Demonstration of Chlamydia trachomatis IgG antibodies in the male partner of the infertile couple is correlated with a reduced likelihood of achieving pregnancy

A. Idahl1, J. Boman2, U. Kumlin2 and J.I. Olofsson1,3

1 Department of Clinical Science/Obstetrics and Gynecology and 2 Department of Clinical Microbiology/Virology, Umeå University, S-901 85 Umeå, Sweden

3 To whom correspondence should be addressed at: Fertilitetscentrum AB, P.O.Box 5418, 402 29 Göteborg, Sweden. e-mail: jan.olofsson{at}fcivf.com


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: The objective of this study was to determine the prevalence of Chlamydia trachomatis among both men and women seeking help at an infertility clinic, and to prospectively follow the effect of previous infection on pregnancy rates and pregnancy outcome after a long follow-up period (mean 37 months). METHODS: A total of 244 infertile couples was tested for C. trachomatis IgG antibodies, and IgG+ couples were also tested for C. trachomatis DNA by PCR in a first-void urine sample. Study parameters were serology, PCR results, clinical diagnoses, treatments, pregnancy rates and pregnancy outcome. As controls, age-matched and spontaneously pregnant women were also tested with serology. RESULTS: The prevalence of IgG antibodies was 24.2, 20.1 and 15.6% among infertile women, infertile men and control women respectively. The prevalence of C. trachomatis DNA was 6.8 and 7.1% among tested women and men respectively. The presence of C. trachomatis IgG antibodies in women was related to tubal factor infertility (TFI) (P = 0.002). Decreased pregnancy rates were seen in couples where the man was IgG+ (P = 0.005) with no relationship to TFI. Among women who achieved pregnancy, there was no difference in pregnancy outcome between IgG+ or negative couples. CONCLUSIONS: C. trachomatis IgG antibodies in the man of the infertile couple was related to decreased pregnancy rates and to the presence of IgG antibodies in the woman. There was a high prevalence of asymptomatic persistent infections among infertile couples.

Key words: Chlamydia trachomatis/infertility/PCR/pregnancy outcome/serology


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chlamydia trachomatis is one of the most prevalent sexually transmitted diseases (STD) worldwide. In Sweden the incidence rate has increased from 157 to 189 per 100 000 from 1997 to 1999 (Gotz et al., 2002Go), and even further during recent years (Ekdahl, 2002Go). An infection with C. trachomatis is known to be difficult to diagnose and can persist in the genital tract for a long period of time (Witkin, 2002Go). Persistence of genital chlamydia infections has recently been demonstrated in 87% of untreated men and women (Joyner et al., 2002Go). The finding of C. trachomatis IgG antibodies, as a marker of previous or persistent infection, shows a high prevalence in women with tubal factor infertility (TFI) (Mol et al., 1997Go). TFI is suggested to reduce pregnancy rates after IVF (Strandell et al., 2000Go) and the clinical demonstration of a Fallopian tube persistently dilated by fluid (hydrosalpinx) is in itself a negative predictor for pregnancy (Strandell et al., 1994Go), but details of the suggested mechanism(s) are as yet unknown. A recent meta-analysis concluded that laparoscopic salpingectomy prior to IVF treatment increases the odds of pregnancy and live birth as compared with no salpingectomy in the short term (Johnson et al., 2002Go).

When it comes to the relevance of testing for C. trachomatis in infertility investigation of the male partner, opinions diverge. Whether C. trachomatis exerts a direct effect on sperm quality or whether the male accessory glands are merely reservoirs for C. trachomatis bacteria that are repeatedly transmitted to the female partner, thereby causing for example tubal damage and diminished pregnancy chances, or a combination of both, remains unknown. There is, to our knowledge, only one previous study that has investigated the relationship between male partner C. trachomatis IgG antibodies and subsequent pregnancy rates. That study reported a lower pregnancy rate after a 6 month follow-up period, among couples where the man was positive for C. trachomatis IgG (Eggert-Kruse et al., 1990Go).

