Endometriosis: Pathogenesis and Treatment 2014 Ed.

20. Biomarkers of Endometriosis

Amelie Fassbender1, 2Dorien O1, 2 Bart De Moor3Etienne Waelkens4Christel Meuleman2Carla Tomassetti2Karen Peeraer2 and Thomas D’Hooghe1, 2, 5  

(1)

Department of Development and Regeneration, Organ systems, KU Leuven, Leuven, Belgium

(2)

Department of Obstetrics and Gynaecology, Leuven University Fertility Centre, University Hospital Leuven, UZ Gasthuisberg, Leuven, Belgium

(3)

Department of Electrical Engineering (ESAT-SCD), KU Leuven, Leuven, Belgium

(4)

Department of Molecular Cell Biology, Campus Gasthuisberg, Leuven, Belgium

(5)

Division of Reproductive Biology, Institute of Primate Research, Karen, Nairobi, Kenya

Thomas D’Hooghe

Email: thomas.dhooghe@uzleuven.be

Abstract

Endometriosis is a benign gynecological disease defined by the ectopic presence of endometrium and associated with pelvic pain and infertility. The etiology and pathogenesis remain unclear. The gold standard of diagnosing endometriosis is laparoscopy followed by histological confirmation, associated with an 8-year delay in the diagnosis of endometriosis. A clinically reliable test for endometriosis can be expected to allow early diagnosis and treatment, with profound impact on the reduction of health care and individual costs. A noninvasive diagnostic test could be developed for serum or plasma, urine, and endometrial or menstrual fluid. A semi-invasive test could be developed for peritoneal fluid and eutopic endometrium. The development of such a test from initial biomarker discovery to be a clinically approved biomarker assay is a long, difficult, and uncertain process and includes four different phases. A review of the existing literature is provided in this chapter. Overall, most endometriosis biomarker studies remain at the level of phase I, with only a few currently in phase II of biomarker development. There is a need for a well-designed multinational study including academic and industrial partners for phase II and phase III trials.

Keywords

Diagnostic testEndometriosisUltrasound negative

*Amelie Fassbender and Dorien O are joint first authors

20.1 Introduction

Endometriosis is one of the most common gynecological disorders, affecting approximately 10 % of women in the reproductive age group and up to 50 % of all infertile women [12]. This estrogen-dependent disease is characterized by the presence of endometrial-like tissue at ectopic sites like the pelvic peritoneum, the ovaries, the rectovaginal septum, and in some cases even the pericardium, the pleura, and the brain [1]. Infertility is frequently associated with endometriosis as it is estimated that up to 50 % of endometriosis patients are subfertile (any form of reduced fertility with prolonged time of unwanted non-conception) [3]. Another important symptom that accompanies endometriosis is pain. Endometriosis-associated pain typically occurs as chronic pelvic pain in 90 % of women suffering from painful menstruations (dysmenorrhea), in 42 % of women suffering from pain during intercourse (dyspareunia), and in 39 % of women suffering from non-menstrual pain [45]. The degree of pain and the severity of endometriosis are poorly related and the exact mechanism of endometriosis-associated pain has yet to be determined [6]. Pain might be caused by pressure of large nodules on visceral organs, pelvic adhesions, tissue damage by infiltration of lesions, or an inflammatory response in the peritoneal cavity triggered by cyclical bleeding of lesions [47]. Recent evidence suggests that endometriosis-associated nerve fibers might be a pain-causing feature. Indeed, in endometriotic lesions, ingrowth of blood vessels along with nerve fibers has been observed [89].

The diagnosis of endometriosis is currently made by means of laparoscopy to identify endometrial-like tissue at ectopic places [1]. The severity of the disease is determined according to the revised American Fertility Society classification system. The classification of the disease stage (stages I to IV or minimal to severe) is based on acquired information during laparoscopic surgery on the morphology and depth of the implants, the presence, place, and type of lesion, and the presence, place, and type of adhesion [10]. Although laparoscopy is considered the golden standard, it can fail to detect very small lesions and lesions might not be noticed because of their location [11]. Furthermore, an average delay of 8 years precedes an accurate diagnosis. Five to six of these years can be attributed to a delay in the search of medical help and the remainder to obtaining the correct diagnosis [12].

The etiology and pathogenesis of endometriosis is still controversial and is almost certainly multifactorial [13]. Despite several established theories such as retrograde menstruation, abnormal immune system, coelomic metaplasia, genetic and epigenetic factors, and the stem cell theory, researchers still have not found the possible mechanism and cause of endometriosis.

