Measurement of serum CA-125 concentrations does not improve the ...

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A correlation between serum CA-125 concentrations and the severity of adnexal ... Key words: CA-125/Chlamydia trachomatis antibody/laparoscopy/tubal ...
Human Reproduction Vol.16, No.4 pp. 775–779, 2001

Measurement of serum CA-125 concentrations does not improve the value of Chlamydia trachomatis antibody in predicting tubal pathology at laparoscopy Ernest Hung Yu Ng1, Oi Shan Tang and Pak Chung Ho Department of Obstetrics and Gynaecology, Queen Mary Hospital, The University of Hong Kong, Pokfulam, Hong Kong Special Administrative Region, People’s Republic of China 1To

whom correspondence should be addressed at Department of Obstetrics and Gynaecology, 6/F, Professorial Block, Queen Mary Hospital, Pokfulam Road, Hong Kong. E-mail: [email protected]

Chlamydia antibody testing (CAT) has been used to predict tubal pathology associated with Chlamydia infection, the leading cause of pelvic inflammatory disease (PID). Tubal pathology not related to C. trachomatis is unlikely to be identified by CAT alone. A correlation between serum CA-125 concentrations and the severity of adnexal inflammation during acute PID was demonstrated. The objectives of this study were to determine the prevalence of C. trachomatis infection in an Asian infertile population and to assess the role of a combination of serum CA-125 and CAT in the prediction of tubal pathology as shown by laparoscopy. A total of 110 consecutive women attending an infertility clinic for work-up were recruited. Blood was taken for CAT and CA-125 on the day of hospital admission and an endocervical swab was taken for culture of C. trachomatis prior to laparoscopy. Two (1.8%) women had C. trachomatis found in the endocervix and 28 (25.5%) women had CAT of ≥1:32. Serum CA-125 concentrations were >35 IU/ml in 11 (10%) women. The discriminative capacity of CAT in the diagnosis of tubal pathology including both proximal and distal obstruction was not improved by measuring serum CA-125, regardless of the threshold values of serum CA-125 concentration. Key words: CA-125/Chlamydia trachomatis antibody/laparoscopy/tubal pathology

Introduction Tubal pathology accounts for 14–36% of female infertility (Cates et al., 1985; Hull et al., 1985). Fallopian tubes can be damaged by different causes, resulting in chronic pelvic pain, tubal infertility and an increased risk of ectopic pregnancy. Diagnostic laparoscopy with dye test (L and D) remains the gold standard for the accurate assessment of tubal patency (Royal College of Obstetricians and Gynaecologists, 1998). L and D also allows pelvic adhesion and endometriosis to be detected. However, it is usually performed under general anaesthesia and can be associated with risks of bleeding and injury to internal organs such as bowel. Chlamydia trachomatis is the major microbiological agent causing pelvic inflammatory disease (PID) (Mårdh, 1980; Moore et al., 1982; Stacey et al., 1992). Chlamydia antibody testing (CAT) has been used for the prediction of tubal pathology associated with Chlamydia infection. A recent metaanalysis (Mol et al., 1997) of 23 studies involving 2729 infertile women who had had both CAT and L and D suggested that the discriminative capacity of CAT was comparable to that of hysterosalpingography in the diagnosis of tubal occlusion. Tubal pathology not related to C. trachomatis is unlikely to be identified by CAT alone. © European Society of Human Reproduction and Embryology

CA-125 is a glycoprotein identified by monoclonal antibody OC-125 (Bast et al., 1981) and has been demonstrated in the epithelium of endometrium and Fallopian tubes and in the mesothelial cells of the peritoneum. Serum CA-125 has been extensively studied in the diagnosis and monitoring of epithelial ovarian carcinoma. The concentrations were also elevated in patients with endometriosis (Koninckx et al., 1992) and PID. The proportion of acute PID with elevated CA-125 concentrations ranged from 32–66% (Halila et al., 1986; Duk et al., 1989; Paavonen et al., 1989; Mozas et al., 1994). A correlation between serum CA-125 concentrations and the severity of adnexal inflammation was also demonstrated during laparoscopy (Paavonen et al., 1989). Little information exists in the literature with regard to the prevalence of C. trachomatis infection in infertile women and the role of CAT in the prediction of tubal pathology in Asian countries. The first objective of this study was to determine the prevalence of C. trachomatis infection in an Asian infertile population. The second objective was to assess the role of CAT and a combination of serum CA-125 and CAT in the prediction of tubal pathology as shown by laparoscopy. The discriminative capacity of combining CAT and serum CA-125 in the diagnosis of tubal pathology has not been studied. The 775

