Abstract
Endometriosis is a common condition characterised by endometrial-like tissue growth outside of the uterus. Endometriosis diagnosis is often delayed due to the limited accuracy of non-invasive methods, particularly for mild disease, leading to a reliance on surgery. Non-invasive biomarkers would transform the diagnostic landscape and are a high priority for endometriosis stakeholders. To this end, this thesis investigated extracellular vesicles (EVs) and cervicovaginal fluid for endometriosis biomarkers.
Chapter 2 presented the first clinical audit of endometriosis surgeries in Aotearoa New Zealand. At Te Whatu Ora – Capital and Coast across 2018 and 2019, endometriosis was confirmed in 68% of laparoscopies for suspected endometriosis, with peritoneal disease the most common phenotype. Encouragingly, repeat laparoscopies were a small proportion of all surgeries. Given around one third of endometriosis surgeries were negative, this study confirmed a clinical need for diagnostic biomarkers in Aotearoa New Zealand.
Next, techniques for studying extracellular vesicles using spectral flow cytometry were developed. Plasma EVs were first characterised according to international guidelines using tunable resistive pulse sensing, transmission electron microscopy and western blotting. Membrane dye performance and antibody multiplexing were thoroughly assessed to establish minimal false positive and negative events. CD10+, CD90+, CD10+CD90+ and CD140b+ plasma EVs were assayed in surgically confirmed endometriosis cases and symptomatic controls (n=30). No differences in either the proportions of positive EVs or antigen density were detected between groups. These findings prompted a shift toward cervicovaginal fluid as a biofluid with greater biological relevance and non-invasive sampling.
In Chapter 4, a comparative study characterised and evaluated the cervicovaginal fluid EVs sampled via matched cervical brushes and vaginal swabs. While cervical brushes showed a trend toward higher particle concentrations, both methods yielded comparable miRNA and protein levels. Critically, the yields of both sampling methods were insufficient for the intended downstream purpose of discovery-based approaches. Given the potential for self-sampling, vaginal swabs were chosen for further study.
Data-independent proteomics was performed on cervicovaginal fluid sampled via vaginal swabs in two separate experiments. Unsupervised analysis revealed a complex data structure, with only around 15% of the total variance explained by the first two principal components. The two experiments showed little consistency in both the differentially abundant proteins and the predicted pathways affected by the observed protein changes. Flow cytometry was employed to analyse cervicovaginal fluid EVs carrying receptors related to the complement cascade (CD55 and CD59) and macrophage polarisation (CCR2 and CD82). No significant differences between groups were identified. However, normalised LGMN concentration was significantly increased in the endometriosis group during the validation stage, as assessed by enzyme-linked immunosorbent assay. Normalised LGMN could discriminate between people with and without endometriosis with a 0.960 sensitivity and a 0.727 specificity, meeting the thresholds for a clinically useful rule-out test.
This thesis has contributed the first audit of surgeries for endometriosis at a public hospital in Aotearoa New Zealand, and cervicovaginal fluid LGMN was identified as a candidate biomarker of endometriosis warranting further investigation. Novel methodologies for studying EVs via spectral flow cytometry were developed and applied to cervicovaginal fluid EVs sampled with vaginal swabs. However, reflections on endometriosis biomarkers prompt the opinion that the methodologies developed in this thesis would best be applied to other clinical challenges in endometriosis.