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Serum IgG and IgA antibodies to chlamydia in ectopic pregnancies
CONTRACEPTION
SERUMIgG ANDIgA ANTIBODIES TO CHLAMYDIA IN ECTOPICPREGNANCIES W. Chaim *, B. Sarov **, I. Sarov ***, B. Piura * A. Cohen * and V. Insler * *
Division of Obstetrics & Gynecology, Soroka Medical Center ** Epidemiology and *** Virology Units, Faculty of Health Sciences Ben Gurion University of the Negev, Beer Sheva, Israel ABSTRACT The possible association of Chlamydia trachomatis with ectopic pregnancies was evaluated in a case-control study, comprising 35 women with ectopic pregnancy and 294 apparently healthy women who served as controls. Chlamydia-specific IgG and IgA antibodies were determined by single serovar (L2) inclusion immunoperoxidase assay (IPA). Socio-demographic characteristics, gynecological history and contraceptive methods were also evaluated. An inverse relationship was found between the educational levels and the prevalence of IgG and IgA antibodies to chlamydia. The prevalence rate of elevated IPA IgG (titer 2128) and IPA IgA (titer 216) specific to chlamydia was significantly higher in women with ectopic pregnancy versus controls (32% vs 8X, respectively, for IgG: odds ratio= 4.9; and 26% vs 4% for IgA : odds ratio = 7.5). Chlamydia trachomatis was not isolated in cell cultures in 10 specimens available from fallopian tubes of women with ectopic pregnancy. Only 9% ‘of the women recall having pelvic inflammatory disease (PID) indicating that most of the infections were asymptomatic. Womenwho did not use IUD had a higher proportion of chlamydia-specific IgG and IgA seropositives, though not statistically significant, as compared to IUD users.This study further supports the hypothesis that subclinical infection of the tube with C. trachomatis underlie ectopic may pregnancies. Submitted for publication October 17, 1988 Accepted for publication March 27, 1989 JULY 1989 VOL. 40 NO. 1
59
CONTRACEPTION
INTRODUCTION Numerous reports are being published in which increasing importance is attributed to Chlamydia trachomatis as the etiological factor in genital infections such as nongonococcal urethritis (NGU), post-gonococcal urethritis endometritis, salpingitis, perihe(PGU), cervicitis, patitis, epididymitis (1) and prepubertal vaginitis (2). Furthermore, a large variety of pathological conditions such as obstructive infertility (3), Reiter’s syndrome, cervical cancer (1)) periappendicitis (4)) have been serologically associated to chlamydia. An episode of salpingitis may increase from 7 to 10 times the risk of ectopic pregnancy (5). Recently it has been demonstrated in a few case-control studies, that women with ectopic pregnancies had a higher prevalence rate of chlamydial IgG antibodies than controls (6-8). In the present study the relationship between chlamydia-specific IgG and IgA antibodies as determined by single serovar inclusion immunoperoxidase assay (IPA) and ectopic pregnancies was determined in a case-control study. Socio-demographic characteristics, gynecological history and contraceptive methods were also evaluated. MATRRIALANDMETHODS The studv group comprised 35 women hospitalized in the gynecological department of the Soroka Medical Center in Beer Sheva due to ectopic pregnancy from February 1985 to July 1986. Socio-demographic characteristics, obstetric and gynecological history, clinical evaluation, duration and type of contraceptives used, as well as surgical findings were recorded on a specially pre-coded questionnaire and analyzed by computer; ethnic origin was defined by father’s country of birth. The control groun consisted of 294 apparently healthy women of reproductive age without history of sexually transmitted diseases, who were tested for chlamydia serology in the community.
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Serology: Serum IgG and IgA antibodies specific to chlamydia were determined by single serovar L2 inclusion immunoperoxidase assay (IPA) (Savyon Diag. Ltd., Beer Sheva). The sensitivity and specificity of immunoblotting against chlamydia 60 Kd structural polypeptide were compared to IPA and found to be 89% and 100% for IgG and 86.3 and 98.1 % for IgA, respectively (9). Blood samples were taken from 21 of the 35 husbands the study group women for serum IgG and IgA evaluation. Titers of 2128 and 216 were considered positive chlamydia-specific IgG and IgA antibodies, respectively.
of for
: At the time of Isolation of Chlamvdia trachomatis surgical intervention, an intraluminal sample of the affected and contralateral tubes by means of a sterile swab placed in SPG buffer ( 0.01 M sodium phosphate, pB 7.2, 0.25 M sucrose, 5 mM L-glutamic acid) was taken in ten of the cases of ectopic pregnancy patients and frozen at -7O’C within 2 hours of collection for further culture of Chlamydia trachomatis. The specimens were inoculated onto cycloheximide-treated McCoy cells and incubated at 36°C for 48 hours (10). Subsequent to incubation, the cells were stained with iodine and examined for typical iodine-stained inclusions.
Statistical Methods: The differences in proportions of seropositives in the various study groups were tested by x2 or Fisher’s exact test as appropriate. Students ‘t’ test was used to compare geometric mean titers. Pearson’s R was used as a test for correlation. RESULTS Chlamvdia
IKG
antibodies
Table I shows that the prevalence rate of chlamydial IgG antibodies was significantly higher ( P=O.OOOl ) in the group of women with ectopic pregnancy than in the control group. The prevalence rates of an IgG titer of 2128 was 32% in patients with ectopic pregnancies and 8% in the control group (odds ratio = 4.9). At the cut-off ,128 of chlamydial IgG antibodies, 73% of women of Afro-Asian origin were positive versus 40% of the women of EuroAmerican origin (p = 0.013 ). JULY 1969 VOL. 40 NO. 1
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Table I DISTRIBUTIONOF CHLAMYDIA-SPECIFIC InG ANTIBODY TITERS IN WOMEN WITHECTOPICPREGNANCY
Study group
Womenwith ECtopic Pregnancy Controls Fisher’s
exact
total tested n
Chlamydia IgG titers 2 128 2 256 n. % n. %
Geometric Mean Titer + In. S.D.
