**1. 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 endometriosis report significant health distress and interference with normal activities including work and leisure time activities all leading to a deleterious effect upon women's social functioning, emotional well-being, employment, and vitality [5].

An important obstacle to the timely diagnosis and effective management of endometriosis is the lack of a simple diagnostic test. Diagnosis has been reported to be delayed by between 6 and 12 years with an average of 9 years from the onset of symptoms to definitive diagnosis [6]. Hence, identification of a clinical tool for the diagnosis of endometriosis has become a high priority research objective [2, 7, 8]. Health care providers and patients face a number of challenges in arriving at a diagnosis of endometriosis including: early age at onset of symptoms, normalization of pain by primary care providers, and suppression of symptoms through intermittent use of oral contraceptive pills [9]. Symptoms of endometriosis are shared with many other diseases including autoimmune diseases, cancer, irritable bowel syndrome (IBS), and musculoskeletal abnormalities*.* Therefore, an ideal biomarker of endometriosis must differentiate between endometriosis and other explanations for patient symptoms. In addition, clinical markers should be as minimally invasive as possible, affordable and convenient to use with the added benefit of providing insight into potential treatment response. Furthermore, the ideal biomarker must provide equivalent or similar outcome measures of sensitivity, specificity, positive, and negative predictive values to laparoscopy.

Currently, the gold standard for diagnosis remains visualization of endometriotic lesions typically by laparoscopy followed by histopathological confirmation of disease [10]. Current trends favoring the non-surgical diagnosis of endometriosis increase the pressure to identify novel clinical markers of endometriosis. Despite a plethora of biochemical differences in the peripheral circulation, peritoneal fluid, and endometrial tissues of women with endometriosis compared to healthy controls, no marker has been found with adequate sensitivity or specificity to challenge laparoscopy for the diagnosis of endometriosis whether used alone or in a panel of clinical markers [11–16]. However, reports of promising results have been brought forward in the literature from which epigenetic markers are potentially the most exciting.

#### **2. 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

**51**

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

[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.

(miRNAs) in the eutopic and ectopic endometrium of women with endometriosis [30–37]. 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, 38–40]. 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].

Several recent studies have documented aberrant expression of multiple microRNAs

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 [29–31, 45–56]. 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 [57–59] and control group characteristics. Moreover, we suggest that different reference material used to

quantify circulating miRNA levels are an additional source of variation.

for normalization of circulating miRNA levels in women with endometriosis.

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 and miR-16-5p are not suitable

*DOI: http://dx.doi.org/10.5772/intechopen.91324*

#### *microRNA and Overcoming the Challenges of Their Use in the Diagnosis of Endometriosis DOI: http://dx.doi.org/10.5772/intechopen.91324*

[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 [30–37]. 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, 38–40]. 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 [29–31, 45–56]. 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 [57–59] 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 and miR-16-5p are not suitable for normalization of circulating miRNA levels in women with endometriosis.

*Endometriosis*

endometriosis report significant health distress and interference with normal activities including work and leisure time activities all leading to a deleterious effect upon women's social functioning, emotional well-being, employment, and vitality [5]. An important obstacle to the timely diagnosis and effective management of endometriosis is the lack of a simple diagnostic test. Diagnosis has been reported to be delayed by between 6 and 12 years with an average of 9 years from the onset of symptoms to definitive diagnosis [6]. Hence, identification of a clinical tool for the diagnosis of endometriosis has become a high priority research objective [2, 7, 8]. Health care providers and patients face a number of challenges in arriving at a diagnosis of endometriosis including: early age at onset of symptoms, normalization of pain by primary care providers, and suppression of symptoms through intermittent use of oral contraceptive pills [9]. Symptoms of endometriosis are shared with many other diseases including autoimmune diseases, cancer, irritable bowel syndrome (IBS), and musculoskeletal abnormalities*.* Therefore, an ideal biomarker of endometriosis must differentiate between endometriosis and other explanations for patient symptoms. In addition, clinical markers should be as minimally invasive as possible, affordable and convenient to use with the added benefit of providing insight into potential treatment response. Furthermore, the ideal biomarker must provide equivalent or similar outcome measures

of sensitivity, specificity, positive, and negative predictive values to laparoscopy. Currently, the gold standard for diagnosis remains visualization of endometriotic lesions typically by laparoscopy followed by histopathological confirmation of disease [10]. Current trends favoring the non-surgical diagnosis of endometriosis increase the pressure to identify novel clinical markers of endometriosis. Despite a plethora of biochemical differences in the peripheral circulation, peritoneal fluid, and endometrial tissues of women with endometriosis compared to healthy controls, no marker has been found with adequate sensitivity or specificity to challenge laparoscopy for the diagnosis of endometriosis whether used alone or in a panel of clinical markers [11–16]. However, reports of promising results have been brought forward in

the literature from which epigenetic markers are potentially the most exciting.

of histone markers in the diagnosis of endometriosis has yet to be explored.

