GYNECOLOGICAL DISEASES
Biomarkers of endometriosis Amelie Fassbender, Ph.D.,a Alexandra Vodolazkaia, Ph.D., M.D.,a Philippa Saunders, Ph.D.,b Dan Lebovic, M.D.,c Etienne Waelkens, Ph.D., M.D.,d Bart De Moor, Ph.D.,e and Thomas D'Hooghe, Ph.D., M.D.a,f,g a Department of Development and Regeneration, Sexual, Pelvic, Reproductive, and Family Studies, University Hospital Gasthuisberg, Leuven, Belgium; b MRC Centre for Reproductive Health, Queen's Medical Research Institute, Edinburgh, Scotland; c Department of Obstetrics and Gynecology, University of Wisconsin-Madison, Middleton, Wisconsin; d Department of Cellular and Molecular Medicine, Campus Gasthuisberg, Leuven, Belgium; e Department of Electrical Engineering (ESAT-SCD), Katholieke Universiteit Leuven, Leuven, Belgium; f Leuven University Fertility Centre, Department of Obstetrics and Gynaecology, University Hospital Gasthuisberg, Leuven, Belgium; and g Division of Reproductive Biology, Institute of Primate Research, Karen, Nairobi, Kenya
A noninvasive test for endometriosis would be useful for the early detection of endometriosis in symptomatic women who have pelvic pain and/or subfertility with normal ultrasound results. This would include nearly all cases of minimal-to-mild endometriosis, some cases of moderate-to-severe endometriosis without a clearly visible ovarian endometrioma, and cases with pelvic adhesions and/or other pelvic pathology that might benefit from surgery to improve pelvic pain and/or subfertility. This overview discusses the diagnostic performance of noninvasive or semi-invasive tests for endometriosis, including panels of known peripheral blood biomarkers, protein/ peptide markers discovered by proteomics, miRNA, and endometrial nerve fiber density. Tests with high sensitivity and acceptable specificity have been developed; some have been validated in independent populations and are therefore promising. To make real progress, international agreement on biobank development is needed for standard operating procedures for the collection, treatment, storage, and analysis of tissue samples and for detailed clinical phenotyping of these samples. Furthermore, it is necessary to validate the diagnostic accuracy of any promising test prospectively in an independent symptomatic patient population with subfertility and/or pain without clear ultrasound evidence of endometriosis and with a clinical indication for surgery, divided into cases with laparoscopically and histologically confirmed endometriosis and conUse your smartphone trols with laparoscopically confirmed absence of endometriosis. (Fertil SterilÒ 2013;99: to scan this QR code 1135–45. Ó2013 by American Society for Reproductive Medicine.) and connect to the Key Words: Biomarker, diagnosis, endometriosis, endometrium, plasma Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/fassbendera-biomarkers-endometriosis/
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ndometriosis, an estrogendependent gynecologic disorder, affects 6% to 10% women of reproductive age from all ethnic and social groups. Endometriosis is defined as the presence of endometrial-like tissue outside the uterine cavity (1). The degree of endometriosis is staged according to the classification system of the American Society of Reproductive Medicine (2) into minimal, mild, moder-
ate, and severe disease. Endometriosis can be associated with infertility and/or pain symptoms, including cyclic pelvic pain, dysmenorrhea, dyspareunia, dysuria, and dyschezia (3, 4). Endometriosis-associated pain can be caused by peritoneal inflammation, adhesion formation, and specific innervation of endometriotic lesions and is correlated with the presence of deep infiltrating disease (5–8). Endometriosis
Received October 19, 2012; revised and accepted January 11, 2013; published online February 13, 2013. A.F. has nothing to disclose. A.V. has nothing to disclose. P.S. has received a grant from the Medical Research Council UK, and is a board member of MRC Physiological Systems and Medicine (unrelated to this work). D.L. has received grants and has grants pending from Abbott Pharmaceutical and royalties from UpToDate (both unrelated to this work). E.W. has nothing to disclose. B.D.M. has nothing to disclose. T.D'H. has received a grant from the World Endometriosis Research Foundation, and reports consultancies with Bayer Schering, Schering Plough (Merck), Merck Serono, Astellas, Arresto, Roche, and Proteomika; and grants/grants pending from Merck Serono, Schering Plough, Ferring, Arresto, Roche, and Proteomika (all unrelated to this work). Reprint requests: Thomas D'Hooghe, Ph.D., M.D., Leuven University Fertility Centre, University Hospital Gasthuisberg, Obstetrics and Gynaecology, Herestraat 49, Leuven B-3000, Belgium (E-mail:
