Lack of association between serum antibodies to Chlamydia trachomatis and a history of recurrent pregnancy loss

FERTILITY AND STERILITY威 VOL. 72, NO. 3, SEPTEMBER 1999 Copyright ©1999 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A.

Lack of association between serum antibodies to Chlamydia trachomatis and a history of recurrent pregnancy loss Maarit Paukku, M.D.,*† Maija Tulppala, M.D., Ph.D.,‡ Mirja Puolakkainen, M.D., Ph.D.,† Tarja Anttila, M.D.,§ and Jorma Paavonen, M.D., Ph.D.* University Central Hospital, University of Helsinki, and The Family Federation of Finland, Helsinki; and National Public Health Institute, Oulu, Findland

Received January 13, 1999; revised and accepted March 31, 1999. Reprint requests: Maarit Paukku, M.D., University of Helsinki, Haartman Institute, Department of Virology, Post Office Box 21, 00014 University of Helsinki, Helsinki, Finland (FAX: 358-09-1912-6491). * Department of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland. † Haartman Institute, Department of Virology, University of Helsinki, and Helsinki University Central Hospital Diagnostics, Department of Virology, University Central Hospital. ‡ Infertility Clinic, The Family Federation of Finland. § National Public Health Institute, Oulu, Finland. 0015-0282/99/$20.00 PII S0015-0282(99)00269-1

Objective: To study the relation between recurrent pregnancy loss (RPL) and infection with Chlamydia trachomatis, and to compare the prevalence of antibodies to C. trachomatis in women with primary and secondary RPL. Design: Prospective comparative study. Setting: University hospital and university student health center. Patient(s): Seventy patients with RPL were selected from women attending an RPL outpatient clinic; 40 normal parous women and 94 asymptomatic sexually active women served as controls. Intervention(s): Blood samples were collected during the clinical examinations for RPL. Main Outcome Measure(s): Serum immunoglobulin (Ig) G and IgA antibodies were detected by two independent methods, a recombinant ELISA specific to the genus Chlamydia and microimmunofluorescence testing specific to the species C. trachomatis. Result(s): There was no statistically significant difference in the frequencies of IgG or IgA between the women with RPL and the controls. The antibody frequencies were similar in the women with primary and secondary RPL. Conclusion(s): The presence of serum antibodies to C. trachomatis is not associated with RPL. Women with primary and secondary RPL do not differ with respect to the prevalence of antichlamydial antibodies. Thus, women with RPL do not benefit from screening for chlamydial IgG or IgA antibodies. (Fertil Steril威 1999; 72:427–30. ©1999 by American Society for Reproductive Medicine.) Key Words: Chlamydia trachomatis, recurrent pregnancy loss, immunoglobulin G and immunoglobulin A antibodies

Recurrent pregnancy loss (RPL) is commonly defined as ⱖ3 consecutive losses of an intrauterine pregnancy that has lasted less than 20 –22 weeks (1). Women with RPL can be divided into those with primary RPL, for whom each successive pregnancy has ended in pregnancy loss, and those with secondary RPL, who have a history of at least one live birth (1). Women with secondary RPL appear to have a lower risk of pregnancy loss with the next subsequent pregnancy compared with women with primary RPL. The cause of RPL has been attributed to reproductive tract infections, immunologic factors, and anatomic, genetic, and endocrine disorders, but in 30%– 40% of the cases, the cause remains unexplained (2, 3). The absence of an

identifiable cause of RPL often causes the patient anxiety during the next pregnancy. When trying to determine the etiologic factors for RPL, some investigators have hypothesized that there is an association between RPL and infection with Chlamydia trachomatis (4, 5). In patients undergoing IVF-ET, a previous infection with C. trachomatis has been suggested to predispose to spontaneous abortion (6 – 8). Chlamydia trachomatis has been shown to persist in a subclinical stage for prolonged periods in the upper genital tract (9 –11). Thus, it has been hypothesized that a persisting chlamydial infection may increase the susceptibility to first trimester pregnancy loss by infecting the fetal tissue or by stimulating an inflammatory response in the fetal compartment (3). 427

However, not all investigators have found evidence to support this hypothesis (12, 13). Thus, the role of C. trachomatis infection in RPL remains unclear. The purpose of this study was to investigate whether RPL is associated with C. trachomatis infection. Because immunoglobulin (Ig) G has been found to persist for years after the initial infection, the short-lived serum IgA antibody has been suggested to be a better marker of an active infection (14 – 17). The detection of not only an IgG response but also an IgA response to C. trachomatis with two independent serologic methods is believed to allow more reliable conclusions about the possible relation. Possible differences in the prevalence of C. trachomatis antibodies between patients with primary and secondary RPL also were explored in this study.