Treatment options are directed against endometriosis itself, against endometriosis-associated pain, or against endometriosis-related infertility. Surgery aims to remove all visible endometriotic lesions [14]. However, as it does not manage the underlying mechanisms of the disease, recurrences of endometriotic lesions are common. Currently, different medical treatments are used in the clinic as well [15]. These treatments focus on altering the hormonal environment, hereby creating a suboptimal milieu for the growth and maintenance of the lesions. Still, these medications have many side effects and their efficiency is not ideal [14]. It has been indicated that, in order to improve diagnostic tools and treatment strategies, the underlying mechanism of endometriosis needs to be clarified [16]. In recent years, new more targeted therapies are being developed, e.g., aromatase inhibitors, angiogenesis inhibitors, and immunomodulating agents [17].

A noninvasive diagnostic test could be developed for serum or plasma, urine, and endometrial or menstrual fluid that can be recovered from the posterior vaginal fornix and from the cervix during speculum examination. A semi-invasive test could be developed in peritoneal fluid, obtained after transvaginal ultrasound-guided aspiration, or in endometrial, obtained after transcervical endometrial biopsy. Whatever method is used, the most important goal of the test is that no women with endometriosis or other significant pelvic pathology are missed who might benefit from surgery [18]. To achieve this, a test with a high sensitivity is needed, which is the probability of a test of being positive when endometriosis is present. At present, such a test does not exist [1920].

Researchers and clinicians need to realize that a diagnostic test may do more harm than good, e.g., by subjecting patients to unnecessary or even potentially harmful procedures [21] since the benefits of treating women with asymptomatic endometriosis are unclear [19]. Therefore, we do not recommend the development or use of a blood test for screening purpose in asymptomatic women. However, up to 45 % of subfertile women with a regular cycle whose partner has normal sperm quality, with or without pelvic pain, and with normal clinical examination and a normal pelvic ultrasound may have endometriosis [2022].

Most gynecologists are not sure if endometriosis is present if a woman has, for example, subfertility, a regular cycle, and a partner with a normal sperm examination and if they have been unsuccessful in trying to conceive for more than 1 year without moderate severe cyclic pelvic pain. In addition, if a woman has chronic pelvic pain (requiring at least cyclic or chronic use of pain killers), combined with a normal clinical examination and a normal pelvic ultrasound, many gynecologists are in doubt about the value of a diagnostic laparoscopy. From a clinical perspective, it is unlikely that these women will have moderate–severe endometriosis, but they may have extensive peritoneal endometriosis with or without adhesions associated with subfertility and possibly mild pain [18]. For this population, a noninvasive or semi-invasive diagnostic test would be useful to discriminate between women without endometriosis who need to avoid having unnecessary surgery and those with endometriosis, most likely minimal–mild disease, who are known to benefit from surgical therapy for both subfertility and pain and from controlled ovarian stimulation in combination with intrauterine insemination for subfertility [15182023]. In summary, a noninvasive test for endometriosis would be useful for women with pelvic pain and/or subfertility with normal ultrasound. This would include nearly all cases of minimal–mild endometriosis, some cases of moderate–severe endometriosis without clearly visible ovarian endometrioma, and cases with pelvic adhesions and/or other pelvic pathology, who might benefit from surgery to improve pelvic pain and/or subfertility [1820].

20.2 Noninvasive Test

The last few years, tremendous work has been published regarding biomarkers. In 2009 and 2013, researchers proposed that the development of reliable noninvasive test of endometriosis is one of the top research priorities in endometriosis [1624]. A clinically reliable test for endometriosis can be expected to have a profound impact on the reduction of health care and individual costs by:

1.

2.

3.

4.

Highly relevant markers known to be involved in the pathogenesis of endometriosis have been studied such as glycoproteins, inflammatory and noninflammatory cytokines, adhesions, and angiogenic and growth factors [20]. At present, neither a single biomarker nor a panel of biomarkers measurable in peripheral blood has been validated as a noninvasive test for endometriosis [19]. The measurement of serum CA-125 levels has no value as a diagnostic tool compared to laparoscopy [15]. Although previous studies have shown that various tumor markers, cytokines, and angiogenic and growth factors show altered levels in peripheral blood (plasma or serum) of women with endometriosis when compared to controls [1926], so far none of them, alone or in combination, have been validated as a noninvasive test for endometriosis [19]. Furthermore, at present there is no consensus on the value of inflammatory factors as biomarkers for endometriosis [20]. Comparable serum IL-6 [2728], IL-8 [2729], TNF-alpha, and IL-1 [272830] levels were previously reported in women with and without endometriosis. However, other investigators reported elevated peripheral levels of IL-6 [3031], IL-8 [3233], TNF-alpha [3134], and IFN-gamma [30] in endometriosis patients compared with controls [20].