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

Long-chain non-coding RNA (lnc-RNAs) are 200–100,000 bp RNA molecules

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

**2. Candidate clinical markers of endometriosis**

**50**


**53**

endometriosis.

**4. Effect of control group definition**

in women with endometriosis merits further investigation.

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

**Cases Controls Reference** 

Plasma 33/20 NR Healthy miR-132 NR ↑15b, 16, 191,

*S = significant effect of menstrual cycle stage, NS = not significant, ND = not determined, NR = not reported, differential miR expression was either increased (*↑*) or decreased (*↓*) in women with endometriosis compared to* 

*Summary of miRNA's differentially expressed by microarray and RT-PCR in women with endometriosis* 

**miRNA**

**Cycle stage**

Healthy RNU6 NR ↓375, 27a-3p,

**Differentially expressed hsa-miR's**

> 195, 1973, 1979, and 4284

and 30d-5p

**Citation**

[45]

[29]

**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

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

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

*DOI: http://dx.doi.org/10.5772/intechopen.91324*

III–IV

*Direction could not be ascertained from the published report.*

*miRNA in bold have been replicated by at least one other group of investigators.*

**Fluid Case/**

*controls. \**

**Table 1.**

*(cases) compared to controls.*

**control (N)**

Plasma 6/4 Stages


*miRNA in bold have been replicated by at least one other group of investigators.*

*S = significant effect of menstrual cycle stage, NS = not significant, ND = not determined, NR = not reported, differential miR expression was either increased (*↑*) or decreased (*↓*) in women with endometriosis compared to controls.*

*\* Direction could not be ascertained from the published report.*

#### **Table 1.**

*Endometriosis*

**Fluid Case/**

**control (N)**

Serum 60/25 Stages

Serum 24/24 Stage

Serum 24/24 Stage

Serum 41/40 Stages

Serum 30/20 Stages

Serum 45/35 Stages

Serum 40/25 Stages

Plasma 23/23 Stage

Plasma 61/30 Stages

Plasma 55/23 Stages

Plasma Variable Stage

Plasma 51/41 Stages

Plasma 60/30 Stages

I–IV

III and IV

III and IV

I–IV

I–II

I–IV

I–IV

III and IV

I–IV

I–IV

I–IV

I–IV

I–IV

**Cases Controls Reference** 

Symptomatic (n = 20) and Healthy (n = 20)

Healthy 18 s

Symptomatic miR-

Symptomatic miR-

Symptomatic miR-28-3p

Symptomatic (n = 35) and Healthy (n = 30)

Symptomatic (n = 8–39) Healthy (n = 8)

mRNA

miR-30e and 99a

103-3p

RNU6 vs. miR-16 vs. miR-30b

106a-5p, 199a-3p, 150-5p, 425-5p, 125a-5p, and 30e-5p

and 423-3p

Symptomatic miR-16 ND ↓17-5p, 20a

**miRNA**

Symptomatic RNU6 NS ↑**199a** and

Symptomatic RNU6 P vs. S ↓let-7b, c, d, e,

Symptomatic RNU6 NS ↓3613-5p

Infertile cel-miR-39 ND ↓30c-5p,

Symptomatic RNU6 ND ↑**122** and **199a** [54]

**Cycle stage**

**Differentially expressed hsa-miR's**

> , **145**\* ,

**122**, ↓9\*

**141**\* , and 542-3p

f (P) and 135a (S)

and 6755-3p ↑18a-5p, 125b-5p, 143-3p, 150-5p 342-3p, **451a** and 500a-3p

127-3p, 99b-5p, 15b-5p, 20a-5p, and ↑424-3p, 185-5p

ND ↓199a-5p [55]

and 22

NS ↓200a-3p and **141-3p**

ND ↑**145** (stages I and II), ↓31(stages I–IV)

NS ↓139-3p, 155 and 574-3p

NS ↓miR154-5p and 378a-3p, ↑196b-5p and 33a-5p

S \*

125b-5p, 28-5p, 29a-3p

RNU6 NS ↑**451a** [48]

**Citation**

[31]

[46]

[47]

[52]

[30]

[49]

[50]

[51]

[53]

[56]

**52**

*Summary of miRNA's differentially expressed by microarray and RT-PCR in women with endometriosis (cases) compared to controls.*
