Hum. Reprod. Advance Access originally published online on May 11, 2007
Human Reproduction 2007 22(7):1861-1865; doi:10.1093/humrep/dem107
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Chlamydia trachomatis infection, Fallopian tube damage and a mannose-binding lectin codon 54 gene polymorphism
1 First Department of Obstetrics and Gynecology, Faculty of Medicine, Semmelweis University, Baross utca 27, H-1088 Budapest, Hungary 2 Division of Immunology and Infectious Diseases, Department of Obstetrics and Gynecology, Weill Medical College of Cornell University, New York, NY, USA
3 Correspondence address. Tel: +36-1-266-04-73; Fax: +36-1-317-61-74; E-mail: sziller{at}noi1.sote.hu
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Abstract |
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BACKGROUND: Mannose-binding lectin (MBL), a component of the innate immune system, provides a first-line defense against invading microorganisms. Polymorphisms in the MBL gene have been associated with increased risk of infection. Chlamydia trachomatis genital tract infections are a major cause of Fallopian tube occlusion. Our objective was to test whether an MBL codon 54 polymorphism might contribute to development of C. trachomatis-associated tubal damage.
METHODS: In a case-control study, 97 women with occluded and 104 women with patent Fallopian tubes were tested for a history of chlamydial infection by serology and for their MBL codon 54 genotype by PCR and restriction fragment length polymorphism analysis. Clinical data were blinded to those performing all laboratory analyses.
RESULTS: Women with tubal occlusion who also had a positive chlamydial serology had the highest rate of variant MBL B allele carriage (P < 0.001). Among women who were chlamydial antibody negative, allele B carriage was also more frequent in those with blocked, as opposed to patent, Fallopian tubes (P < 0.01).
CONCLUSIONS: Wild-type allele A homozygosity is protective against, while carriage of the variant allele B is a risk factor for, Fallopian tube occlusion in women who are seropositive or seronegative for C. trachomatis.
Key words: Chlamydia trachomatis/gene polymorphism/mannose-binding lectin/serology/tubal occlusion
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Introduction |
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Chlamydia trachomatis is the major sexually transmitted bacterial pathogen in developed countries and is a primary cause of infertility, ectopic pregnancy and pelvic inflammatory disease (PID) (Paavonen and Lehtinen, 1996
; Sziller et al., 1998; Debattista et al., 2003). A unique aspect of a C. trachomatis reproductive tract infection is the absence of identifiable clinical symptoms in the majority of affected women. Most women who are diagnosed with infertility due to damaged Fallopian tubes, or who suffer an ectopic pregnancy, never had symptoms of an upper genital tract infection (Cates et al., 1993) and their exposure to this microorganism is only retrospectively determined by detection of anti-chlamydial antibodies (Tiitinen et al., 2006). The factors associated with chlamydial evasion of host immune defense mechanisms and migration from the lower to the upper female genital tract remain incompletely determined (Neuer et al., 2000; Debattista et al., 2003). The ability to predict which women with a C. trachomatis infection are at greatest risk for migration of the organism to the Fallopian tubes, microbial persistence at that site and developing subsequent tubal pathology would aid in determining optimal treatment protocols which may differ for individual women.
The innate immune system is the first line of defense against microbial invasion. Prior to acquisition of microbe- and antigen-specific antibody and cell-mediated immune responses, components of innate immunity recognize and respond to conserved structures present on different microorganisms. Mannose-binding lectin (MBL) is a vital component of this early anti-microbial protection system (Turner, 2003). By binding to carbohydrate residues, chiefly mannose, fucose and N-acetylglucosamine, present on microbial cell surfaces microorganisms are tagged for opsonization by phagocytic cells. In addition, MBL binding to microorganisms triggers activation of the complement cascade, resulting in cell killing as well as providing additional sites for opsonization (Neth et al., 2000; Jack and Turner, 2003). Deficiencies in MBL levels have been associated with increased susceptibility to, and modulated course and outcome of, infectious diseases (Eisen and Minchinton, 2003).
Glycoprotein components on the surface of C. trachomatis are rich in mannose, galactose, N-acetylglucosamine and fucose (Swanson and Kuo, 1991), and are known as ligands mediating attachment and infectivity of the pathogen (Kuo et al., 1997). In HeLa cell culture, MBL has been shown to inhibit infection by all chlamydial strains (Swanson et al., 1998). As a pattern recognition molecule in the first-line host defense, binding of MBL to sugar groups of the chlamydial major outer membrane protein blocks the attachment of organisms to host cells (Swanson et al., 1998). The inhibitory effect was shown to be abrogated by mannose or high-mannose type molecules.