The present study was undertaken to determine the prevalence of C. trachomatis antibodies and ongoing infections in couples attending the gynaecological outpatient clinic because of infertility. An additional aim was to correlate the presence of C. trachomatis antibodies in the male and female partner to pregnancy rates and pregnancy outcome, whether achieved spontaneously or after assisted reproduction techniques. For controls, age-matched women attending the maternity screening programme and reporting spontaneous pregnancies were tested for the prevalence of IgG-antibodies to C. trachomatis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study was a prospective cohort study and the Human Ethics Committee of the Medical Faculty, Umeå University, approved of all parts.

Patients and specimen sampling
During the period October 1997 through February 2001 all consecutive couples (n = 244) attending the gynaecological outpatient clinic at Umeå University Hospital due to infertility (unfulfilled desire for pregnancy for >1 year) were included in the study. Blood was drawn from both partners upon their first visit, and tested for the occurrence of IgG antibodies to C. trachomatis after informed consent. Both partners were tested for HIV and hepatitis B and C. The female was tested for rubella immunity, and for ovulation by using mid-luteal serum progesterone. Semen analysis (using criteria according to the World Health Organization as modified by NAFA/ESHRE; Kvist and Björndahl, 2002Go) of the man was undertaken. The couple received general information about the investigation to come. If one of the partners of the couple had C. trachomatis IgG antibodies (threshold level 1/20), a second blood test for confirmation of the result was drawn and a first-void urine sample for detection of C. trachomatis DNA was collected. If at least one of the partners was positive in the DNA test for C. trachomatis, they were both treated with doxycycline 100 mg x2 the first day and thereafter doxycycline 100 mg x1 for 9 more days. None of the partners who had a positive DNA test admitted to extramarital sexual activity.

Chlamydia trachomatis serology and DNA testing
C. trachomatis-specific IgG antibodies were determined by the microimmunofluorescence (MIF) test (MRL Diagnostics/Focus, USA). Serum dilutions starting at 1/40 up to 1/640 were used. A weak specific immunofluorescence in the 1/40 dilution was reported as positive in 1/20. Antibody incubations were performed at 37°C for 60 min. Positive and negative controls from the kit were included. Upon receipt, the urine specimens were analysed for C. trachomatis DNA by means of a commercially available PCR test (Cobas Amplicor C. trachomatis test; Roche Diagnostics, Switzerland) according to the instructions from the manufacturer. For each specimen an internal control was included. A negative test result was reported as negative only if the sample was negative for C. trachomatis and the internal control was positive. A sample positive for C. trachomatis was re-analysed and reported as positive if the re-analysis was also positive.

Clinical investigation
Within 1–3 months after their first visit, when all test results were available, the couple returned to the clinic for a further consultation. This visit was conducted according to routine infertility work-up including clinical examination and ultrasonography, and further investigations such as hysterosalpingosonography (HSS) (n = 87), laparoscopy (n = 66), or both (n = 20) and additional hormonal assays were planned. From here on the couples were advised about treatment according to the findings. TFI was defined as one or both tubes occluded or dilated at HSS or laparoscopic chromopertubation.

Follow-up
After a follow-up period of 14–54 months (mean 37 months), the medical records of all couples included were studied with respect to C. trachomatis IgG antibodies and C. trachomatis DNA, findings at HSS and laparoscopy, clinical diagnoses, treatments and reported pregnancies. Primary endpoints of the study were pregnancy and pregnancy outcome (passed 28 weeks of gestation, spontaneous abortion or ectopic pregnancy). All pregnancies were routinely screened by ultrasound at gestation week 15–17. Those couples who had not achieved pregnancy by the time the medical records were studied were contacted by mail with information about the study and a request to contact them by telephone. During a structured telephone interview (carried out by A.I.) they were asked about any treatments not found in the medical records (some couples were treated elsewhere) and pregnancies and pregnancy outcome that had not previously been recorded. Six couples (2.5%) were lost to follow-up, of whom three couples had emigrated and the remaining three did not respond or were unreachable. Thus, pregnancy data were available from 238/244 (97.5%) of the couples.