[email protected]). Fertility and Sterility® Vol. 99, No. 4, March 15, 2013 0015-0282/$36.00 Copyright ©2013 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2013.01.097 VOL. 99 NO. 4 / MARCH 15, 2013
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can appear as peritoneal lesions, ovarian superficial implants or endometriotic cysts, and/or deeply infiltrative disease with extension to bowel, bladder, and ureter; it is often associated with pelvic adhesions (9). So far, it has not been possible to predict the presence of endometriosis based on symptoms, clinical examination, imaging techniques or blood tests. A symptom-based model study (10) has found that menstrual dyschezia and a history of benign ovarian cysts predict both any and stage III–IV endometriosis. Although stage III–IV disease was predicted with a good accuracy (area under the curve [AUC] ¼ 84.9, sensitivity 82.3% and specificity 75.8%), any-stage endometriosis was predicted relatively poorly (AUC ¼ 68.3, sensitivity 84.8% and specificity 43.5%) (10). At present, the gold standard for diagnosis of endometriosis is laparoscopic inspection with 1135
GYNECOLOGICAL DISEASES histologic confirmation after retrieval of lesions (11). However, laparoscopy is a surgical procedure with rare but significant potential risks for the patients (12). It is interesting that there is no correlation between severity of endometriosis (revised American Fertility Society [AFS] classification) and the type or severity of pain symptoms (11). As endometriosis can be progressive in up to 50% of women (13), early noninvasive diagnosis has the potential to offer early treatment and prevent progression. Transvaginal ultrasound (TVU) is an adequate diagnostic method to detect ovarian endometriotic cysts but does not rule out peritoneal endometriosis, endometriosis-associated adhesions (11, 14), or some locations of deep infiltrating endometriosis (DIE) (15–17). Furthermore, routine vaginal examination alone may be insufficient to detect endometriosis before laparoscopy (18, 19). A noninvasive test would be particularly welcome; recent evidence suggests that significant biologic differences exist between eutopic endometrium from women with and without endometriosis (1), so this may offer a basis for a semi-invasive diagnostic test based on the analysis of an endometrial biopsy sample. Despite extensive research, no reliable blood tests currently exist for the diagnosis of endometriosis. A biomarker is a measurable ‘‘biologic marker’’ that correlates with a specific outcome or state of the disease (20). The biomarker hypothesis states that changes in levels of analytes, proteins, microRNA (miRNA), genes, or other markers could be a specific characteristic of the disease state (20). Although CA-125, cytokines, and angiogenic and growth factors all show altered levels in the peripheral blood of women with endometriosis when compared with controls (21, 22), thus far neither a single biomarker nor a panel of biomarkers has been validated for clinical use as a diagnostic test in women with endometriosis (22, 23). The development of a noninvasive diagnostic test—from initial biomarker discovery to a clinically approved biomarker assay—is a long, difficult, and uncertain process (24).
BENEFITS OF A CLINICAL DIAGNOSTIC TEST According to a panel of internationally respected endometriosis experts (25), the development of a noninvasive diagnostic test for endometriosis is one of the top research priorities. However, researchers and clinicians need to realize that a diagnostic test may do more harm than good by subjecting patients to unnecessary or even potentially harmful procedures (26), as the benefits of treating women with asymptomatic endometriosis is unclear (22). Therefore, we do not recommend the development or use a blood test for screening purposes in asymptomatic women. However, up to 45% of subfertile women, with or without pelvic pain, who have regular cycles, a partner with normal sperm quality, and normal clinical examination and pelvic ultrasound results may have endometriosis (27).