E.H.Y.Ng, O.S.Tang and P.C.Ho

Table I. Tubal pathology at laparoscopy in women with positive and negative Chlamydia antibody testing (CAT) CAT

Positive Negative Total

Tubal pathology Present

Absent

Total

17 14 31

11 68 79

28 82 110

P ⬍ 0.001 (χ2 test). Sensitivity ⫽ 54.8%; specificity ⫽ 86.1%; LR(⫹) ⫽ 3.94; LR(–) ⫽ 0.53; OR ⫽ 7.51 (2.90–19.45); LR(⫹) ⫽ likelihood ratio of a positive test result; LR(–) ⫽ likelihood ratio of a negative test result; OR ⫽ odds ratio.

Table II. Tubal pathology at laparoscopy in women with abnormal and normal combination tests (CAT⫹CA-125) CAT ⫹ CA-125 (⬎35 IU/l) Tubal pathology

Abnormal Normal Total

Present

Absent

Total

19 12 31

19 60 79

38 72 110

P ⬍ 0.001 (χ2 test). Sensitivity ⫽ 61.3%; specificity ⫽ 75.9%; LR(⫹) ⫽ 2.54; LR(–) ⫽ 0.51; OR ⫽ 4.99 (2.06–12.15).

hypothesis was that the discriminative capacity of CAT in the diagnosis of tubal pathology would be improved by measuring serum CA-125 at the same setting. Materials and methods Women were recruited from those attending the infertility clinic at the Department of Obstetrics and Gynaecology, The University of Hong Kong. They were usually referred by gynaecologists from our own hospital or other local hospitals, Family Planning Association and the private sector. For the infertility work-up, they underwent a standard protocol of history taking, physical examination and investigations including conventional semen analysis on at least on two occasions and hormonal assessment of the women for mid-luteal progesterone concentrations and early follicular FSH concentrations on day 2 of the cycle. Only women who were advised to undergo L and D with or without hysteroscopy to assess tubal patency were approached. Exclusion criteria included: (i) history of any pelvic surgery other than L and D and (ii) need for IVF/embryo transfer treatment because of severe male factors. Every patient was extensively counselled and gave an informed consent prior to participating in the study, which was approved by the Ethics Committee, Faculty of Medicine, The University of Hong Kong. Blood was taken for CAT and CA-125 on the day of admission into hospital. After the patient was put under general anaesthesia, an endocervical swab was taken for culture of C. trachomatis. L and D was performed as day surgery in the usual manner and tubal patency was confirmed with free spillage of Methylene blue dye from the fimbrial end. Surgeons were not aware of the results of CAT and serum CA-125 at the time of operation. Sera were processed blindly and without knowledge of the clinical data of the women. Serum CA-125 was measured using a commercially available

