Hum. Reprod. Advance Access published March 10, 2005
Human Reproduction Page 1 of 7
doi:10.1093/humrep/deh796
A predictive model for endometriosis M.M.Wo¨lfler1,4, F.Nagele1, A.Kolbus1, S.Seidl2, B.Schneider3, J.C.Huber1 and W.Tschugguel1,5 1
Department of Obstetrics and Gynaecology, Division of Gynaecological Endocrinology and Reproductive Medicine, 2Department of Internal Medicine I, Division of Oncology, Medical University of Vienna, Waehringer Gu¨rtel 18–20, 1090 Vienna, 3Department of Medical Statistics, Vienna University, Schwarzspanierstrasse 6, 1090 Vienna, Austria and 4Department of Obstetrics and Gynaecology, Medical Faculty of the Technical University of Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany 5
To whom correspondence should be addressed. E-mail:
[email protected]
Key words: aromatase/endometriosis/endometrium/menstrual characteristics/predictive model
Introduction Endometriosis is characterized by the presence of endometrial glands and stroma within the pelvic peritoneum and other extrauterine sites. Although the exact prevalence remains unclear, 5 –10% of all women in the reproductive age group are estimated to be affected (Olive and Schwartz, 1993; Vessey et al., 1993). Endometriosis is associated with an array of gynaecological disorders including dysmenorrhoea, chronic pelvic pain and unexplained infertility (Olive and Pritts, 2001). A definitive diagnosis of endometriosis is based on direct visualization of peritoneal and ovarian implants at laparoscopy or laparotomy. The extent of pelvic endometriosis can be classified according to the revised American Society for Reproductive Medicine (1997) classification (rAFS). Endometriosis is regarded as a polygenically inherited disease of complex multifactorial aetiology (Olive and Schwartz, 1993; Bulun et al., 1997; Arvanitis et al., 2003). Among all theories, transplantation of endometrial tissue via retrograde menstruation is the most widely accepted hypothesis (Sampson, 1927). Although retrograde menstruation can be observed in up to 90% of all cycling women, the prevalence of endometriosis is far lower (Halme et al., 1984; Lebovic et al., 2001). Hence, besides a massive exposure to
menstrual efflux, a defect of clearance from pelvic peritoneum suggests that other factors, such as aberrant expression of certain cytokines and tissue matrix metalloproteinases, as well as aberrant expression of aromatase and deficiency of 17b-hydroxysteroid dehydrogenase, may contribute to the development of the disease (Osteen et al., 1996; Zeitoun et al., 1998; Zeitoun and Bulun, 1999; Bergqvist et al., 2001). Eutopic endometrium of patients seems to share alterations with ectopic endometrial implants. There is evidently a difference between eutopic endometrium of women suffering from endometriosis and disease-free women (Bulun et al., 2002; Leyendecker et al., 2002). Hence, a predisposing factor for endometriosis might be found within eutopic endometrium, probably leading to the establishment of ectopic disease. The key enzyme converting C19 steroids into estrogen is cytochrome P450 (CYP 19) aromatase, converting androstendione into estrone and testosterone into estradiol. Aberrant aromatase expression has been clearly documented in ectopically located endometriotic lesions (Noble et al., 1997; Fang et al., 2002) as well as in eutopic endometrium of women suffering from endometriosis or other estrogen-dependent diseases (Noble et al., 1996; Kitawaki et al., 1997). First reports on the usefulness of eutopic aromatase as a target for
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BACKGROUND: Aromatase is the key enzyme in the process of estrogen biosynthesis from the precursor androgen. Recently, aromatase has been found to be aberrantly expressed in eutopic endometrium of patients suffering from endometriosis. This finding has prompted speculation about the contribution of this enzyme to the prediction of this disease. METHODS: We prospectively aimed to evaluate whether endometrial biopsy, prior to laparoscopy in symptomatic women to screen for the presence of aromatase by real-time RT –PCR and immunohistochemistry, combined with select patients’ characteristics, is of value to predict endometriosis. RESULTS: Of 48 consecutive symptomatic and eligible patients, 25 (52.1%) exhibited endometriosis and 23 (47.9%) were disease-free. A multiple logistic regression model revealed that 95.5% of patients whose eutopic endometrium was found to be positive for aromatase mRNA as well as immunohistochemically detected protein and who were additionally suffering from moderate to severe dysmenorrhoea (visual analogue scale score >4/10) exhibited endometriosis at laparoscopy. CONCLUSIONS: These findings provide direct evidence that screening for eutopic endometrial aromatase in combination with clinical data could be of discriminative value in the prediction of disease.