Fertile controls
Retrospectively, serum was analysed from women attending the Antenatal Care programme at the local health care centres caring for women in the same region as the women attending the gynaecological outpatient clinic. At their first visit, blood had been collected for analysis of rubella immunity and an additional aliquot of serum was banked. Using the database from the computerized medical records, samples from these women were matched to the women seeking help for infertility with respect to age (birth year) and the year when blood samples were collected. Only women with spontaneous pregnancies (as recorded in the medical records) were included (n = 244). Serum was analysed with respect to the presence of IgG antibodies to C. trachomatis by the same method as for the study population.

Statistics
The statistical analyses were performed using SPSS software version 10.0.7. For significance of difference between two independent groups, the {chi}2-test, and, in applicable cases, Fisher’s exact test, were used. P < 0.05 was considered significant. Spearman’s non-parametric rank correlation was used for calculating correlations. Odds ratios (OR) and confidence intervals (95% CI) were calculated by multiple logistic regression and multivariate analysis was performed. To minimize the chance of getting a Type 1 error when making multiple comparisons, a Bonferroni–Holm adjustment of P-values was done.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patients
Mean age of the women was 31.1 ± 4.7 (mean ± SD) years and of the men 34.0 ± 5.4 years. Duration of infertility was ≥1 year but in isolated cases ranging up to 10 years. For the female partner the infertility was primary in 138/244 (56.6%) of the women and secondary in 106/244 (43.4%). In those women where the infertility was secondary, the woman had either had a child or had a history of spontaneous abortion, extrauterine  pregnancy or induced abortion.

Clinical diagnoses, Chlamydia trachomatis IgG and DNA prevalence
For distribution of the principal diagnoses, see Table I. Nineteen per cent of the women had TFI as one of three diagnoses. In the subgroup that underwent IVF/ICSI treatment, the proportion that had TFI as one of three diagnoses was 22/57 (39%). For details of C. trachomatis IgG prevalence and DNA positivity, see Table II. None of the patients complained of symptoms indicating ongoing STD. In the control group of pregnant women the prevalence of C. trachomatis IgG antibodies was 38/244 (15.6%), which was significantly different from the prevalence in the group of infertile women (OR 1.73, 95% CI 1.10–2.72, P = 0.023). The finding of a positive C. trachomatis DNA amplification test using PCR was equally distributed in both sexes and in only one couple were both partners positive (Table II). All nine couples where either one or both partner(s) were positive in the C. trachomatis PCR test were treated with antibiotics. One month after treatment, all couples were PCR-tested again with a negative test result. Four out of these nine couples had TFI as the principal cause of infertility. During the follow-up period, three of nine treated couples achieved pregnancies, all spontaneously.


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Table I. Diagnoses, treatments and pregnancies
 

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Table II. Prevalence of IgG and DNA positivity
 
Chlamydia trachomatis IgG and DNA among women and relationship to TFI and pregnancy rates
The finding of C. trachomatis IgG in the women was significantly related to the finding of TFI (Table III). The presence of C. trachomatis IgG antibodies in the woman among all couples tested (independent of diagnosis) was not related to pregnancy (Table III). In the small group of IVF-treated couples, the relationship between the woman’s C. trachomatis IgG and pregnancy did not reach statistical significance (n = 32, OR 4.2, 95% CI 0.9–19.4, P = 0.13). In the C. trachomatis IgG group, 15/20 were able to establish a pregnancy as compared with only 5/12 in the C. trachomatis IgG+ group. There was a significant, albeit weak, correlation between the level of the women’s C. trachomatis IgG titres and the finding of a positive PCR test (r = 0.3, P = 0.026).


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Table III. Analyses of correlations
 
Chlamydia trachomatis IgG and DNA among men and relationship to pregnancy rates
The finding of C. trachomatis IgG in the male partners was a highly significant outcome predictor with an OR of 2.6 (95% CI 1.3–4.9, P = 0.005) (Table III) of not achieving pregnancy. In the subgroup of couples that did not go through IVF or ICSI treatment, the OR of not achieving pregnancy if the man was C. trachomatis IgG+ was even higher (OR 3.9, 95% CI 1.4–10.9, P = 0.012). However, there was no significant relationship between C. trachomatis IgG antibodies in the man and male factor infertility, the latter as judged by sperm analysis. There was no relationship between C. trachomatis IgG antibodies in the man and TFI in the female partner. No other significant relationships between male IgG positivity and possible confounding variables were seen (i.e. endometriosis, partner’s age, treatments etc.). In the IVF or icsi group the C. trachomatis IgG men achieved pregnancy in 28/43 (65.1%) of the cases as compared with 6/14 (42.9%) among C. trachomatis IgG+ men. There was a highly significant relationship between C. trachomatis IgG-positivity in the man and the woman (Table III) and also a significant correlation between their respective IgG titre levels (r = 0.4, P < 0.001).

