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Serum and peritoneal fluid CA-125 levels in patients with endometriosis*
FERTILITY AND STERILITY Copyright
©
Vol. 61, No.3, March 1994
1994 The American Fertility Society
Printed on acid-free paper in U. S. A.
Serum and peritoneal fluid CA-125 levels in patients with endometriosis·
Antonella Barbati, Ph.D. t Ermelando Vinicio Cosmi, M.D. Rita Spaziani, M.D.
Rocco Ventura, M.D. Gennaro Montanino, M.D.
2nd Institute of Obstetrics and Gynecology, University of Rome, "La Sapienza," Rome, Italy
Objective: To evaluate CA-125 in peritoneal fluid (PF) as an indicator of endometriosis. Design: CA-125 levels in paired serum and PF were determined by the one-step immunoradiometric assay. For peritoneal samples, high dilution of the sample (1:100) was used to avoid false low results, caused by the "hook effect" phenomenon. Patients: Forty-one women with and without endometriosis, undergoing laparoscopy or laparotomy during the follicular phase of the menstrual cycle, were selected. Setting: 2nd Institute of Obstetrics and Gynecology, University of Rome "La Sapienza," Rome, Italy. Main Outcome Measure: Peritoneal fluid CA-125 levels obtained using diluted samples were significantly higher than those found using undiluted ones. Results: CA-125 levels in PF were approximately 100 times higher than those found in paired serum, ranging from 970 to 10,636 U /mL. In patients with endometriosis, CA-125 levels in PF were significantly elevated when compared with the control group. In serum, CA-125 levels increased only in advanced stages of endometriosis. Conclusions: The sensitivity of the CA-125 test for endometriosis in PF is greater than in serum. Therefore, the measurement of CA -125 levels in PF could be useful in the detection of early stage endometriosis, which tends to be overlooked by the CA-125 serum test. Fertil Steril 1994;61:438-42 Key Words: CA-125, peritoneal fluid, endometriosis, immunoradiometric assay, hook effect
CA-125 is a cell surface antigen that is expressed in tissues derived from embryonic celomic epithelium such as the endometrium, endocervix, fallopian tubes, peritoneum, pleura, and pericardium (1). High concentrations of CA -125 have been dem0nstrated in a variety of normal biological fluids such as cervical mucus, human milk, saliva, and amniotic and peritoneal fluids (PF) (2-6). Serum antigen levels in women are low, exhibiting a variable expression during the normal menstrual cycle with
Received June 4, 1993; revised and accepted October 28, 1993. * Supported by grant 92.02130.39 ACRO from the National Research Counc,il, Rome, Italy. t Reprint requests: Antonella Barbati, Ph.D., 2nd Institute of Obstetrics and Gynecology, University of Rome, "La Sapienza," Policlinico Umberto I, 00161 Rome, Italy. 438
Barbati et a1.
Peritoneal fluid CA-125 in endometriosis
peak serum levels during menses (7). Increased serum levels occur in ~80% of women with epithelial ovarian carcinoma (8), in a low percentage of other malignant and nonmalignant conditions (9) and in pregnancy (10). To date, the clinical use of CA-125 antigen has been limited to the detection and management of ovarian cancer. Since Barbieri et al. (11) demonstrated elevated serum concentrations of CA -125 in patients with advanced endometriosis, various investigators (6, 12) have attempted to use this antigen in the preoperative diagnosis of endometriosis. Because an endometrial origin for CA-125 has been well documented (13), the antigen has been investigated also in the PF to evaluate whether PF levels are a more sensitive indicator of disease than measurements in serum (14, 15). The values obtained were approximately 10-fold higher Fertility and Sterility
than serum levels. No differences in CA-125 levels were found between women with and without endometriosis. Most of the studies employed the same assay standardized to quantify CA-125 in serum: an immunoradiometric "one-step" assay (IRMA) (8). This test, based on the sandwich principle, uses the OC-125 monoclonal antibody (mAb) as capture (polystyrene beads on which the OC-125 are fixed) and as tracer antibody (OC-125 radioiodinated) in a simultaneous incubation, because multiple CA-125 antigenic determinant are associated with the molecule. Recently it was noted that in this assay, CA125 levels in diluted samples of PF (1:2 to 1:10) do not showed the expected decrease in binding (14, 16). The so-called "hook effect," i.e., a decreased response in samples containing a large excess of antigen, causes falsely low results. This phenomenon is usually associated with one-step sandwich assay. High levels of the antigen in PF were found when the hook effect was eliminated using the IRMA modified by the two-step assay or by high dilutions of the sample, ;;:::1:50 (17). The aims ofthe present study were as follows: [1] to determine reliable values of CA-125 in PF of women with endometriosis, using an assay not invalided by the hook effect; [2] to investigate whether PF CA-125 levels in women with endometriosis were increased when compared with controIs; and [3] to evaluate its possible role as a marker for endometriosis.
