Review
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Michigan University on 10/18/14 For personal use only.
Noninvasive biomarkers of endometriosis: myth or reality? Expert Rev. Mol. Diagn. 14(3), 365–385 (2014)
Tea Lanisˇnik Rizˇner Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia Tel.: +386 1 5437 657 Fax: +386 1 5437 641 [email protected]
Endometriosis affects 10% of premenopausal women and 35–50% of women with infertility, pelvic pain, or both. At present, endometriosis can only be diagnosed with surgery, where laparoscopy is considered a gold standard. Noninvasive biomarkers are thus urgently needed. In 2010, the peripheral biomarkers of endometriosis were systematically reviewed by May et al. However, with the introduction of ‘-omics’ technologies, we have witnessed immense progress in biomarker discovery, which now calls for an overview of recent studies. This report looks at potential blood and urine biomarkers of endometriosis published in the last 3 years. The current status of noninvasive diagnostic biomarkers of endometriosis is discussed, with the limitations of these studies identified and recommendations for future biomarker discovery provided. KEYWORDS: biomarkers • endometriosis • metabolomics • proteomics • transcriptomics
transplantation, and especially on retrograde menstruation, are relatively widely accepted [8,9].
Endometriosis is a common gynecological disease
Endometriosis is a frequent benign gynecological disease that is characterized by the presence of endometrial tissue outside the uterine cavity. In the general population of women of reproductive age, the predicted prevalence of endometriosis is 6–10%, but the frequency increases to 30–50% in women with pain, infertility or both [1,2]. Worldwide, endometriosis affects an estimated 176 million women [3]. Ectopic endometrial tissue can be found in different parts of the peritoneal cavity, with these locations defining three different entities with different etiologies and pathogenesis: ovarian, peritoneal and deep infiltrating endometriosis [4,5]. In rare cases, endometrium-like tissue can be found outside the peritoneal cavity in other sites, such as the pleura, and even in the brain [6,7]. The pathogenesis of endometriosis is very complex and involves enhanced cell adhesion, degradation of the extracellular matrix, angiogenesis, proliferation, aberrant apoptosis, disturbed cell communication, disturbances of the immune system, loss of differentiation capacity and other pathophysiological processes [8]. There is no single theory that can explain all of the aspects of endometriosis although the theories on its in situ development (i.e., the metaplastic theory) and informahealthcare.com
10.1586/14737159.2014.899905
Why do we need noninvasive diagnostic biomarkers for endometriosis?
The current diagnostics of endometriosis is problematic because the symptoms are nonspecific and they can be associated with a number of different conditions (e.g., irritable bowel syndrome, pelvic inflammatory disease) [10]. At present, the disease can only be diagnosed with surgery: the gold standard is a laparoscopic procedure, which is a surgical visual inspection of the pelvic organs. This requires an experienced surgeon and general anesthesia, and it also carries surgery-associated risks like major vessel or bowel injury. However, laparoscopic visualization of the peritoneal cavity also has its limitations and should be combined with histological analysis. Indeed, only 54–67% of suspected lesions are confirmed histologically [6,11], while biopsies of normal appearing peritoneum followed by histology reveals endometriosis in 6% of infertile women [6,7,12]. As a result of the nonspecific symptoms and sometimes inadequate invasive diagnosis, there is an average delay of 6.7 years between the onset of symptoms and the surgical diagnosis of endometriosis [1]. Indeed, in certain centers, it can take up to 11 years
2014 Informa UK Ltd
ISSN 1473-7159
365
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Michigan University on 10/18/14 For personal use only.
