was to characterise the differential response in NETosis between neutrophils from endometriosis patients and asymptomatic controls when exposed to β-oestradiol and progesterone.
Peripheral venous blood was collected from those undergoing surgery for endometriosis at Wellington and Kenepuru hospitals, and healthy volunteers (who formed the asymptomatic control group). Based on histological results, surgical participants were assigned to either the endometriosis or symptomatic control group. Participants were also grouped by their menstrual phase. Neutrophils were isolated using either negative magnetic bead separation for flow cytometry, or density gradient centrifugation for hormonal cell culture. Flow cytometry identified neutrophils through expression of CD15 and CD66b. Dead neutrophils were excluded through staining for Zombie NIR. NET expression on live neutrophils was defined as staining positive for extracellular DNA, neutrophil elastase, myeloperoxidase. NET neutrophils were also found to largely stain positive for citrullinated histones. Neutrophils isolated by density gradient centrifugation from endometriosis patients and healthy controls were cultured with optimised concentrations of progesterone or β-oestradiol and ionomycin. NETosis was expressed as the fold change in SYTOX Green fluorescence relative to hormonally naïve neutrophils.
NET burden was significantly elevated in the symptomatic control group compared to both the endometriosis group and asymptomatic control group, but did not significantly vary between the endometriosis and asymptomatic control group. Due to insufficient distribution of participants throughout menstrual phases, it was not possible to characterise NET burden throughout the menstrual cycle. Overall, neutrophils from healthy controls appeared to produce a larger amount of NETs and had a greater variation in NET response when exposed to progesterone and β-oestradiol.
While the use of NETs as a diagnostic biomarker for endometriosis cannot be ruled out, the results of this study did not support their use as one. The unexpected, elevated NET burden in the symptomatic control group may indicate the presence of a novel NET-mediated inflammatory pathology in this group which is yet to be characterised. There appeared to be a dysregulation of NET response in endometriosis neutrophils when exposed to sex hormones. Overall, this study indicates a role of NETs in both endometriosis and non-endometriosis related chronic pelvic pain.