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
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Abstract |
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Key words: Chlamydia trachomatis/10-kDa heat shock protein/hydrosalpinx/IVF outcome
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Introduction |
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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., 1994; Vandromme et al., 1995
; Katz et al., 1996
). However, these studies were retrospective and another investigation yielded differing results (Sharara et al., 1996
). Nevertheless, prophylactic salpingectomy for patients with hydrosalpinges has been recommended (Lessey et al., 1995
; Mukherjee et al., 1996
).
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., 1994). 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., 1995
).
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., 1994, 1995
; Neuer et al., 1998
). 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 hsp60hsp10 expression. While immunopathogenesis associated with Chsp60 is well documented (Morrison et al. 1989
), 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.
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Materials and methods |
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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, 1996).
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 3060 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 (0.6033.187) 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, 1996). 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 (330010 000 IU) when a minimum of two follicles reached at least 1618 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, 1996). Progesterone was administered on day 3 after HCG administration (2550 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 MannWhitney U-test, Student's t-test, Fisher's exact test, 2 test, and analysis of variance where appropriate. P < 0.05 was considered significant.
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Results |
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Discussion |
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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., 1993, 1994
). 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., 1994; Vandromme et al., 1995
; Katz et al., 1996
), 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., 1996
). 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., 1994), 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., 1996
). 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., 1994), no association with systemic immunity to C. trachomatis or heat shock proteins and IVF outcome was demonstrated.
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Acknowledgments |
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Notes |
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References |
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Submitted on March 13, 1998; accepted on September 24, 1998.