Pregnancy outcome
For women who achieved pregnancy during the study period, whether spontaneous or treatment-related, there were no statistically significant differences between C. trachomatis IgG+ or negative men and/or women concerning pregnancy outcome (Table IV). Furthermore, there were no differences in pregnancy outcome following different assisted reproduction treatments provided to C. trachomatis IgG+ or negative couples (data not shown). Although the diagnosis of a male factor seemingly was related to a higher incidence of spontaneous abortions (P = 0.025), this was found to be not significant when Bonferroni–Holm correction was applied.


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Table IV. Chlamydia trachomatis IgG and pregnancy outcome in spontaneous or treatment- related pregnancies
 
Multivariate analysis
In a multivariate analysis, only one parameter was found to significantly influence the primary study outcome. The finding of C. trachomatis IgG antibodies in the male partner was related to a decreased likelihood of achieving pregnancy (Table III).


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As anticipated, we found a raised prevalence of C. trachomatis IgG antibodies in the infertile population as compared with a proven fertile population. The presence of C. trachomatis IgG antibodies in the male partner of the infertile couple was significantly and inversely correlated to the overall pregnancy rate, with an OR of 2.6 of not achieving pregnancy. An interesting finding in this study is that while a decreased pregnancy rate was seen in IgG+ couples, there were no differences in pregnancy outcome between IgG+ and IgG couples. There was also a high prevalence of PCR-detected C. trachomatis infections among couples exhibiting C. trachomatis IgG antibodies (13.1% of IgG+ couples or 3.7% of the entire study group). Arguably, it should be cost-beneficial to screen infertile C. trachomatis IgG+ patients using PCR to detect and treat a possible persistent infection, since this has been suggested to be cost-effective in populations with a prevalence of C. trachomatis of ≥4% (Paavonen et al., 1998Go).

The strength of this study was the relatively long follow-up period (mean 37 months) with few patients lost to follow-up (2.5%). However, the cohort was too small to make any firm conclusions as to the possible impact of circulating antibodies to C. trachomatis in some of the study subgroups. DNA testing was only performed if one of the partners exhibited C. trachomatis IgG antibodies. Hence it is not possible to comprehensively evaluate the predictive value of a positive C. trachomatis IgG test and the chance of detecting an ongoing C. trachomatis infection. Nevertheless, the C. trachomatis IgG titre levels were significantly correlated to DNA findings, suggesting that there were very few—if any—DNA+ cases among those that were C. trachomatis IgG.

Punnonen et al. (1979Go) clearly demonstrated an association between C. trachomatis seropositivity and TFI (Mol et al., 1997Go). Similar to that finding, in the present study we found an OR of 3.6 of demonstrating tubal occlusion in a subsequent HSS or laparoscopy following a positive C. trachomatis IgG test of the female partner. There are diverging results from published studies, whether or not C. trachomatis IgG antibodies are a negative predictor for IVF outcome. Several studies have demonstrated a negative relationship (Rowland et al., 1985Go; Lunenfeld et al., 1989Go; Sharara and Queenan, 1999Go), while a few (smaller) studies did not find such a relationship (Torode et al., 1987Go; Tasdemir et al., 1994Go). In one study, doxycycline treatment may have masked the negative effect of C. trachomatis antibodies (Sharara et al., 1997Go). Such antibiotic treatment may cure a hidden residual infection since a correlation between C. trachomatis antibodies and findings of antigen in ovarian, tubal and endometrial samples (Czerwenka et al., 1994Go) has been demonstrated. However, C. trachomatis antigen has been identified in Fallopian tubes, ovaries and endometrium of culture-negative women with tubal factor infertility (Toth et al., 2000Go) and ectopic pregnancies (Barlow et al., 2001Go). Nonetheless, mechanisms other than residual C. trachomatis infections may reduce the pregnancy rate in spontaneous or assisted reproductive cycles. Repeated or persistent C. trachomatis infections are suggested to elicit an autoimmune response to human heat shock proteins (HSP) due to the structural similarities to chlamydial HSP (Neuer et al., 1997Go; Witkin et al., 1998Go). This may in turn exert a negative influence on embryo development and implantation at vital stages when human HSP are expressed, resulting in decreased reproductive outcome (Witkin, 1999Go; Neuer et al., 2000Go).