Which Patients Should Be Targeted for a Clinical Test of Endometriosis? In the presence of subfertility combined with a history of cyclic or chronic pelvic pain, a clinical examination with positive results for pain, and an ultrasound with positive 1136
results for ovarian endometriotic cysts or deep endometriotic nodules, the probability of endometriosis is so high that most gynecologists will offer the patient a laparoscopy combined with excision of all visible endometriotic lesions as well as a histologic examination of at least one implant to confirm the presence of endometrial glands and stroma (19). Most gynecologists are not sure whether endometriosis is present when a woman has, for example, subfertility since more than 1 year, a regular cycle, no or limited dysmenorrhea, and a partner with a normal sperm. 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 remain in doubt about the value of a diagnostic laparoscopy. From a clinical perspective, it is unlikely that these women have moderate-to-severe endometriosis; however, they may have extensive peritoneal endometriosis with or without adhesions associated with subfertility and possibly mild pain (19). For this population, a noninvasive or semiinvasive diagnostic test would be useful to discriminate between women without endometriosis (for whom the surgery would be unnecessary) and those with endometriosis, most likely minimal-to-mild disease, who may benefit from surgical therapy for both subfertility and pain and from controlled ovarian stimulation in combination with intrauterine insemination for subfertility (11, 19, 28). If such a test were also diagnostic for women with pelvic adhesions after previous surgery or after pelvic inflammatory disease (PID), these women would also benefit from surgical laparoscopic adhesiolysis to improve fertility and to reduce pain. In fact, such a test would then be ‘‘false positive’’ for endometriosis but still ‘‘true positive’’ for pelvic adhesions that can be managed by surgery. For the same reasons, such a test would be highly valuable for women or adolescents who do not desire to conceive but have pelvic pain that does not respond well to medical therapy in conjunction with normal clinical examination and pelvic ultrasound results. In summary, a noninvasive test for endometriosis would be useful for women with pelvic pain and/or subfertility with normal ultrasound results. This would include nearly all cases of minimal-to-mild endometriosis, some cases of moderateto-severe endometriosis without clearly visible ovarian endometrioma, and women with pelvic adhesions and/or other pelvic pathology, who might benefit from surgery to improve their pelvic pain and/or subfertility (19). A noninvasive diagnostic test could be developed for serum, plasma, urine, or menstrual fluid that can be recovered from the posterior vaginal fornix or the cervix during speculum examination. A semi-invasive test could be developed for peritoneal fluid obtained via transvaginal ultrasound–guided aspiration or for endometrium obtained via 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 (19). To achieve this goal, a test with a high sensitivity is needed, which is the probability of a test of being positive when endometriosis is present. In addition, the test needs to have a high specificity, to ensure VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility® a high probability of the test being negative when endometriosis is absent. Sensitivity and specificity are statistical measures of performance of a binary classification test, which could be a confounder in data analyses. The predictive value of any diagnostic test is influenced by its sensitivity and specificity as well as the prevalence of the target disease in the population being evaluated. Thus, the predictive value of a diagnostic test includes information about both the test itself and the tested population to give a more useful clinical measure (29, 30). A rule of thumb is that the sensitivity and specificity of a good test should add to at least 1.50, and those of a very good test should add to at least 1.80 (30, 31). At present, such a test does not exist.