MATERIALS AND METHODS Patients and Samples Collection
Paired serum and PF were obtained from women undergoing laparotomy or diagnostic laparoscopy for infertility and/or pelvic pain: 18 women had untreated endometriosis, (aged 23 to 41 years); 20 had benign gynecological disease (aged 16 to 55 years) including pelvic adhesions, pelvic inflammatory disease (PID), leiomyoma, and ovarian cysts; 7 women, who at laparoscopy presented a normal pelvis without signs of present or previous inflammation, served as control group (aged 26 to 39 years). The endometriosis group, staged according to the revised American Fertility Society classification, included patients with stages I and II (n = 12) and patients with stages III and IV (n = 6) (18). In this study, only women undergoing laparotomy or laparoscopy during the midfollicular phase (cycle days 8 to 12), were included to eliminate the Vol. 61, No.3, March 1994
variations of CA-125 concentration due to menstrual cycle. The day of the cycle was determined by patient menstrual history. None ofthe women received any hormonal treatment during the 3 months preceding the intervention. Blood sample was obtained by venipuncture from all patients immediately before induction of anesthesia. Peritoneal fluid was aspirated from the pelvis at the beginning of the laparoscopic examination or laparotomy. Fluids transferred to the laboratory were centrifuged (1000 X g for 10 minutes) and the supernatants stored at -20°C until assayed. In six patients the paired PFs were not available for the assay. CA-125 Assay
CA-125 levels in serum and in PF were measured in duplicate by an immunoradiometric "one-step sandwich" assay (IRMA CA-125 II K; Sorin Biomedica, Saluggia, Italy), used according the manufacturer's instructions. It is a second-generation assay utilizing polystyrene beads coated with a new capture mAb, Mll (19), which reacts with an epitope of the CA-125 antigen different from those recognized by the mAb OC-125. The tracer, composed of radioiodinated OC-125, quantifies the CA-125 antigenic determinants. The standard points ranged from 15 to 500 U /mL. The minimal detectable concentration is 1.4 U /mL. To evaluate the validity of CA-125 measurements in PF, using the new kit assay, we assayed a pool (n = 5) of PF and a pool of serum samples at dilutions 1:2, 1:5, 1:10, 1:50, 1:100, and 1:200 with saline solution. All PF samples were reassayed at dilution 1:100 to obtain valid results. The intra-assay and interassay coefficients of variation were 7.5% and 8.7%, respectively. Statistical analysis was performed by using Student's t-test and linear-regression analysis. RESULTS
CA-125 values obtained for serum (range: 7.5 to 181 U /mL) and PF (range: 970 to 10,636 U /mL) are shown in Figure 1. They represent the mean of duplicate measurements of each sample. The concentration of CA-125 in PF was significantly greater than the corresponding serum in all subjects (t-test, P < 0.0001). Serum CA-125
When women with endometriosis were combined to constitute two groups, one with stages I and II Barbati et al.
Peritoneal fluid CA-125 in endometriosis
439
Table 2 Reliability of Serum and Peritoneal Fluid CA-125 Levels for the Diagnosis of Endometriosis Parameter
Serum*
Peritoneal fiuidt
Sensitivity Specificity Positive predictive value Negative predictive value
0.44 0.88 0.72 0.70
0.83 0.64 0.57 0.88
* CA-125 >35 U/mL. t CA-125 >2,500 U/mL.
increased levels (25%). In patients with benign gynecological disease, moderate increases of CA-125 levels were observed in 3 of 20 patients (15%). As a cut-off, we considered the value of 35 U jmL, which is usually accepted by various authors (6, 12). Statistical parameters are reported in Table 2. Peritoneal Fluid CA-125 Levels
Undiluted PF
and one with stages III and IV (18), the patients with advanced endometriosis showed serum CA125 levels significantly higher than those found in the other groups (Table 1). CA-125 levels were elevated in 5 of 6 patients (83 %). Patients with stage I and II endometriosis showed serum CA-125 levels similar to the controls. Only 3 of 12 patients had
In undiluted PF, the CA-125 levels ranged between 41 and 207 UjmL. The mean ± SD for endometriosis, control, and benign gynecological disease groups were 132 ± 57, 118 ± 42.9,122 ± 54.6 UjmL, respectively. No difference was found between the mean values reported for each group. Although the CA-125 values of undiluted sample were within the linearity range of the assay, the samples assayed at dilutions 1:2 to 1:200 did not show any dilutional effect to 1:10 so that the values obtained can be considered invalid. Only at dilutions oLd:50, parallelism with the serum dilutional curve was observed, and the results can be considered valid. These results demonstrate that, also with the new assay using the Mll monoclonal antibody, CA-125 determination in PF exhibited the hook effect. Diluted PF
Table 1
CA·125 Levels in Serum and PF Samples*
Group
Serum CA-125
PF CA-125
U/mL
Control Endometriosis Stage I and II Stage III and IV Ben Gyn Disease
15.0 41.9 20.5 77.9 17.4
± ± ± ± ±
4.2 42.2 15.2 55.3+ 12.4
U/mL
1695 5971 5537 6752 3726
± ± ± ± ±
439 2,695t 2,590t 3,000t 2,807
* Values are means ± SD. t P < 0.05 compared with the control group. P < 0.01 compared with the control group.
+ 440
Barbati et al.