The present study draws attention to the interesting finding that male chlamydia IgG positivity confers a decreased fecundity in affected couples. Although as yet speculative, since they were not included as parameters in the present study, decreased sperm motility or concurrent or undetected infection may play a role. It is known that infection with Mycoplasma genitalium, which is also sexually transmitted, is an established cause of acute and probably also persistent non-gonococcal urethritis (NGU) (Taylor-Robinson, 2002Go). Use of serological tests in women attending an infertility clinic has suggested that M. genitalium may be an independent risk factor in the development of tubal damage (Clausen et al., 2001Go). The present study was not aimed to evaluate a possible effect of C. trachomatis IgG on semen characteristics of the male partner. However, no significant relationship between C. trachomatis IgG seropositivity and the diagnosis on conventional spermiogram parameters indicative of male factor infertility, was found. In one study comprising 491 asymptomatic males of infertile couples (Eggert-Kruse et al., 1990Go) a negative influence of male C. trachomatis IgG antibodies on pregnancy rates after 6 months was reported. The authors concluded that the effect was probably due to sexual transmission and a negative influence on tubal function of the female partner, since male C. trachomatis IgG positivity was correlated to female TFI. There was no such correlation between C. trachomatis IgG antibodies in the male and TFI in the present study, suggesting that there might be alternative or additional mechanism(s) that can reduce fertility. Several studies have addressed the relationship between C. trachomatis IgG and IgA antibodies in serum and semen and sperm quality but few significant relationships have been shown (Bjercke and Purvis, 1992Go; Dieterle et al., 1995Go; Eggert-Kruse et al., 1996Go; Weidner et al., 1996Go; Habermann and Krause, 1999Go; Ochsendorf et al., 1999Go). However, Gdoura et al. (2001Go) found an association between C. trachomatis DNA in semen and reduced sperm motility. In this context it is interesting to note that a recent study has demonstrated that co-incubation of sperm for 1–6 h with C. trachomatis serovar E increased the proportion of immotile and dead sperm (Hosseinzadeh et al., 2001Go).

In conclusion, we suggest that C. trachomatis antibody testing of both the male and female partner of the couple should be included in the routine infertility work-up in order to enable a more adequate prognosis for the likelihood of a spontaneous pregnancy. It remains to be seen in a randomized controlled study if antibiotic treatment to C. trachomatis IgG positive infertile patients and/or to all, as suggested by Land et al. (2002Go), will increase the couple’s likelihood to achieve pregnancy in spontaneous and/or assisted reproductive cycles.


    Acknowledgements
 
The authors wish to express their gratitude to nurse coordinator Åsa Hampusson, laboratory work by Iréne Eriksson and statistical advice from Nils-Gunnar Pehrsson. This work was supported by grants from the Swedish Medical Research Council 13144 (J.I.O.) and by the Medical Faculty at Umeå University.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Barlow RE, Cooke ID, Odukoya O, Heatley MK, Jenkins J, Narayansingh G, Ramsewak SS and Eley A (2001) The prevalence of Chlamydia trachomatis in fresh tissue specimens from patients with ectopic pregnancy or tubal factor infertility as determined by PCR and in-situ hybridisation. J Med Microbiol 50,902–908.[Abstract/Free Full Text]

Bjercke S and Purvis K (1992) Chlamydial serology in the investigation of infertility. Hum Reprod 7,621–624.[Abstract]

Clausen HF, Fedder J, Drasbek M, Nielsen PK, Toft B, Ingerslev HJ, Birkelund S and Christiansen G (2001) Serological investigation of Mycoplasma genitalium in infertile women. Hum Reprod 16,1866–1874.[Abstract/Free Full Text]

Czerwenka K, Heuss F, Hosmann J, Manavi M, Jelincic D and Kubista E (1994) Salpingitis caused by Chlamydia trachomatis and its significance for infertility. Acta Obstet Gynecol 73,711–715.