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a screening test are contradictory (Kitawaki et al., 1999; Dheenadayalu et al., 2002). However, apart from other screening markers suggested (Bedaiwy et al., 2002; Harada et al., 2002; Brosens et al., 2003), patients’ histories provide valuable information about clinical symptoms typically associated with endometriosis (Cramer et al., 1986; Darrow et al., 1993; Kuohung et al., 2002). The aim of this study was to prospectively evaluate whether screening for the presence of CYP 19 aromatase in eutopic endometrium in combination with select patients’ characteristics might assist in developing a predictive model for endometriosis. Materials and methods
RNA extraction and real-time RT– PCR analysis For RNA extraction, frozen tissue samples were triturated and total RNA was extracted using the TRI Reagent method by MRC Table I. Indication for operative procedure Indication
Case (n ¼ 25)
Control (n ¼ 23)
Unexplained infertility Dysmenorrhoea Dyspareunia Chronic pelvic pain
14 5 1 5
8 (35) 8 (35) 0 (0) 7 (30)
(56) (20) (4) (20)
Values in parentheses are percentages. Forty-eight of 64 women attending the tertiary care centre for diagnosis and/or treatment were eligible for analysis.
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Immunohistochemical analysis Frozen tissue samples were cut into 6 mm sections and fixed using acetone, air-dried and washed in phosphate-buffered saline (pH 7.4). To block endogenous peroxidase activity, the sections were treated with 3% H2O2 in methanol for 15 min at room temperature. To block non-specific protein binding the sections were incubated with Dako Protein Block Serum-Free (Dako, USA) for 10 min at room temperature. For the detection of human aromatase cytochrome P450 we used the monoclonal mouse anti-human antibody MAb 2-C-2 (Hauptmann – Woodward Medical Research Inc., USA) in a 1:500 dilution. All sections were incubated overnight at 4 8C. Visualization of the immunoreaction was carried out with the ChemMate Antibody Detection Kit (Dako) according to the manufacturer’s instructions. Gill’s haematoxylin (Merck, Germany) was applied as a nuclear counterstain. As a positive control for aromatase expression, human placental tissue was obtained from a patient at 39 weeks of gestation with an uneventful obstetric history. Sections serving as negative control for aromatase cytochrome P450 were incubated with a 1:500 dilution of non-immunized mouse immunoglobulin G serum (Dako). Six sections of each tissue sample were examined microscopically by three independent trained physicians who were unaware of the patients’ history. Each slide was graded into negative (0) or positive (1). Due to small sample sizes, no further analysis of subgroups was performed. Western blot analysis Whole cell extracts were prepared from endometrial tissue, homogenized in lysis buffer [10 mmol/l Tris, 30 mmol/l Na4P2O7, 50 mmol/l NaCl, 50 mmol/l NaF, 1% Triton X-100, pH 7.1; 1 mmol/l phenylmethylsulphonyl fluoride; 0.1 mmol/l Na3VO4 and protease inhibitor cocktail (1:25; Boehringer Ingelheim GmbH, Germany)]. Insoluble material was removed by centrifugation (14 000 rpm for 5 min) and the protein concentration was determined by the Bradford assay (Bio-Rad Laboratories Inc., USA). Thirty micrograms of protein extract were separated on 10% SDS– PAGE gels in Mini-Protean 3 Gel Tank (Bio-Rad) at 200 V for 1 h, followed by electrophoretic transfer to nitrocellulose membranes (Amersham Pharmacia, Germany). Membranes were blocked for 1 h at room temperature using 5% non-fat milk powder dissolved in Tris-buffered saline containing 0.02% Tween 20 and incubated overnight at 4 8C with the goat polyclonal antiserum directed against human aromatase at a 1:400 dilution (Santa Cruz Biotechnology Inc., USA). Membranes were then incubated for 1 h at room