Peritoneal fluid CA -125 in endometriosis
CA-125 levels obtained using high dilutions (1:100) of the samples were significantly higher than values obtained from undiluted ones. The values ranged from 970 to 10,636 U jmL. The mean value ± SD of each group are reported in Table 1. The PF levels of CA-125 in women with endometriosis were significantly higher than those found in control and benign gynecological disease groups. Unlike CA-125 serum levels in patients with stages I and II endometriosis, PF levels in this group were significantly different from controls. Seven of 9 patients (77%) displayed increased CA-125 concenFertility and Sterility
trations. All PFs from patients with stages III and IV endometriosis had increased levels ofCA-125. In patients with benign gynecological disease, CA-125 levels were increased in 9 of 18 patients (50%). The highest (CA-125 = 10,636 U/mL) value was observed in a patient with PID. Amounts of CA -125 in PF appear to be a more sensitive indicator of endometriosis than serum concentrations of the antigen, although its specificity was lower. In fact, in benign gynecological diseases group, the CA -125 levels in PF were elevated in 50% of patients, whereas in their respective serum, moderate increased values were detectable only in 15% of cases. Sensitivity, specificity and other statistical parameters (Table 2) were calculated for endometriosis using the cut-off of 2,500 U /mL as previously determined (16). Regression analysis performed for CA -125 values in diluted PF and paired serum samples showed a significant correlation (r = 0.75, P = 0.01; n = 39). DISCUSSION
The diagnosis of endometriosis is usually established by laparoscopy or laparotomy with direct visualization of implants and preferably confirmed by histological examination of biopsies. Noninvasive diagnostic methods for evaluation of treatment and for the detection of recurrence are currently being sought (20, 21). Using CA-125 as a marker for endometriosis, we found, as did others (6, 11, 12), that significantly elevated levels of CA-125 in serum become evident only in advanced stages of the disease (stages III and IV [18]). In stages I and II endometriosis the serum levels of CA-125 are low, precluding its measurement as a diagnostic test of disease. Nevertheless, the serum test seems to be useful in monitoring patients during and after medical or surgical therapy, because a significant correlation between CA-125 levels and the clinical course of the disease was observed (22). With regard to CA-125 levels in PF, the previous determinations (14, 15) failed to show any difference between women with and without endometriosis. One study (23) gave evidence of increased amounts of PF CA-125 in endometriosis, but only because of the higher volume of the fluid in these patients. The authors considered these results valid, because they were not awared of the hook effect associated with CA-125 determination in PF using the simultaneous "one-step" IRMA assay. The generally accepted cause of this phenomenon is an excess of antigen, which progressively saturates Vol. 61, No.3, March 1994
both the solid-phase and the detection antibodies, thus preventing formation of the sandwich. Nevertheless, because this phenomenon was not operative in amniotic fluid containing similar high levels of CA-125 (10), others causes as the presence of different CA-125 molecular forms in PF may be implicated. Recent improvements to this assay have been the design of a two-step assay procedure and the usage of higher sample dilutions. Using the first modification, Kruitwagen et al. (24) recently found that CA-125 levels in PF, throughout the normal menstrual cycle, ranged from 630 to 12,000 U /mL, with values significantly higher during the early follicular phase compared with the luteal phase. Using high sample dilutions, we found (16) significant differences in CA-125 levels between women with ovarian and endometrial cancer and women of the control group (mean values: 9,184 versus 2,278 U/mL, respectively). We now have been able to demonstrate a significant increase in CA-125 levels in the PF of women with endometriosis compared with women of the control group. Levels of CA-125 in PF were a more sensitive indicator of disease than the serum test (0.83 versus 0.44), especially for minimal and mild endometriosis (0.77 versus 0.25). In fact, most of the patients with stages I and II endometriosis, who did not show increased CA-125 levels in serum, had high levels in PF (CA-125 > 2,500 U/mL). Employing measurement of this antigen for the diagnosis of endometriosis is, however, limited because of its low specificity. At least, detection of elevated levels of CA-125 in serum or in PF should increase the clinical suspicion of endometriosis in infertile patients or in patients presenting classical symptoms (dysmenorrhea, dyspareunia, or pelvic pain) supporting the need of laparoscopy, so that delays in diagnosis can be avoid. The increased levels of CA-125 that we found in PF of patients with endometriosis along with previous observations of antigen variations throughout the menstrual cycle (24) confirm that the endometrium is the main source and suggest that CA-125 levels in PF are directly influenced by endometrial modification. In serum, the increased levels might be caused by impaired peritoneum, which normally acts as a barrier allowing only a limited diffusion ofCA-125 antigen from the peritoneal cavity into the circulation. In those women with more advanced stages of endometriosis, the peritoneum is damaged at the sites of endometriotic implants, resulting in a higher release ofCA-125 antigen into the circulation. Additionally, there may be, as Barbati et al.