Review
Lanisˇnik Riz ˇner
before affected women are diagnosed and receive the appropriate treatment [13]. Endometriosis is also associated with high healthcare costs, which have been estimated as an annual e9,579 per woman, which includes the costs of both the direct healthcare and the loss of productivity [14]. The economic burden associated with endometriosis is similar to other chronic diseases such as diabetes, Crohn disease and rheumatoid arthritis. Women who are affected lose 10.8 h/week of work on average, which, as calculated for Italy, translates into costs as high as US$231 per woman per week [13]. There is a large potential for improving the cost–effectiveness of the management of endometriosis by reducing the time of diagnosis, so that the disease is under control before the onset of the various complications and infertility [1]. The development of a noninvasive diagnostic test for endometriosis would have a groundbreaking effect on the quality of life of these patients. Earlier diagnosis and treatment would reduce the discomfort of endometriosis patients, and the disease progression that is associated with infertility and other complications. A European Endometriosis Alliance survey carried out with 7025 women with endometriosis revealed that 65% of these patients were misdiagnosed and 46% had to see five or more doctors to get a correct diagnosis [15]. The recurrence rate of endometriosis is high as well; estimates show that following surgery, 21.5% of patients experience recurrence within 2 years and 40–50% within 5 years [16]. Currently, there are no known diagnostic biomarkers, and even less so, biomarkers of recurrence. Although imaging techniques, such as ultrasound, computed tomography and MRI, are being explored, their current diagnostic accuracy is still inferior to direct laparoscopic visualization [1,17,18]. Other issues related to these technologies include the required training level to achieve acceptable sensitivity and specificity rates and the costs of these procedures [1]. According to a consensus workshop that was held at the World Congress on Endometriosis in 2011, a noninvasive test for reliable diagnosis of endometriosis, and particularly of early endometriosis, is of paramount importance [1]. Up to 45% of subfertile patients with a regular cycle might have endometriosis, and they would benefit from a blood test that could identify the symptomatic patients who would need surgical therapy for subfertility and pain [19,20]. Recent reports on potential noninvasive biomarkers of endometriosis
A biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to therapeutic intervention [21]. May et al. [10] systematically reviewed peripheral biomarkers of endometriosis and assessed the quality of individual studies using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) criteria [22,23]. Although over 100 potential biomarkers of endometriosis met the selection criteria, neither a single biomarker nor a panel of biomarkers have so far been unequivocally shown to be clinically useful [10]. However, with the introduction of the ‘-omics’ technologies, we 366
have witnessed immense progress in the field of biomarker identification, which now calls for an overview of recent studies. This manuscript includes the reports on potential biomarkers for noninvasive diagnostics of endometriosis, which have been published since the last systematic review in 2010 [10]. Relevant studies were identified by searching the Pubmed database using endometriosis and biomarkers as the key words, for the period from August 2009 to November 2013. Only manuscripts in English were included. Studies comparing endometriosis with infertile patients, healthy controls and patients with benign ovarian cysts were included; studies comparing endometriosis with ovarian cancer were excluded. From the 36 published studies initially included, the following information was extracted: sample analyzed (e.g., whole blood, plasma, serum, urine), analytes (e.g., proteins, metabolites, RNA molecules, blood cells, panels of biomarkers); characteristics of the case group and of the enrolled patients (e.g., mean age, BMI, type of endometriosis, stage of endometriosis, menstrual phase), characteristics of the control group and of the control patients or healthy women (e.g., mean age, BMI, diagnosis, menstrual phase), methodology used (ELISA, MALDI–TOF–MS including statistical methods), main findings, and if available, the proposed statistical model (TABLES 1–8). Based on the modified QUADAS criteria [10], the quality of these studies was assessed and their limitations were identified. The majority of case groups comprised healthy women or patients with other gynecological diseases, not women with endometriosis-like symptoms. The absence of endometriosis was generally confirmed by laparoscopy and only five of the studies reported that histology was also performed. In several studies, the patients were not stratified based on their menstrual phase, or it was not clear whether this potential confounding effect was taken into consideration. The stage of disease was usually reported, but more than half of the studies did not stratify patients according to different stages, or they did not report whether this potential confounding effect was examined. Although different types of endometriosis represent different entities, the type of endometriosis was reported in only one-third of all of these studies. Different approaches in search of noninvasive biomarkers of endometriosis