35
11
32
7
21
119.5
L 1.29
294
25
8
15
5
98.6
+ 0.652
test:
P=0.0001
P=O .004
t-test
N.S.
An inverse statistical relation between educational level and chlamydial IgG antibody titers of 2128 has also been observed. Seventy-eight percent of the women with 8 to 12 years of schooling were positives as compared to 35% of positives among those with 212 years of schooling (p=O.O13). No significant differences of chlamydial seropositives were found by crowding index of living (number of persons/number of rooms), or by age. Only 3 of the women of our study group (9%) recalled a history of previous PID. Chlamydial IgG antibodies in the male partner of 21 of the women studied, highly correlated with those of their spouses (Pearson’s R = .61660; P=O.O015). In 43% of the cases both partners were positive, in 33% both partners were negative. Chlamvdia InA antibodies Table II shows that the prevalence rate of chlamydial IgA antibodies at titer 2 16 was significantly higher (p=O.OOOl) in the ectopic pregnancy group as compared to the control group, 26% vs 4% (odds ratio= 7.5).
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CONTRACEPTION
Table
II
DISTRIBUTIONOF CHLAMYDIASPECIFICIRA ANTIBODYTITERS IN WOMEN WITH ECTOPICPREGNANCY
Study group total tested n Women with Ectopic Pregnancy
35
Controls Fisher’s
exact
Chlamydia IgA titers L 16 2 32 n. % n. %
9
Geometric Titer + In.
Mean
S.D.
26
3
9
13.9
+
0.5
4
6
2
22
+
0.7
294
13
test:
P=0.0001
P=O.O59
t-test
N.S.
As was the case for IgG, if the woman’s birthplace was Afro-Asian, the positivity was 64% and if Euro-American, 10%. Similarly, if the father’s birthplace was AfroAsian the positivity was 65% and if Euro-American, 17% (P= 0.003). The higher the educational level, the lower the IgA positivity rate (P= 0.004). A correlation of chlamydial IgA antibodies at titers 18 has been observed between sex partners. In 29% of couples both partners were positive and in 57% both were negative. Fifteen of the 35 women of the study group were IUD and one a the women used pills, three of users, diaphragm. Forty-seven percent of the women using IUD to chlamydial IgG antibodies, as compared were positive of IUD to 70% among the non-users. Twenty-seven percent users were positive for IgA antibodies as were 50% of the The difference between IUD users and non-users non-users. chlamydial IgG and IgA proportions of specific in antibody seropositives was found to be of no statistical to the small sample size significance, probably due (Table III). JULY
1989 VOL.
40 NO. 1
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Table III CHLAMYDIA-SPECIFIC IgG ANDIgA ANDTHE USE OF IUD Use of IUD Total No.
No.
IgG
2128
%
No.
IgA ‘216
%
Yes
15
7
47
4
27
No
20
14
70
10
50
The cultures of the intraluminal samples taken during the surgical intervention in all 10 cases of ectopic pregnancies.
of the tubes were negative
DISCUSSION The incidence of ectopic pregnancies has increased dramatically in recent years, with an annual increase varying from 4% to 8% in different parts of the world (6,11, 12). The two most probable factors which have been pointed out as potential determinants of ectopic pregnancies are tubal infection and contraceptive practices (6). Westrom, cited by Svensson et al. (7), incriminates acute salpingitis as the main factor leading to ectopic pregnancy. The major etiological pathogen in cases of ectopic pregnancy as in tubal infertility has been suggested to be Chlamydia trachomatis (13) which has also been considered as the major cause of salpingitis in young women in the Western world (14). These facts constitute a heavy economic burden for many today the countries. Recently, Washington et al.(15) estimated annual cost of Chlamydia trachomatis infections in men, women and children in the United States to be over $1.4 billion per year, and that chlamydial PID causes some 280 deaths a year.
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CONTRACEPTION
In the present study the cultures of samples taken in 10 cases with ectopic pregnancy from within the affected tube as well as from the contralateral one were all negative. The difficulty in chlamydia isolation from tubes of ectopic pregnancy cases is well known and has been experienced by others (6,16); it may be that at the time of ectopic pregnancy occurrence, only very small amounts of chlamydia are present in the tube, or that Chlamydia tubal tissue has been damaged by previous trachomatis infections, or that chlamydia is present in there other sites such as the endometrium or cervix, stimulating the production of elevated titers of IgG and IgA. Specific IgM is not a good marker of chronic salpingitis are not regularly found even in because IgM antibodies the first episode of chlamydial infection (7). Elevated IgG has been suggested as a potential marker of persistent or active chlamydial infection (1,17). Particularly elevated chlamydial IgG titers were found in diseases such as salpingitis, mechanical infertility, peri-hepatitis ( Curtis - Pitz - Hugh syndrome ) and pneumonitis (18-24). Furthermore, Guderian and Trobough (25) have shown by laparoscopy and/or laparotomy that in nearly all of the infertile women with IgG titers of 2512 by single L2 serovar inclusion immunofluorescent assay, a wide spectrum of pelvic inflammatory residues from severe periadnexal adhesions and tubal occlusions to mild adhesions and minimal tubal epithelium damage could be detected. Recently a correlation of IgG to chlamydia as determined by microimmunofluorescent assay (MU) and isolation of C. trachomatis from biopsy specimens in epididymitispatients has also been found (26). In the present study we demonstrated that significantly elevated (P-0.0001) IgG antibodies were more frequently found in ectopic pregnancies than in controls. These results are in agreement with those of Svensson et al. in Sweden (7) and Brunham et al. (6) in Canada who present data documenting that women with ectopic pregnancies had significantly more often Chlamydia trachomatis IgG antibody than did control women.