Peritoneal fluid CA -125 in endometriosis
441
suggested, direct shedding of cell-surface antigens into the systemic circulation as a result of endometriotic lesions (13). To date, the utility of the CA-125 test in monitoring endometriosis was documented only for serum, in PF the antigen failed to show any significant variations during disease or treatment (15). We now can affirm that in PF measurements, the values were invalided by the hook effect. Because we found a correlation between serum and PF values (r = 0.75), we speculate that the fluctuations of CA-125 in serum levels, seen when monitoring the course of the disease during and after treatment, should be detectable also in PF. This is supported by similar variations of CA -125 concentrations in serum (7) and PF (24, 25) throughout the menstrual cycle. Furthermore, while the serum test is useful in monitoring endometriosis mainly in patients with advanced disease, the CA-125 test in PF should be useful as indicator of treatment efficacy in a greater number (83%) of women with endometriosis, including those with stages I and II. For a diagnostic test, the PF can be aspirated by culdocentesis from the posterior cul-de-sac so that repeated laparoscopies are avoided. Further investigations, using an IRMA assay not invalided by the hook effect, are needed to determine whether CA-125 levels in PF are a useful marker for monitoring endometriosis.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19. REFERENCES 1. Kabawat SE, Bast RC Jr, Bhan AK, Welch WR, Knapp RC, Colvin RB. Tissue distribution of a coelomic-epithelium antigen recognized by the monoclonal antibody OC 125. Int J Gynecol Pathol 1983;2:275-85. 2. de Bruijn HWA, Jager CS, Duk JM, Aalders JG, Fleuren GJ. The tumor marker CA-125 is a common constituent of normal cervical mucus. Am J Obstet Gynecol 1986;154: 1088-91. 3. Fuith LC, Daxenbichler G, Marth C. CA-125 in human milk and serum. Gynecol Obstet Invest 1989;28:11-3. 4. Di-Xia C, Schwartz PE, Fan-qin L. Saliva and serum CA125 assays for detecting malignant ovarian tumors. Obstet GynecoI1990;75:701-4. 5. O'Brien TJ, Hardin JW, Bannon GA, Norris JS, Quirk JG. CA-125 antigen in human amniotic fluid and fetal membranes. Am J Obstet Gynecol 1986;155:50-5. 6. Moretuzzo RW, DiLauro S, Jenison E, Chen SL, Reindollar RH, McDonough PG. Serum and peritoneal lavage fluid CA-125 levels in endometriosis. Fertil Steril1988;50:430-3. 7. Pittaway DE, Fayez JA. Serum CA-125 antigen levels increase during menses. Am J Obstet GynecoI1987;156:75-6. 8. Bast RC, Khig TL, St. John E, Jenison E, Niloff JM, Lazarus H. A radioimmunoassay using monoclonal antibody to
442
Barbati et al.
Peritoneal fluid CA -125 in endometriosis
20.
21.
22.
23.
24.
25.
monitor the course of epithelial ovarian cancer. N Engl J Med 1983;309:883-9. Niloff JM, Klug PL, Schaetzl E, Zurawski VR, Knapp RC, Bast RC. Elevation of serum CA-125 in carcinomas of Fallopian tube, endometrium and endocervix. Am J Obstet GynecoI1984;148:1057-9. Barbati A, Anceschi M, Alberti P, Porn iii G, Di Renzo GC, Cosmi EV. Ontogeny of CA-125 in pregnancy: immunoradiometric determination in amniotic fluid and immunohistochemical localization in fetal membranes. Am J Obstet Gynecol 1989;160:514-8. Barbieri RL, Niloff JM, Bast RC Jr, Schaetzl E, Kistner RW, Knapp RC. Elevated serum concentrations ofCA-125 in patients with advanced endometriosis. Fertil Steril 1986;45:630-4. Hornstein MD, Thomas PP, Gleason RE, Barbieri RL. Menstrual cyclicity of CA-125 in patients with endometriosis. Fertil Steril 1992;58:279-83. Bischof P, Tseng L, Brioschi PA, Herrmann WL. Cancer antigen CA-125 is produced by human endometrial stromal cells. Hum Reprod 1986;1:423-6. Williams RS, Rao CV, Yussman MA. Interference in the measurement of CA-125 in peritoneal fluid. Fertil Steril 1988;49:547-50. Moen MH, Hagen B, Onsrud M. CA-125 in peritoneal fluid from patients with endometriosis. Hum Reprod 1991;6: 1400-3. Barbati A, Anceschi M, Di Renzo GC, Cosmi EV. CA-125 in peritoneal fluid: reliable values at high dilutions. Obstet GynecoI1992;79:1011-5. Barbati A, Di Renzo GC, Cosmi EV. Hook effect in immunoradiometric Assay of CA-125 in peritoneal fluid: evidence of high CA-125 concentrations by a comparative immunoblotting analysis. Clin Chern 1993;39:1548-9. The American Fertility Society. Revised American Fertility Society Classification of endometriosis: 1985. Fertil Steril 1985;43:351-2. O'Brien TJ, Raymond LM, Bannon GA, Ford DH, Hardardottir H, Miller FC, et al. New monoclonal antibodies identify the glycoprotein carrying the CA-125 epitope. Am J Obstet GynecoI1991;165:1857-64. Oosterlynck DJ, Meuleman C, Waer M, Vandeputte M, Koninckx PRo The natural killer activity of peritoneal fluid lymphocytes is decreased in women with endometriosis. Fertil Steril 1992;58:290-5. Confino E, Harlow L, Gleicher N. Peritoneal fluid and serum autoantibody levels in patients with endometriosis. Fertil Steril 1990;53:242-5. Pittaway DE, Fayez JA. The use ofCA-125 in the diagnosis and management of endometriosis. Fertil Steril 1986;46: 790-5. Fisk NM, Tan CE. CA-125 in peritoneal fluid and serum of patients with endometriosis. Eur J Obstet Gynecol Reprod Bioi 1988;29:166-70. Kruitwagen RFMP, Thomas C, Poels LG, Koster AM, Willemsen WNP, Rolland R. High CA-125 concentrations in peritoneal fluid of normal cyclic women with various infertility-related factors as demonstrated with two-step immunoradiometric assay. Fertil Steril 1991;56:863-9. Koninckx PR, Riittinen L, Seppala M, Cornillie FJ. CA-125 and placental protein 14 concentrations in plasma and peritoneal fluid of women with deeply infiltrating pelvic endometriosis. Fertil Steril 1992;57:523-30.
