microRNA and Overcoming the Challenges of Their Use in the Diagnosis of Endometriosis

Endometriosis is a common estrogen dependent and inflammatory disease affecting approximately 176 million women worldwide. Currently, the time between onset of symptoms and a definitive diagnosis has been reported by several international studies to range from 6 to 12 years. Presently, laparoscopic surgery followed by histopathological confirmation of lesions remains the gold standard for diagnosis. In part because of cost and invasiveness, current trends favor reduced laparoscopic surgeries in preference of the non-surgical diagnosis of endometriosis. However, the search for a clinical marker or markers of endometriosis that provide equal or similar sensitivity and specificity to laparoscopy has remained elusive. Thus, the search for a diagnostic test for the diagnosis of endometriosis continues to be a high priority research and clinical issue. Recent studies have reported favorable results with microRNA; however, lack of replication and absence of validation suggest that circulating miRNA may not be reliable for clinical use. Use of different screening platforms together with divergent methods may account for some of the lack or reproducibility in the literature. Herein we critically assess the recent literature and explore sources for discrepant findings. We suggest that prospective studies using validated reference miRNA to normalize results together with improved study design may yet reveal a suitable diagnostic marker or panel of markers for the diagnosis of endometriosis.


Background
Endometriosis is a common estrogen dependent and progesterone resistant disease of unknown cause characterized by growth of endometrial cells outside the uterine cavity [1]. It is estimated that 6-11% of all women are affected by endometriosis reaching an estimated 176 million women globally [2]. A chronic painful disease [3], endometriosis causes substantial health distress and interference with normal activities including work resulting in an average loss of 10.8 h/ week from work [2] all leading to diminished quality of life (QOL) for affected women and their families. Chronic pelvic pain and infertility are common symptoms of endometriosis that bring women with this disease to seek medical attention. Approximately 71-87% of all women experiencing chronic pelvic pain and 50% of infertile women are diagnosed with endometriosis [4]. Thus, women with

Candidate clinical markers of endometriosis
Multiple gene and protein expression levels have been documented in women with endometriosis compared to controls; however, none have yielded reliable clinical markers of disease. Recent studies investigating the mechanisms controlling gene expression have produced promising results. Several histone modifications have been associated with endometriosis. For example, endometriotic stromal cells (ESC) have a lower global acetylation level of H3, and histone deacetylases 1 and 2 (HDAC1 and HDAC2) were upregulated compared to women without endometriosis [17]. Furthermore, histone deacetylase inhibitor (HDACI) treatment promoted apoptosis by reactivating the silenced chromatin [18]. G-protein-coupled estrogen receptor (GPER) expression and proliferation of endometriotic cells was inhibited by treatment with the HDACI's romidepsin and vorinostat [19]. These data suggest that histone modifications are involved in the pathophysiology of endometriosis and that HDACI's are promising agents for endometriosis treatment. However, use of histone markers in the diagnosis of endometriosis has yet to be explored.
Long-chain non-coding RNA (lnc-RNAs) are 200-100,000 bp RNA molecules which do not encode for protein, but are involved in transcriptional and posttranscriptional regulation of gene expression [20]. They are the most common noncoding RNAs and are involved in cell proliferation, differentiation, and apoptosis; all processes central in the pathobiology of endometriosis [21]. Some lnc-RNAs proposed as diagnostic markers of endometriosis include: H19 [22], CHL1-AS2  [23,24], AC002454.1 [25], lncRNA SRA (steroid receptor RNA activator) [26], MALAT-1 [27], and LINC01279 [28]. Results of a recent study revealed the lnc-RNA are carried in circulating extracellular vesicles in women with endometriosis [29]. However, use of lnc-RNAs in the diagnosis of endometriosis has not been evaluated in a prospective study of women with symptoms suggestive of endometriosis with an independent validation step and thus their clinical utility remains uncertain.
Several recent studies have documented aberrant expression of multiple microRNAs (miRNAs) in the eutopic and ectopic endometrium of women with endometriosis [30][31][32][33][34][35][36][37]. miRNAs are short non-coding RNAs, 19-25 nucleotides long, that negatively regulate mRNA translation by repressing the protein translational machinery or degrading their target transcripts. Greater than 2000 mature human miRNA sequences have been identified and are thought to regulate approximately 50% of all protein coding genes. Multiple recent studies have documented differential microRNA (miRNA) expression in endometriotic tissues compared to eutopic endometrium of women with endometriosis and controls [33,[38][39][40]. miRNA are thought to hold promise as diagnostic biomarkers of disease because they are post-transcriptional regulators of gene expression that are stably expressed over time in bodily fluids and tissues [41]. Briefly, miRNA regulate protein expression through binding to and inhibiting the translation of mRNA transcripts into protein [42]. miRNAs are synthesized in the cytoplasm from nucleic hairpin intermediates (pre-miRNA) [43] which are then processed to yield mature miRNA that resist RNase degradation [41]. miRNA form an RNA-induced silencing complex (RISC) with Argonaute, Dicer, TAR RNA binding protein (TRBP) and protein activator of PKR (PACT) to post-transcriptionally regulate genes by binding to the 3′ region of the mRNA transcript and inhibiting translation [44].
In the early 2000s, several studies proposed that circulating levels of miRNA are differentially expressed in women with endometriosis compared to controls and thus could have diagnostic value [30,31,45]. Different methods including in situ hybridization, targeted RT-PCR and several different screening platforms including miRNA based microarrays, next generation sequencing and bio-informatics followed by RT-PCR validation have subsequently revealed a broad spectrum of miRNAs that are differentially expressed in women with endometriosis compared to control groups [29][30][31][45][46][47][48][49][50][51][52][53][54][55][56]. However, to date, only the results for hsa-miR-451a [47,48], 199a-5p [31,54] and hsa-miR-141-3p [31,49] have been successfully replicated in more than one study ( Table 1). For the vast majority of miRNAs, differential expression has only been reported in a single study or the results for a few miRNAs have not been replicated by other investigators. For example, circulating levels of hsa-miR-145 were lower in women with endometriosis compared to controls [31] whereas hsa-miR-145 levels did not differ [47] or were higher in women with endometriosis compared to the control groups [50]. We postulate that divergent results may be the consequence of different screening platforms and technologies used to identify candidate miRNA markers of disease [57][58][59] and control group characteristics. Moreover, we suggest that different reference material used to quantify circulating miRNA levels are an additional source of variation.
While RNU6 has been widely used in the general miRNA literature to normalize miRNA expression in tissue, abundance and stability of expression have not been evaluated for circulating miRNA expression in women with endometriosis. Furthermore, RNU6 has low stability and abundance that is greatly influenced by sample storage and processing and the Cp values of RNU6 are highly variable from miRNA Cp values [51,60,61]. Similarly, the abundance and stability of miR-16-5p levels in the serum of women with endometriosis is uncertain but variable from the Cp values of miRNA targets [51]. Furthermore, circulating levels of miR-16-5p are altered by inflammation and stress [62,63] and thus we suggest that both RNU6

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][47][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 hsa-miR-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 hsa-miR-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 hsa-miR-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.

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.

Future directions
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 differentially expressed in women with endometriosis compared to controls.
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 suggest therapeutic targets for additional research.

Summary and conclusions
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 © 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.