**Acknowledgements**

*Endometriosis*

ate reference miRNA.

**5. Future directions**

endometriosis [51], we propose that usefulness of miRNA for the diagnosis of endometriosis cannot be evaluated without appropriate determination of appropri-

differentially expressed in women with endometriosis compared to controls.

suggest therapeutic targets for additional research.

**6. Summary and conclusions**

Having identified candidate miRNA for the diagnosis of endometriosis it will be important to determine their relationship with pelvic pain as well as response to treatment. In the absence of this data the potential prognostic value of candidate markers of endometriosis remains uncertain. We also propose that future studies with robust sample size will be needed to clarify the relationship between circulating miRNA levels and menstrual cycle phase. Studies reporting menstrual cycle stage and circulating miRNA levels are thus far have produced equivocal results [31, 46–49, 51, 53, 56]. Furthermore, lesion type (endometrioma, peritoneal endometriosis, deep infiltrating endometriosis) are biologically distinct and thus a single clinical marker is unlikely to be dysregulated in all lesion types and thus a panel of markers may be more relevant. Furthermore, duration of disease and age of lesion may also present with functional differences. Therefore, discovery of clinical markers should describe the lesion types present in study participants. The influence of study participant age and body mass index are also important variables associated with pelvic pain and disease severity that are frequently not considered in analyses of clinical markers of endometriosis. Finally, the functional role of candidate markers in endometriosis has the potential to

Use of reference miRNA that may not be ideal for normalization of results may account for noted weaknesses in the literature. Use of validated reference miRNA markers and careful control of sample condition for potential confounders should improve study replication. Finally, although circulating miRNA levels have low variability in women with endometriosis, it will be necessary for discovery phases to include a large number of study participants to control for participant age,

Although identification of clinical markers of endometriosis has long been sought, none has so far been suitable to displace laparoscopy as the gold standard for diagnosis. Endometriosis is a complex heterogenous disease with variable presentation whose symptoms are easily confused with other clinical problems. Since endometriosis is detectable with high frequency amongst asymptomatic women [68] surgical exclusion of disease in the control group is essential to prevent biasing results towards the null. Consequently, we suggest that control or reference group definition is important. Numerous prior studies reporting differential miRNA expression in women with endometriosis have employed asymptomatic women as their reference population [29, 45, 48, 49, 51, 55]. However, healthy women without symptoms of endometriosis and without evidence of endometriosis by laparoscopy (asymptomatic control) and symptomatic women without evidence of disease at the time of laparoscopy (symptomatic control) are functionally different, yet both groups continue to be employed as controls in contemporary studies. Results from our laboratory suggest that inclusion of asymptomatic controls can produce misleading results and thus speculate that restricting the control group to symptomatic controls in future studies may improve reproducibility of results. In addition to control group, we propose that the use of validated reference miRNA to normalize results also affects detection of levels of miRNA

**54**

Funding support for this project was provided by a research operating grant (MOP142230) from the Canadian Institutes of Health Research to WGF. The authors are grateful to Ms. Annette Bullen for her assistance in study participant recruitment, access to clinical data, and sample collection. The assistance of the Endo@Mac surgical team (Drs. Nick Leyland, Sarah Scatollon, and Dustin Costescu) with access to study participants, staging of disease, and collection of blood samples is greatly appreciated.