Fertility and Sterility
©
Vol. 61, No.3, March 1994
1994 The American Fertility Society
Printed on acid-free paper in U. S. A.
Serum and peritoneal fluid CA-125 levels in patients with endometriosis·
Antonella Barbati, Ph.D. t Ermelando Vinicio Cosmi, M.D. Rita Spaziani, M.D.
Rocco Ventura, M.D. Gennaro Montanino, M.D.
2nd Institute of Obstetrics and Gynecology, University of Rome, "La Sapienza," Rome, Italy
Objective: To evaluate CA-125 in peritoneal fluid (PF) as an indicator of endometriosis. Design: CA-125 levels in paired serum and PF were determined by the one-step immunoradiometric assay. For peritoneal samples, high dilution of the sample (1:100) was used to avoid false low results, caused by the "hook effect" phenomenon. Patients: Forty-one women with and without endometriosis, undergoing laparoscopy or laparotomy during the follicular phase of the menstrual cycle, were selected. Setting: 2nd Institute of Obstetrics and Gynecology, University of Rome "La Sapienza," Rome, Italy. Main Outcome Measure: Peritoneal fluid CA-125 levels obtained using diluted samples were significantly higher than those found using undiluted ones. Results: CA-125 levels in PF were approximately 100 times higher than those found in paired serum, ranging from 970 to 10,636 U /mL. In patients with endometriosis, CA-125 levels in PF were significantly elevated when compared with the control group. In serum, CA-125 levels increased only in advanced stages of endometriosis. Conclusions: The sensitivity of the CA-125 test for endometriosis in PF is greater than in serum. Therefore, the measurement of CA -125 levels in PF could be useful in the detection of early stage endometriosis, which tends to be overlooked by the CA-125 serum test. Fertil Steril 1994;61:438-42 Key Words: CA-125, peritoneal fluid, endometriosis, immunoradiometric assay, hook effect
CA-125 is a cell surface antigen that is expressed in tissues derived from embryonic celomic epithelium such as the endometrium, endocervix, fallopian tubes, peritoneum, pleura, and pericardium (1). High concentrations of CA -125 have been dem0nstrated in a variety of normal biological fluids such as cervical mucus, human milk, saliva, and amniotic and peritoneal fluids (PF) (2-6). Serum antigen levels in women are low, exhibiting a variable expression during the normal menstrual cycle with
Received June 4, 1993; revised and accepted October 28, 1993. * Supported by grant 92.02130.39 ACRO from the National Research Counc,il, Rome, Italy. t Reprint requests: Antonella Barbati, Ph.D., 2nd Institute of Obstetrics and Gynecology, University of Rome, "La Sapienza," Policlinico Umberto I, 00161 Rome, Italy. 438
Barbati et a1.
Peritoneal fluid CA-125 in endometriosis
peak serum levels during menses (7). Increased serum levels occur in ~80% of women with epithelial ovarian carcinoma (8), in a low percentage of other malignant and nonmalignant conditions (9) and in pregnancy (10). To date, the clinical use of CA-125 antigen has been limited to the detection and management of ovarian cancer. Since Barbieri et al. (11) demonstrated elevated serum concentrations of CA -125 in patients with advanced endometriosis, various investigators (6, 12) have attempted to use this antigen in the preoperative diagnosis of endometriosis. Because an endometrial origin for CA-125 has been well documented (13), the antigen has been investigated also in the PF to evaluate whether PF levels are a more sensitive indicator of disease than measurements in serum (14, 15). The values obtained were approximately 10-fold higher Fertility and Sterility
than serum levels. No differences in CA-125 levels were found between women with and without endometriosis. Most of the studies employed the same assay standardized to quantify CA-125 in serum: an immunoradiometric "one-step" assay (IRMA) (8). This test, based on the sandwich principle, uses the OC-125 monoclonal antibody (mAb) as capture (polystyrene beads on which the OC-125 are fixed) and as tracer antibody (OC-125 radioiodinated) in a simultaneous incubation, because multiple CA-125 antigenic determinant are associated with the molecule. Recently it was noted that in this assay, CA125 levels in diluted samples of PF (1:2 to 1:10) do not showed the expected decrease in binding (14, 16). The so-called "hook effect," i.e., a decreased response in samples containing a large excess of antigen, causes falsely low results. This phenomenon is usually associated with one-step sandwich assay. High levels of the antigen in PF were found when the hook effect was eliminated using the IRMA modified by the two-step assay or by high dilutions of the sample, ;;:::1:50 (17). The aims ofthe present study were as follows: [1] to determine reliable values of CA-125 in PF of women with endometriosis, using an assay not invalided by the hook effect; [2] to investigate whether PF CA-125 levels in women with endometriosis were increased when compared with controIs; and [3] to evaluate its possible role as a marker for endometriosis.