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Patients, tissue sampling and processing This study was designed as a prospective cohort trial and was approved by the local Ethics Committee according to the Declaration of Helsinki. All participating patients signed an informed consent prior to enrolment. Sixty-four women aged 21–48 years (mean: 32.9 years) attending this tertiary care centre for diagnosis and/or treatment of unexplained infertility, dysmenorrhoea, dyspareunia or chronic pelvic pain were selected (Table I). Prior to enrolment, estrogen-dependent diseases (EDD) other than endometriosis were clinically excluded by performing gynaecological exploration as well as transvaginal ultrasound examination. None of the patients was diagnosed with endometriosis or another EDD prior to inclusion or received any endocrine therapy such as GnRH analogues or danazol at that time. Five women using oral contraceptives were excluded from this trial due to possibly altered endocrine regulation and aromatase activity in the endometrium. Menstrual characteristics as well as other demographic data were collected in a standardized interview. Furthermore, all subjects completed a questionnaire including visual analogue scales (VAS) to assess the level of dysmenorrhoea, dyspareunia and pelvic pain irrespective of menstrual symptoms and sexual intercourse during a period of 30 days. Prior to laparoscopy, endometrial biopsies were taken using a sharp curette. Endometriosis was diagnosed according to the current gold standard via laparoscopic visualization followed by histopathological assessment of putative lesions (Brosens, 1997). The stage of endometriosis was assigned during laparoscopy according to the rAFS. Endometrial tissue samples were snap-frozen in liquid nitrogen immediately after biopsy and stored at 280 8C until further analysis.
(Molecular Research Centre, Inc., USA). RNA concentration was determined by measuring the optical density at 260 nm. RNA was reverse-transcribed into first strand cDNA using Superscript (Invitrogen Ltd, UK). The resulting cDNA was amplified and quantified using the TaqManw Gene Expression Assay for the detection of exon 9 of the human CYP 19 aromatase gene in real-time RT– PCR according to the standardized protocol (Applied Biosystems, USA). The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified in parallel reactions as a housekeeping reference gene serving as an internal control for the quantity and quality of the cDNA. The quantification of the target gene expression (CYP 19 aromatase) was performed according to the delta-delta CT method for relative quantification (Livak and Schmittgen, 2001): expression levels of aromatase were related to the expression of a reference housekeeping gene (GAPDH) in the same sample. The difference in expression (¼ ratio) was calculated to explore the relative quantity of aromatase expression in each endometrial biopsy specimen.
Prediction of endometriosis
temperature in peroxidase-conjugated donkey anti-goat secondary antibody (Jackson Immuno Research, USA). Proteins were detected by using an enhanced chemiluminescence detection system (Pierce Chemical Co., USA).
Results Of the biopsy specimens taken from 64 patients, seven samples could not be used for further analysis because of inferior quality due to deviation from the freezing procedure. Two other specimens had to be discarded because of insufficient RNA extraction and failure of cDNA reverse transcription. Moreover, samples from five patients using oral contraceptives were not subjected to further molecular and statistical analysis due to altered hormonal regulation of the endometrium to prevent biasing results. For two samples, we did not have sufficient material for the repetition of the experiment. Therefore, the final analysis using real-time RT – PCR for the quantification of aromatase expression was performed with a total number of 48 samples. The mean age of all patients was 32.9 years (^ 6.4 years), 25 out of 48 (52.1%) women were diagnosed with endometriosis; 23 out of 48 (47.9%) had no evidence of disease. Diagnosis of endometriosis was performed according to the current gold standard (Brosens, 1997). Consequently, three out of 48 patients, who revealed putative endometriotic lesions at laparoscopy that could not be confirmed during further histopathologic assessment, were considered to be disease-free. Additional pelvic disease was discovered in two patients; one patient was diagnosed with a mild myomatosis of the uterus (which was not detected by pelvic ultrasound prior to surgery) in coexistence with endometriosis. The other patient undergoing laparoscopy for diagnostic infertility work-up and treatment of an ovarian cyst appeared to have a borderline tumour in one ovary, whereas endometriosis could not be confirmed.