So far, studies evaluating panels of biomarkers [3538] have been limited with respect to the number of biomarkers analyzed, the statistics used, and the lack of validation in an independent test set of patients [20]. One study proposed two panels of four biomarkers (annexin V, VEGF, CA-125, and glycodelin or sICAM-1) measured in plasma samples obtained during menstruation allowed the detection of ultrasound-negative endometriosis with high sensitivity (82 %) and acceptable specificity (63–75 %) in an independent test data set [39]. In the same study, three biomarkers (VEGF, annexin V, and CA-125) present in plasma obtained during menstruation allowed the diagnosis of endometriosis (minimal–severe endometriosis, both with and without ultrasound evidence) with 85–94 % sensitivity and 62–75 % specificity in an independent test data set [39]. These results are promising but need to be validated in a prospective study. Surprisingly, inflammatory molecules did not emerge as biomarkers in this study [39].

Recently, a diagnostic model including patient-reported clinical data derived from a validated questionnaire predicted any-stage endometriosis poorly, but stages III and IV accurately, with menstrual dyschezia and a history of benign ovarian cysts as the strongest predictive factors [40]. More research is needed to add clinical factors to diagnostic models based on plasma or endometrial analysis.

The development of a noninvasive diagnostic test, from initial biomarker discovery to a clinically approved biomarker assay, is a long, difficult, and uncertain process [41] and occurs in four different phases as described below:

·               Phase I—Preclinical discovery phase. This phase consists of exploratory preclinical studies aiming to identify potential biomarkers. In endometriosis research, the state of the art in this field has recently been reviewed by May et al. [19].

·               Phase II—Preclinical assay development and validation of a clinically useful noninvasive diagnostic test in the preclinical setting, as has been done in the context of endometriosis in our most recent paper [39].

·               Phase III—Prospective clinical validation and determination of clinical utility. This phase establishes the diagnostic accuracy and predictive value in the target population, but this phase has not yet been reached in endometriosis biomarker research so far.

·               Phase IV—Commercialization: product development by industry, which has not yet been done successfully for noninvasive endometriosis biomarkers.

Overall, most endometriosis biomarker studies remain at the level of phase I [19] and only a few have made it to phase II studies. There is a need for well-designed phase II and phase III trials to make progress in this field.

20.2.1 Nerve Fibers

The most promising efforts in developing a semi-invasive diagnostic test reported an increased nerve fiber density in the functional layer of eutopic endometrium of women with endometriosis, uniting the concept of an alternation of eutopic endometrium with the presence of nerve fibers provoking pelvic pain [42].

The first study to investigate the presence of nerve fibers in eutopic endometrium as a potential endometriosis biomarker reported a higher density of small nerve fibers in the functional layer of women with endometriosis compared with women without endometriosis [43]. Uterine curettage (endometriosis n = 25 and control n = 47) and hysterectomy samples (endometriosis n = 10 and control n = 35) were immunostained with antibodies against protein gene product 9.5 (PGP 9.5) as pan-neural marker for both unmyelinated and myelinated nerve fibers and neurofilament (NF) for myelinated nerve fibers. The most outspoken difference could be found in the functional layer of the endometrium where PGP 9.5-positive nerve fibers were observed in all endometriosis cases (on average for all cycle phases 11 ± 5 mm−2 for hysterectomy specimens and 10 ± 5 mm−2 for curettage specimens), but never in control patients (P < 0.001). The difference was the most obvious in the secretory phase of the cycle, but was also significant in the menstrual and proliferative phases [43].

To determine the type of small nerve fibers in the endometrium, the same research group published an additional study [44] in which uterine blocks were obtained after hysterectomy from women with endometriosis (n = 10) and controls (n = 35). Positive staining of the nerve fibers in the functional layer for SP (1.0 ± 0.2 mm−2), CGRP (1.7 ± 0.4 mm−2), VIP (8.5 ± 2.3 mm−2), and NPY (9.6 ± 2.8 mm−2) and the absence of staining with TH or VAChT antibodies indicated the presence of different types of sensory C nerve fibers in women with endometriosis. This finding might be relevant to the mechanism of pain generation in women with endometriosis.

