Previously undetected Chlamydia trachomatis infection, immunity to heat shock proteins and tubal occlusion in women undergoing in-vitro fertilization

S.D. Spandorfer1, A. Neuer1, D. LaVerda2, G. Byrne2, H-C. Liu1, Z. Rosenwaks1 and S.S. Witkin1,3

1 Department of Obstetrics and Gynecology, Cornell University Medical College, 515 East 71st Street, New York, NY 10021 and 2 University of Wisconsin, Madison, WI, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The relationship between a previously undetected Chlamydia trachomatis infection, tubal infertility, immunity to heat shock proteins and subsequent in-vitro fertilization (IVF) outcome was evaluated. Women with tubal occlusion, with or without hydrosalpinges, and no history of C. trachomatis infection were tested for circulating antibodies to the human 60-kDa heat shock protein (Hhsp60), the C. trachomatis 10-kDa heat shock protein (Chsp10) and C. trachomatis surface antigens prior to their initial IVF cycle. Sera were obtained from 50 women whose male partners were infertile, 58 women with tubal occlusion but no hydrosalpinx and 39 women with tubal occlusions plus hydrosalpinx. Clinical pregnancies were documented in 68% of the women with male factor infertility. This was higher than the 43.1% rate in women with tubal occlusions (P = 0.04) and the 41% rate in women with hydrosalpinx (P = 0.02). C. trachomatis antibodies were present in one (2%) woman with male factor infertility as opposed to 15 (25.9%) women with tubal occlusion (P = 0.003) and 13 (33%) with hydrosalpinx (P < 0.0001). Antibodies to Chsp10 were more prevalent in women with hydrosalpinx (46.8%) than in women with male factor infertility (P < 0.0001, 6%) or tubal occlusion (P = 0.0009, 15.5%). Hhsp60 antibodies were equally more prevalent in women with tubal occlusion plus (46.8%) or minus hydrosalpinx (41.4%) than in women with male factor infertility (P < 0.0002). Hhsp60 was more prevalent in those women positive for Chsp10 (P = 0.02) or C. trachomatis (P = 0.04) antibodies than in women lacking these antibodies. There was no relationship between any of the antibodies measured in sera and IVF outcome.

Key words: Chlamydia trachomatis/10-kDa heat shock protein/hydrosalpinx/IVF outcome


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infertility due to Fallopian tube obstruction is a common indication for in-vitro fertilization (IVF) embryo transfer. Most women with tubal infertility have had `silent infections' but no history of a symptomatic pelvic infection or acquisition of a sexually transmitted disease (STD). The majority of these women, however, have serological evidence of exposure to Chlamydia trachomatis (Jones et al., 1982Go; Cates et al., 1993Go; Witkin et al., 1994Go; Eggert-Kruse et al., 1997Go).

The tubal obstruction may be unilateral or bilateral, proximal or distal, and/or include the presence of hydrosalpinges. Recent reports have indicated a worse prognosis for IVF embryo transfer in women with hydrosalpinges as compared with that of patients with occluded tubes but no hydrosalpinges (Strandell et al., 1994Go; Vandromme et al., 1995Go; Katz et al., 1996Go). However, these studies were retrospective and another investigation yielded differing results (Sharara et al., 1996Go). Nevertheless, prophylactic salpingectomy for patients with hydrosalpinges has been recommended (Lessey et al., 1995Go; Mukherjee et al., 1996Go).

Possible mechanism(s) whereby hydrosalpinges might negatively affect IVF outcome remain unclear. Direct flow of toxins, microorganisms or microbial products from the dilated tube into the uterus may exert a detrimental effect on the endometrium and/or embryo (Strandell et al., 1994Go). Irreversible damage to the endometrium during the acute infectious tubal insult could also diminish implantation rates. An alteration in endometrial integrin production associated with hydrosalpinges has also been proposed (Lessey et al., 1995Go).