Figure 1. Aromatase expression in human eutopic endometrium. Left box blot: aromatase mRNA expression in human eutopic endometrium in patients with no endometriosis (range 1.0 – 19.49, median 4.41, SD 4.3). Right box blot: aromatase mRNA expression in human eutopic endometrium in patients with endometriosis (range 2.41 – 100.71, median 11.8, SD 36.73).
Expression and localization of aromatase in eutopic endometrium According to real-time RT – PCR, aromatase mRNA expression was found in all the 48 samples, ranging from 1- to 100.7-fold expression after relative quantification according to the delta-delta CT method (Figure 1). The quantity of CYP 19 aromatase transcripts was strongly associated with the presence of endometriosis (Pearson’s correlation coefficient r ¼ 0.49, P , 0.001). When calculated with regard to the stage of disease, the correlation coefficient increased to r ¼ 0.51 (P , 0.001). To localize aromatase protein, immunohistochemistry (IHC) was performed. Fifteen out of 25 (60.0%) cases and three out of 23 (13.1%) controls were aromatase positive (rAFS I and II: 50.0%; rAFS III and IV: 63.9%). There was a clear correlation between aromatase protein in endometrial stromal and/or endometrial epithelial cells and the presence of endometriosis (r ¼ 0.48, P , 0.001). The sensitivity of IHC testing for aromatase expression in endometrial epithelial and stromal cells was 60.0%, the specificity 86.9%; the positive and negative predictive values were 83.3% and 66.7% respectively. Representative sections are shown in Figure 2. We found aromatase protein to be localized in both endometrial epithelial and stromal cells with a strong association of its presence between the epithelial and stromal cells (r ¼ 0.50, P , 0.001). There was a strong association between the presence of aromatase protein in epithelial and stromal cells (r ¼ 0.50, P , 0.001) in the 48 patients examined. To confirm the specificity of our IHC data, we performed western blot analyses in a series of select samples. The sample size was limited to the residual material available following generation of data that pertain to the model. Hence, in 21 of the 64 samples we had sufficient material to perform western Page 3 of 7
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Statistical analysis For the comparison of metric variables between groups we used the Student’s t-test, whereas for categorical variables the x2-test or Fisher’s exact test was applied. To evaluate associations between continuous variables, Pearson’s correlation coefficient was computed. The prevalence of disease according to the study sample was 52.1%. For the assessment of a predictive model for endometriosis, stepwise logistic regression was performed for clinically relevant patients’ characteristics and the applied testing methods; the significance level for a variable to enter and stay in this model was 15%. The discriminating power of the resulting multiple logistic model outcome (linear combination of the selected explanatory variables in the logistic model) was visualized by a receiver operating characteristics curve (ROC) and quantified by the area under the curve (AUC). P , 5% was considered to be statistically significant. All computations were performed using SAS 8.2 software (SAS Institute, USA).
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Figure 3. Western blot analysis. CYP 19 expression in endometrial tissue. Lane 1 demonstrates placental whole tissue extract used as a positive control. Whole tissue extracts were prepared from endometrium of patients suffering from endometriosis (lanes 2 – 6) and of patients with no endometriosis (lanes 7 – 10).
analyses. We found aromatase protein in 12 of the 21 samples (57.1%). Ten of these 12 patients suffered from endometriosis whereas in two patients showing aromatase protein in western blot analysis endometriosis was not confirmed (sensitivity: 83.3%) (Figure 3). Of 10 endometriosis patients, in nine aromatase activity was found concordantly in IHC. In the two controls, one showed no signal for aromatase in IHC in stromal and epithelial cells whereas in the other patient aromatase protein was found in endometrial stromal cells in IHC. Page 4 of 7
On the other hand, in nine of the 21 samples (42.9%) no aromatase protein was found in western blot analysis. Of those nine patients, six had no endometriosis (specificity 66.7%). Of those six patients, four were concordantly negative in IHC and two showed aromatase activity in endometrial epithelial cells. Of the three endometriosis patients with no signal in western blot analysis, only one showed aromatase activity in endometrial stromal cells in IHC, the other two were negative in IHC.