KNOWN BIOMARKERS Previous studies have focused on the glycoproteins, inflammatory and noninflammatory cytokines, adhesion molecules, and angiogenic and growth factors that are known to be highly relevant to the pathogenesis of endometriosis and the development of endometriotic lesions. However, neither a single biomarker nor a panel of biomarkers has been validated as a reliable noninvasive test for endometriosis (22). Cancer antigen 125 (CA-125), the most extensively investigated and widely used peripheral biomarker of endometriosis (32), is produced by endometrial and mesothelial cells and gains entry into the circulation via the endothelial lining of capillaries in response to inflammation (32). However, CA125 levels in peripheral blood lack diagnostic power as a single biomarker of endometriosis due to low sensitivity (11, 33). Although previous studies have shown that tumor markers, cytokines, and angiogenic and growth factors show altered levels in peripheral blood (plasma or serum) of women with endometriosis when compared with controls (21, 22), so far none of them, alone or in combination, have been validated as a noninvasive test for endometriosis (22), possibly because most studies have included a limited number of patients, limited assessment of different cycle phases and endometriosis stages, a limited number of biomarkers analyzed, limited statistical analysis (mostly univariate statistical analysis only), and have lacked validation in an independent test set of patients. Because of the different types (superficial, deep, cyst) and locations of endometriosis, it is possible that a different subset of biomarkers may be required for the diagnosis of different stages of endometriosis (25). That is, women with peritoneal endometriosis may have different markers than those with rectovaginal endometriosis (22). In a study evaluating 28 biomarkers, multivariate analysis of four/five biomarkers—annexin V, vascular endothelial growth factor (VEGF), CA-125, and soluble intercellular adhesion molecule-1 (sICAM-1)/or glycodelin—in plasma samples enabled the diagnosis of endometriosis in women (n ¼ 175) who had disease undetectable by ultrasound with a sensitivity of 81% to 90% and a specificity of 63% to 81% compared with controls who had a laparoscopically confirmed absence of endometriosis (n ¼ 121) (23). These data, which were obtained in a training set and were validated in an independent test set, are therefore promising. The next step is to apply these models for preoperative prediction of VOL. 99 NO. 4 / MARCH 15, 2013
endometriosis in an independent set of patients with infertility and/or pain without ultrasound evidence of endometriosis who are scheduled for laparoscopy. It is interesting to look at the biologic relevance of the biomarkers (annexin V, VEGF, CA-125, and sICAM-1/or glycodelin) that performed best in that study, which evaluated 28 biomarkers (23). Vascular endothelial growth factor, one of the main stimuli for angiogenesis and increased vessel permeability, contributes to the development of endometriotic lesions (34, 35). However, there is no consensus regarding the value of VEGF as biomarker of endometriosis. Indeed, peripheral blood VEGF levels have been reported to be either increased (36–38) or to be similar (39–41) in women with endometriosis when compared with controls, probably due to differences in study design and methodology (such serum and plasma collection, time of sample collection, and processing). Glycodelin, an endometrium-derived protein with known angiogenic, immunosuppressive, and contraceptive effects, could contribute to the development of endometriosis and endometriosis-related infertility (42). Moreover, glycodelin is not only produced in the glandular epithelium of secretory endometrium (43, 44) but also is shed from endometriotic lesions into the peritoneal fluid and serum (42, 44, 45). Increased plasma glycodelin levels have been observed in patients with endometriosis (44, 45). Soluble intercellular adhesion molecule-1, one of the major adhesion molecules that inhibits natural killer cell–mediated cytotoxicity (46), resulting in defective immune surveillance, is involved in the implantation and development of endometriotic lesions (47). To date, studies have shown an increase (48, 49) and a decrease (23, 50) of sICAM-1 levels (plasma/serum) in women with endometriosis compared with controls. In addition, no significant differences of sICAM 1 levels have been reported (51–53). This discrepancy may be due to differing study designs, enzyme-linked immunosorbent assay (ELISA) kits, or types of blood specimens, or to varying phases of the menstrual cycle. Annexin V, a marker of apoptosis, has been reported to be a promising semi-invasive endometrial biomarker for diagnosis of minimal-to-mild endometriosis (54). Indeed, alterations in the regulation of apoptosis in eutopic and ectopic endometrium from women with endometriosis could contribute to the survival of endometrial