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immunoassay kit (Chiron Diagnostics Corporation, East Walpole, MA, USA) and the threshold value for elevated concentrations was ⬎35 IU/ml. CAT was detected using a micro-immunofluorescence test (MRI Diagnostics, Cypress, CA, USA) according to the manufacturer’s instructions. Sera were screened at 1:32 (Land et al., 1998) and positive specimens were titrated to the end point. In brief, serum diluted in phosphate buffered saline (PBS) was incubated in a moist chamber for 30 min at 37°C on wells containing micro-dots of the elementary bodies of C. trachomatis, C. psittaci, C. pneumonia and control antigen. The wells were washed, anti-human immunoglobulin (Ig) G conjugate added, and the slides re-incubated as before. Finally, the slides were washed free of unbound conjugate, mounted in glycerol-PBS under a cover-slip and examined for fluorescing elementary bodies in a fluorescent microscope. Each run contained positive and negative controls. Tubal pathology could be either distal or proximal obstruction. Distal obstruction was diagnosed when there was mild to severe peritubal/adnexal adhesion scored according to The American Fertility Society classification (1988) or no spillage of dye from the fimbrial end with or without hydrosalpinx. Proximal obstruction was considered only when there was no filling of the tube and absence of hydrosalpinx and peritubal/adnexal adhesion. Pathology even on one side was considered to be abnormal. Endometriosis, if present, was graded according to revised American Society for Reproductive Medicine classification of endometriosis (1997) into four stages: minimal, mild, moderate and severe. Statistical analysis The sensitivity, specificity, likelihood ratios and odds ratio for CAT alone and the combination were determined. The likelihood ratio of a positive test result (LR⫹) indicated the likelihood of a positive test in a patient with the disease over the likelihood of a positive test in a patient without the disease. The likelihood ratio of a negative test result (LR–) indicated the likelihood of a negative test in a patient with the disease over the likelihood of a negative test in a patient without the disease. The LR⫹ was calculated as sensitivity/ (1 – specificity) and the LR– was calculated as (1 – sensitivity)/ specificity. An LR⫹ between two and five indicates a fair test, between 5–10 is good and ⬎10 is excellent. A LR– between 0.5 and 0.2 indicates a fair test, between 0.2 and 0.1 is good and ⬍0.1 is excellent. The odds ratio (OR) is given by LR⫹/LR– and reflects the probability of a patient with an abnormal test having the diseased state. Statistical comparison was carried out by χ2 test and Fisher’s exact test, where appropriate. P value (two-tailed) of ⬍ 0.05 was taken as significant.

Results A total of 110 consecutive women aged 22–43 years (median 33.0 years) were recruited and 28 (25.5%) women had secondary infertility. The median duration of infertility was 3 years (range: 1–12 years). Two (1.8%) women were found to have C. trachomatis present in the endocervix: one with positive CAT and another with negative CAT. CAT was positive in 28 (25.5%) women (1:32 titre in 15; 1:128 titre in nine and 1:512 titre in four). Eleven (10%) women had serum CA-125 concentrations ⬎35 IU/l and only one woman had both serum CA-125 concentration ⬎35 IU/l and positive CAT. Tubal pathology was found in 31 (28.2%) women. Of the 28 women with positive CAT, 17 had tubal pathology whereas 14 of 82 women with negative CAT had tubal pathology. The difference was statistically significant (P ⬍ 0.001, χ2 test).

Serum CA-125 and C. trachomatis antibody in tubal pathology prediction

Table III. Use of CAT alone and the combination test with different serum CA-125 threshold values in the prediction of tubal pathology, distal obstruction and proximal obstruction at laparoscopy Sensitivity (%)

Specificity (%)

LR(⫹)

LR(–)

OR (95% CI)

Tubal pathology CAT

54.8

86.1

3.94

0.53

CAT ⫹ CA-125 (⬎35 IU/l)

61.3

75.9

2.54

0.51

CAT ⫹ CA-125 (⬎25 IU/l)

74.2

68.4

2.35

0.38

7.51 (2.90–19.45) 4.99 (2.06–12.15) 6.18 (2.44–15.80)

Distal obstruction CAT

71.4

85.4

4.89

0.33

CAT ⫹ CA-125

76.2

75.3

3.09

0.32

CAT ⫹ CA-125 (⬎25 IU/l)

85.7

66.3

2.54

0.22

Proximal obstruction CAT

20.0

74.0

0.77

1.08

CAT ⫹ CA-125 (⬎35 IU/l)

30.0

65.0

0.86

0.92

CAT ⫹ CA-125 (⬎25 IU/l)