A prolonged and undetected infection with C. trachomatis could induce hydrosalpinx formation as well as the production of antibodies to heat shock proteins. Cervical immunity to the chlamydial 60-kDa heat shock protein (Chsp60) has been associated with poor IVF outcome, possibly due to induction of autoimmunity to the homologous human 60-kDa heat shock protein (Hhsp60; Witkin et al., 1994Go, 1995Go; Neuer et al., 1998Go). Recently, the chlamydial 10-kDa heat shock protein (Chsp10) has been cloned and antibodies to Chsp10 became available (La Verda and Byrne, 1997). The chlamydial 10-kDa heat shock protein is genetically linked to Chsp60; the two proteins bind to each other and prevent incorrect protein folding and denaturation. Thus, the pathogen's ability to survive stressful environmental conditions and persist in the host is maximized by hsp60–hsp10 expression. While immunopathogenesis associated with Chsp60 is well documented (Morrison et al. 1989Go), this is unknown for Chsp10. To expand our studies on the relationship between tubal infertility, immunity to C. trachomatis and heat shock proteins, and IVF outcome, we evaluated women with tubal occlusion with or without hydrosalpinges for antibodies to Hhsp60 and Chsp10.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Sera were obtained from three groups of women during their initial IVF embryo transfer cycle. These included patients with tubal disease without the presence of a hydrosalpinx (group I; n = 58) and tubal infertility patients with a hydrosalpinx detected by ultrasound examination during their stimulation (group II; n = 39). Detection of hydrosalpinx occurred at any time during the stimulation and was noted by characteristic findings of a cystic, tubular structure outside of the ovary. A third group, which comprised wives of male factor infertility patients with documented patent tubes by hysterosalpingogram, was utilized as a control group (group III; n = 50). Groups I and III were obtained retrospectively from consecutive patients in August 1996. Patients in group II reflect a longer time period of January 1992–August 1996, although most of the patients were from 1995–1996.

Male factor patients were defined as patients undergoing IVF for the sole indication of male factor infertility. Male factor patients were only included if the female partner had no evidence of any STD and had normal tubal function as exhibited by a hysterosalpingogram. These patients were chosen to represent a control group that would be expected to have had a low prevalence of exposure to C. trachomatis. For the male infertility patients, the mean (± SD) of the sperm count, motility and normal forms by strict criteria were 26.9x106/ml (± 28.7), 29.8% (± 22.1), and 1.6% (± 1.8) respectively (Davis and Rosenwaks, 1996Go).

Blood was collected during follicular stimulation by venipuncture and sera obtained and stored at –20°C until assayed.

Detection of antibodies to Hhsp60
Recombinant human hsp60 (StressGen, Victoria, BC, Canada) was diluted to 10 µg/ml in 0.1M carbonate buffer, pH 9.8 and 0.1 ml added to wells of a microtitre plate. After an overnight incubation at 4°C, the wells were washed four times with 0.1% phosphate-buffered saline (PBS)–Tween. Aliquots (0.1 ml) of serum diluted 1:200 in PBS-Tween were added to the wells and the plate floated on a 37°C water bath for 60 min. The wells were then washed and incubated with a 1:200 dilution of alkaline phosphatase (AP)-conjugated goat antibody to human immunoglobulin G (IgG). Following an additional 37°C 60-min incubation, the wells were washed as above and the colourless AP substrate, p-nitrophenylphosphate in 10% diethanolamine buffer was added. After 30–60 min room-temperature incubation, the appearance of a yellow colour in the wells was quantified at 4 or 5 nm. Known positive and negative samples were assayed in parallel to the test samples. Inter- and intra-assay variation was <10%. A positive sample was defined as one yielding an optical density value that was at least two standard deviations above the mean value obtained with a panel of samples from subjects of proven fertility and no history of abortions or genital tract infections. Each sample was run twice in parallel and the results were averaged if agreement was within 10%. If results were not in agreement, then the assay was repeated.