MATERIALS AND METHODS Patients

All the patients had regular menstrual cycles and were otherwise healthy. The baseline investigations included parental karyotyping, glucose tolerance testing, determination of hormonal levels and autoantibodies, hysteroscopy, and vaginal ultrasonography. Cervical swabs had been taken previously for culture of C. trachomatis, Neisseria gonorrhoeae, herpes simplex virus, and Listeria monocytogenes. Serum antibodies to Toxoplasma gondii and cytomegalovirus also had been tested. Eleven patients had a luteal phase defect, two patients had cardiolipin antibodies, four patients had elevated blood prolactin concentrations, and one patient had uterine septum (18). None of these findings was regarded as the cause of the patients’ RPL. Forty parous women who had no history of abortion served as control group A (mean age, 36 years). Control group B (mean age, 25 years) consisted of 94 asymptomatic women who attended the Student Health Center at the University of Helsinki for routine gynecologic examinations or contraceptive counseling. A first-void urine polymerase chain reaction analysis and a cervical swab antigen test showed that none of the controls in group B had C. trachomatis infection (19). The study was approved by the local ethics committee.

Microimmunofluorescence Testing A microimmunofluorescence test was performed for chlamydial species–specific antibodies, with modifications, at the National Public Health Institute, Oulu, Finland, accordPaukku et al.

Antibodies to Chlamydia trachomatis in women with recurrent pregnancy loss and in controls. Test used to determine chlamydial antibodies MIF

rELISA

Study group

IgG (1:32)

IgA (1:16)

IgG

IgA

Primary RPL (n ⫽ 42) Secondary RPL (n ⫽ 28) Control group A (n ⫽ 40) Control group B (n ⫽ 94)

6 (14) 3 (11) 11 (28) 16 (17)

2 (5) 2 (7) 4 (10) 2 (2)

13 (31) 11 (39) 21 (53) 26 (28)

7 (17) 2 (7) 8 (20) 11 (12)

Note: Ig ⫽ immunoglobulin; MIF ⫽ microimmunofluorescence; rELISA ⫽ recombinant ELISA; RPL ⫽ recurrent pregnancy loss. Values are actual numbers of patients with percentages in parentheses. Paukku. C. trachomatis. Fertil Steril 1999.

The patients consisted of 70 women with at least three consecutive pregnancy losses who had been referred to our RPL outpatient clinic. Blood samples were collected during a prospective study performed from 1988 –1991. Forty-two women (mean age, 32 years) who had never had a successful pregnancy were classified as having primary RPL. Twentyeight women (mean age, 35 years) who had had pregnancy losses after having given birth to 1–3 children were classified as having secondary RPL.

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TABLE 1

Chlamydia trachomatis and RPL

ing to the procedure of Wang and Grayston (20). The C. trachomatis antigen consisted of the antigen pools BED, CHIJ, and GFK (Washington Research Foundation, Seattle, WA). Chlamydia pneumoniae strain K6 (National Public Health Institute, Oulu, Finland) was used as a control antigen (21). The sera were screened at a dilution of 1:32 for IgG and 1:16 for IgA, followed by further analysis of the positive samples by titration at twofold dilutions to the end point.

Recombinant ELISA Chlamydial genus–specific antibodies were determined with a recombinant ELISA (Medac GmbH, Hamburg, Germany) according to the manufacturer’s instructions. Immunoglobulin G and IgA class antibodies were detected with the use of microtiter plates coated with recombinant glycoconjugate antigens. The results were interpreted as suggested by the manufacturer.

Statistical Methods

The ␹2 test with Yates’ correction was used to estimate the significance of the differences in the antibody prevalences of the patient and control groups.

RESULTS No statistically significant differences in the frequencies of IgG or IgA antibodies were observed between the RPL and control groups. In addition, no statistically significant difference was observed in the antibody prevalence of the women with primary and secondary RPL. The frequency of antibodies to C. trachomatis in the patients and controls is shown in Table 1. The titers of antichlamydial IgG antibodies were generally low; the geometric mean titer was 1:87 for the women with RPL. The patients with primary RPL had a geometric mean titer of 1:64 and those with secondary RPL had a titer of 1:161, although the small numbers of patients Vol. 72, No. 3, September 1999

TABLE 2 Geometric mean titers of the immunoglobulin G antibodies to different Chlamydia trachomatis serotypes. Geometric mean titers of immunoglobulin G antibody to indicated C. trachomatis serotype Study group

BED

CJHI

GFK

74 81

59 58

78 70

Patients with recurrent pregnancy loss (n ⫽ 9) Controls (n ⫽ 27) Paukku. C. trachomatis. Fertil Steril 1999.

did not allow statistical analysis of the significance of the difference. The geometric mean titers for control groups A and B were 1:71 and 1:81, respectively. The distribution of antibody specificities to different serotypes were similar for the patients and controls. Fifty-six percent of the sera from the patients and 68% of the sera from the controls showed specificity for CJHI serotypes; the figures were 44% and 52%, respectively, for GFK serotypes and 33% and 35%, respectively, for BED serotypes. Although the difference in the geometric mean titers of the IgG antibodies to different chlamydial serotypes was not statistically significant, the antibody titers were lowest to CJHI serotypes and highest to GFK serotypes among the patients with RPL (Table 2). Similarly, among the controls, the lowest geometric mean titer was for antibodies to CJHI serotypes; however, the highest titer was detected for antibodies to BED serotypes. Immunoglobulin G antibodies to C. pneumoniae were found in 60% of the patients with RPL and in 80% and 55% of control groups A and B, respectively (data not shown).