MATERIALS AND METHODS Patients and Samples Collection
Paired serum and PF were obtained from women undergoing laparotomy or diagnostic laparoscopy for infertility and/or pelvic pain: 18 women had untreated endometriosis, (aged 23 to 41 years); 20 had benign gynecological disease (aged 16 to 55 years) including pelvic adhesions, pelvic inflammatory disease (PID), leiomyoma, and ovarian cysts; 7 women, who at laparoscopy presented a normal pelvis without signs of present or previous inflammation, served as control group (aged 26 to 39 years). The endometriosis group, staged according to the revised American Fertility Society classification, included patients with stages I and II (n = 12) and patients with stages III and IV (n = 6) (18). In this study, only women undergoing laparotomy or laparoscopy during the midfollicular phase (cycle days 8 to 12), were included to eliminate the Vol. 61, No.3, March 1994
variations of CA-125 concentration due to menstrual cycle. The day of the cycle was determined by patient menstrual history. None ofthe women received any hormonal treatment during the 3 months preceding the intervention. Blood sample was obtained by venipuncture from all patients immediately before induction of anesthesia. Peritoneal fluid was aspirated from the pelvis at the beginning of the laparoscopic examination or laparotomy. Fluids transferred to the laboratory were centrifuged (1000 X g for 10 minutes) and the supernatants stored at -20°C until assayed. In six patients the paired PFs were not available for the assay. CA-125 Assay
CA-125 levels in serum and in PF were measured in duplicate by an immunoradiometric "one-step sandwich" assay (IRMA CA-125 II K; Sorin Biomedica, Saluggia, Italy), used according the manufacturer's instructions. It is a second-generation assay utilizing polystyrene beads coated with a new capture mAb, Mll (19), which reacts with an epitope of the CA-125 antigen different from those recognized by the mAb OC-125. The tracer, composed of radioiodinated OC-125, quantifies the CA-125 antigenic determinants. The standard points ranged from 15 to 500 U /mL. The minimal detectable concentration is 1.4 U /mL. To evaluate the validity of CA-125 measurements in PF, using the new kit assay, we assayed a pool (n = 5) of PF and a pool of serum samples at dilutions 1:2, 1:5, 1:10, 1:50, 1:100, and 1:200 with saline solution. All PF samples were reassayed at dilution 1:100 to obtain valid results. The intra-assay and interassay coefficients of variation were 7.5% and 8.7%, respectively. Statistical analysis was performed by using Student's t-test and linear-regression analysis. RESULTS
CA-125 values obtained for serum (range: 7.5 to 181 U /mL) and PF (range: 970 to 10,636 U /mL) are shown in Figure 1. They represent the mean of duplicate measurements of each sample. The concentration of CA-125 in PF was significantly greater than the corresponding serum in all subjects (t-test, P < 0.0001). Serum CA-125
When women with endometriosis were combined to constitute two groups, one with stages I and II Barbati et al.
Peritoneal fluid CA-125 in endometriosis
439
Table 2 Reliability of Serum and Peritoneal Fluid CA-125 Levels for the Diagnosis of Endometriosis Parameter
Serum*
Peritoneal fiuidt
Sensitivity Specificity Positive predictive value Negative predictive value
0.44 0.88 0.72 0.70
0.83 0.64 0.57 0.88
* CA-125 >35 U/mL. t CA-125 >2,500 U/mL.
increased levels (25%). In patients with benign gynecological disease, moderate increases of CA-125 levels were observed in 3 of 20 patients (15%). As a cut-off, we considered the value of 35 U jmL, which is usually accepted by various authors (6, 12). Statistical parameters are reported in Table 2. Peritoneal Fluid CA-125 Levels
Undiluted PF
and one with stages III and IV (18), the patients with advanced endometriosis showed serum CA125 levels significantly higher than those found in the other groups (Table 1). CA-125 levels were elevated in 5 of 6 patients (83 %). Patients with stage I and II endometriosis showed serum CA-125 levels similar to the controls. Only 3 of 12 patients had
In undiluted PF, the CA-125 levels ranged between 41 and 207 UjmL. The mean ± SD for endometriosis, control, and benign gynecological disease groups were 132 ± 57, 118 ± 42.9,122 ± 54.6 UjmL, respectively. No difference was found between the mean values reported for each group. Although the CA-125 values of undiluted sample were within the linearity range of the assay, the samples assayed at dilutions 1:2 to 1:200 did not show any dilutional effect to 1:10 so that the values obtained can be considered invalid. Only at dilutions oLd:50, parallelism with the serum dilutional curve was observed, and the results can be considered valid. These results demonstrate that, also with the new assay using the Mll monoclonal antibody, CA-125 determination in PF exhibited the hook effect. Diluted PF
Table 1
CA·125 Levels in Serum and PF Samples*
Group
Serum CA-125
PF CA-125
U/mL
Control Endometriosis Stage I and II Stage III and IV Ben Gyn Disease
15.0 41.9 20.5 77.9 17.4
± ± ± ± ±
4.2 42.2 15.2 55.3+ 12.4
U/mL
1695 5971 5537 6752 3726
± ± ± ± ±
439 2,695t 2,590t 3,000t 2,807
* Values are means ± SD. t P < 0.05 compared with the control group. P < 0.01 compared with the control group.
+ 440
Barbati et al.