Dieterle S, Mahony JB, Luinstra KE and Stibbe W (1995) Chlamydial immunoglobulin IgG and IgA antibodies in serum and semen are not associated with the presence of Chlamydia trachomatis DNA or rRNA in semen from male partners of infertile couples. Hum Reprod 10,315–319.[Abstract]

Eggert-Kruse W, Gerhard I, Naher H, Tilgen W and Runnebaum B (1990) Chlamydial infection—a female and/or male infertility factor? Fertil Steril 53,1037–1043.[Medline]

Eggert-Kruse W, Buhlinger-Gopfarth N, Rohr G, Probst S, Aufenanger J, Naher H and Runnebaum B (1996) Antibodies to chlamydia trachomatis in semen and relationship with parameters of male fertility. Hum Reprod 11,1408–1417.[Abstract/Free Full Text]

Ekdahl K (ed) (2002) Annual Report. Swedish Institute for Infectious Disease Control, Stockholm, Sweden, pp. 8–10.

Gdoura R, Keskes-Ammar L, Bouzid F, Eb F, Hammami A and Orfila J (2001) Chlamydia trachomatis and male infertility in Tunisia. Eur J Contracept Reprod Health Care 6,102–107.[Medline]

Gotz H, Lindback J, Ripa T, Arneborn M, Ramsted K and Ekdahl K (2002) Is the increase in notifications of Chlamydia trachomatis infections in Sweden the result of changes in prevalence, sampling frequency or diagnostic methods? Scand J Infect Dis 34,28–34.[CrossRef][Medline]

Habermann B and Krause W (1999) Altered sperm function or sperm antibodies are not associated with chlamydial antibodies in infertile men with leucocytospermia. J Eur Acad Dermatol Venereol 12,25–29.[CrossRef][Medline]

Hosseinzadeh S, Brewis IA, Eley A and Pacey AA (2001) Co-incubation of human spermatozoa with Chlamydia trachomatis serovar E causes premature sperm death. Hum Reprod 16,293–299.[Abstract/Free Full Text]

Johnson NP, Mak W and Sowter MC (2002) Laparoscopic salpingectomy for women with hydrosalpinges enhances the success of IVF: a Cochrane review. Hum Reprod 17,543.[Abstract/Free Full Text]

Joyner JL, Douglas JM, Jr, Foster M and Judson FN (2002) Persistence of Chlamydia trachomatis infection detected by polymerase chain reaction in untreated patients. Sex Transm Dis 29,196–200.[Medline]

Kvist U and Björndahl L (eds) (2002) Manual on Basic Semen Analysis. ESHRE Monographs. Oxford University Press, Oxford, UK.

Land JA, Gijsen AP, Evers JL and Bruggeman CA (2002) Chlamydia trachomatis in subfertile women undergoing uterine instrumentation: screen or treat? Hum Reprod 17,525–527.[Abstract/Free Full Text]

Lunenfeld E, Shapiro BS, Sarov B, Sarov I, Insler V and Decherney AH (1989) The association between chlamydial-specific IgG and IgA antibodies and pregnancy outcome in an in vitro fertilization program. J In Vitro Fertil Embryo Transfer 6,222–227.

Mol BW, Dijkman B, Wertheim P, Lijmer J, van der Veen F and Bossuyt PM (1997) The accuracy of serum chlamydial antibodies in the diagnosis of tubal pathology: a meta-analysis. Fertil Steril 67,1031–1037.[CrossRef][Medline]

Neuer A, Lam KN, Tiller FW, Kiesel L and Witkin SS (1997) Humoral immune response to membrane components of Chlamydia trachomatis and expression of human 60 kDa heat shock protein in follicular fluid of in-vitro fertilization patients. Hum Reprod 12,925–929.[CrossRef][Medline]

Neuer A, Spandorfer SD, Giraldo P, Dieterle S, Rosenwaks Z and Witkin SS (2000) The role of heat shock proteins in reproduction. Hum Reprod 6,149–159.[CrossRef]

