Endocervical chlamydial deoxyribonucleic acid in infertile women*

FERTILITY AND STERILITY

Vol. 54, No.5, November 1990

Printed on acid-free paper in U.S.A.

Copyright© 1990 The American Fertility Society

Endocervical chlamydial deoxyribonucleic acid in infertile women*

Yung-Kuei Soong, M.D.t Shu-Min Kao, B.S.:j: Chang-Jen Lee, M.D.t

Pei-Shun Lee, R.N.t Chia C. Pao, Ph.D.:j:§

Chang Gung Memorial Hospital and Medical College, Taipei, Taiwan, Republic of China

The presence of chlamydia! deoxyribonucleic acid (DNA) was evaluated by DNA hybridization in endocervical cells of infertile and normal fertile women. Chlamydia! DNA was detected in 49 of 186 (26.3%) infertile patients, which is significantly more common than in fertile control individuals (12.5%, or 8 of 64 individuals). Among infertile patients, 49.3% (33 of 67) of those with tubal factors as cause of infertility and 13.4% (16 of 119) of those with nontubal factors were found to contain chlamydia! DNA in their endocervical cells. The results show that chlamydia! DNA could be found significantly more frequently in endocervical cells of infertile patients with tubal factor than those without tubal factors or in normal controls. Fertil Steril 54:815, 1990

Chlamydia trachomatis infection is the most prevalent sexually transmitted disease and has been shown to be associated with a number of diseases-urethritis, cervicitis, conjunctivitis, salpingitis, Reiter's, and Fitz-Hugh-Curtis syndromes. 1- 3 One of the most important sequelae of chlamydia! infection in women is pelvic inflammatory disease (PID). A number of studies have suggested that damage to the fallopian tubes resulting from chlamydia! infection can lead to ectopic pregnancies (EPs) or tubal infertility. 4- 8 Chlamydia! infection can be diagnosed by immunological, cytologic, or cell culture methods. 1•2 All of these methods have their advantages as well as their limitations. We have shown that deoxyribo-

Received November 1, 1989; revised and accepted July 24, 1990. *Supported by grant CMRP-145 from The Chang Gung Memorial Hospital (Y.-K.S.) and by grants CMRP-235 and -286 from Chang Gung Medical College, Taipei, Taiwan, Republic of China (C.C.P.). t Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital. :j: Department of Biochemistry, Chang Gung Medical College. §'Reprint requests: Professor Chia C. Pao, Ph.D., Department of Biochemistry, Chang Gung Medical College, 199 Tun Hwa North Road, Taipei 10560, Taiwan, Republic of China. Vol. 54, No.5, November 1990

nucleic acid (DNA) hybridization method can be a sensitive and specific alternative for the direct detection of C. trachomatis in clinical specimens. 9 In this study, we reported the results of using DNA hybridization technique in the investigation of the presence of C. trachomatis DNA in endocervical cells of both normal fertile women and infertile women with or without tubal factors. MATERIALS AND METHODS

A total of 186 patients who visited the infertility outpatient clinics of the Department of Obstetrics and Gynecology ofthe Chang Gung Memorial Hospital were chosen and recruited for this study. Major causes for the infertility among the 127 patients with primary infertility who experienced no conception after 1 year of unprotected coitus were male factor (17 patients), tubal factor (41 patients), cervical factor (3 patients), ovulation abnormalities (65 patients), and unknown factor (28 patients). The possible causes or contributing factors for the remaining 59 secondary infertile patients were recurrent abortions (24 patients), tubal factors (22 patients), ovulation abnormalities (4 patients), uterine synaechiae (2 patients), and others (7 patients). One hundred seventeen of the 186 Soong et al.