Detection of antibodies to the Chsp10
Chsp10 was detected utilizing recombinant chsp10 (La Verda and Byrne, 1997) in an enzyme-linked immunosorbent assay (ELISA) format. The purified recombinant protein was bound to the wells of a microtitre plate (1µg/well), incubated with serum as above at a dilution of 1:200, and the presence of bound IgG detected with AP-conjugated anti-human IgG. Positive and negative controls were assayed in parallel to the test samples. Duplicates differing by more than 10% were retested. Samples were assayed without knowledge of IVF outcome or cause of infertility. A positive sample was defined as an optical density of 0.6 or more, which was two standard deviations above the mean value of that obtained with the samples from the wives of men with male infertility. The median and range of positive sera were 0.829 (0.603–3.187) optical density units.

Detection of serum antibodies (IgG) to C. trachomatis
Antibodies to surface antigens of C. trachomatis were detected by ELISA using the SeroCt IgG kit (Savyon Diagnostics, Ashdod, Israel). This assay measures antibodies to the major structural antigens of C. trachomatis. Positive samples were defined by the manufacturer.

IVF protocol
Patients were treated with standard ovulation induction protocols and underwent IVF embryo transfer according to previously published guidelines (Davis and Rosenwaks, 1996Go). In brief, women treated with luteal-phase leuprolide acetate were desensitized with gonadotrophin-releasing hormone (GnRH-a; Lupron; Tap Pharmaceuticals, Deerfield, IL, USA), 1 mg s.c. daily until ovarian suppression was achieved. Most patients were treated with luteal-phase leuprolide acetate [tubal without hydrosalpinx 57/58, tubal with hydrosalpinx (36/39) and male factor (50/50]. Women not treated with luteal-phase leuprolide acetate began stimulation on day 3 of their treatment cycle. Ovarian stimulation was then carried out using a combination of gonadotrophins [human menopausal gonadotrophin; pure follicle stimulating hormone (Pergonal or Metrodin; Serono, Waltham, MA, USA)], following our standard step-down protocol. Human chorionic gonadotrophin (HCG) was administered (3300–10 000 IU) when a minimum of two follicles reached at least 16–18 mm in mean diameter as measured by transvaginal ultrasound. Oocytes were harvested by transvaginal ultrasound-guided puncture around 35 h after HCG administration under i.v. sedation.

Oocyte insemination and the use of micromanipulation were utilized on a case-by-case need. Intracytoplasmic sperm injection was performed in 9/58 tubal patients without hydrosalpinx, 8/39 patients with hydrosalpinx and in 47/50 patients with male factor infertility. Morphologically good embryos were transferred back into the uterine cavity approximately 72 h after the retrieval. The number of embryos transferred was dependent on maternal age according to our standard protocol. In general, patients under 35 years of age had three embryos transferred, patients 35 to 40 years of age had four embryos transferred, and patients over 40 had up to five embryos transferred. As a standard protocol, starting on the day of oocyte retrieval, methylprednisolone (16 mg/day) and tetracycline (250 mg every 6 h) were administered for 4 days to all patients in case of the need for assisted hatching (Davis and Rosenwaks, 1996Go). Progesterone was administered on day 3 after HCG administration (25–50 mg i.m./day) and was continued until the assessment of pregnancy. A clinical pregnancy was defined as a positive fetal heart detected at ultrasound.

Statistical analysis included the use of the Mann–Whitney U-test, Student's t-test, Fisher's exact test, {chi}2 test, and analysis of variance where appropriate. P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient demographics, and numbers of oocytes retrieved and embryos transferred are presented in Table IGo. Only a minority of women with tubal disease reported a previous history of a STD. Patients with tubal disease without a hydrosalpinx were no more likely to have had a detected STD than were women who had not conceived due to their partner's infertility. Similarly, only one-quarter of patients with hydrosalpinges reported having had an STD. This was greater than the incidence of self-reported STD in patients in group I or in group III (P < 0.05), but still only involved a minority of patients. Patients with male factor infertility or tubal occlusion with hydrosalpinges were of similar age and younger than patients with tubal occlusion and no hydrosalpinges. There was no relation between the numbers of oocytes retrieved or fertilized, embryos transferred and the cause of infertility.