While the previous studies [4344] focused on full endometrial curettage and full uterine blocks after hysterectomy, clinical relevance of an endometrial biomarker is only valid if an endometrial biopsy can generate the same sensitivity and specificity [45]. Endometrial curettage requires general anesthesia and the need for hospitalization, while endometrial biopsy sampling can be done in an outpatient setting [46]. A pilot study including 20 endometriosis patients and 17 controls compared the diagnostic value of an endometrial biopsy and a full curettage [45]. Much care was given to the correct sampling of the endometrial biopsy, using a cannula to take a tissue column representing full thickness of the endometrium. Despite the wide range in nerve fiber densities observed in the functional layer of the endometrium, previous findings of increased nerve fiber density in endometrium obtained in hysterectomy specimens were confirmed in endometrium obtained by endometrial sampling (26.7 ± 55.9 mm−2) or by curettage (20.4 ± 33.1 mm−2), suggesting equal diagnostic capacity of the two collection methods. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were all 100 %.

To further prove the diagnostic relevance of the detection of nerve fibers in an endometrial biopsy, a double-blind study [47] was conducted using endometrial biopsies from 99 consecutive patients who received a laparoscopy for pelvic pain and/or infertility, including 64 cases with endometriosis and 35 controls without endometriosis. Immunohistochemical staining with PGP 9.5 showed a mean nerve fiber density of 2.7 ± 3.4 nerve fibers per mm2 in the functional layer of endometrium from nearly all endometriosis patients and an absence of nerve fibers in most control patients (except in six cases), resulting in a statistically significant difference between the two groups (P < 0.001). The specificity and sensitivity of the test were 83 and 98 %. PPV and NPV were 91 and 96 %, respectively.

The accuracy of making an endometrial-based diagnosis of endometriosis was also investigated by two other independent research groups [4849]. In a paper from Bokor et al. [49], endometrial samples from only the secretory phase were included, because the initial Tokushige pilot study had reported a higher nerve fiber density in this phase [43]. Only patients with minimal (n = 10) or mild (n = 10) endometriosis were recruited, as they have the greatest need of a semi-invasive diagnostic test [49]. Another 20 patients with a normal pelvis were included as controls. Prevalence of pain symptoms was comparable in both groups. PGP 9.5, NF, SP, VIP, NPY, and CGRP expression was detected by immunohistochemistry and showed a 14-fold higher density of small nerve fibers in the functional layer of women with endometriosis (1.96 ± 2.73 mm−2) compared with the control group (0.14 ± 0.46 mm−2) (P < 0.0001). Multivariate analysis showed that the combined assessment of VIP, PGP 9.5, and SP had the greatest diagnostic accuracy to predict endometriosis with a sensitivity of 95 %, a specificity of 100 %, a PPV of 100 %, and an NPV of 95 %. In contrast to the Bokor study (84), results were not controlled for cycle phase or disease severity in another study [48]. In spite of this limitation, nerve fibers positive for PGP 9.5 or NF were present in all 12 endometriosis cases (mean 13.1 ± 3.3 mm−2) and only in 3 of 15 patients without endometriosis (mean 2.2 ± 4.7 mm−2) resulting in a significant difference between the two groups, although no exact p-value was mentioned [48]. Sensitivity and specificity were not mentioned, but can be calculated based on the published data to be 100 and 80 %, respectively. No significant differences in the pain characteristics of both groups were reported.

An alternative study showed the added value of detection of endometrial PGP 9.5-stained nerve fibers to IL-6 measurement in serum for diagnosis of minimal/mild endometriosis [50] in endometriosis patients (n = 35) compared with controls (n = 40). Specificity and sensitivity of the test for predicting minimal/mild endometriosis regarding the presence of nerve fibers were 92 and 80 %, respectively (PPV and NPV were 81 and 91 %). These values raised to 100 and 92.5 % (PPV and NPV 92.7 and 100 %) when IL-6 was added. The exact nerve fiber densities in endometriosis patients were not mentioned.

A meta-analysis of several studies [434749] included 131 women with endometriosis and 152 controls [51]. Mean nerve fiber density per mm2 was significantly higher in endometriosis patients (5.67 ± 12.08) versus controls (0.78 ± 3.39, P < 0.005) [51]. This meta-analysis demonstrated that PGP 9.5 staining of eutopic endometrium can be used as a semi-invasive test and could reduce in the delay of diagnosis; however, further research is necessary [51].

Despite the promising data shown in the previously discussed papers, nerve fiber density in eutopic endometrium is not yet ready to be used in clinical practice. The abovementioned data were not confirmed by Newman and coworkers, comparing 20 patients with endometriosis (minimal–mild n = 20, moderate n = 4, and severe n = 4) to 25 controls [52]. No significant difference was observed between the disease and control group in PGP 9.5-positive nerve fibers, visualized by immunohistochemistry [52]. Using western blotting as a quantitative method to detect the neural markers, all three markers were expressed in endometrium from both endometriosis cases and controls (PGP 9.5: P = 0.0991, VR I: P = 0.0621, NGFp75: P = 0.2586) [52].