Reliable biological markers for noninvasive diagnostics have been searched for in peripheral blood, whole blood (5 studies), serum samples (20 studies) and plasma samples (8 studies), as well as in urine samples (4 studies) (TABLES 1–8). Serum is still the most common biological sample analyzed, although it can be problematic due to inherent variability in composition, depending on the blood-clotting process [24]. Surprisingly, in the last few years, there have been no reports on the evaluation of biomarkers for endometriosis in menstrual blood, even though this material most closely reflects the physiological and molecular environment of the pathologically altered eutopic endometrium of endometriosis patients [8]. To date, different methodologies have been used in the search for biomarkers for endometriosis [10,19]. By targeted Expert Rev. Mol. Diagn. 14(3), (2014)
informahealthcare.com
HE-4
Urocortin
CRP
PEDF
CD-23
Huhtinen et al. (2009)
Tokmak et al. (2011)
Vodolazkaia et al. (2011)
Chen et al. (2012)
Ramos et al. (2012)
Serum before surgery; >8 h fasting; stored at -20˚C
Serum before laparoscopy, (after >12 h fasting)
Plasma before surgery, stored at -80˚C
Plasma before surgery, stored at -70˚C or -80˚C
Serum before surgery, stored at -20˚C or -80˚C
Sample
C/E stages III-IV (>0.71 mg/l) SEN: 80.7% SP: 63.9% SEN + SP = 1.45 NA
NA
" CRP levels E/C (L phase) 0.89 mg/l/0.54 mg/l
# PEDF non-E/E (mean ± SD) 16.3 ± 6.6 ng/ml vs 24.5 ± 7.3 ng/ml, p < 0.001 # PEDF in E patients with pain
~ CD-23 levels
High sensitivity CRP assay (Roche, Belgium) Mann–Whitney test ELISA (P056-9, Groundwork Biotechnology Diagnosticate, USA) Univariate analysis
ELISA (Bender MedSystems, Austria); Mann–Whitney test
91 subfertile women (19 M, 36 F, 36 L)
28 patients without endometriosis, indications: infertility (3), pelvic mass (17; 12 mesosalpinx cyst, 5 paraovarian cyst), elective tubal sterilization (8) 17 F phase, 11 L phase Age: 31.3 ± 6.6, BMI: 21.1 ± 2.3 58 women without endometriosis; age between 18 and 45, in M and F phase
43 patients (30 OE); 28 F phase 15 L phase Confirmed by laparoscopy and histology Age: 31.1 ± 5.3, BMI: 20.8 ± 2.4
44 patients confirmed by laparoscopy and histology; age between 18 and 45, in M and F phase
Urocortin (4.16 ng/ml)/ CA-125 (21.38 U/l) SEN: 76.2/ 88.1% SP: 45.7/ 63% SEN + SP = 1.51
~ Urocortin OE: 4.8 ± 1.0 ng/ml controls: 4.5 ± 1.0 ng/ml
Urocortin EIA kit (Phoenix Pharmaceuticals, USA) Mann-Whitney test
46 patients with benign ovarian cysts, 23 serous, 8 teratoma, 5 mucinous, 10 functional ovarian cysts Age: 33.2 ± 11.8 BMI: 23.9 ± 2.6 Early F
No
No
No
[28]
[33]
[27]
[29]
[25]
No
OE/H HE-4+CA-125 SEN: 62% SP: 95% SEN + SP = 1.57
HE-4: E: 45.5 pM OE: 46.0 pM H: 40.5 pM
ELISA (Fujirebio Diagnostics Inc., USA) Tukey’s multiple comparisons
66 healthy women (tubal sterilization) Laparoscopically proven Age: 38.5
No
Ref.
Validation
Model
Findings
Methodology
Control group
204 patients; stages: 135 I-II, 69 III-IV; 41 M, 83 F, 80 L
42 OE patients, stages: 22 III and 20 IV Age: 34.3 ± 7.7 BMI: 23.9 ± 2.5 Early F
129 patients (stages: 16 I/II, 17 II, 33 III, 63 IV); 69 OE patients (stages: 24 III, 45 IV) Laparoscopically and histologically proven Age: 31.8
Endometriosis group
C: Control group; CRP: C-reactive protein; DIE: Deep infiltrating endometriosis; E: Endometriosis group; F: Folicular phase; H: Healthy women; HE-4: Human epididymal secretory protein; L: Luteal phase; M: Menstrual phase; NA: Not available; OE: Ovarian endometriosis; P: Proliferative phase; PE: Peritoneal endometriosis, PEDF: Pigment epithelium derived factor; S: Secretory phase, SEN: Sensitivity; SP: Specificity.
Analyte
Study (year)
Table 1. Individual proteins as biomarkers of endometriosis.
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Noninvasive biomarkers of endometriosis
Review
367
368
Activin A and follistatin
CA-125
Glycodelin A
VEGF-A
Reis et al. (2012)
Szubert et al. (2012)
Kocbek et al. (2013)
Mohamed et al. (2013)
Serum before surgery; postoperation in the third menstrual cycle; stored at -70˚C
Serum before laparoscopy; stored at -80˚C
Serum samples before surgery
Serum before surgery, stored at -80˚C
Sample C/OE activin A x folistatin SEN: 41.0% SP: 90.0% SEN + SP = 1.31
E/C CA-125 >11.0 U/ml SEN: 68.3% SP: 66.7% E III/IV/controls CA-125 >14.7 U/ml SEN: 91.7% SP: 86.7% SEN + SP = 1.78 OE/C Glycodelin A + age + BMI SEN: 82.1% SP: 78.4% SEN + SP = 1.60 VEGF-A in E/C (680 pg/ml) SEN: 93.3% SP: 96.7% SEN + SP = 1.90 CA-125 (35 mg/ml) SEN: 70.0% SP: 90.0% SEN + SP = 1.60
~ folistatin " activin A in OE/H (0.22 ± 0.01 ng/ml vs 0.17 ± 0.01 ng/ml, p twofold # 5 ~12 fold " Cytokeratin-19 only in patients (Western blotting) " 22 proteins, 5 > 10-fold " prealbumin, enolase-1, alpha1 antitrypsin, chain A solution structure of Bb’ domains of human protein disulfide isomerase, vitamin D binding protein (VDBP) ELISA E/C: 111.9 ± 74.6, 69.9 ± 43.8 ng/mg creatine
MB-WCX, Anchor Chip target plate, MALDI–TOF– MS; support vector machine
2D PAGE, MALDI–TOF–MS Western blotting
2D PAGE, (4E, 4C) LC–MS/MS Western blotting (4E, 4C); ELISA (57 E, 38 C) (ALPCO Diagnostics, USA); Student’s t test, Kruskal– Wallis test
16 women without endometriosis with subfertility, abdominal pelvic pain; age: 35 (26–50); BMI: 27 (20–35); menstrual phase reported
6 women without endometriosis based on laparoscopy and histology Age: 38.6 years 3P, 3 S 38 patients without endometriosis (laparoscopically proven) 18 dermoid cysts, 20 serous cystadenoma and paratubal cysts Age: 32.7 ± 10.3 BMI: 17.7 ± 7.0; Menstrual phase reported
23 patients, age: 35 (20– 46); BMI: 25 (19–39); stages I–IV; menstrual phase reported
11 patients; confirmed by laparoscopy and histology Age: 32.8 years 6 P, 5 S Stage I–IV 57 patients (histologically and laparoscopically proven) 5 stages I–II, 52 stages III–IV Age: 34.2 ± 6.9 BMI: 18.1 ± 7.3 Menstrual phase reported
No
Validation
Model
Findings
Methodology
Control group
Endometriosis group
C: Control group; E: Endometriosis group; F: Folicular phase; LC–MS: Liquid chromatography–mass spectrometry; L: Luteal phase; MALDI–TOF–MS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MB WCX: Magnetic beads weak cation exchange; OE: Ovarian endometriosis; P: Proliferative phase; S: Secretory phase.