cells in the peritoneal cavity and the development of endometriosis (55). Annexin V was included in a panel of biomarkers in a possible noninvasive test for endometriosis (23). To the best of our knowledge, no other groups have investigated annexin V as a biomarker for endometriosis. To our surprise, in the study evaluating 28 biomarkers, inflammatory molecules did not emerge as biomarkers in a panel with the best diagnostic performance (23). At present, there is no consensus regarding the value of inflammatory factors as biomarkers of endometriosis. Comparable serum interleukin-6 (IL-6) (56, 57), IL-8 (56, 58), tumor necrosis factor a (TNF-a) and IL-1 (40, 56, 57) levels had been previously reported in women with and without endometriosis. However, other investigators reported elevated peripheral levels of IL-6 (40, 59), IL-8 (60, 61), TNF-a (37, 59), and interferon-g (IFN-g) (40) in endometriosis patients compared with controls. 1137
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NERVE FIBERS An increasing body of evidence suggests that endometriosis may be diagnosed on the basis of an increased density of nerve fibers in the endometrium of women with endometriosis as compared with controls who lack the disease (62–65). Indeed, sensory nerve fibers have been identified in the functional layer of human endometrium by immunohistochemical analysis, and they result in the endometrial presence of various neural transmitters such as substance P (SP), vasoactive intestinal polypeptide (VIP), and neural proteins such as protein gene product 9.5 (PGP9.5), neurofilament (NF), neuropeptide Y (NPY), and calcitonin gene-related protein (CGRP) (64). In a proteomic study, researchers found that NT-4/5 and brain-derived neurotrophic factor proteins were significantly higher in the eutopic endometrium from endometriosis patients (n ¼ 18) than in controls (n ¼ 15), whereas nerve growth factor levels were similar (66). A higher density of small unmyelinated nerve fibers has been observed in the functional layer of endometrium from women with confirmed endometriosis when compared with women without endometriosis (65, 67). On this basis, the detection of endometrial nerve fibers has been proposed by the Fraser group as a diagnostic tool for endometriosis in a pilot study (62), and was confirmed in a blinded study (63). Our own group found that endometrial nerve density was 14 times higher in women with minimalto-mild endometriosis (n ¼ 20) than in controls with a laparoscopically confirmed absence of endometriosis and normal pelvis (n ¼ 20); the combined analysis of neural markers PGP9.5, VIP, and SP could predict the presence of minimalto-mild endometriosis with 95% sensitivity, 100% specificity, and 97.5% accuracy (64). Only three patients with endometriosis had dysmenorrhea (n ¼ 3); three patients without endometriosis had dysmenorrhea (n ¼ 2) or dyspareunia (1) (64). These data now need to be confirmed by other research groups and to be validated in a symptomatic patient population with pain and/or subfertility and a 30% prevalence of endometriosis. Indeed, other investigators have disputed these findings and reported that the presence of nerve fibers in the functional layer of endometrium does not depend on the presence of endometriosis but rather is associated with a diagnosis of pelvic pain (68–70). Furthermore, similar endometrial innervation and NGF and NT-3 expression has been reported in women with adenomyosis (without endometriosis) and in women with combined adenomyosis and endometriosis (68), suggesting that further, more detailed studies in a larger number of women are now required before diagnosis on the basis of nerve fibers can be adopted as a reliable diagnostic tool.
DISCOVERY OF POTENTIALLY NEW BIOMARKERS Biomarker Discovery by Proteomics Proteomics studies have been used in biomarker discovery to analyze patients with and without endometriosis. To date, proteomic studies using two-dimensional gel electrophoresis (2DIGE) have shown differences in the eutopic endometrium and serum of patients with and without endometriosis but 1138
have yet to identify a biomarker or a panel of biomarkers for endometriosis (71–75). Proteomic analysis of eutopic endometrium and blood samples analyzed on surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) ProteinChip technology combined with bioinformatics analysis have taken a first step toward developing a diagnostic test for minimal-tosevere endometriosis (54, 76–78). Analysis with SELDI-TOF MS is known to have a poor mass accuracy and reproducibility, which makes it difficult to obtain reliable protein identification (79). In an effort to remediate these weaknesses, our recent endometriosis research using SELDI-TOF MS included clearly defined sample characteristics, robust statistical approaches, pretest depletion of abundant proteins, acceptable intraassay and interassay variability, and protein identification for selected peaks after endometrial plasma analysis (78, 80).