In another study, no statistical difference was observed in endometrial nerve fiber density, determined by PGP 9.5 staining, between women with (n = 47) and without endometriosis (n = 21) [53]. This study, however, was marked by several methodological drawbacks. Firstly, threshold for endometrial biopsy quality was low (one low-power field of well-oriented endometrial mucosa sample perceived to be sufficient to being classified as “satisfactory”). Secondly, the exact endometrial nerve fiber density was not mentioned, since data were classified as positive or negative depending on the presence or absence of nerve fibers [53]. This study highlighted that methodological factors such as the method of endometrial biopsy sampling and image analysis might influence the consistency of results for this semi-invasive diagnostic test [53]. Taking into account the uneven distribution of the nerve fibers in a biopsy, reliable results may require inspection of more than one section per biopsy [45]. Furthermore, the whole surface of an endometrial section should ideally be examined [49] as opposed to counting endometrial nerve fibers in randomly chosen fields [45]. Additionally, it is crucial that background staining during immunohistochemistry is reduced as much as possible, which can be challenging. In a research context, results should be analyzed by an experienced pathologist. While it is essential to refine methodology, it is equally important to select and phenotype the study population. All cases in the endometriosis group should have laparoscopically confirmed endometriosis, preferably with histological confirmation of the presence of endometrial glands and stroma in the lesions [49]. In some of the studies, this criterion was not met, as only in a part of the cases histological confirmation of endometriosis was available [4547]. Further, according to the modified QUADAS (Quality Assessment of Diagnostic Accuracy Studies) criteria [19], detailed information should be available regarding the demographics such as disease stage and cycle phase [49]. Study patients ideally have not received hormonal medication 3 months prior to surgery, because systemic exposure to hormonal medication might reduce the presence of nerve fibers in eutopic endometrium [43]. However, this medication-free period was as short as 1 month in one study [50] and could have been even shorter in another study stating that patients were not on medication only at the time of laparoscopy [45]. Additionally, one study included three patient groups (one on current hormonal therapy (n = 11), one without hormonal therapy (n = 44), and a group with unknown status (n = 13)) and found an overall poor sensitivity for the test that was independent of the treatment status [53]. This lack of significance might have been due to the low sample size in each group. The control group should be equally well characterized and should have absence of endometriosis confirmed by laparoscopy (QUADAS criteria [19]). Included controls were only described in detail in a number of studies [4344]. They consisted of women undergoing tubal sterilization, assessment prior to tubal reanastomosis or investigation of infertility [43] and women with uterine fibroids, uterine prolapse, abdominal adhesions, and abnormal menstrual bleeding [44]. Two studies included only controls with a normal pelvis [4950]. Pain characteristics should be comparable in case and control groups, although this did not always seem to be the case. In the double-blind study by Al-Jefout, patients with pain symptoms were present in 87.5 % of the endometriosis cases, but only in 45.7 % of controls [47]. This could influence the results as previous studies have postulated a link between pain and nerve fiber ingrowth in ovarian or deep infiltrating endometriosis [5455]. Interestingly, the association between pain and the presence of nerve fibers in endometrium exists in women with other gynecological disorders such as adenomyosis or uterine fibroids, suggesting that the presence of nerve fibers is linked to the diagnosis of pelvic pain rather than to the occurrence of endometriosis [2056].

Implemented statistics were comparable in most studies with the use of univariate analysis such as the Mann–Whitney U-test as a nonparametric test to compare two groups [434447] and the Kruskal–Wallis chi-square test for the comparison of multiple groups [47]. The Kolmogorov–Smirnov test [48] was used by one group to determine normality, before performing a student t-test or related nonparametric test to evaluate nerve fibers [48]. The use of the Kolmogorov–Smirnov test is discouraged, because of its poor performance in assessing normality [57]. Other groups mentioned using the student t-test, but did not state whether they had verified a normal data distribution first [5053]. Advanced multivariate statistical analysis was only performed in one study by Bokor and coworkers, using multivariate logistic regression and leave-one-out cross-validation (LOO-CV) analysis with least-squares support vector machines (LS-SVM) modeling [49].

In conclusion, it can be stated that the determination of nerve fiber density in the endometrium as a diagnostic test for endometriosis is a promising prospect. However, the techniques and study populations should be randomized and further validation with larger patient groups should be performed before this test can be used in the clinic. Up to now, none of the studies evaluating nerve fibers in endometrial biopsies have reached phase III of biomarker development. To make progress in this field, there is a need for well-designed phase II and III studies. Validation should be carried out in a patient population experiencing pain/infertility with a 30 % prevalence of endometriosis.