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Figure 2. Immunohistochemistry. Representative immunostaining for CYP 19 aromatase in eutopic endometrium. Positive staining for aromatase in epithelial and stromal cells (A), stromal cells only (B), epithelial cells only (C) and no staining for aromatase (D). Placental tissue was used for positive and negative control sections (E and F). For the immunostaining, a monoclonal mouse antibody (brown cytoplasmatic staining) was applied. The original magnification used was £ 400 (bar ¼ 20 mm).
Prediction of endometriosis Table II. Distribution of clinical parameters
Table III. Individual probability for disease a
Variable
Case (n ¼ 25)
Control (n ¼ 23)
P Pa
Age at menarche #13 years Length of cycle #28 days Duration of menses $5 days Mean no. of pregnancies Pregnancies $1 Parity $1 Miscarriage $1 Mean VAS scoreb for Dysmenorrhoea Dyspareunia Pelvic pain
21 (84) 11 (44) 14 (56) 0.8 11 (44) 10 (40) 3 (12)
12 (52) 9 (39) 8 (34) 1.2 14 (62) 10 (43) 5 (23)
, 0.05 NS , 0.05 , 0.05 , 0.05 NS NS
6.7 0.2 2.6
3.3 0.9 1.8
, 0.001 NS NS
a
For calculation of P-values, see Statistics section. Visual analogue scales (VAS) (ranging from 0 to 10) were completed at the time of enrolment prior to laparoscopy. NS ¼ not significant. b
b
Real-time RT–PCR IHC (stroma)c VAS dysmenorrhoead Value of the linear predictor (VLP)e Individual probability of disease (IPD)f
1
2
3
4
5.7 0 3 23.43
10.4 0 4 20.58
6.6 1 3 2.02
80.7 1 7 40.87
0.031
0.358
0.860
0.999
a
The patients’ characteristics listed above are fictitious in order to show that the individual probability of disease can be calculated for any patient. b Values for real-time RT–PCR range from 1- to 100.7-fold mRNA expression. c Immunohistochemistry (IHC) is graded as 0 ¼ negative and 1 ¼ positive for aromatase protein. d Visual analogues scales (VAS) range from 0 to 10. e,f For calculation of the individual probability of disease, see formulas in Results section.
according to the following mathematical function using Euler’s constant (e < 2.718282): Individual probability of disease ðIPDÞ ¼ evalue of linear predictor =1 þ evalue of linear predictor The linear predictor is an artificial variable calculated by the regression model that facilitates the estimation of the individual probability of disease for each patient. The value of the linear predictor is calculated as follows: Value of the linear predictor ðVLPÞ ¼ 27:78 þ 0:5 £ ðvalue of quantitative real–time RT–PCRÞ þ 4:8 £ ð0 or 1 according to IHC instromal cellsÞ þ 0:5 £ ð0 to 10 according to the VAS score for dysmenorrhoeaÞ The individual probability of disease for each patient and the value of the linear predictor result in the sigmoid curve. Examples for the calculation of the individual probability of disease in four fictitious patients are listed in Table III. For instance, patient 1 [low levels of aromatase mRNA (5.7), no aromatase protein, VAS score for dysmenorrhoea ¼ 3] would have a VLP of 2 3.43 and an IPD of 0.031 indicating a probability of endometriosis of 3.1%. In contrast, patient 4 [high levels of aromatase mRNA expression (80.7), presence of aromatase protein in IHC, severe dysmenorrhoea (VAS score: 7)] would have a VLP and an IPD of 40.87 and 0.999 respectively. Thus, for this patient the confidence of prediction of endometriosis would be 99.9%.