Peritoneal fluid CA -125 in endometriosis
CA-125 levels obtained using high dilutions (1:100) of the samples were significantly higher than values obtained from undiluted ones. The values ranged from 970 to 10,636 U jmL. The mean value ± SD of each group are reported in Table 1. The PF levels of CA-125 in women with endometriosis were significantly higher than those found in control and benign gynecological disease groups. Unlike CA-125 serum levels in patients with stages I and II endometriosis, PF levels in this group were significantly different from controls. Seven of 9 patients (77%) displayed increased CA-125 concenFertility and Sterility
trations. All PFs from patients with stages III and IV endometriosis had increased levels ofCA-125. In patients with benign gynecological disease, CA-125 levels were increased in 9 of 18 patients (50%). The highest (CA-125 = 10,636 U/mL) value was observed in a patient with PID. Amounts of CA -125 in PF appear to be a more sensitive indicator of endometriosis than serum concentrations of the antigen, although its specificity was lower. In fact, in benign gynecological diseases group, the CA -125 levels in PF were elevated in 50% of patients, whereas in their respective serum, moderate increased values were detectable only in 15% of cases. Sensitivity, specificity and other statistical parameters (Table 2) were calculated for endometriosis using the cut-off of 2,500 U /mL as previously determined (16). Regression analysis performed for CA -125 values in diluted PF and paired serum samples showed a significant correlation (r = 0.75, P = 0.01; n = 39). DISCUSSION
The diagnosis of endometriosis is usually established by laparoscopy or laparotomy with direct visualization of implants and preferably confirmed by histological examination of biopsies. Noninvasive diagnostic methods for evaluation of treatment and for the detection of recurrence are currently being sought (20, 21). Using CA-125 as a marker for endometriosis, we found, as did others (6, 11, 12), that significantly elevated levels of CA-125 in serum become evident only in advanced stages of the disease (stages III and IV [18]). In stages I and II endometriosis the serum levels of CA-125 are low, precluding its measurement as a diagnostic test of disease. Nevertheless, the serum test seems to be useful in monitoring patients during and after medical or surgical therapy, because a significant correlation between CA-125 levels and the clinical course of the disease was observed (22). With regard to CA-125 levels in PF, the previous determinations (14, 15) failed to show any difference between women with and without endometriosis. One study (23) gave evidence of increased amounts of PF CA-125 in endometriosis, but only because of the higher volume of the fluid in these patients. The authors considered these results valid, because they were not awared of the hook effect associated with CA-125 determination in PF using the simultaneous "one-step" IRMA assay. The generally accepted cause of this phenomenon is an excess of antigen, which progressively saturates Vol. 61, No.3, March 1994
both the solid-phase and the detection antibodies, thus preventing formation of the sandwich. Nevertheless, because this phenomenon was not operative in amniotic fluid containing similar high levels of CA-125 (10), others causes as the presence of different CA-125 molecular forms in PF may be implicated. Recent improvements to this assay have been the design of a two-step assay procedure and the usage of higher sample dilutions. Using the first modification, Kruitwagen et al. (24) recently found that CA-125 levels in PF, throughout the normal menstrual cycle, ranged from 630 to 12,000 U /mL, with values significantly higher during the early follicular phase compared with the luteal phase. Using high sample dilutions, we found (16) significant differences in CA-125 levels between women with ovarian and endometrial cancer and women of the control group (mean values: 9,184 versus 2,278 U/mL, respectively). We now have been able to demonstrate a significant increase in CA-125 levels in the PF of women with endometriosis compared with women of the control group. Levels of CA-125 in PF were a more sensitive indicator of disease than the serum test (0.83 versus 0.44), especially for minimal and mild endometriosis (0.77 versus 0.25). In fact, most of the patients with stages I and II endometriosis, who did not show increased CA-125 levels in serum, had high levels in PF (CA-125 > 2,500 U/mL). Employing measurement of this antigen for the diagnosis of endometriosis is, however, limited because of its low specificity. At least, detection of elevated levels of CA-125 in serum or in PF should increase the clinical suspicion of endometriosis in infertile patients or in patients presenting classical symptoms (dysmenorrhea, dyspareunia, or pelvic pain) supporting the need of laparoscopy, so that delays in diagnosis can be avoid. The increased levels of CA-125 that we found in PF of patients with endometriosis along with previous observations of antigen variations throughout the menstrual cycle (24) confirm that the endometrium is the main source and suggest that CA-125 levels in PF are directly influenced by endometrial modification. In serum, the increased levels might be caused by impaired peritoneum, which normally acts as a barrier allowing only a limited diffusion ofCA-125 antigen from the peritoneal cavity into the circulation. In those women with more advanced stages of endometriosis, the peritoneum is damaged at the sites of endometriotic implants, resulting in a higher release ofCA-125 antigen into the circulation. Additionally, there may be, as Barbati et al.