20.2.2 MicroRNAs and Endometriosis

Since endometriosis is a multifactorial and polygenic disease, aberrant expression of microRNA (miRNA) has been proposed as a potential pathogenic mechanism [2058]. Essentially, miRNAs are short (~22 nucleotides) single-stranded noncoding RNAs with the capacity to regulate gene expression at a posttranscriptional level through translational repression or messenger RNA (mRNA) degradation [59]. One miRNA may regulate the expression of several hundreds of mRNAs [60]. Diagnostic use of miRNA signatures has been proposed for various diseases, such as cancer, cardiovascular diseases, rheumatic diseases, and neurological disorders [61]. In cancer, miRNAs perform better than mRNAs to classify poorly differentiated tumors [62].

20.2.2.1 Endometrium

Between eutopic endometrium and endometriotic lesions, differential expression of a number of miRNAs has been shown and some of their mRNA targets, as predicted by in silico algorithms, have previously been identified as dysregulated in endometriosis [6366]. A number of cellular events involved in the development of endometriotic lesions has been linked to miRNA dysfunction, such as hypoxic injury, inflammation, tissue repair, disrupted cell cycling, extracellular matrix remodeling, angiogenesis, cellular movement, and DNA methylation [6568].

More importantly, differences in miRNA expression between eutopic endometrium of endometriosis patients and controls have been found, providing a potential role for miRNAs as biomarkers or therapeutic tools in endometriosis [20]. In a study conducted by Toloubeydokhti et al., miR-17-5p, miR-23a, miR-23b, and miR-542-3p were upregulated in eutopic endometrium (n = 5) of patients with endometriosis, compared with controls (n = 5) [69]. These miRNAs have previously been linked to cancer [70]. The predicted downstream targets were known to be involved in endometriosis (steroidogenic acute regulatory protein (StAR), aromatase, and COX-2) and were confirmed to be upregulated, using RT-PCR [69]. However, an independent research group found reduced expression of miR-23a and miR-23b in eutopic endometrium of endometriosis cases compared with disease-free controls [71]. This reduction in miRNA expression correlated with a 2.26-fold increase (P = 0.041) in steroidogenic factor 1 (SF-1) expression [71]. In another study, endometrial miR-9 and miR-34 were significantly reduced in patients with moderate/severe endometriosis (n = 4) compared with controls with uterine leiomyomata (n = 3) [72]. Their targets were predicted to enhance the proliferative capacity of the endometrium [72]. In another study, an increased miR-21 expression throughout the menstrual cycle was reported to allow distinction between severe and mild endometriosis (controls: n = 12, mild endometriosis: n = 19, severe endometriosis: n = 44) and associated with a downregulation of tumor suppressor genes such as PTEN [73]. In another study, it was found that miR135a/b expression was significantly upregulated in eutopic endometrium of endometriosis patients (n = 32) compared with controls (n = 50) in the proliferative phase (miR135a and miR135b) and the secretory phase (miR135b) [74]. This upregulation of miR135a/b was marked by a downregulation of HOX10 mRNA and protein (a regulator of endometrial receptivity) which was reversible by the addition of miR-135a/b inhibitors [74]. In yet another paper, the downregulation of miR-126 was observed in ectopic endometrium (n = 16) and in eutopic endometrium (n = 31) from women with endometriosis (n = 31) when compared with controls (n = 27) [75]. This downregulation of miRNA was inversely correlated with the expression of Crk, an oncogene [75].

20.2.2.2 Peripheral Blood

Recently, several studies of miRNA in peripheral blood have been performed in the context of endometriosis [7678]. One study, investigating circulating miRNAs in plasma, showed a significant downregulation of miR-17-5p (p = 0.011), miR-20a (p = 0.0020), and miR-22 (p = 0.0002) in women with endometriosis (n = 23) compared with women without endometriosis (n = 23) [78]. Receiver operating characteristic (ROC) curve analysis permitted the calculation of the area under the curve (AUC) (0.74, 0.79, and 0.85 for miR-17-5p, miR-20a, and miR-22, respectively) after which a cutoff value could be set to determine sensitivity (70.0, 60.0, 90.0 % for miR-17-5p, miR-20a, and miR-22, respectively) and specificity (70.0, 90.0, 80.0 % for miR-17-5p, miR-20a, and miR-22, respectively) to differentiate between women with and without endometriosis [78]. Combined assessment of the three miRNAs resulted in an AUC of 0.90 [78]. Additionally, another study showed that discrimination was possible between plasma of healthy controls (n = 20) and endometriosis patients (n = 33) with 88 % sensitivity and 60 % specificity (AUC = 0.90), based on the assessment of miR-16, miR-191, and miR-195 which were all highly expressed in endometriosis [77]. Endometriosis and endometriosis-associated ovarian cancer (EAOC) (n = 14) could be distinguished from each other through a combination of miR-21, miR-362-5p, and miR-1274a with 57 % sensitivity and 91 % specificity and an AUC of 0.92. Distinction of endometriosis and serous ovarian cancer (SOC) (n = 21) was possible with 90 % sensitivity and 73 % specificity (AUC 0.88), based on the assessment of miR-362-5p, miR-628-3p, and miR-1915 [77]. A trend of elevated plasma miRNA expression compared with healthy controls was found in endometriosis cases and even more so in EAOC cases. This suggests that endometriosis might be a precursor stage of EAOC and the possibility for miRNA signatures to act as disease progression markers [77].