Analytes
Study (year)
Table 5. Urine biomarkers of endometriosis identified by proteomics approach.
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Michigan University on 10/18/14 For personal use only.
[54]
[53]
[49]
Ref.
Review Lanisˇnik Riz ˇner
Expert Rev. Mol. Diagn. 14(3), (2014)
informahealthcare.com
VEGFA, MMP-3, MMP-9 mRNA levels
VEGFA, MMP-3, MMP-9 and survivin mRNA levels
Discovery phase 1113 miRNA Validation phase: 24 miRNA
Discovery phase 765 miRNA Validation phase 12 miRNA
De Sanctis et al. (2011)
Mabrouk et al. (2012)
Suryawanshi et al. (2013)
Wang et al. (2013)
Sera samples collected 1–3 days before surgery, stored at -80˚C
Plasma samples stored at -80˚C
Discovery cohort: Pool of 10 patients Validation cohort: 60 patients (41 OE, 19 PE, 18 with additional DIE) Confirmed by laparoscopy and histology BMI: 20.6 (14.5–24.9) Age: 34.4 (20–58) 42 F phase;18 L phase; stages: 17 I, 5 II, 14 III, 24 IV
Discovery cohort: 7 patients with endometriosis 7 patients with endometriosis associated ovarian cancer (EAOC) 5 with endometriosis Validation cohort: 33 endometriosis, 14 EAOC (6 endometrioid, 7 clear cell, 1 mixed histology); histologically confirmed Discovery cohort: Pool of 10 controls Validation cohort: 25 controls, confirmed by laparoscopy (22 infertile, due to fallopian tube disease); BMI: 20.6 (16.7–27.9); Age: 30.0 (24–39); 22 F phase, 3 L phase
Total RNA extraction, TaqMan microRNA array; normalized to U6 Mann–Whitney, discriminant analysis
miRNA isolation, miRNome Profiler kit, qPCR, normalized to miR-132; univariate comparison, ANOVA, linear discrimination analysis
Discovery cohort: 6 healthy controls Validation cohort: 20 healthy controls, 21 serous ovarian cancer
E/H: miR-16, miR-191, miR-195 SEN: 88% SP: 60% SEN + SP = 1.48 E/EAOC: miR-21, miE-362-5p, miR.1274a SEN: 57% SP: 91% SEN + SP = 1.48 miR-199a, miR-122, miR-145*, miR-542-3p SEN: 93.2% SP: 96.0% SEN + SP = 1.89
23 miRNA differentially expressed, E/H: " miR-16, miR-195, miR-191, miR1974, miR-4284, miR-15b, miR-1978, miR1979, miR-362-5p, miR-1973
" miR-199a, miR-122 # miR-145*, miR-141*, miR-542-3p, miR-9*; miR-199a and miR-122 discriminate between (III–IV) and (I–II) endometriosis
CA125 + Ca 19–9 + mRNA survivin SEN: 87%
Automated RNA extraction, qPCR normalized to GAPDH Kruskal–Wallis and Dunn post hoc test Logistic regression
20 patients without endometriosis, confirmed laparoscopically (myoma, tubal ligations, ovarian biopsy) Mean age: 30 (28–35), BMI: 22.6 (19.5–27) F phase
" mRNA for MMP-3 # mRNA for survivin No difference between OE and DIE
20 patients with OE Mean age: 29 (28–40), BMI: 21.5 (19–25.5) 20 patients with DIE Mean age: 31 (26–40), BMI: 21.5 (18–25) Laparoscopically confirmed; F phase
NA
" mRNA for MMP-3 ~ mRNA for MMP-9, VEGFA
Automated RNA extraction, qPCR normalized to GAPDH and control pooled mRNA, Kruskal–Wallis and Dunn post hoc test
20 endometriosis-free women, laparoscopically confirmed, (infertility, myoma, tubal ligations, ovarian biopsy) Mean age: 31, range 26–34; BMI: >17 and 17 and
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Michigan University on 10/18/14 For personal use only.