Peripheral Blood Studies In peripheral blood, several proteomic studies using SELDITOF MS (81–85) reported protein/peptides with altered levels in women with and without endometriosis. Only one study made an effort to identify the protein/peptide peaks with altered levels (78) after analysis of 254 plasma samples (training set 70%; test set 30%) from women with (n ¼ 165) and without (n ¼ 89) endometriosis. Ultrasonographynegative endometriosis was best predicted (sensitivity 88%; specificity 84%) using a model based on five protein/peptide peaks (2.058 m/z, 2,456 m/z, 3.883 m/z, 14.694 m/z, and 42.065 m/z) in plasma samples obtained during the menstrual phase. One of the peptide peaks (2189 m/z), which was decreased in women with moderate-to-severe endometriosis compared with controls, was identified as fibrinogen b-chain by use of MALDI-TOF/TOF MS (78). This peptide was previously patented (www.patentstorm.us/patents/7794958) for endometriosis by Fazleabas and coworkers, who found that fibrinogen b-chain was significantly decreased in uterine flushing fluid from induced endometriosis in the baboon. In another study, a peak of 5,830 m/z was found to be upregulated in presurgical versus postsurgical serum samples from women with endometriosis and was absent in the spectra of healthy volunteers (81). However, in a prospective cohort study, investigators reported a low sensitivity and specificity for serum analysis of proteins patterns by SELDITOF MS, suggesting that serum proteomic analysis may not lead to a quick fix diagnostic test (84).
Endometrial Studies In an exploratory study, a panel of four mass peaks (two upregulated: 90.675 kd and 35.956 kd; and two down-regulated: 1.9 kd and 2.5 kd) allowed the identification of endometriosis with maximal sensitivity (100%) and specificity (100%) (54). The 90.675 kd and 35.956 kd mass peaks were identified as T-plastin and annexin V proteins, respectively (54). Annexin has a role in proliferation and/or cell mobility, has metastatic potential, and may promote the pathogenesis of endometriosis by stimulating early invasion of endometrial cells into the mesothelium after initial attachment to the peritoneal VOL. 99 NO. 4 / MARCH 15, 2013
Fertility and Sterility® wall (54). T-plastin plays a role in cellular motility, formation of the actin bundles required for cell locomotion, and maintenance of the cellular architecture (54). A similar sensitivity (92%) and specificity (90%) was reported by another study that used SELDI-TOF MS in a similar patient population (86), which found that the five peaks selected for diagnosis had a MW of 5–7 kd (6,898 m/ z, 5,891 m/z, 5,385 m/z, 6,448 m/z, and 5,425 m/z). In yet another study, three combined potential peptides, with mass-tocharge ratios (m/z) of 15,334, 15,128, and 16,069, could be used effectively to distinguish endometriosis samples from control samples with a specificity of 86.2% and a sensitivity of 87.5% (87). Similarly, proteomic analysis of endometrium samples from women with and without endometriosis (n ¼ 49) resulted in five peptide peaks—2,072 mass/charge (m/z); 2,973 m/z; 3,623 m/z; 3,680 m/z, and 21,133 m/z— which allowed the diagnosis of endometriosis with a high sensitivity and specificity (91%; 80%) (80). A proteomic endometriosis study conducted to identify specific endometrial antigens used one-dimensional and two-dimensional Western blots followed by further downstream analysis of five immunoreactive spots with the aid of MALDI-TOF/TOF MS (88). Enzyme-linked immunosorbent assays (ELISAs) were established for specific epitopes, and autoantibody titers were estimated in an independent cohort for validation. The sensitivity and specificity of serum antigens (28% to 78% and 89% to 96%, respectively) were better than those of serum CA-125 levels (21% and 89%, respectively) for the detection of early stages of endometriosis (88). Overall, proteomics research is promising, but to make real progress it is necessary to increase the reproducibility of the techniques before judging whether peptide fingerprinting can be used for the diagnosis of endometriosis. Furthermore, it is important to identify the most promising peptide peaks in order to generate ELISA assays for their detection in body fluids. Newly discovered proteins could be added to the promising ‘‘biomarker panels’’ as previously discussed and could improve their diagnostic performance. Finally, it is necessary to validate the diagnostic accuracy of any promising protein/peptide peaks prospectively in an independent symptomatic patient population who have subfertility and/or pain without ultrasound evidence of endometriosis and with a clinical indication for surgery.