In serum of patients with endometriosis (n = 60) and controls (n = 25) the combination of miR-199a, miR-122, miR-145*, and miR-542-3p could predict endometriosis with 93.22 % sensitivity and 96.00 % specificity (AUC = 0.994) [76].

Currently, there is no agreement on which circulating miRNA can be used for data normalization. One group selected U6 as a normalization control because of its use as internal control in other studies [76]. However, other investigators preferred miR-16 as endogenous control because it is more stable and less variable in circulation than other miRNAs [78]. Yet another research group used miR-132 for data normalization, as they found this miRNA to be homogeneously expressed across all samples [77].

It is also important to distinguish the presence of miRNAs in either plasma or serum. Although higher miRNA concentrations were observed in plasma than in serum in one study [79], this was not confirmed in another study [80]. This discrepancy might have been due to pre-analytical variability concerning blood tube type or differences in sample-processing protocols [79]. Conversely, other studies have shown an increased concentration of miRNAs in serum samples when compared to plasma samples of the same patient, although they conclude that plasma might be the sample of choice because miRNAs that are released during the coagulation process in serum samples may interfere with the true miRNA profile [81].

Interestingly, miRNAs are exceptionally stable in plasma, serum, and tissue samples, making them excellent candidates for biomarker research [80]. Possibly miRNA is protected from RNases in blood by being packaged in vesicles or bound to RNA-binding proteins [82].

Despite the potential advantages of a miRNA-based blood test for endometriosis, some possible pitfalls should be taken into consideration. It should be noted that apart from their best known function as transcriptional repressor, miRNAs can act as translational activators [83]. Therefore the relationship between miRNA and target mRNA is not necessarily an inverse one [72]. Additionally, in most miRNA studies, specific mRNA targets are often not experimentally verified, but only predicted by various computational algorithms [6572]. Beside the influence of the choice of mathematical algorithm, one mRNA can be the target of multiple miRNAs that might not all be altered, rendering validation in an in vitro setup essential to determine the effect of the dysregulation of the miRNA of interest [6572].

Since miRNA patterns change under the influence of reproductive hormones and are thus altered in different phases of the menstrual cycle [84], an adequate control group should be chosen, preferably without any other illness as this could also influence the miRNA profile [78].

The discrepancies observed in different miRNA results can be explained by differences in patient selection, microarray protocol, and choice of housekeeping genes for data normalization. Therefore a standardized methodological approach needs to be determined and controls and patients need to be selected according to the QUADAS criteria. The rationale for implementing next-generation sequencing is that a larger amount of miRNAs can be examined, without being dependent on the availability of probes [66]. Exceptionally important is the role of advanced statistical methodology in studies where many variables are compared. Therefore, all data should be controlled for multiple testing [85].

In conclusion, miRNAs are interesting subjects in the further development of biomarker research, although more extensive research in a larger population and validation in independent test sets should be conducted with full awareness of methodological issues and the complexity of miRNA mechanisms.

20.3 Conclusions

Despite its gradual progress, the biomarker research field still faces the challenge of successfully developing a clinically approved biomarker assay for endometriosis [20]. Up to now, no semi- or noninvasive diagnostic test exists for endometriosis [19]. Most studies so far have included limited numbers of patients, limited assessment of different cycle phases and endometriosis stages, limited number of biomarkers analyzed, limited statistical analysis (mostly univariate statistical analysis only), and the lack of validation in an independent test set of patients [20].

In the past, collaborations between research centers have been limited and standard operating procedures (SOPs) are different among centers [20]. Most biomarker studies remain at the level of phase I, the preclinical discovery phase, as reviewed by May et al. [1942]. Only a few biomarkers make it to phase II, the preclinical assay development and validation [394786].