50.0

57.0

1.16

0.88

The LR(⫹), LR(–) and OR of CAT in prediction of tubal pathology were 3.94, 0.53 and 7.51 respectively (Table I). In 38 women with abnormal combination tests (CAT ⫹ CA-125, ⬎35 IU/l), 19 had tubal pathology shown. Out of 72 women with normal tests, 12 had tubal pathology. The difference was also statistically significant (P ⬍ 0.001, χ2 test). The LR(⫹), LR(–) and OR of the combination test in prediction of tubal pathology were 2.54, 0.51 and 4.99 respectively (Table II). Twenty-one women had distal blockage (eight unilateral; 13 bilateral) and co-existing proximal obstruction was present in four of them. Proximal obstruction only was found in another 10 women (six unilateral; four bilateral). The sensitivity, specificity, LR(⫹), LR(–) and OR of CAT alone and the combination test in predicting tubal pathology, distal obstruction and proximal obstruction are summarized in Table III. Different threshold values for elevated serum CA-125 concentration (⬎35 IU/l and ⬎25 IU/l) were evaluated. Endometriosis was noted in 43 women (27 stage I; four stage II; six stage III; six stage IV). Of 11 women with serum CA-125 concentrations ⬎35 IU/l, eight had endometriosis whereas 35 of 99 women with normal CA-125 concentrations were also found to have endometriosis. The difference was statistically significant (P ⬍ 0.023, Fisher’s exact test). The LR(⫹), LR(–) and OR of serum CA-125 concentration in predicting endometriosis were 4.13, 0.85 and 4.85 respectively (Table IV). Discussion PID is by far the most important cause of tubal damage. Westman (1950) found that tuberculosis, gonorrhoea, infection resulting from abortion and childbirth, and infections of unknown origin occurred with equal frequency in women with PID (Westman, 1950). C. trachomatis appears to be the major microbiological agent identified in more recent studies (Mårdh,

14.6 (4.79–44.55) 9.76 (3.20–29.68) 11.54 (3.22–43.25) 0.71 (0.14–3.57) 0.93 (0.19–3.27) 1.32 (0.36–4.87)

Table IV. Endometriosis at laparoscopy in women with abnormal and normal serum CA-125 concentrations CA-125 (⬎35 IU/l)

Abnormal Normal Total

Endometriosis Present

Absent

Total

8 35 43

3 64 67

11 99 110

P ⬍ 0.023 (Fisher’s exact test). Sensitivity ⫽ 18.6%; specificity ⫽ 95.5%; LR(⫹) ⫽ 4.13; LR(–) ⫽ 0.85; OR ⫽ 4.85 (1.22–19.57).

1980; Moore et al., 1982; Stacey et al., 1992). Current Chlamydia infections can be detected by culturing or staining endocervical swabs whereas past infections can be revealed by the serology test. In this study, only two (1.8%) out of 110 infertile women were found to have C. trachomatis in the endocervix prior to laparosocpy. The prevalence of current Chlamydia infection in our infertile population is quite similar to other reports (ranging from 0.8–1.0%) in Western countries using the culture technique (Åestad et al., 1987; Eggert-Kruse et al., 1997). The low prevalence in the infertile population can be partly explained by the older age of these women (Macmillan and Templeton, 1999). Another reason for the low prevalence may be a more stable relationship in the infertile population. As the majority of C. trachomatis infections remain clinically silent but result in severe tubal damage (Bevan et al., 1995), CAT has been used as a screening test for the tubal pathology prior to L and D as it is simple, inexpensive and non-invasive. Despite the low prevalence of current Chlamydia infections in our population, 28 (25.5%) women had Chlamydia antibody titre of 艌1:32. Twenty to 44% of infertile women in Western 777