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Table I. Patient demographics, oocyte retrieval and embryo transfer in in-vitro fertilization patients
 
Table IIGo demonstrates the relationship between serum antibodies to C. trachomatis, Chsp10, and Hhsp60 and the aetiology of infertility. Patients with tubal disease with or without hydrosalpinges were equally likely to have had an elevated prevalence of circulating IgG antibodies to C. trachomatis as compared with that of male factor patients (P = 0.003). Similarly, patients with tubal disease with or without hydrosalpinges were more likely to have serum antibodies to Hhsp60 than were patients with male factor infertility (P <= 0.0004). However, patients whose tubal disease included hydrosalpinx formation had an increased prevalence of antibodies to the Chsp10 than did patients with either male factor infertility (P < 0.0001) or tubal disease but without the presence of a hydrosalpinx (P = 0.0009). Women with antibodies to the Hhsp60 were more likely than women without sensitization to Hhsp60 to be positive for antibodies to C. trachomatis (15/45 versus 15/102; P = 0.01) and to the Chsp10 (16/45 versus 15/102; P = 0.01).


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Table II. Relationship between cause of infertility and circulating immunoglobulin G antibodies to Chlamydia trachomatis surface antigens, chlamydial 10-kDa heat shock protein (Chsp10) and the human 60-kDa heat shock protein (Hhsp60)
 
Figure 1Go demonstrates the clinical pregnancy outcome based on the aetiology of infertility. Patients whose husbands had male factor infertility were more likely to have had a clinical pregnancy than patients with tubal disease (P < 0.004). No difference in outcome was noted, however, for tubal patients based on the presence or absence of a hydrosalpinx.



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Figure 1. The relationship between the cause of infertility and in-vitro fertilization outcome. Women with a diagnosis of male factor infertility were more likely to achieve a clinical pregnancy than women with tubal factor infertility ± hydrosalpinx. Clinical pregnancies are defined as an ongoing pregnancy beyond the first trimester. Results are reported as a percentage of clinical pregnancies per patient undergoing embryo retrieval.

 
Figure 2Go demonstrates the relationship between IVF outcomes and antibodies to C. trachomatis, Chsp10, and Hhsp60. The percentage of women in each category whose IVF cycle resulted in a term birth did not differ between those positive or negative for any of these antibodies either singly or in combination.



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Figure 2. The relationship between in-vitro fertilization (IVF) outcome and antibodies to Chlamydia trachomatis (Ct IgG), Chsp10 IgG and Hhsp60 IgG. Sera from women undergoing IVF who were infertile due to male factor or tubal ± hydrosalpinx were assayed for antibodies to C. trachomatis, Chsp10 and Hhsp60 by enzyme-linked immunosorbent assay. No relationship was found between the presence of these antibodies and clinical pregnancy in an IVF cycle. Clinical pregnancies are defined as an ongoing pregnancy beyond the first trimester. Results are reported as a percentage of clinical pregnancies per patient undergoing embryo retrieval. IgG = immunoglobulin G; Chsp10 = chlamydial 10-kDa heat shock protein; Hhsp60 = human 60-kDa heat shock protein.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
At least 75% of women in the present study who were infertile due to Fallopian tubal occlusion had no history of a prior STD. However, tubal occlusion was strongly associated with circulating antibodies to both C. trachomatis structural antigens and to the chlamydial hsp10. This highlights the asymptomatic character of C. trachomatis female genital tract infections and the relation between `silent' chlamydial pelvic inflammatory disease (PID) and tubal infertility. The results emphasize the need for C. trachomatis screening of asymptomatic sexually active women at risk of this infection as well as couples seeking an infertility evaluation. Identification and prompt treatment of asymptomatic carriers of C. trachomatis genital tract infections and their sexual partners will decrease the prevalence of tubal factor infertility.