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The geometric mean titer (GMT) of IgG antibodies in women with ectopic pregnancies in the present study were lower than those found in salpingitis patients by IPA (119.5 vs 256) (11). This can be explained by the fact that salpingitis being an infectious state develops a higher immune response than ectopic pregnancy, which is a sequela of such infection. Since the half-life of IgA in human sera is 5 to 6 days (27),it has been recently suggested that it might be used viral and active and persistent as a marker for chlamydial infection (28). In previous studies a higher prevalence rate of specific antibodies was found in Chlamydia trachomatis IgA isolation positive non-gonococcal urethritis patients, patients with acute conjunctivitis, and in asymptomatic women seeking than in abortion isolation negative patients (29-32). Patients with salpingitis and epididymitis and women with obstructive infertility and invasive cervical cancer also had significantly higher prevalence of chlamydial IgA antibodies than controls The present study demonstrates that women with (17). ectopic pregnancy have significantly (P=O.OOOl) higher prevalence rate of chlamydial IgA antibodies than controls (26% vs 4%, odds ratio = 7.5). Only 9% of the women of our study group revealed a his tory of previous PID which indicates that most of the infections were asymptomatic. Similar findings were observed by other investigators (33-35). The percentage of women with elevated IgG and IgA antibody titres using copper IUDs was lower than in non-IUD users. Svensson et al. also found that IUD users have significantly lower prevalence rate of elevated IgG antibodies to than chlamydia non-users (7). The possibility that the use of copper IUDs may act as a kind of protection against Chlamydia trachomatis infection or reactivation is supported by recent studies by Kleinman et al- (36) who have demonstrated (in vitro) that copper concentrations (comparable to those found in human uteri of copper IUD users) caused inhibition of chlamydial replication in human endometrial culture. 66
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CONTRACEPTION
In summary, the present serological study further supports the hypothesis that current or prior chlamydial infection might be associated with ectopic pregnancies. Both elevated chlamydial serum IgG and IgA antibodies markedly differentiate between women with ectopic pregnancy and healthy controls. Further clinical trials are required to evaluate whether antichlamydial therapy in ectopic pregnancy with elevated IgG and IgA antibodies to chlamydia might reduce the appearance of another tubal pregnancy in the future. The growing proportions in which Chlamydia trachomatis infections appear in the community transform them into a considerable problem whose best solution is early detection and adequate antibiotic treatment to prevent possible complications such as tubal infertility, salpingitis and ectopic pregnancy.
REFERENCES 1.
Ladany S and Sarov I. Recent advances trachomatis. Eur J Epidemiol 4: 235-256,
2.
Bump RC. Chlamydia trachomatis as a cause of bertal vaginitis. Obstet and Gynec 65:384-388,
3.
G. et al. Specific Kleinman D , Holcberg Sarov I, to Chlamydia trachomatis in IgG and IgA antibodies 31: 193-197, 1986. infertile women. Int J Fertil
4.
Mardh PA and Wolner-Hanssen Chlamydia trachomatis salpingitis. 160: 304-306, 1985.
5.
Westrom L, Bengtsson LP and Mardh PA. trends and risks of ectopic pregnancy in a of women. Br Med J 282: 15-18, 1981.
6.
Brunham RC, Binns B , McDowell J and Paraskevas M. Chlamydia trachomatis infection in women with ectopic pregnancy. Obstet and Gynec 67: 722-726, 1986.
7.
F. Svennsson L, Mardh PA, Ahlgren M and Nordeskjold and antibodies to Chlamydia Ectopic pregnancy trachomatis. Pert Steril 44: 313-317, 1985.
JULY 1969 VOL. 40 NO. 1
in Chlamydia 1985.
P. Periappendicitis Surg Gynec
Prepu1985.
and Obstet
Incidence, population
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8.
Walters MD, Carlton EA, Gibbs RS, Schachter J, Holden MA and Pauerstein CJ. Antibodies to Chlamydia trachomatis and risk for tubal pregnancy. J Obstet Gynecol 159: 942-946, 1988.
9.
Hanuka N, Glasner M and Sarov I. Detection of IgG and IgA antibodies to Chlamydia trachomatis in sera of patients with chlamydial infections: use of immunoblotting and immunoperoxidase assays. Sex Transm Dis 15: 93-99, 1988
10. Ripa KT and Mardh PA. Cultivation trachomatis in cycloheximide-treated Clin Microbial 6: 328-331, 1970.
of Chlamydia McCoy cells. J
11. Piura B, Sarov I, Sarov B, Kleinman D, Chaim W and Insler V. Serum IgG and IgA antibodies specific for Chlamydia trachomatis in salpingitis patients as determined by the immunoperoxidase assay. Eur J Epidemiol 1: 110-116, 1985. 12. Moss TR. Is the incidence to ectopic pregnancy rising? Br Med Journal 291: 1199-1200, 1985. 13. Russel JB. The etiology of ectopic Obstet and Gynecol 30: 181-190, 1987.
pregnancy.