Noninvasive biomarkers of endometriosis: myth or reality? Expert Rev. Mol. Diagn. 14(3), 365–385 (2014)
Tea Lanisˇnik Rizˇner Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia Tel.: +386 1 5437 657 Fax: +386 1 5437 641 [email protected]
Endometriosis affects 10% of premenopausal women and 35–50% of women with infertility, pelvic pain, or both. At present, endometriosis can only be diagnosed with surgery, where laparoscopy is considered a gold standard. Noninvasive biomarkers are thus urgently needed. In 2010, the peripheral biomarkers of endometriosis were systematically reviewed by May et al. However, with the introduction of ‘-omics’ technologies, we have witnessed immense progress in biomarker discovery, which now calls for an overview of recent studies. This report looks at potential blood and urine biomarkers of endometriosis published in the last 3 years. The current status of noninvasive diagnostic biomarkers of endometriosis is discussed, with the limitations of these studies identified and recommendations for future biomarker discovery provided. KEYWORDS: biomarkers • endometriosis • metabolomics • proteomics • transcriptomics
transplantation, and especially on retrograde menstruation, are relatively widely accepted [8,9].
Endometriosis is a common gynecological disease
Endometriosis is a frequent benign gynecological disease that is characterized by the presence of endometrial tissue outside the uterine cavity. In the general population of women of reproductive age, the predicted prevalence of endometriosis is 6–10%, but the frequency increases to 30–50% in women with pain, infertility or both [1,2]. Worldwide, endometriosis affects an estimated 176 million women [3]. Ectopic endometrial tissue can be found in different parts of the peritoneal cavity, with these locations defining three different entities with different etiologies and pathogenesis: ovarian, peritoneal and deep infiltrating endometriosis [4,5]. In rare cases, endometrium-like tissue can be found outside the peritoneal cavity in other sites, such as the pleura, and even in the brain [6,7]. The pathogenesis of endometriosis is very complex and involves enhanced cell adhesion, degradation of the extracellular matrix, angiogenesis, proliferation, aberrant apoptosis, disturbed cell communication, disturbances of the immune system, loss of differentiation capacity and other pathophysiological processes [8]. There is no single theory that can explain all of the aspects of endometriosis although the theories on its in situ development (i.e., the metaplastic theory) and informahealthcare.com
10.1586/14737159.2014.899905
Why do we need noninvasive diagnostic biomarkers for endometriosis?
The current diagnostics of endometriosis is problematic because the symptoms are nonspecific and they can be associated with a number of different conditions (e.g., irritable bowel syndrome, pelvic inflammatory disease) [10]. At present, the disease can only be diagnosed with surgery: the gold standard is a laparoscopic procedure, which is a surgical visual inspection of the pelvic organs. This requires an experienced surgeon and general anesthesia, and it also carries surgery-associated risks like major vessel or bowel injury. However, laparoscopic visualization of the peritoneal cavity also has its limitations and should be combined with histological analysis. Indeed, only 54–67% of suspected lesions are confirmed histologically [6,11], while biopsies of normal appearing peritoneum followed by histology reveals endometriosis in 6% of infertile women [6,7,12]. As a result of the nonspecific symptoms and sometimes inadequate invasive diagnosis, there is an average delay of 6.7 years between the onset of symptoms and the surgical diagnosis of endometriosis [1]. Indeed, in certain centers, it can take up to 11 years
2014 Informa UK Ltd
ISSN 1473-7159
365
Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Michigan University on 10/18/14 For personal use only.