Figure 4. Receiver operating characteristics (ROC) curve. The discriminating power of this multiple logistic model is visualized by the ROC and quantified by the area under the curve (AUC).
Discussion The objective of this study was to establish a model to predict pelvic endometriosis by means of endometrial aromatase Page 5 of 7
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Devising a predictive model for endometriosis In a multivariate analysis using the applied testing methods and clinical data from patients’ histories, a series of gynaecological characteristics was different between cases and controls (Table II). The presence of endometriosis clearly correlated with the severity of dysmenorrhoea (r ¼ 0.44, P , 0.01) whereas for pelvic pain (r ¼ 0.23, P . 0.05) and for dyspareunia (r ¼ 0.11, P . 0.05) no significant association was found. Of all parameters investigated, the results of quantitative real-time RT – PCR analysis and IHC analysis of endometrial stromal cells as well as the severity of dysmenorrhoea were selected according to stepwise logistic regression. These three entities then entered the final multiple logistic regression model showing a ROC as depicted in Figure 4 with a 95.5% AUC, indicating a high discriminatory ability between patients with and without disease. Furthermore, this model provides formulas whereby the individual probability of endometriosis can be calculated
Patienta
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However, our preliminary data need to be confirmed by a larger series before routine clinical application can be proposed. The limitations of our screening method comprise a possible dependency on the phase of the menstrual cycle for endometrial biopsy and the influence of concomitant medication [i.e. oral contraceptives (Meresman et al., 2002) and GnRH analogues (Ishihara et al., 2003)]. Additionally, a further confounding influence might occur by using a subjective pain score (VAS) to describe the severity of dysmenorrhoea. In order to minimize this limitation, we used mean pain scores of the complete phase of menstrual efflux as described (Chapron et al., 2003; Walsh et al., 2003) and found them indicative for disease prediction. In conclusion, this model, based on cytochrome P450 aromatase screening of eutopic endometrial biopsy specimens combined with select patients’ characteristics, represents a novel, non-surgical diagnostic method which can easily be performed in every outpatient infertility or chronic pelvic pain clinic.
Acknowledgements The authors wish to thank Alexander Dangl and Ladislaus Szabo for their skilful technical assistance. This study would not have been possible without the generous contribution of all the women who have participated in this study.
References Abbott JA, Hawe J, Clayton RD and Garry R (2003) The effects and effectiveness of laparoscopic excision of endometriosis: a prospective study with 2–5 year follow-up. Hum Reprod 18,1922–1927. American Society for Reproductive Medicine (1997) Revised American Society for Reproductive Medicine classification of endometriosis. Fertil Steril,817–821. Arvanitis DA, Koumantakis GE, Goumenou AG, Matalliotakis IM, Koumantakis EE and Spandidos DA (2003) CYP1A1, CYP19, and GSTM1 polymorphisms increase the risk of endometriosis. Fertil Steril 1,702–709. Bedaiwy MA, Falcone T, Sharma RK, Goldberg JM, Attaran M, Nelson DR and Agarwal A (2002) Prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. Hum Reprod 17, 426–431. Bergqvist A, Bruse C, Carlberg M and Carlstrom K (2001) Interleukin 1beta, interleukin-6, and tumor necrosis factor-alpha in endometriotic tissue and in endometrium. Fertil Steril 75,489– 495. Brosens I (1997) Diagnosis of endometriosis. Semin Reprod Endocrinol 15, 229–233. Brosens I, Puttemans P, Campo R, Gordts S and Brosens J (2003) Non-invasive methods of diagnosis of endometriosis. Curr Opin Obstet Gynecol 15,519–522. Brosens J, Verhoeven H, Campo R, Gianaroli L, Gordts S, Hazekamp J, Hagglund L, Mardesic T, Varila E, Zech J and Brosens I (2004) High endometrial aromatase P450 mRNA expression is associated with poor IVF outcome. Hum Reprod 19,352–356. Bulun SE, Noble LS, Takayama K, Michael MD, Agarwal