Clin
14. Westrom L and Mardh PA. Genital chlamydial infections in the female. In: Mardh PA, Holmes KK, Piot P, (eds). Chlamydial Schachter J Oriel JD and Elsevier Biomedical Press, Amsterdam, pp infections. 121-139, 1982. 15. Washington AE, Johnson RE and Sanders LL. Chlamydia trachomatis infections in the United States. What are they costing us ? JAMA 257: 2070-2072, 1987. 16. Hartford SL, Silva PD, dizerega GS and Yonekura ML. evidence of prior chlamydial infection in Serologic and pregnancy ectopic with tubal patients Fert Steril 47: 118contralateral tubal disease. 121, 1986.
68
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17. Sarov I, Sarov B, Lunenfeld E, Hagai 2, Chaim W and Piura B. The significance of chlamydia-specific serum IgA antibodies in Chlamydia trachbmatis- infections; of the European Society for Chlamydia in Proceedings Research 1: 234-238, 1988. 18. Henry-Suchet H, Catalan F, Paris X and Loffredo V. Antibody titer to C. trachomatis in acute salpingitis and obstructive sterilities. 1n:Mardh PA, Holmes KK, Oriel JD, Piot P and Schachter J (eds). Chlamydial infections. Elsevier Biomedical Press, Amsterdam, pp.183-187, 1982 19. Jones BR, Ardery BR, Hui SL and Cleary RE. Correlation between serum antichlamydial antibodies and tubal factor as a cause of infertility. Fertil Steril 38: 553-558, 1982. 20. Robertson JN , Ward ME, Conway D and Caul EO. Chlamydial and gonococcal antibodies in sera of infertile women with tubal obstruction. J Clin Path01 40: 377-383, 1987. 21. Moore DE, Foy HM, Daling JR et al. Chlamydia trachomatis in infertility due to distal tubal diseases. Lancet 2: 574-577, 1982. 22. Puolakkainen M, Saikku R Leinonen M et Chlamydial pneumonitis and its serodiagnosis infants. J Infect Dis 149: 598-604,1984.
al. in
23. Schachter .J, Grossman M and Azimi PH. Serology of Chlamydia trachomatis in infants. J Infect Dis 146: 530-535,1982. 24. Simmons D , Forsey T , Thin RN et al. Antichlamydial antibodies in PID. Brit J Vener Dis 55: 419-421, 1979. 25. Guderian AMand Trobough GE. Residues of pelvic inflammatory disease in IUD users: a result of the intrauterine device or Chlamydia trachomatis infection? Am J Obstet Gynec 154: 497-503, 1986.
JULY 1989 VOL. 40 NO. 1
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CONTRACEPTION
26.
Doble A, Harris JRW, Witherow RONand Taylor-Robinson Acute epididymitis: a microbiological and ultrasonographic study. Proceedings of the European Society for Chlamydia Research 1: 161, 1988
27.
Tomasi TB and Grey HM. Structure and function immunoglobulin A. Progr Allergy 16: 81-113, 1972.
28.
Sarov I, Insler V, Sarov B et al. Specific serum IgA antibodies in the diagnosis of active viral and chla1n:New Horizons in Microbiology, mydial infections. Sanna A and Morace G (eds). Elsevier Biomedical Press Amsterdam, p. 157-168, 1984.
29.
Hagay 2, Sarov B, Sachs J, Shaked 0 and Sarov I. Detection of Chlamydia trachomatis in male patients isolation in cell with urethritis; serology vs. culture. Genitourin Med in press, 1989.
30.
Csango PA, Sarov B, Schiotz H and Sarov I. Chlamydia trachomatis isolation and the comparative diagnostic J Clin value of serology in women seeking abortion. Path01 91: 89-92, 1988.
31.
Osborne NG, Hecht Y, Gorsline J et al. A comparison of culture, direct fluorescent antibody test, and a quantitative indirect immunoperoxidase assay for pregnant of Chlamydia trachomatis in detection women. Obstet Gynecol 71: 412-415, 1988.
32.
Hermann B , Dannevig L, Stenberg K and Mardh PA. The immune response and its value in the diagnosis of chlamydial conjunctivitis compared to culture, ELISA and immunofluorescence tests. Proceedings of for Chlamydia Research the European Society 1: 265, 1988.
33.
et al. ChlamydiaSarov I; Lunenfeld E, Sarov B specific IgG and IgA in women with obstructive and infertility as determined by immunoblotting Eur J Epidemiol 4: 216immunoperoxidase assays. 233,
70
of
1988.
JULY 1989 VOL. 40 NO. 1
D.
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34.
Punnonen R, Terho P, Nikkanen V et al. Chlamydial serology in infertile women by immunofluorescence. Fertil Steril 31: 656-659, 1979.
35.
of prior Gump DW, Gibson M, and Asikaga T. Evidence disease and its relationship to pelvic inflammatory Chlamydia trachomatis antibody and IUD use in infertile women. Am J Obstet Gynecol 146: 153-159, 1983.
36.
Kleinman D, Insler V and Sarov I. Inhibition of Chlamydia trachomatis growth in endometrial cells by copper: possible relevance for the use of the copper IUD. for Proceedings of the European Society Chlamydial Research 1: 129, 1988.