As compared with women whose tubal occlusions were not accompanied by hydrosalpinx formation, women with a tubal hydrosalpinx had an increased incidence of a prior diagnosed symptomatic STD. Microorganisms other than C. trachomatis which are likely to evoke symptoms in the infected host might, therefore, be associated with those cases of tubal blockage that lead to hydrosalpinx formation. Alternatively, hydrosalpinx formation itself might evoke symptoms, whether due to C. trachomatis or some other microorganism. A comparative microbial analysis using gene amplification technology of Fallopian tubes of women whose blockage is or is not associated with hydrosalpinx will be necessary to evaluate these possibilities.

Antibodies to the Chsp10, but not antibodies to chlamydial structural components, were more prevalent in women with blocked tubes plus hydrosalpinges. The increased immunogenicity of this protein in association with hydrosalpinx formation suggests a greater or more prolonged expression of hsp10 in these women. Perhaps the likelihood of hydrosalpinx formation is increased under as yet undefined conditions, when Chsp10 gene expression is increased.

Antibodies to Hhsp60 were strongly associated with tubal infertility but their prevalence did not depend on the presence or absence of a hydrosalpinx. The relationship between C. trachomatis associated tubal infertility and development of antibodies that recognize the Hhsp60 has been noted previously (Witkin et al., 1993Go, 1994Go). The present results suggest that this cross-reactive immunity is independent of hydrosalpinx formation. We were unable in the present study to evaluate our patients for antibodies reactive with human hsp10. These studies are being planned.

Unlike previous retrospective studies (Strandell et al., 1994Go; Vandromme et al., 1995Go; Katz et al., 1996Go), we did not find a difference in pregnancy outcome in tubal infertility patients based on the occurrence of hydrosalpinges. The high rate of successful IVF outcome in the present study minimizes the occurrence of confounding factors that could have interfered with the analysis of the association between outcome and hydrosalpinges in previous studies. The present finding is in accordance with a previous observation (Sharara et al., 1996Go). The tubal factor patients without hydrosalpinges were, on average, 2 years older than patients with hydrosalpinges. However, this would not solely account for the lack of a difference in pregnancy outcomes. Furthermore, this study is weakened by the fact that the hydrosalpinx patients reflect a longer time period which, given that IVF success has improved with time, would be expected to have a detrimental effect on outcome. Most importantly, inconsistencies exhibited in IVF prognosis associated with the presence of hydrosalpinges in tubal factor patients highlight the need for a well-designed, prospective study.

Tubal factor infertility, with or without hydrosalpinges, was associated with a poorer prognosis after IVF than male factor infertility. However, unlike the association between cervical antibodies to C. trachomatis or Hhsp60 and poor IVF outcome (Witkin et al., 1994Go), the reduced pregnancy rates in tubal factor patients were unrelated to these antibodies in patients' sera. This confirms that systemic immunity differs from local cervical immunity. The lack of an effect of systemic immunity to C. trachomatis on IVF outcome is in accordance with a previous study (Claman et al., 1996Go). Unfortunately, cervical samples from the subjects in our study were not available for analysis.

In conclusion, we have demonstrated an association between tubal factor infertility and immunity to C. trachomatis. Furthermore, the presence of hydrosalpinges was associated with an increased prevalence of a prior symptomatic STD and with antibodies to chlamydial hsp10, as compared with that of tubal factor infertility patients without hydrosalpinges. However, unlike the association with local immunity to C. trachomatis and IVF outcome (Witkin et al., 1994Go), no association with systemic immunity to C. trachomatis or heat shock proteins and IVF outcome was demonstrated.


    Acknowledgments
 
Supported in part by Deutsche Forschungsgemeinschaft grant Ne 602/1–1.


    Notes
 
3 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Davis, O.K. and Rosenwaks, Z. (1996) In vitro fertilization. In Adashi, E., Rock, J.A., Rosenwaks, Z. (eds), Reproductive Endocrinology, Surgery, and Technology. Lippincott-Raven, Philadelphia, pp. 2319–2334.

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Submitted on March 13, 1998; accepted on September 24, 1998.