V, Fisher C, Zhao Y, Hinshelwood MM, Ito Y and Simpson ER (1997) Endocrine disorders associated with inappropriately high aromatase expression. J Steroid Biochem Mol Biol 61,133–139. Bulun SE, Gurates B, Fang Z, Tamura M, Sebastian S, Zhou J, Amin S and Yang S (2002) Mechanisms of excessive estrogen formation in endometriosis. J Reprod Immunol 55,21–33. Chapron C, Fauconnier A, Dubuisson JB, Barakat H, Vieira M and Breart G (2003) Deep infiltrating endometriosis: relation between severity of dysmenorrhoea and extent of disease. Hum Reprod 18,760–766. Cramer DW, Wilson E, Stillman RJ, Berger MJ, Belisle S, Schiff I, Albrecht B, Gibson M, Stadel BV and Schoenbaum SC (1986) The relation of
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detection in combination with select patients’ characteristics. We found the screening on aberrant aromatase in eutopic endometrium and menstrual symptoms, i.e. the severity of dysmenorrhoea, indicative for endometriosis prediction. Histological confirmation of endometriosis by laparoscopy revealed a prevalence of 52.1% for the disease among symptomatic patients attending our tertiary care centre for dysmenorrhoea, dyspareunia, chronic pelvic pain and unexplained infertility. This figure is similar to recently published data about epidemiological facts on endometriosis (Mahmood and Templeton, 1991; Spaczynski and Duleba, 2003). The present study confirms that high levels of CYP 19 aromatase in eutopic endometrium are conclusively associated with the presence of endometriosis. Several earlier studies have shown that endometriosis and other pelvic pathologies are strongly associated with aberrantly high aromatase expression (Noble et al., 1996; Bulun et al., 1997; Kitawaki et al., 1997). For instance, Kitawaki et al. (1999) found aromatase expression and activity in endometrial specimens from patients with EDD such as endometriosis, adenomyosis and leiomyoma of the uterus, but not in disease-free women. In their retrospective analysis of 105 patients using non-quantitative RT – PCR and IHC analysis, they found a sensitivity and specificity of 91 and 100% respectively. A prospective trial with 56 patients showed a far lower sensitivity and specificity using non-quantitative PCR techniques (82 and 59% respectively) (Dheenadayalu et al., 2002). These differences in sensitivity and specificity of aromatase testing might be due to a series of limitations, such as small sample sizes in both trials and a lack of comparability due to polymorbid patients in the retrospective trial (Kitawaki et al., 1999) as well as selection bias, or the restriction to non-quantitative PCR techniques in the prospective trial (Dheenadayalu et al., 2002). However, recently, aromatase was found at particularly low levels in any endometrial tissue, rising to aberrantly high levels in association with EDD (Brosens et al., 2004). Along with these data, our results showed aromatase expression— although at very low levels—in each single endometrial sample ranging from 1- to 100.7-fold expression. Therefore, efforts to predict endometriosis on the basis of non-quantitative aromatase detection might not be sustainable. Despite advanced attempts to generate laboratory tests or appropriate imaging techniques for the diagnosis of endometriosis, to date no simple non-invasive tests have been available to reduce the number of uninformative laparoscopies. The use of clinical symptoms and signs together with laboratory markers appears to be a potentially effective method to predict endometriosis (Eskenazi et al., 2001; Falcone and Mascha, 2003; Gagne et al., 2003). Early diagnosis and treatment of endometriosis is crucial for the management of this chronic disease (Donnez et al., 2003). While some women suffering from pelvic pain possibly associated with endometriosis might benefit from an initial medical rather than from a surgical therapy (Olive and Pritts, 2001), others might benefit from immediate excision of lesions by laparoscopic surgery (Abbott et al., 2003). The statistical formula proposed here may help the clinician to identify patients suffering from endometriosis to reconcile appropriate treatment.
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