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SERUMIgG ANDIgA ANTIBODIES TO CHLAMYDIA IN ECTOPICPREGNANCIES W. Chaim *, B. Sarov **, I. Sarov ***, B. Piura * A. Cohen * and V. Insler * *
Division of Obstetrics & Gynecology, Soroka Medical Center ** Epidemiology and *** Virology Units, Faculty of Health Sciences Ben Gurion University of the Negev, Beer Sheva, Israel ABSTRACT The possible association of Chlamydia trachomatis with ectopic pregnancies was evaluated in a case-control study, comprising 35 women with ectopic pregnancy and 294 apparently healthy women who served as controls. Chlamydia-specific IgG and IgA antibodies were determined by single serovar (L2) inclusion immunoperoxidase assay (IPA). Socio-demographic characteristics, gynecological history and contraceptive methods were also evaluated. An inverse relationship was found between the educational levels and the prevalence of IgG and IgA antibodies to chlamydia. The prevalence rate of elevated IPA IgG (titer 2128) and IPA IgA (titer 216) specific to chlamydia was significantly higher in women with ectopic pregnancy versus controls (32% vs 8X, respectively, for IgG: odds ratio= 4.9; and 26% vs 4% for IgA : odds ratio = 7.5). Chlamydia trachomatis was not isolated in cell cultures in 10 specimens available from fallopian tubes of women with ectopic pregnancy. Only 9% ‘of the women recall having pelvic inflammatory disease (PID) indicating that most of the infections were asymptomatic. Womenwho did not use IUD had a higher proportion of chlamydia-specific IgG and IgA seropositives, though not statistically significant, as compared to IUD users.This study further supports the hypothesis that subclinical infection of the tube with C. trachomatis underlie ectopic may pregnancies. Submitted for publication October 17, 1988 Accepted for publication March 27, 1989 JULY 1989 VOL. 40 NO. 1
59
CONTRACEPTION
INTRODUCTION Numerous reports are being published in which increasing importance is attributed to Chlamydia trachomatis as the etiological factor in genital infections such as nongonococcal urethritis (NGU), post-gonococcal urethritis endometritis, salpingitis, perihe(PGU), cervicitis, patitis, epididymitis (1) and prepubertal vaginitis (2). Furthermore, a large variety of pathological conditions such as obstructive infertility (3), Reiter’s syndrome, cervical cancer (1)) periappendicitis (4)) have been serologically associated to chlamydia. An episode of salpingitis may increase from 7 to 10 times the risk of ectopic pregnancy (5). Recently it has been demonstrated in a few case-control studies, that women with ectopic pregnancies had a higher prevalence rate of chlamydial IgG antibodies than controls (6-8). In the present study the relationship between chlamydia-specific IgG and IgA antibodies as determined by single serovar inclusion immunoperoxidase assay (IPA) and ectopic pregnancies was determined in a case-control study. Socio-demographic characteristics, gynecological history and contraceptive methods were also evaluated. MATRRIALANDMETHODS The studv group comprised 35 women hospitalized in the gynecological department of the Soroka Medical Center in Beer Sheva due to ectopic pregnancy from February 1985 to July 1986. Socio-demographic characteristics, obstetric and gynecological history, clinical evaluation, duration and type of contraceptives used, as well as surgical findings were recorded on a specially pre-coded questionnaire and analyzed by computer; ethnic origin was defined by father’s country of birth. The control groun consisted of 294 apparently healthy women of reproductive age without history of sexually transmitted diseases, who were tested for chlamydia serology in the community.
60
JULY 1989 VOL. 40 NO. 1
CONTRACEPTION
Serology: Serum IgG and IgA antibodies specific to chlamydia were determined by single serovar L2 inclusion immunoperoxidase assay (IPA) (Savyon Diag. Ltd., Beer Sheva). The sensitivity and specificity of immunoblotting against chlamydia 60 Kd structural polypeptide were compared to IPA and found to be 89% and 100% for IgG and 86.3 and 98.1 % for IgA, respectively (9). Blood samples were taken from 21 of the 35 husbands the study group women for serum IgG and IgA evaluation. Titers of 2128 and 216 were considered positive chlamydia-specific IgG and IgA antibodies, respectively.
of for
: At the time of Isolation of Chlamvdia trachomatis surgical intervention, an intraluminal sample of the affected and contralateral tubes by means of a sterile swab placed in SPG buffer ( 0.01 M sodium phosphate, pB 7.2, 0.25 M sucrose, 5 mM L-glutamic acid) was taken in ten of the cases of ectopic pregnancy patients and frozen at -7O’C within 2 hours of collection for further culture of Chlamydia trachomatis. The specimens were inoculated onto cycloheximide-treated McCoy cells and incubated at 36°C for 48 hours (10). Subsequent to incubation, the cells were stained with iodine and examined for typical iodine-stained inclusions.
Statistical Methods: The differences in proportions of seropositives in the various study groups were tested by x2 or Fisher’s exact test as appropriate. Students ‘t’ test was used to compare geometric mean titers. Pearson’s R was used as a test for correlation. RESULTS Chlamvdia
IKG
antibodies
Table I shows that the prevalence rate of chlamydial IgG antibodies was significantly higher ( P=O.OOOl ) in the group of women with ectopic pregnancy than in the control group. The prevalence rates of an IgG titer of 2128 was 32% in patients with ectopic pregnancies and 8% in the control group (odds ratio = 4.9). At the cut-off ,128 of chlamydial IgG antibodies, 73% of women of Afro-Asian origin were positive versus 40% of the women of EuroAmerican origin (p = 0.013 ). JULY 1969 VOL. 40 NO. 1
61
CONTRACEPTION
Table I DISTRIBUTIONOF CHLAMYDIA-SPECIFIC InG ANTIBODY TITERS IN WOMEN WITHECTOPICPREGNANCY
Study group
Womenwith ECtopic Pregnancy Controls Fisher’s
exact
total tested n
Chlamydia IgG titers 2 128 2 256 n. % n. %
Geometric Mean Titer + In. S.D.