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suggested, direct shedding of cell-surface antigens into the systemic circulation as a result of endometriotic lesions (13). To date, the utility of the CA-125 test in monitoring endometriosis was documented only for serum, in PF the antigen failed to show any significant variations during disease or treatment (15). We now can affirm that in PF measurements, the values were invalided by the hook effect. Because we found a correlation between serum and PF values (r = 0.75), we speculate that the fluctuations of CA-125 in serum levels, seen when monitoring the course of the disease during and after treatment, should be detectable also in PF. This is supported by similar variations of CA -125 concentrations in serum (7) and PF (24, 25) throughout the menstrual cycle. Furthermore, while the serum test is useful in monitoring endometriosis mainly in patients with advanced disease, the CA-125 test in PF should be useful as indicator of treatment efficacy in a greater number (83%) of women with endometriosis, including those with stages I and II. For a diagnostic test, the PF can be aspirated by culdocentesis from the posterior cul-de-sac so that repeated laparoscopies are avoided. Further investigations, using an IRMA assay not invalided by the hook effect, are needed to determine whether CA-125 levels in PF are a useful marker for monitoring endometriosis.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19. REFERENCES 1. Kabawat SE, Bast RC Jr, Bhan AK, Welch WR, Knapp RC, Colvin RB. Tissue distribution of a coelomic-epithelium antigen recognized by the monoclonal antibody OC 125. Int J Gynecol Pathol 1983;2:275-85. 2. de Bruijn HWA, Jager CS, Duk JM, Aalders JG, Fleuren GJ. The tumor marker CA-125 is a common constituent of normal cervical mucus. Am J Obstet Gynecol 1986;154: 1088-91. 3. Fuith LC, Daxenbichler G, Marth C. CA-125 in human milk and serum. Gynecol Obstet Invest 1989;28:11-3. 4. Di-Xia C, Schwartz PE, Fan-qin L. Saliva and serum CA125 assays for detecting malignant ovarian tumors. Obstet GynecoI1990;75:701-4. 5. O'Brien TJ, Hardin JW, Bannon GA, Norris JS, Quirk JG. CA-125 antigen in human amniotic fluid and fetal membranes. Am J Obstet Gynecol 1986;155:50-5. 6. Moretuzzo RW, DiLauro S, Jenison E, Chen SL, Reindollar RH, McDonough PG. Serum and peritoneal lavage fluid CA-125 levels in endometriosis. Fertil Steril1988;50:430-3. 7. Pittaway DE, Fayez JA. Serum CA-125 antigen levels increase during menses. Am J Obstet GynecoI1987;156:75-6. 8. Bast RC, Khig TL, St. John E, Jenison E, Niloff JM, Lazarus H. A radioimmunoassay using monoclonal antibody to
442
Barbati et al.
Peritoneal fluid CA -125 in endometriosis
20.
21.
22.
23.
24.
25.
monitor the course of epithelial ovarian cancer. N Engl J Med 1983;309:883-9. Niloff JM, Klug PL, Schaetzl E, Zurawski VR, Knapp RC, Bast RC. Elevation of serum CA-125 in carcinomas of Fallopian tube, endometrium and endocervix. Am J Obstet GynecoI1984;148:1057-9. Barbati A, Anceschi M, Alberti P, Porn iii G, Di Renzo GC, Cosmi EV. Ontogeny of CA-125 in pregnancy: immunoradiometric determination in amniotic fluid and immunohistochemical localization in fetal membranes. Am J Obstet Gynecol 1989;160:514-8. Barbieri RL, Niloff JM, Bast RC Jr, Schaetzl E, Kistner RW, Knapp RC. Elevated serum concentrations ofCA-125 in patients with advanced endometriosis. Fertil Steril 1986;45:630-4. Hornstein MD, Thomas PP, Gleason RE, Barbieri RL. Menstrual cyclicity of CA-125 in patients with endometriosis. Fertil Steril 1992;58:279-83. Bischof P, Tseng L, Brioschi PA, Herrmann WL. Cancer antigen CA-125 is produced by human endometrial stromal cells. Hum Reprod 1986;1:423-6. Williams RS, Rao CV, Yussman MA. Interference in the measurement of CA-125 in peritoneal fluid. Fertil Steril 1988;49:547-50. Moen MH, Hagen B, Onsrud M. CA-125 in peritoneal fluid from patients with endometriosis. Hum Reprod 1991;6: 1400-3. Barbati A, Anceschi M, Di Renzo GC, Cosmi EV. CA-125 in peritoneal fluid: reliable values at high dilutions. Obstet GynecoI1992;79:1011-5. Barbati A, Di Renzo GC, Cosmi EV. Hook effect in immunoradiometric Assay of CA-125 in peritoneal fluid: evidence of high CA-125 concentrations by a comparative immunoblotting analysis. Clin Chern 1993;39:1548-9. The American Fertility Society. Revised American Fertility Society Classification of endometriosis: 1985. Fertil Steril 1985;43:351-2. O'Brien TJ, Raymond LM, Bannon GA, Ford DH, Hardardottir H, Miller FC, et al. New monoclonal antibodies identify the glycoprotein carrying the CA-125 epitope. Am J Obstet GynecoI1991;165:1857-64. Oosterlynck DJ, Meuleman C, Waer M, Vandeputte M, Koninckx PRo The natural killer activity of peritoneal fluid lymphocytes is decreased in women with endometriosis. Fertil Steril 1992;58:290-5. Confino E, Harlow L, Gleicher N. Peritoneal fluid and serum autoantibody levels in patients with endometriosis. Fertil Steril 1990;53:242-5. Pittaway DE, Fayez JA. The use ofCA-125 in the diagnosis and management of endometriosis. Fertil Steril 1986;46: 790-5. Fisk NM, Tan CE. CA-125 in peritoneal fluid and serum of patients with endometriosis. Eur J Obstet Gynecol Reprod Bioi 1988;29:166-70. Kruitwagen RFMP, Thomas C, Poels LG, Koster AM, Willemsen WNP, Rolland R. High CA-125 concentrations in peritoneal fluid of normal cyclic women with various infertility-related factors as demonstrated with two-step immunoradiometric assay. Fertil Steril 1991;56:863-9. Koninckx PR, Riittinen L, Seppala M, Cornillie FJ. CA-125 and placental protein 14 concentrations in plasma and peritoneal fluid of women with deeply infiltrating pelvic endometriosis. Fertil Steril 1992;57:523-30.
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