DISCUSSION We found no relation between active C. trachomatis infection and RPL on the basis of the prevalences of IgA and IgG antibodies. This was the first study to compare the prevalence of antibodies to C. trachomatis in women with primary and secondary RPL. In addition, both the cases and the controls were well characterized. The IgG prevalence of 11%–28% detected in this study for both the patients with RPL and the controls is within the range of 7%– 44% reported to be the baseline prevalence for women between the ages of 20 and 35 years (7, 22). Some investigators previously suggested an association between antibodies to C. trachomatis and pregnancy loss, whereas the findings of other investigators have not supported such an association (4, 5, 12, 13). The serologic methods used in these earlier studies were based on a lipopolysaccharide antigen, which is a common structure for all FERTILITY & STERILITY威

chlamydial species and thus was able to detect antibodies to C. pneumoniae as well. Moreover, the conclusions of the previous studies were based on the determination of only the IgG class of antibodies, which may reflect merely a past exposure to C. trachomatis (4, 5, 12–15). Chlamydia trachomatis–specific IgG antibodies detected by microimmunofluorescence testing represent both antibodies to an active infection and antibodies persisting months or years after the disappearance of an infection (23). The detection of only IgG antibodies is not an ideal method for evaluating the association between C. trachomatis and RPL. Because IgA appears early in active genital infection, and because its half-life has been reported to be only 5– 6 days, IgA antibody was chosen to serve as a potential marker of active infection (16, 17, 24). Our finding of statistically similar IgA prevalences in patients and controls indicates that an active chlamydial infection plays no role in the etiology of RPL. In conclusion, this study shows that there is no correlation between RPL and the presence of IgG or IgA antibodies to C. trachomatis. The absence of an identifiable cause of RPL usually leads to intensive investigations for possible causes whose association with RPL has not yet been proven. The importance of our findings lies in our demonstration that women with RPL do not benefit from screening for chlamydial IgG or IgA antibodies, and thus there is no reason to add these tests to the burden of routine clinical investigations. References 1. Stirrat GM. Recurrent pregnancy loss I: definition and epidemiology. Lancet 1990;336:673–5. 2. Katz VL, Kuller JA. Recurrent pregnancy loss. Am J Perinatol 1994; 11:386 –97. 3. Stirrat GM. Recurrent pregnancy loss II: clinical associations, causes, and management. Lancet 1990;336:728 –33. 4. Quinn PA, Petric M, Barkin M, Butany J, Derzko C, Gysler M, et al. Prevalence of antibody to Chlamydia trachomatis in spontaneous abortion and infertility. Am J Obstet Gynecol 1987;156:291– 6. 5. Witkin SS, Ledger WJ. Antibodies to Chlamydia trachomatis in sera of women with recurrent spontaneous abortion. Am J Obstet Gynecol 1992;167:135–9. 6. Licciardi F, Grifo JA, Rosenwaks Z, Witkin SS. Relation between antibodies to Chlamydia trachomatis and spontaneous abortion following in vitro fertilization. J Assist Reprod Genet 1992;9:207–10. 7. Lunenfeld E, Shapiro BS, Sarov B, Sarov I, Insler V, Decherney AH. The association between chlamydial-specific IgG and IgA antibodies and pregnancy outcome in an in vitro fertilization program. J In Vitro Fert Embryo Transfer 1989;6:222–7. 8. Witkin SS, Kligman I, Grifo JA, Rosenwaks Z. Chlamydia trachomatis detected by polymerase chain reaction in cervices of culture-negative women correlates with adverse in vitro fertilization outcome. J Infect Dis 1995;171:1657–9. 9. Campbell LA, Patton DL, Moore DE, Cappuccio AL, Mueller BA, Wang S. Detection of Chlamydia trachomatis deoxyribonucleic acid in women with tubal infertility. Fertil Steril 1993;59:45–50. 10. Cates W, Wasserheit JE. Genital chlamydial infections: epidemiology and reproductive sequelae. Am J Obstet Gynecol 1991;164: 1771– 81. 11. Cleary RE, Jones RB. Recovery of Chlamydia trachomatis from the endometrium in infertile women with serum antichlamydial antibodies. Fertil Steril 1985;44:233–5. 12. Rae R, Smith IW, Liston WA, Kilpatrick DC. Chlamydial serologic studies and recurrent spontaneous abortion. Am J Obstet Gynecol 1994;170:782–5. 13. Plouffe L, White EW, Tho SP, Sweet CS, Layman LC, Whitman GF, et al. Etiologic factors of recurrent abortion and subsequent reproduc-

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