35
11
32
7
21
119.5
L 1.29
294
25
8
15
5
98.6
+ 0.652
test:
P=0.0001
P=O .004
t-test
N.S.
An inverse statistical relation between educational level and chlamydial IgG antibody titers of 2128 has also been observed. Seventy-eight percent of the women with 8 to 12 years of schooling were positives as compared to 35% of positives among those with 212 years of schooling (p=O.O13). No significant differences of chlamydial seropositives were found by crowding index of living (number of persons/number of rooms), or by age. Only 3 of the women of our study group (9%) recalled a history of previous PID. Chlamydial IgG antibodies in the male partner of 21 of the women studied, highly correlated with those of their spouses (Pearson’s R = .61660; P=O.O015). In 43% of the cases both partners were positive, in 33% both partners were negative. Chlamvdia InA antibodies Table II shows that the prevalence rate of chlamydial IgA antibodies at titer 2 16 was significantly higher (p=O.OOOl) in the ectopic pregnancy group as compared to the control group, 26% vs 4% (odds ratio= 7.5).
62
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CONTRACEPTION
Table
II
DISTRIBUTIONOF CHLAMYDIASPECIFICIRA ANTIBODYTITERS IN WOMEN WITH ECTOPICPREGNANCY
Study group total tested n Women with Ectopic Pregnancy
35
Controls Fisher’s
exact
Chlamydia IgA titers L 16 2 32 n. % n. %
9
Geometric Titer + In.
Mean
S.D.
26
3
9
13.9
+
0.5
4
6
2
22
+
0.7
294
13
test:
P=0.0001
P=O.O59
t-test
N.S.
As was the case for IgG, if the woman’s birthplace was Afro-Asian, the positivity was 64% and if Euro-American, 10%. Similarly, if the father’s birthplace was AfroAsian the positivity was 65% and if Euro-American, 17% (P= 0.003). The higher the educational level, the lower the IgA positivity rate (P= 0.004). A correlation of chlamydial IgA antibodies at titers 18 has been observed between sex partners. In 29% of couples both partners were positive and in 57% both were negative. Fifteen of the 35 women of the study group were IUD and one a the women used pills, three of users, diaphragm. Forty-seven percent of the women using IUD to chlamydial IgG antibodies, as compared were positive of IUD to 70% among the non-users. Twenty-seven percent users were positive for IgA antibodies as were 50% of the The difference between IUD users and non-users non-users. chlamydial IgG and IgA proportions of specific in antibody seropositives was found to be of no statistical to the small sample size significance, probably due (Table III). JULY
1989 VOL.
40 NO. 1
63
CONTRACEPTION
Table III CHLAMYDIA-SPECIFIC IgG ANDIgA ANDTHE USE OF IUD Use of IUD Total No.
No.
IgG
2128
%
No.
IgA ‘216
%
Yes
15
7
47
4
27
No
20
14
70
10
50
The cultures of the intraluminal samples taken during the surgical intervention in all 10 cases of ectopic pregnancies.
of the tubes were negative
DISCUSSION The incidence of ectopic pregnancies has increased dramatically in recent years, with an annual increase varying from 4% to 8% in different parts of the world (6,11, 12). The two most probable factors which have been pointed out as potential determinants of ectopic pregnancies are tubal infection and contraceptive practices (6). Westrom, cited by Svensson et al. (7), incriminates acute salpingitis as the main factor leading to ectopic pregnancy. The major etiological pathogen in cases of ectopic pregnancy as in tubal infertility has been suggested to be Chlamydia trachomatis (13) which has also been considered as the major cause of salpingitis in young women in the Western world (14). These facts constitute a heavy economic burden for many today the countries. Recently, Washington et al.(15) estimated annual cost of Chlamydia trachomatis infections in men, women and children in the United States to be over $1.4 billion per year, and that chlamydial PID causes some 280 deaths a year.
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In the present study the cultures of samples taken in 10 cases with ectopic pregnancy from within the affected tube as well as from the contralateral one were all negative. The difficulty in chlamydia isolation from tubes of ectopic pregnancy cases is well known and has been experienced by others (6,16); it may be that at the time of ectopic pregnancy occurrence, only very small amounts of chlamydia are present in the tube, or that Chlamydia tubal tissue has been damaged by previous trachomatis infections, or that chlamydia is present in there other sites such as the endometrium or cervix, stimulating the production of elevated titers of IgG and IgA. Specific IgM is not a good marker of chronic salpingitis are not regularly found even in because IgM antibodies the first episode of chlamydial infection (7). Elevated IgG has been suggested as a potential marker of persistent or active chlamydial infection (1,17). Particularly elevated chlamydial IgG titers were found in diseases such as salpingitis, mechanical infertility, peri-hepatitis ( Curtis - Pitz - Hugh syndrome ) and pneumonitis (18-24). Furthermore, Guderian and Trobough (25) have shown by laparoscopy and/or laparotomy that in nearly all of the infertile women with IgG titers of 2512 by single L2 serovar inclusion immunofluorescent assay, a wide spectrum of pelvic inflammatory residues from severe periadnexal adhesions and tubal occlusions to mild adhesions and minimal tubal epithelium damage could be detected. Recently a correlation of IgG to chlamydia as determined by microimmunofluorescent assay (MU) and isolation of C. trachomatis from biopsy specimens in epididymitispatients has also been found (26). In the present study we demonstrated that significantly elevated (P-0.0001) IgG antibodies were more frequently found in ectopic pregnancies than in controls. These results are in agreement with those of Svensson et al. in Sweden (7) and Brunham et al. (6) in Canada who present data documenting that women with ectopic pregnancies had significantly more often Chlamydia trachomatis IgG antibody than did control women.