Chlamydia/ DNA in infertile women

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Table 2 Comparison of Endocervical Chlamydia! DNA, Antigen, Culture, and Serum Antichlamydial Antibody in Infertile and Normal Control Women

Table 1 Characteristics of Patients and Prevalence of Endocervical Chlamydia! DNA

Age a Married/ single History of PID D&C EP IUD users Chlamydia! DNA a

b

Normal control (n = 64)

Pregnant (n = 147)

Infertile (n = 186)

30.3 ± 2.2 55/9

25 ± 8.2 147/0

29.6 ± 5.7 179/7

2 (3.1)b 14 (21.9) 3 (4.7) 5 (7.8) 8 (12.5)

15 21 8 12 9

38 (20.4) 34 (18.3) 6 (3.2) 15 (8.1) 49 (26.3)

(10.2) (14.3) (5.4) (8.2) (6.1)

Ages are means± SD. Values in parentheses are percents.

infertile patients underwent laparoscopic evaluation. Tubal patency, phimosed fimbrae, peritubal adhesions, cornual blockage, hydrosalpinx, and other findings such as endometriosis, ovarian cyst, or congenital uterine abnormalities were all noted. Tubal patency was diagnosed by observing the passage of methylene blue dye beyond the fimbriae after the dye solution was injected through the uterine cervix with a Spackman cannula. Another 64 patients who were matched in age and socioeconomic status and who visited the clinics for routine physical examinations were selected as control group. One hundred forty-seven pregnant women (32, 70, and 45 women in their first, second, and third trimesters, respectively) were also included in this study. Informed consent was obtained from all patients. The characteristics ofthe patients in each group as well as prior history of PID, dilation and curettage (D & C), and EP were summarized in Table 1. Endocervical cells were collected with STD-EZE swabs (Abbott Laboratories Inc., North Chicago, IL). Deoxyribonucleic acid was extracted from the endocervical cells with phenol and chloroform and purified by ethanol precipitation. Positivity of chlamydia! DNA in endocervical cells was determined with molecular DNA hybridization method we have described before. 9 The limitation of detection was determined to be approximately 1 pg of chlamydia! DNA, and the sensitivity and specificity of the DNA hybridization method were 92% and 95%, respectively. 9 Cell culture for chlamydia diagnosis was performed with cycloheximide-treated McCoy cell monolayers as described elsewhere. 10 Chlamydia! antigen was determined by enzymelinked immunoassay kit "Chlamydiazyme," also obtained commercially from Abbott Laboratories Inc. Antichlamydial antibody was also determined 816

Soong et al.

Chlamydia/ DNA in infertile women

DNA Antigen Culture Antibody

Infertile (n = 104)

Control (n = 64)

30 16 12 17

8 (12.5) 6 (9.4) 4 (6.3) 3 (4.7)

(29.0)a (15.4) (19.2) (16.3)

x2 = x2= x2= x2 =

6.08b 1.28, Nsc 1.30, Nsc 5.10b

Values in parentheses are percents. P<0.05. c NS, not significant. a b

with enzyme-linked immunoassay kits obtained from Exdevelop Pharma (Taby, Sweden). Statistical analysis was performed with x2 test.

RESULTS Chlamydia! DNA was found in endocervical cells of 49 of 186 (26.3%) infertile patients, which is significantly more frequent than that of normal control or the pregnant women group (x 2 = 5.19, p < 0.05 when compared with normal control individuals; x2 = 23.3, P < 0.001 when compared with pregnant women) (Table 1). Among infertile patients, chlamydia! DNA was detected in 29 of 127 (22.8%) and 20 of 59 (33.9%) of patients with primary and secondary infertility, respectively. Complete sets of endocervical chlamydia! DNA, antigen, culture, and serum antichlamydial antibody data were available from 104 infertile and 64 normal women, and the results were shown in Table 2. The positive rates of DNA and antichlamydial antibodies but not chlamydia! antigen and chlamydia! cell culture were significantly higher in infertile than in normal control group. The prevalence of chlamydia! DNA among infertile women with the following signs and prior history were: PID (29.0% or 11 of 38), therapeutic D & C (23.5% or 8 of 34), and EP (33.3% or 2 of 6). The positive rates of chlamydia! DNA relative to tubal conditions of infertile patients were summarized in Table 3. Among infertile women, 49.3% (33 Table 3 Prevalence of Chlamydia! DNA in Infertile Women With Tubal and Non tubal Factors Subjects

Cases

Chlamydia! DNA positive

Tubal Non tubal

67 119

33 (49.3)a 16 (13.4) a

Total

186

49

a Chlamydia! DNA positive percentages are significantly different between tubal and nontubal patients. X2 = 28.1, P < 0.001. Values in parentheses are percents.