Ochsendorf FR, Ozdemir K, Rabenau H, Fenner T, Oremek R, Milbradt R and Doerr HW (1999) Chlamydia trachomatis and male infertility: chlamydia-IgA antibodies in seminal plasma are C. trachomatis specific and associated with an inflammatory response. J Eur Acad Dermatol Venereol 12,143–152.[CrossRef][Medline]

Paavonen J, Puolakkainen M, Paukku M and Sintonen H (1998) Cost-benefit analysis of first-void urine Chlamydia trachomatis screening program. Obstet Gynecol 92,292–298.[Abstract/Free Full Text]

Punnonen R, Terho P, Nikkanen V and Meurman O (1979) Chlamydial serology in infertile women by immunofluorescence. Fertil Steril 31,656–659.[Medline]

Rowland GF, Forsey T, Moss TR, Steptoe PC, Hewitt J and Darougar S (1985) Failure of in vitro fertilization and embryo replacement following infection with Chlamydia trachomatis. J In Vitro Fertil Embryo Transfer 2,151–155.

Sharara FI and Queenan JT, Jr (1999) Elevated serum Chlamydia trachomatis IgG antibodies. Association with decreased implantation rates in GIFT. J Reprod Med 44,581–586.[Medline]

Sharara FI, Queenan JT Jr, Springer RS, Marut EL, Scoccia B and Scommegna A (1997) Elevated serum Chlamydia trachomatis IgG antibodies. What do they mean for IVF pregnancy rates and loss? J Reprod Med 42,281–286.[Medline]

Strandell A, Waldenstrom U, Nilsson L and Hamberger L (1994) Hydrosalpinx reduces in-vitro fertilization/embryo transfer pregnancy rates. Hum Reprod 9,861–863.[Abstract]

Strandell A, Bergh C and Lundin K (2000) Selection of patients suitable for one-embryo transfer may reduce the rate of multiple births by half without impairment of overall birth rates. Hum Reprod 15,2520–2525.[Abstract/Free Full Text]

Tasdemir I, Tasdemir M, Kodama H, Sekine K and Tanaka T (1994) Effect of chlamydial antibodies on the outcome of in vitro fertilization (IVF) treatment. J Assist Reprod Genet 11,104–106.[Medline]

Taylor-Robinson D (2002) Mycoplasma genitalium—an up-date. Int J STD AIDS 13,145–151.[CrossRef][Medline]

Torode HW, Wheeler PA, Saunders DM, McPetrie RA, Medcalf SC and Ackerman VP (1987) The role of chlamydial antibodies in an in vitro fertilization program. Fertil Steril 48,987–990.[Medline]

Toth M, Patton DL, Campbell LA, Carretta EI, Mouradian J, Toth A, Shevchuk M, Baergen R and Ledger W (2000) Detection of chlamydial antigenic material in ovarian, prostatic, ectopic pregnancy and semen samples of culture-negative subjects. Am J Reprod Immunol 43,218–222.[CrossRef][Medline]

Weidner W, Floren E, Zimmermann O, Thiele D and Ludwig M (1996) Chlamydial antibodies in semen: search for ‘silent’ chlamydial infections in asymptomatic andrological patients. Infection 24,309–313.[Medline]

Witkin SS (1999) Immunity to heat shock proteins and pregnancy outcome. Infect Dis Obstet Gynecol 7,35–38.[CrossRef][Medline]

Witkin SS (2002) Immunological aspects of genital chlamydia infections. Best Pract Res Clin Obstet Gynaecol 16,865–874.[CrossRef][Medline]

Witkin SS, Askienazy-Elbhar M, Henry-Suchet J, Belaisch-Allart J, Tort-Grumbach J and Sarjdine K (1998) Circulating antibodies to a conserved epitope of the Chlamydia trachomatis 60 kDa heat shock protein (hsp60) in infertile couples and its relationship to antibodies to C.trachomatis surface antigens and the Escherichia coli and human HSP60. Hum Reprod 13,1175–1179.[Abstract]

Submitted on July 5, 2002; resubmitted on November 18, 2003; accepted on December 4, 2003.