Review
Lanisˇnik Riz ˇner
before affected women are diagnosed and receive the appropriate treatment [13]. Endometriosis is also associated with high healthcare costs, which have been estimated as an annual e9,579 per woman, which includes the costs of both the direct healthcare and the loss of productivity [14]. The economic burden associated with endometriosis is similar to other chronic diseases such as diabetes, Crohn disease and rheumatoid arthritis. Women who are affected lose 10.8 h/week of work on average, which, as calculated for Italy, translates into costs as high as US$231 per woman per week [13]. There is a large potential for improving the cost–effectiveness of the management of endometriosis by reducing the time of diagnosis, so that the disease is under control before the onset of the various complications and infertility [1]. The development of a noninvasive diagnostic test for endometriosis would have a groundbreaking effect on the quality of life of these patients. Earlier diagnosis and treatment would reduce the discomfort of endometriosis patients, and the disease progression that is associated with infertility and other complications. A European Endometriosis Alliance survey carried out with 7025 women with endometriosis revealed that 65% of these patients were misdiagnosed and 46% had to see five or more doctors to get a correct diagnosis [15]. The recurrence rate of endometriosis is high as well; estimates show that following surgery, 21.5% of patients experience recurrence within 2 years and 40–50% within 5 years [16]. Currently, there are no known diagnostic biomarkers, and even less so, biomarkers of recurrence. Although imaging techniques, such as ultrasound, computed tomography and MRI, are being explored, their current diagnostic accuracy is still inferior to direct laparoscopic visualization [1,17,18]. Other issues related to these technologies include the required training level to achieve acceptable sensitivity and specificity rates and the costs of these procedures [1]. According to a consensus workshop that was held at the World Congress on Endometriosis in 2011, a noninvasive test for reliable diagnosis of endometriosis, and particularly of early endometriosis, is of paramount importance [1]. Up to 45% of subfertile patients with a regular cycle might have endometriosis, and they would benefit from a blood test that could identify the symptomatic patients who would need surgical therapy for subfertility and pain [19,20]. Recent reports on potential noninvasive biomarkers of endometriosis
A biomarker is defined as a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to therapeutic intervention [21]. May et al. [10] systematically reviewed peripheral biomarkers of endometriosis and assessed the quality of individual studies using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS) criteria [22,23]. Although over 100 potential biomarkers of endometriosis met the selection criteria, neither a single biomarker nor a panel of biomarkers have so far been unequivocally shown to be clinically useful [10]. However, with the introduction of the ‘-omics’ technologies, we 366
have witnessed immense progress in the field of biomarker identification, which now calls for an overview of recent studies. This manuscript includes the reports on potential biomarkers for noninvasive diagnostics of endometriosis, which have been published since the last systematic review in 2010 [10]. Relevant studies were identified by searching the Pubmed database using endometriosis and biomarkers as the key words, for the period from August 2009 to November 2013. Only manuscripts in English were included. Studies comparing endometriosis with infertile patients, healthy controls and patients with benign ovarian cysts were included; studies comparing endometriosis with ovarian cancer were excluded. From the 36 published studies initially included, the following information was extracted: sample analyzed (e.g., whole blood, plasma, serum, urine), analytes (e.g., proteins, metabolites, RNA molecules, blood cells, panels of biomarkers); characteristics of the case group and of the enrolled patients (e.g., mean age, BMI, type of endometriosis, stage of endometriosis, menstrual phase), characteristics of the control group and of the control patients or healthy women (e.g., mean age, BMI, diagnosis, menstrual phase), methodology used (ELISA, MALDI–TOF–MS including statistical methods), main findings, and if available, the proposed statistical model (TABLES 1–8). Based on the modified QUADAS criteria [10], the quality of these studies was assessed and their limitations were identified. The majority of case groups comprised healthy women or patients with other gynecological diseases, not women with endometriosis-like symptoms. The absence of endometriosis was generally confirmed by laparoscopy and only five of the studies reported that histology was also performed. In several studies, the patients were not stratified based on their menstrual phase, or it was not clear whether this potential confounding effect was taken into consideration. The stage of disease was usually reported, but more than half of the studies did not stratify patients according to different stages, or they did not report whether this potential confounding effect was examined. Although different types of endometriosis represent different entities, the type of endometriosis was reported in only one-third of all of these studies. Different approaches in search of noninvasive biomarkers of endometriosis