endometrium of women with endometriosis compared to controls (n ¼ 50) in the proliferative and secretory phases. Transfection of endometrial stromal cells with mir135a/b or miR135a/b inhibitors resulted in the altered expression of HOXA10 mRNA and protein (95). A study of miRNA in peripheral blood would be interesting, but this has yet to be done in endometriosis research.
THE FUTURE OF BIOMARKERS FOR ENDOMETRIOSIS Research of biomarkers for endometriosis is emerging, but still we have no available clinical test. Biomarker research in endometriosis has been generally marked by unclear patient characterization with respect to cycle phase, endometriosis stage, and control group as well as a lack of validation in an independent set of patients, no or limited reproducibility studies, and no identification of protein/peptide peaks or robust statistical approaches. Studies evaluating panels of biomarkers (39, 96–99) also have been limited with respect to the number of biomarkers analyzed, the statistics used (univariate statistical analysis), and the lack of validation in an independent test set of patients. The challenge is to validate findings and move to a clinically affordable, cost-effective test (Fig. 1). Future biobanking in endometriosis is based on both standardized operating procedures (SOPs) and standardized clinical phenotyping. It is hard to anticipate what number of samples is needed for biomarker validation for a noninvasive diagnosis of endometriosis. As a rule of thumb, we propose to perform biomarker validation studies in an equal or higher number of samples when compared with biomarker discovery studies, with a sample distribution reflecting a realistic prevalence of endometriosis in the populations tested (i.e., 30% in women with infertility, 30% to 50% in women with pelvic pain). The Wellcome Trust Case Control Consortium has provided compelling evidence that sample numbers of around 2,000 are the minimum requirement to detect associations with modest genetic effects (http://www.wellcome.ac.uk).
FIGURE 1
MICRO RNAs AND ENDOMETRIOSIS Endometriosis is believed to be multifactorial and polygenic disease, and emerging data provide evidence that dysregulation of miRNA expression may be involved (89). The miRNAs, a novel class of regulatory molecules with the ability to control gene expression at the posttranscriptional level through degradation, repression, and silencing (90), could be used as biomarkers/therapeutic tools in endometriosis (91, 92). In eutopic endometrium, miRNA 17–5p, 23/a–b, 542–3p (93); miR 9, miR 34 (91), and miRNA 21 (94) were found to be dysregulated in patients with endometriosis when compared with controls. The investigators found that miR135b levels were significantly increased in the VOL. 99 NO. 4 / MARCH 15, 2013
Recommendations for the improvement of biobank-based research in endometriosis. (SOPs ¼ standard operating procedures.) Fassbender. Biomarkers of endometriosis. Fertil Steril 2013.
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Standard operating procedures (SOPs) for sample collection and storage at the biobank of the Leuven University Endometriosis Research Group. Type of samplea EDTA plasma
Heparin plasma
Peritoneal fluidb
Details
Biopsy 1. Endometrial biopsy 2. Peritoneum biopsy Note: Specify exact size before biopsy, exact location, and relationship to nearest endometriotic lesion, if present. 3. Endometriotic lesion biopsy
VOL. 99 NO. 4 / MARCH 15, 2013
Handling and storage
After blood collection, the EDTA tube is gently inverted 10 times to prevent the formation of blood clot. The tube is centrifuged at 1,400 g for 10 min at 4 C. 500 mL of plasma is placed in labeled Eppendorf tubes and stored at 80 C until use.