To develop a diagnostic test for endometriosis, semi-invasive techniques utilizing endometrial biopsies have been explored [87]. An increased amount of small unmyelinated nerve fibers in the functional layer of eutopic endometrium of endometriosis patients has been reported [454988]. However, prospective validation of this method in a blinded fashion is needed in a larger patient population, using a standardized biopsy sampling technique and standard immunohistochemical and statistical methods. Validation should be carried out in a patient population experiencing pain/infertility with a 30 % prevalence of endometriosis.

At present, neither a single biomarker nor a panel of biomarkers measurable in peripheral blood has been validated as a noninvasive test for endometriosis [19], although panels with reasonable specificity and sensitivity have recently been published [39]. Panels of biomarkers that have been proposed as diagnostic tests in phase I and II trials should be validated in prospective phase III studies [20]. In this phase, diagnostic accuracy of the proposed panel needs to be confirmed in an independent test population with infertility/pain scheduled for surgery [20]. The predicted outcome should be compared with the actual presence of endometriosis. Future studies should also focus on the large and clinically relevant population of endometriosis patients using hormonal medication, which is underrepresented in published biomarker discovery studies.

In order to improve the specificity and sensitivity of previously proposed diagnostic models, several choices are possible: using advanced protein technology to discover new and unknown biomarkers, combining a blood test with a semi-invasive test, or adding clinical factors to a model.

Advanced protein technologies such as antibody-based large-scale protein arrays, allowing concurrent detection of up to 1,000 proteins, might be required to improve the diagnostic power of a noninvasive blood test for endometriosis. Antibody-based microarrays have already been applied in biomarker screening for cancer research [89]. Combined assessment of a blood sample and an endometrial biopsy is another option to increase the power of a diagnostic test. A study has been published, combining a noninvasive test measuring IL-6 serum levels with a semi-invasive test for the presence of nerve fibers [50]. Despite the high sensitivity and specificity obtained by this method, it might not be suitable for clinical use due to the need for two interventions (blood and endometrial biopsy sampling). Additionally, the diagnostic accuracy of a test might be enhanced by the incorporation of clinical factors into a non- or semi-invasive test. A diagnostic method published by Nnoaham et al. predicted any-stage endometriosis poorly, but stages III and IV accurately [40]. The most predictive factors for the presence of endometriosis were menstrual dyschezia and a history of benign ovarian cysts [40].

While genomics, transcriptomics, and proteomics have already been covered extensively in endometriosis research, metabolomics is an underdeveloped field with only one study published up to date [90]. More extensive research in this topic should clarify whether the alteration of the metabolomic profile in the blood of women with endometriosis could serve as a diagnostic test for endometriosis. Another field of interest could be stem cell markers and endometriosis. Stem progenitor cells may serve as early markers and also detect recurrence [91]. The endometrium contains endometrial/stem progenitor cells that have been reported to be present in the peritoneal cavity in women that have retrograde menstruation [91]. Positive immunostaining for stem cell markers such as CD9, CD34, c-Kit, Oct-4, and Musashi-1 was detected in isolated epithelial and stromal cells in eutopic and ectopic endometrium [92]. The expression of stemness-related markers suggested that endometriosis arises as a clonal proliferation with the putative involvement of stem cells (2). The limitation of this study was the small sample size [20].

In conclusion, biomarker research will have to proceed from the discovery phase to the validation phase, focusing on successfully conducting phase III and IV trials [20]. To achieve phase III biomarker validation in both semi- and noninvasive testing, independent patient populations should be investigated, preferably with a realistic prevalence of endometriosis (30 % in women with infertility and 30–50 % in women with pelvic pain) [20]. According to one of the QUADAS criteria, patient characterization regarding cycle phase and endometriosis stage should be clearly stated and the control group should be standardized [1920]. Patients should have laparoscopically confirmed presence or absence (for controls) of endometriosis [19]. The different stages and clinical classifications of endometriosis should be taken into account in further research, treating peritoneal, ovarian, and rectovaginal septum endometriosis as different entities [9394]. Because of the different types (superficial, deep, cyst) and location of endometriosis, it is possible that a different subset of biomarkers may be required for the diagnosis of different stages of endometriosis [16], i.e., women with peritoneal endometriosis may have different markers when compared to those with rectovaginal endometriosis [1920]. This requires more genomic analysis of the different lesion types and different locations.

Statistical approaches should be multivariate, instead of the univariate approaches used in many studies now, and the involvement of biostatisticians is crucial to extract a diagnostic test from raw data [20]. Standardized protocols for sample collection, processing, and storage along with reporting uniform clinical information are crucial to compare results between studies and to enable a multicenter biobanking approach [1695]. Collaborations are essential to obtain the large sample sizes that are required for the statistical power of validation studies [16].

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