Reliable biological markers for noninvasive diagnostics have been searched for in peripheral blood, whole blood (5 studies), serum samples (20 studies) and plasma samples (8 studies), as well as in urine samples (4 studies) (TABLES 1–8). Serum is still the most common biological sample analyzed, although it can be problematic due to inherent variability in composition, depending on the blood-clotting process [24]. Surprisingly, in the last few years, there have been no reports on the evaluation of biomarkers for endometriosis in menstrual blood, even though this material most closely reflects the physiological and molecular environment of the pathologically altered eutopic endometrium of endometriosis patients [8]. To date, different methodologies have been used in the search for biomarkers for endometriosis [10,19]. By targeted Expert Rev. Mol. Diagn. 14(3), (2014)
informahealthcare.com
HE-4
Urocortin
CRP
PEDF
CD-23
Huhtinen et al. (2009)
Tokmak et al. (2011)
Vodolazkaia et al. (2011)
Chen et al. (2012)
Ramos et al. (2012)
Serum before surgery; >8 h fasting; stored at -20˚C
Serum before laparoscopy, (after >12 h fasting)
Plasma before surgery, stored at -80˚C
Plasma before surgery, stored at -70˚C or -80˚C
Serum before surgery, stored at -20˚C or -80˚C
Sample
C/E stages III-IV (>0.71 mg/l) SEN: 80.7% SP: 63.9% SEN + SP = 1.45 NA
NA
" CRP levels E/C (L phase) 0.89 mg/l/0.54 mg/l
# PEDF non-E/E (mean ± SD) 16.3 ± 6.6 ng/ml vs 24.5 ± 7.3 ng/ml, p < 0.001 # PEDF in E patients with pain
~ CD-23 levels
High sensitivity CRP assay (Roche, Belgium) Mann–Whitney test ELISA (P056-9, Groundwork Biotechnology Diagnosticate, USA) Univariate analysis
ELISA (Bender MedSystems, Austria); Mann–Whitney test
91 subfertile women (19 M, 36 F, 36 L)
28 patients without endometriosis, indications: infertility (3), pelvic mass (17; 12 mesosalpinx cyst, 5 paraovarian cyst), elective tubal sterilization (8) 17 F phase, 11 L phase Age: 31.3 ± 6.6, BMI: 21.1 ± 2.3 58 women without endometriosis; age between 18 and 45, in M and F phase
43 patients (30 OE); 28 F phase 15 L phase Confirmed by laparoscopy and histology Age: 31.1 ± 5.3, BMI: 20.8 ± 2.4
44 patients confirmed by laparoscopy and histology; age between 18 and 45, in M and F phase
Urocortin (4.16 ng/ml)/ CA-125 (21.38 U/l) SEN: 76.2/ 88.1% SP: 45.7/ 63% SEN + SP = 1.51
~ Urocortin OE: 4.8 ± 1.0 ng/ml controls: 4.5 ± 1.0 ng/ml
Urocortin EIA kit (Phoenix Pharmaceuticals, USA) Mann-Whitney test
46 patients with benign ovarian cysts, 23 serous, 8 teratoma, 5 mucinous, 10 functional ovarian cysts Age: 33.2 ± 11.8 BMI: 23.9 ± 2.6 Early F
No
No
No
[28]
[33]
[27]
[29]
[25]
No
OE/H HE-4+CA-125 SEN: 62% SP: 95% SEN + SP = 1.57
HE-4: E: 45.5 pM OE: 46.0 pM H: 40.5 pM
ELISA (Fujirebio Diagnostics Inc., USA) Tukey’s multiple comparisons
66 healthy women (tubal sterilization) Laparoscopically proven Age: 38.5
No
Ref.
Validation
Model
Findings
Methodology
Control group
204 patients; stages: 135 I-II, 69 III-IV; 41 M, 83 F, 80 L
42 OE patients, stages: 22 III and 20 IV Age: 34.3 ± 7.7 BMI: 23.9 ± 2.5 Early F
129 patients (stages: 16 I/II, 17 II, 33 III, 63 IV); 69 OE patients (stages: 24 III, 45 IV) Laparoscopically and histologically proven Age: 31.8
Endometriosis group
C: Control group; CRP: C-reactive protein; DIE: Deep infiltrating endometriosis; E: Endometriosis group; F: Folicular phase; H: Healthy women; HE-4: Human epididymal secretory protein; L: Luteal phase; M: Menstrual phase; NA: Not available; OE: Ovarian endometriosis; P: Proliferative phase; PE: Peritoneal endometriosis, PEDF: Pigment epithelium derived factor; S: Secretory phase, SEN: Sensitivity; SP: Specificity.
Analyte
Study (year)
Table 1. Individual proteins as biomarkers of endometriosis.
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Noninvasive biomarkers of endometriosis
Review
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368
Activin A and follistatin
CA-125
Glycodelin A
VEGF-A
Reis et al. (2012)
Szubert et al. (2012)
Kocbek et al. (2013)
Mohamed et al. (2013)
Serum before surgery; postoperation in the third menstrual cycle; stored at -70˚C
Serum before laparoscopy; stored at -80˚C
Serum samples before surgery
Serum before surgery, stored at -80˚C
Sample C/OE activin A x folistatin SEN: 41.0% SP: 90.0% SEN + SP = 1.31
E/C CA-125 >11.0 U/ml SEN: 68.3% SP: 66.7% E III/IV/controls CA-125 >14.7 U/ml SEN: 91.7% SP: 86.7% SEN + SP = 1.78 OE/C Glycodelin A + age + BMI SEN: 82.1% SP: 78.4% SEN + SP = 1.60 VEGF-A in E/C (680 pg/ml) SEN: 93.3% SP: 96.7% SEN + SP = 1.90 CA-125 (35 mg/ml) SEN: 70.0% SP: 90.0% SEN + SP = 1.60
~ folistatin " activin A in OE/H (0.22 ± 0.01 ng/ml vs 0.17 ± 0.01 ng/ml, p twofold # 5 ~12 fold " Cytokeratin-19 only in patients (Western blotting) " 22 proteins, 5 > 10-fold " prealbumin, enolase-1, alpha1 antitrypsin, chain A solution structure of Bb’ domains of human protein disulfide isomerase, vitamin D binding protein (VDBP) ELISA E/C: 111.9 ± 74.6, 69.9 ± 43.8 ng/mg creatine
MB-WCX, Anchor Chip target plate, MALDI–TOF– MS; support vector machine
2D PAGE, MALDI–TOF–MS Western blotting
2D PAGE, (4E, 4C) LC–MS/MS Western blotting (4E, 4C); ELISA (57 E, 38 C) (ALPCO Diagnostics, USA); Student’s t test, Kruskal– Wallis test
16 women without endometriosis with subfertility, abdominal pelvic pain; age: 35 (26–50); BMI: 27 (20–35); menstrual phase reported
6 women without endometriosis based on laparoscopy and histology Age: 38.6 years 3P, 3 S 38 patients without endometriosis (laparoscopically proven) 18 dermoid cysts, 20 serous cystadenoma and paratubal cysts Age: 32.7 ± 10.3 BMI: 17.7 ± 7.0; Menstrual phase reported
23 patients, age: 35 (20– 46); BMI: 25 (19–39); stages I–IV; menstrual phase reported
11 patients; confirmed by laparoscopy and histology Age: 32.8 years 6 P, 5 S Stage I–IV 57 patients (histologically and laparoscopically proven) 5 stages I–II, 52 stages III–IV Age: 34.2 ± 6.9 BMI: 18.1 ± 7.3 Menstrual phase reported
No
Validation
Model
Findings
Methodology
Control group
Endometriosis group
C: Control group; E: Endometriosis group; F: Folicular phase; LC–MS: Liquid chromatography–mass spectrometry; L: Luteal phase; MALDI–TOF–MS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MB WCX: Magnetic beads weak cation exchange; OE: Ovarian endometriosis; P: Proliferative phase; S: Secretory phase.