After blood collection, the heparin tube is gently inverted 10 times to prevent the formation of blood clot. The tube is centrifuged at 1,400 g for 10 min at 4 C. 300 mL of plasma is placed in labeled Eppendorf tubes and stored at 80 C until use.
The tube containing PF is centrifuged at 1,400 g for 10 min at 4 C. 500 mL of PF supernatant are prepared into labeled Eppendorf tubes and stored at –80 C until use The PF pellet is washed in PBS, supernatant is discarded, and the pellet is then stored in a cryovial at 80 C.
The endometrial, peritoneum, or endometriotic lesion biopsy is rinsed in sterile PBS. One piece of tissue is placed in a labeled cryovial and snap frozen immediately during/after surgery in liquid nitrogen and stored at 80 C until use. One piece of tissue is placed in a labeled tube with formalin fixative.
Rules
Average time interval between patient entering the OR and blood draw should be 45 to 50 min. Plasma should be processed and stored within a maximum time interval of 1 h after sampling. Sample data must be entered into an electronic database such as Filemaker.c
Average time interval between patient entering the OR and blood draw should be 45 to 50 min. Plasma should be processed and stored within a maximum time interval of 1 h after sampling. Sample data must be entered into an electronic database such as Filemaker.c
PF should be processed within a maximum of 1 hour. Sample data must be entered into an electronic database such as Filemaker.c
Tissue is kept for 24 h in buffered formalin followed by transfer to a phosphate/sucrose buffer for a minimum of 1 night (maximum 1 w) and then transferred to 70% ethanol until it is paraffin embedded. Sample data must be entered into an electronic database such as Filemaker.c
Note: OR ¼ operation room; PBS ¼ phosphate-buffered saline; PF ¼ peritoneal fluid. a Signed informed consent must be obtained for surgery and for blood and tissue sample collection for research purposes. b If no PF or a very small amount of PF is found, the pelvis is washed with 20-mL sterile normal saline solution using a laparoscopic needle followed by manual aspiration using a syringe under direct visual control. This peritoneal lavage fluid (PLF) can be processed as PF, the PF pellet can be stored, and the supernatant can be stored (PLF) or can be discarded. c Any deviations that may occur in the protocol must be recorded. Fassbender. Biomarkers of endometriosis. Fertil Steril 2013.
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TABLE 1
Fertility and Sterility®
TABLE 2
TABLE 2
Clinical phenotyping information collected for each sample stored at the biobank of the Leuven University Endometriosis Research Group (patient information, and preoperative and intraoperative information).
Continued.
Patient information Category
Responses
Demographics Name File number Date of birth Ethnic group
DD/MM/YYYY Asian/Oriental, Black/African American/Caribbean, White (North/West European), White (East European), White (South European), White (other), mixed race, other, unknown
Specifics Body mass index Smoking
Clinical information Category Endometriotic nodule If yes, specify: Number Size Surgical data Date of operation Patient age at time of surgery First day of last menstruation Phase of cycle
Phase of cycle according to
No, occasionally, daily, unknown
Infertility history Infertility Duration infertility Current medication used Medication(s) used Specifics Previous endometriosis surgery Previous surgery Date of surgery Pain history Pelvic pain Specifics Obstetrics history Gravida Parity Spontaneous abortions Mother with children Previous diseases Specifics Menstrual history Regularity of menses Time interval between menses Duration of flow
Primary, secondary, none n (mo) Yes/no/unknown Oral contraception, GnRH, pain medication, other Yes/no DD/MM/YYYY Yes/no Dysmenorrhea, dyspareunia, nonmenstrual pelvic pain, dyschezia n n n n
Absent, regular (variation 2–20 d), irregular (variation >20 d), unknown Frequent (>24 d); normal (24– 38 d), infrequent (>38 d), unknown Prolonged (>8 d); normal (4.5– 8.0 d); shortened (