E.H.Y.Ng, O.S.Tang and P.C.Ho

countries were shown to have elevated Chlamydia antibody titres detected by the micro-immunofluorescence test (Dabekausen et al., 1994; Eggert-Kruse et al., 1997; Land et al., 1998). The prevalence of positive CAT depends on the techniques used. Using an enzyme-linked immunosorbent assay, 51 (39%) of 131 Japanese women attending an infertility clinic were positive for Chlamydia antibodies (Tanikawa et al., 1996). Seven (14.2%) of 48 infertile Indian women had elevated Chlamydia antibody detected by indirect immunoperoxidase assay (Chaudhry et al., 1997). The sensitivity and specificity of CAT in the prediction of tubal pathology in our population were 54.8 and 86.1% respectively. These were within the range in a meta-analysis (Mol et al., 1997), which showed that the sensitivity and specificity of CAT varied between 21–90% and between 29– 100% respectively. As shown in Table III, CAT predicts distal obstruction much better than proximal obstruction. This finding agrees with the results of other studies (Mol et al., 1997; Land et al., 1998). The sensitivity and specificity of CAT for distal obstruction were 71.4 and 85.4% respectively while the corresponding values for proximal obstruction were only 20.0 and 74.0% respectively. The proximal tubal obstruction may be more likely due to non-Chlamydia origin. Tubal pathology not related to C. trachomatis is unlikely to be identified by CAT. Other organisms including Mycoplasma hominis, Ureaplasma urealyticum and group A streptococci may also be implicated in PID. Acute salpingitis can also occur as a result of adjacent inflammatory processes such as appendicitis. Moderate to severe endometriosis characterized by dense adhesion in the pelvic cavity may lead to distortion of tubo-ovarian relationship and tubal obstruction. Endometriosis was shown to be present in 72.2% of patients with proximal tubal occlusion diagnosed by selective salpingography and laparoscopic methylene dye studies (Woolcott et al., 1999). In the present study, 1/10 (10%) of patients with proximal tubal occlusion diagnosed by laparoscopic dye studies alone had visible endometriosis. Serum CA-125 is an important marker in the diagnosis and monitoring of epithelial ovarian carcinoma. Elevated concentrations were also found in some benign gynaecological conditions including endometriosis (Koninckx et al., 1992) and acute PID (Halila et al., 1986; Duk et al., 1989; Paavonen et al., 1989; Mozas et al., 1994). This study attempted to assess the role of serum CA-125 concentration in the prediction of tubal pathology, when combined with CAT. Serum CA-125 concentrations were elevated in only three patients with tubal pathology at laparoscopy (two with distal obstruction and one with proximal obstruction). The sensitivity of the combination test (CAT and CA-125) in the prediction of tubal pathology was better than that of CAT alone but the LR(⫹), LR(–) and OR of CAT were not improved by measuring serum CA-125 concentration (Table III). The same trend was also shown in the prediction of distal and proximal obstruction at laparoscopy. The discriminative capacity of CAT was not improved by lowering the cut-off value of serum CA-125 from 35 IU/l to 25 IU/l (Table III). Proliferative mesothelial cells of the peritoneum showed intense staining with OC-125 whereas normal peritoneal cells 778

had negative staining. It was assumed that a rapid drainage of even small amounts of peritoneal fluid containing CA-125 by the lymphatic capillaries could cause a significant increase in the serum CA-125 concentrations (Feldman and Knapp, 1974; Fleuren et al., 1987). Tubal pathology resulting from chronic PID may be associated with minimal or localized inflammatory responses of the basement membrane of the epithelial lining of the Fallopian tubes and the surrounding peritoneum. This could well explain the observation that in this study only a small number (n ⫽ 3) of women with tubal pathology had elevated serum CA-125 concentrations found as well. The sensitivity and specificity of serum CA-125 for the diagnosis of endometriosis, were 18.6 and 95.5% respectively (Table IV). The high specificity of serum CA-125 suggests that in patients with elevated CA-125 concentrations a laparoscopy should be arranged early in the infertility work-up, even in the absence of clinical signs of endometriosis. It was suggested that a higher specificity and sensitivity could be achieved for the diagnosis of deeply infiltrating endometriosis because these secreted CA-125 preferentially toward the bloodstream (Koninckx et al., 1992). In conclusion, two (1.8%) of 110 infertile women had C. trachomatis found in the endocervix prior to diagnostic laparoscopy for tubal assessment and 28 (25.5%) women had Chlamydia antibody titres of 艌1:32 detected by a microimmunofluorescence test. The prevalence of current and past C. trachomatis infection was similar to other Western countries. Serum CA-125 concentrations were ⬎35 IU/l in 11 (10%) women. The discriminative capacity of Chlamydia antibody testing in the diagnosis of tubal pathology including both proximal and distal obstruction was not improved by measuring serum CA-125, regardless of the threshold values of serum CA-125 concentration.

Acknowledgements This work was supported in part by a grant from The Hong Kong Obstetrical and Gynaecological Trust Fund.

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