Analytes
Study (year)
Table 5. Urine biomarkers of endometriosis identified by proteomics approach.
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[54]
[53]
[49]
Ref.
Review Lanisˇnik Riz ˇner
Expert Rev. Mol. Diagn. 14(3), (2014)
informahealthcare.com
VEGFA, MMP-3, MMP-9 mRNA levels
VEGFA, MMP-3, MMP-9 and survivin mRNA levels
Discovery phase 1113 miRNA Validation phase: 24 miRNA
Discovery phase 765 miRNA Validation phase 12 miRNA
De Sanctis et al. (2011)
Mabrouk et al. (2012)
Suryawanshi et al. (2013)
Wang et al. (2013)
Sera samples collected 1–3 days before surgery, stored at -80˚C
Plasma samples stored at -80˚C
Discovery cohort: Pool of 10 patients Validation cohort: 60 patients (41 OE, 19 PE, 18 with additional DIE) Confirmed by laparoscopy and histology BMI: 20.6 (14.5–24.9) Age: 34.4 (20–58) 42 F phase;18 L phase; stages: 17 I, 5 II, 14 III, 24 IV
Discovery cohort: 7 patients with endometriosis 7 patients with endometriosis associated ovarian cancer (EAOC) 5 with endometriosis Validation cohort: 33 endometriosis, 14 EAOC (6 endometrioid, 7 clear cell, 1 mixed histology); histologically confirmed Discovery cohort: Pool of 10 controls Validation cohort: 25 controls, confirmed by laparoscopy (22 infertile, due to fallopian tube disease); BMI: 20.6 (16.7–27.9); Age: 30.0 (24–39); 22 F phase, 3 L phase
Total RNA extraction, TaqMan microRNA array; normalized to U6 Mann–Whitney, discriminant analysis
miRNA isolation, miRNome Profiler kit, qPCR, normalized to miR-132; univariate comparison, ANOVA, linear discrimination analysis
Discovery cohort: 6 healthy controls Validation cohort: 20 healthy controls, 21 serous ovarian cancer
E/H: miR-16, miR-191, miR-195 SEN: 88% SP: 60% SEN + SP = 1.48 E/EAOC: miR-21, miE-362-5p, miR.1274a SEN: 57% SP: 91% SEN + SP = 1.48 miR-199a, miR-122, miR-145*, miR-542-3p SEN: 93.2% SP: 96.0% SEN + SP = 1.89
23 miRNA differentially expressed, E/H: " miR-16, miR-195, miR-191, miR1974, miR-4284, miR-15b, miR-1978, miR1979, miR-362-5p, miR-1973
" miR-199a, miR-122 # miR-145*, miR-141*, miR-542-3p, miR-9*; miR-199a and miR-122 discriminate between (III–IV) and (I–II) endometriosis
CA125 + Ca 19–9 + mRNA survivin SEN: 87%
Automated RNA extraction, qPCR normalized to GAPDH Kruskal–Wallis and Dunn post hoc test Logistic regression
20 patients without endometriosis, confirmed laparoscopically (myoma, tubal ligations, ovarian biopsy) Mean age: 30 (28–35), BMI: 22.6 (19.5–27) F phase
" mRNA for MMP-3 # mRNA for survivin No difference between OE and DIE
20 patients with OE Mean age: 29 (28–40), BMI: 21.5 (19–25.5) 20 patients with DIE Mean age: 31 (26–40), BMI: 21.5 (18–25) Laparoscopically confirmed; F phase
NA
" mRNA for MMP-3 ~ mRNA for MMP-9, VEGFA
Automated RNA extraction, qPCR normalized to GAPDH and control pooled mRNA, Kruskal–Wallis and Dunn post hoc test
20 endometriosis-free women, laparoscopically confirmed, (infertility, myoma, tubal ligations, ovarian biopsy) Mean age: 31, range 26–34; BMI: >17 and 17 and