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The geometric mean titer (GMT) of IgG antibodies in women with ectopic pregnancies in the present study were lower than those found in salpingitis patients by IPA (119.5 vs 256) (11). This can be explained by the fact that salpingitis being an infectious state develops a higher immune response than ectopic pregnancy, which is a sequela of such infection. Since the half-life of IgA in human sera is 5 to 6 days (27),it has been recently suggested that it might be used viral and active and persistent as a marker for chlamydial infection (28). In previous studies a higher prevalence rate of specific antibodies was found in Chlamydia trachomatis IgA isolation positive non-gonococcal urethritis patients, patients with acute conjunctivitis, and in asymptomatic women seeking than in abortion isolation negative patients (29-32). Patients with salpingitis and epididymitis and women with obstructive infertility and invasive cervical cancer also had significantly higher prevalence of chlamydial IgA antibodies than controls The present study demonstrates that women with (17). ectopic pregnancy have significantly (P=O.OOOl) higher prevalence rate of chlamydial IgA antibodies than controls (26% vs 4%, odds ratio = 7.5). Only 9% of the women of our study group revealed a his tory of previous PID which indicates that most of the infections were asymptomatic. Similar findings were observed by other investigators (33-35). The percentage of women with elevated IgG and IgA antibody titres using copper IUDs was lower than in non-IUD users. Svensson et al. also found that IUD users have significantly lower prevalence rate of elevated IgG antibodies to than chlamydia non-users (7). The possibility that the use of copper IUDs may act as a kind of protection against Chlamydia trachomatis infection or reactivation is supported by recent studies by Kleinman et al- (36) who have demonstrated (in vitro) that copper concentrations (comparable to those found in human uteri of copper IUD users) caused inhibition of chlamydial replication in human endometrial culture. 66
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In summary, the present serological study further supports the hypothesis that current or prior chlamydial infection might be associated with ectopic pregnancies. Both elevated chlamydial serum IgG and IgA antibodies markedly differentiate between women with ectopic pregnancy and healthy controls. Further clinical trials are required to evaluate whether antichlamydial therapy in ectopic pregnancy with elevated IgG and IgA antibodies to chlamydia might reduce the appearance of another tubal pregnancy in the future. The growing proportions in which Chlamydia trachomatis infections appear in the community transform them into a considerable problem whose best solution is early detection and adequate antibiotic treatment to prevent possible complications such as tubal infertility, salpingitis and ectopic pregnancy.
REFERENCES 1.
Ladany S and Sarov I. Recent advances trachomatis. Eur J Epidemiol 4: 235-256,
2.
Bump RC. Chlamydia trachomatis as a cause of bertal vaginitis. Obstet and Gynec 65:384-388,
3.
G. et al. Specific Kleinman D , Holcberg Sarov I, to Chlamydia trachomatis in IgG and IgA antibodies 31: 193-197, 1986. infertile women. Int J Fertil
4.
Mardh PA and Wolner-Hanssen Chlamydia trachomatis salpingitis. 160: 304-306, 1985.
5.
Westrom L, Bengtsson LP and Mardh PA. trends and risks of ectopic pregnancy in a of women. Br Med J 282: 15-18, 1981.
6.
Brunham RC, Binns B , McDowell J and Paraskevas M. Chlamydia trachomatis infection in women with ectopic pregnancy. Obstet and Gynec 67: 722-726, 1986.
7.
F. Svennsson L, Mardh PA, Ahlgren M and Nordeskjold and antibodies to Chlamydia Ectopic pregnancy trachomatis. Pert Steril 44: 313-317, 1985.
JULY 1969 VOL. 40 NO. 1
in Chlamydia 1985.
P. Periappendicitis Surg Gynec
Prepu1985.
and Obstet
Incidence, population
67
CONTRACEPTION
8.
Walters MD, Carlton EA, Gibbs RS, Schachter J, Holden MA and Pauerstein CJ. Antibodies to Chlamydia trachomatis and risk for tubal pregnancy. J Obstet Gynecol 159: 942-946, 1988.
9.
Hanuka N, Glasner M and Sarov I. Detection of IgG and IgA antibodies to Chlamydia trachomatis in sera of patients with chlamydial infections: use of immunoblotting and immunoperoxidase assays. Sex Transm Dis 15: 93-99, 1988
10. Ripa KT and Mardh PA. Cultivation trachomatis in cycloheximide-treated Clin Microbial 6: 328-331, 1970.
of Chlamydia McCoy cells. J
11. Piura B, Sarov I, Sarov B, Kleinman D, Chaim W and Insler V. Serum IgG and IgA antibodies specific for Chlamydia trachomatis in salpingitis patients as determined by the immunoperoxidase assay. Eur J Epidemiol 1: 110-116, 1985. 12. Moss TR. Is the incidence to ectopic pregnancy rising? Br Med Journal 291: 1199-1200, 1985. 13. Russel JB. The etiology of ectopic Obstet and Gynecol 30: 181-190, 1987.
pregnancy.
