**3. Effect of reference miRNA used to normalize results**

While serum RNU6 has been widely used as the reference miRNA in prior endometriosis studies [29, 31, 46–48, 54], its levels have previously been reported to be unstable, unreliable, and a poor reference for miRNA since it is not processed or protected in the same way as miRNA [61, 63]. Therefore, we suggest that choice of reference miRNA can influence ability to detect significant differences and the direction of significant differences elicited. Previous studies report that hsamiR-451a is upregulated in women with endometriosis compared to symptomatic controls [47] and compared to both symptomatic and asymptomatic (healthy) control groups [48]. Both prior studies employed RNU6 as a reference. While hsamiR-451a has been found to act as a tumor suppressor [64, 65], it is also a marker of hemolysis [66] and thus we suggest that care should be employed to exclude samples with hemolysis before analysis. The miRNA ratio of hsa-miR-451a and hsamiR-23a-3p has been employed by others [56, 67] to monitor for sample hemolysis. Therefore, we suggest that hsa-miR-451a has limited value as a candidate marker of endometriosis.

### **4. Effect of control group definition**

Several studies have employed healthy women as their control population [29, 45, 48, 49, 51, 55], thus allowing circulating miRNA levels in women with endometriosis to be compared to symptomatic and asymptomatic healthy control populations. While the majority of previous reports employed symptomatic controls [30, 31, 46–51, 53, 54, 56], hsa-miR-16-5p [30, 51] RNU6 (the most common) reference material used to normalize miRNA expression [31, 46–48, 51, 54]; reference materials that are unsuitable for normalizing serum miRNA expression. In our experience, differential miRNA expression was dependent upon whether comparisons were made with asymptomatic compared to symptomatic controls. Therefore, we suggest that control group characteristics on the differential expression of candidate miRNA in women with endometriosis merits further investigation.

While, lack of replication, absence of validation of results, and poor sensitivity and specificity currently limit the value of miRNA as diagnostic markers of

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