Fertility and Sterility

of 67) of those with tubal factors as the major contributing factor for infertility and 13.4% (16 of 119) of women with infertility not associated with tubal factors were found to contain chlamydia! DNA in their endocervical cells (x 2 = 28.1, P < 0.001). Significantly more infertile patients presenting with tubal factor were positive for endocervical chlamydia! DNA than those infertile patients without tubal factors. The chlamydia! DNA positive rate of 13.4% in infertile women with nontubal factors was not significantly different from the 12.5% chlamydia! DNA positive rate found in normal fertile women. DISCUSSION

Previous studies have reported that antichlamydial antibodies could be found more frequently in infertile women with tubal factors than their fertile control group.U- 13 Kane et al. 7 reported that antichlamydial antibodies could be found in 11% of either normal control group or infertile women with normal patent fallopian tubes at laparoscopy. This prevalence of antichlamydial antibodies was almost identical to the chlamydia! DNA positive rate we have found in our normal control group (Table 1) and in infertile women with nontubal factors (Table 3). There are a number of methods to determine the presence of C. trachomatis. Cell culture of clinical specimens for C. trachomatis is generally considered as a sensitive and specific method of diagnosis. However, it is usually expensive, time-consuming, and requires laboratory expertise that is not always available. Antibody-based methodologies for chlamydia detection have certain definite advantages and have been widely used recently. However, use of antigen detection for screening for chlamydia! infection can be problematic, especially in populations with low prevalence. Cross-reacting antigens produced by other micro-organisms yield false-positive results in a percentage of tests. Serologic methods are also available, but their relative utility in a specific situation depends on many factors, and their effectiveness is limited by their sensitivity and lack of direct correlation with the disease activity under certain circumstances. Molecular DNA hybridization test has been shown to be a sensitive and specific method to detect C. trachomatis independent of the viability of the microorgfinisms.9 Our data differ from others in that we did not find a significant correlation between the prevaVol. 54, No.5, November 1990

lences of chlamydia! antigen and cell culture and infertility. Small sample size may have contributed to their differences. However, results of this investigation did indicate a strong association between chlamydia! infection, as shown by the presence of chlamydia! DNA in endocervical cells, and presumed tubal factor infertility. The proportion of patients with chlamydia! DNA was twice as high in the infertile as in the control group. In particular, chlamydia! DNA was found in as many as one third of infertile patients with either past PID or history of EP. The fact that chlamydia! DNA positive rate was significantly higher in infertile women with tubal factors than those without tubal factors (Table 3) supports the important role of chlamydia! infection in the pathogenesis of tubal infertility.s,7,B,9,14 The results from this study also suggest thatchlamydia! DNA may be a useful marker for the identification of chlamydia infections and for the identification of patients with historical factors who are at high risk of becoming infertile because of chlamydia! infection-associated tubal diseases. With that in mind, detection of chlamydia! DNA can be further facilitated by polymerase chain reaction, an in vitro DNA amplification technique. 15 Polymerase chain reaction is expected to be even more sensitive and can be completed within a matter of hours and may be useful in making rapid detection of chlamydia! DNA in clinical specimens. In conclusion, the data presented here in conjunction with that available in the literature confirm that C. trachomatis infection is a major cause of tubal infertility, and endocervical detection of chlamydia! DNA may be a useful marker for the presence of chlamydia! infection. Acknowledgment. We acknowledge the constant encouragement and support of Chau · Hsiung Chang, M.D., of Chang Gung Memorial Hospital and Delon Wu, M.D., of Chang Gung Medical College. Ms. Lin-Feng Yao's assistance in preparing this manuscript is also appreciated.

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