Clin
14. Westrom L and Mardh PA. Genital chlamydial infections in the female. In: Mardh PA, Holmes KK, Piot P, (eds). Chlamydial Schachter J Oriel JD and Elsevier Biomedical Press, Amsterdam, pp infections. 121-139, 1982. 15. Washington AE, Johnson RE and Sanders LL. Chlamydia trachomatis infections in the United States. What are they costing us ? JAMA 257: 2070-2072, 1987. 16. Hartford SL, Silva PD, dizerega GS and Yonekura ML. evidence of prior chlamydial infection in Serologic and pregnancy ectopic with tubal patients Fert Steril 47: 118contralateral tubal disease. 121, 1986.
68
JULY 1989 VOL. 40 NO. 1
CONTRACEPTION
17. Sarov I, Sarov B, Lunenfeld E, Hagai 2, Chaim W and Piura B. The significance of chlamydia-specific serum IgA antibodies in Chlamydia trachbmatis- infections; of the European Society for Chlamydia in Proceedings Research 1: 234-238, 1988. 18. Henry-Suchet H, Catalan F, Paris X and Loffredo V. Antibody titer to C. trachomatis in acute salpingitis and obstructive sterilities. 1n:Mardh PA, Holmes KK, Oriel JD, Piot P and Schachter J (eds). Chlamydial infections. Elsevier Biomedical Press, Amsterdam, pp.183-187, 1982 19. Jones BR, Ardery BR, Hui SL and Cleary RE. Correlation between serum antichlamydial antibodies and tubal factor as a cause of infertility. Fertil Steril 38: 553-558, 1982. 20. Robertson JN , Ward ME, Conway D and Caul EO. Chlamydial and gonococcal antibodies in sera of infertile women with tubal obstruction. J Clin Path01 40: 377-383, 1987. 21. Moore DE, Foy HM, Daling JR et al. Chlamydia trachomatis in infertility due to distal tubal diseases. Lancet 2: 574-577, 1982. 22. Puolakkainen M, Saikku R Leinonen M et Chlamydial pneumonitis and its serodiagnosis infants. J Infect Dis 149: 598-604,1984.
al. in
23. Schachter .J, Grossman M and Azimi PH. Serology of Chlamydia trachomatis in infants. J Infect Dis 146: 530-535,1982. 24. Simmons D , Forsey T , Thin RN et al. Antichlamydial antibodies in PID. Brit J Vener Dis 55: 419-421, 1979. 25. Guderian AMand Trobough GE. Residues of pelvic inflammatory disease in IUD users: a result of the intrauterine device or Chlamydia trachomatis infection? Am J Obstet Gynec 154: 497-503, 1986.
JULY 1989 VOL. 40 NO. 1
69
CONTRACEPTION
26.
Doble A, Harris JRW, Witherow RONand Taylor-Robinson Acute epididymitis: a microbiological and ultrasonographic study. Proceedings of the European Society for Chlamydia Research 1: 161, 1988
27.
Tomasi TB and Grey HM. Structure and function immunoglobulin A. Progr Allergy 16: 81-113, 1972.
28.
Sarov I, Insler V, Sarov B et al. Specific serum IgA antibodies in the diagnosis of active viral and chla1n:New Horizons in Microbiology, mydial infections. Sanna A and Morace G (eds). Elsevier Biomedical Press Amsterdam, p. 157-168, 1984.
29.
Hagay 2, Sarov B, Sachs J, Shaked 0 and Sarov I. Detection of Chlamydia trachomatis in male patients isolation in cell with urethritis; serology vs. culture. Genitourin Med in press, 1989.
30.
Csango PA, Sarov B, Schiotz H and Sarov I. Chlamydia trachomatis isolation and the comparative diagnostic J Clin value of serology in women seeking abortion. Path01 91: 89-92, 1988.
31.
Osborne NG, Hecht Y, Gorsline J et al. A comparison of culture, direct fluorescent antibody test, and a quantitative indirect immunoperoxidase assay for pregnant of Chlamydia trachomatis in detection women. Obstet Gynecol 71: 412-415, 1988.
32.
Hermann B , Dannevig L, Stenberg K and Mardh PA. The immune response and its value in the diagnosis of chlamydial conjunctivitis compared to culture, ELISA and immunofluorescence tests. Proceedings of for Chlamydia Research the European Society 1: 265, 1988.
33.
et al. ChlamydiaSarov I; Lunenfeld E, Sarov B specific IgG and IgA in women with obstructive and infertility as determined by immunoblotting Eur J Epidemiol 4: 216immunoperoxidase assays. 233,
70
of
1988.
JULY 1989 VOL. 40 NO. 1
D.
CONTRACEPTION
34.
Punnonen R, Terho P, Nikkanen V et al. Chlamydial serology in infertile women by immunofluorescence. Fertil Steril 31: 656-659, 1979.
35.
of prior Gump DW, Gibson M, and Asikaga T. Evidence disease and its relationship to pelvic inflammatory Chlamydia trachomatis antibody and IUD use in infertile women. Am J Obstet Gynecol 146: 153-159, 1983.
36.
Kleinman D, Insler V and Sarov I. Inhibition of Chlamydia trachomatis growth in endometrial cells by copper: possible relevance for the use of the copper IUD. for Proceedings of the European Society Chlamydial Research 1: 129, 1988.
JULY 1989 VOL. 40 NO. 1
71