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
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Abstract |
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Key words: Chlamydia trachomatis/infertility/PCR/pregnancy outcome/serology
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Introduction |
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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., 1990).
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.
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Materials and methods |
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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, 2002) 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 13 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 1454 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 1517. 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 2-test, and, in applicable cases, Fishers exact test, were used. P < 0.05 was considered significant. Spearmans 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 BonferroniHolm adjustment of P-values was done.
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Results |
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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 1.102.72, 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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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 BonferroniHolm correction was applied.
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Discussion |
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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 fewif anyDNA+ cases among those that were C. trachomatis IgG.
Punnonen et al. (1979) clearly demonstrated an association between C. trachomatis seropositivity and TFI (Mol et al., 1997
). 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., 1985
; Lunenfeld et al., 1989
; Sharara and Queenan, 1999
), while a few (smaller) studies did not find such a relationship (Torode et al., 1987
; Tasdemir et al., 1994
). In one study, doxycycline treatment may have masked the negative effect of C. trachomatis antibodies (Sharara et al., 1997
). 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., 1994
) 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., 2000
) and ectopic pregnancies (Barlow et al., 2001
). 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., 1997
; Witkin et al., 1998
). 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, 1999
; Neuer et al., 2000
).
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, 2002). 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., 2001
). 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., 1990
) 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, 1992
; Dieterle et al., 1995
; Eggert-Kruse et al., 1996
; Weidner et al., 1996
; Habermann and Krause, 1999
; Ochsendorf et al., 1999
). However, Gdoura et al. (2001
) 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 16 h with C. trachomatis serovar E increased the proportion of immotile and dead sperm (Hosseinzadeh et al., 2001
).
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. (2002), will increase the couples likelihood to achieve pregnancy in spontaneous and/or assisted reproductive cycles.
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Acknowledgements |
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Submitted on July 5, 2002; resubmitted on November 18, 2003; accepted on December 4, 2003.