The human MBL gene (MBL2) is located on chromosome 10 (q21–24), and functional polymorphisms have been identified in exon 1 (Garred et al., 2003). Codon 54 is the site of the most common MBL polymorphism in white populations (Babovic-Vuksanovic et al., 1999). The polymorphisms result in production of an unstable MBL polymeric protein that is rapidly degraded, resulting in greatly reduced concentrations of MBL in the circulation (Madsen et al., 1994; Babovic-Vuksanovic et al., 1999; Babula et al., 2004) and the vaginal fluid (Babula et al., 2004). Carriage of the variant allele B has been associated with repeated respiratory infections (Gomi et al., 2004). Moreover, two studies have reported that the extent of C. pneumoniae-mediated damage in coronary artery disease (Hegele et al., 2000; Rugonfalvi-Kiss et al., 2002) or asthma (Nagy et al., 2003) varied according to the MBL genotype. The presence of a variant genotype, associated with reduced MBL concentrations, was related to a more severe disease.
Analogous to these observations, we evaluated whether possession of the wild-type allele A or variant allele B at codon 54, exon 1 in the MBL-2 gene influenced the occurrence of Fallopian tube occlusion in women with or without evidence of prior C. trachomatis infection.
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Material and Methods |
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Between January 2004 and June 2005, consecutive patients scheduled for a diagnostic laparoscopy were asked to participate. Since single nucleotide polymorphisms (SNP) might depend on ethnic background, only native Hungarian Cuacasian women were included. Possible candidates for enrollment into the study were recruited from: (1) women who previously underwent hysterosalpingography as part of the infertility work-up, and had high suspicion for, but not evidence of, Fallopian tube blockage uni- or bilaterally (n = 77); (2) those attending for evaluation of their tubal patency by laparoscopic chromopertubation (n = 90) and (3) reproductive-aged women attending for legally approved tubal sterilization during the study period (n = 50). Tubal patency was tested before the procedure in each woman seeking tubal sterilization to rule out asymptomatic tubal blockage. Of these 217 women, blocked Fallopian tubes were diagnosed in 107 patients (cases), and patent Fallopian tubes without macroscopic evidence of tubal disease in 110 attendants (controls). Sixteen of the subjects, 10 with blocked tubes and 6 with open tubes, were diagnosed as having pelvic endometriosis, and were excluded from the analysis. Thus, the final number of case patients was 97, and that of the control women 104.
The study was approved by the Ethics Committees at Semmelweis University and Weill Medical College, and each participant gave her informed consent.
Both cases and controls were asked to fill out a questionnaire containing demographic parameters and their medical history including any kind of prior abdominal surgery, for example, appendectomy, pelvic surgery for benign diseases and cholecystectomy (laparotomy). At the time of surgery, peripheral blood was obtained from patients and sera stored at – 20°C until utilized. In addition, cells from the buccal mucosa were taken by rotating a cotton swab against the inside of the cheek. Specimens were collected and stored at 4°C for further testing.
Serum samples were assayed for immunoglobulin G (IgG) and IgA antibodies to C. trachomatis surface antigens by the commercially available C. trachomatis-pELISA (Medac GmbH, Hamburg, Germany) as well as for antibodies against the 60 kDa heat shock protein (HSP) of C. trachomatis (cHSP60-IgG-ELISA, Medac GmbH, Hamburg, Germany). The tests were performed and results interpreted according to the instructions of the manufacturer.
For the MBL gene polymorphism analysis, DNA was extracted by suspending the cells in a 1% solution of the non-ionic detergent, Brij 35 in Tris buffer of pH 8.3 containing 5 mg/ml proteinase K followed by incubation at 56°C for 60 min. Proteinase K was subsequently inactivated by incubation at 95°C for 10 min. The extracts were diluted 1:5 in 10 mmol/l Tris–HCl of pH 8.3 that contained 1.5 mmol/l MgCl2, 50 mmol/l KCl, 0.2 mmol/l each of dATP, dCTP, dGTP and TTP, 1.25 units of Taq DNA polymerase and 30 pmol of oligonucleotide primers that amplified the codon 54 polymorphic region of the MBL gene (Madsen et al., 1994; Babula et al., 2004) [ref. SNP ID: 17287498]. The final volume was 0.05 ml. Samples were incubated in a thermal cycler for 2 min at 94°C, followed by 35 cycles of 94°C for 50 s, 58°C for 1.5 min and 72°C for 15 s, and then by a final incubation at 72°C for 5 min.
The PCR amplicons were digested with Ban I endonuclease (New England BioLabs, Beverly, MA) by incubation at 37°C for 18 h. Fragments were analysed on 2% agarose gels and stained with ethidium bromide. Ban I digestion resulted in either formation of two bands, at 260 and 89base pairs (wild type, allele A), or a single uncut 349 base pair fraction (variant, allele B). Duplicate analysis of a subset of samples always yielded identical results.
Parameters with continuous distribution were compared using Student t-test. Genotype and allele frequencies were determined by direct counting and then divided by the number of chromosomes to obtain allele frequency and by the number of women to obtain genotype frequency. Associations between MBL genotypes or alleles and tubal status were analysed by the chi-square test or Fisher's exact test if appropriate. Goodness of fit to Hardy–Weinberg equilibrium was determined by comparing the expected genotype frequencies with the observed values, using the chi-square test (Chakravarti, 1999). A P-value < 0.05 was considered as significant. In addition, odds ratios and 95% confidence intervals were calculated.
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Results |
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Demographic characteristics of study population
The demographic characteristics and medical history of the study subjects with occluded and patent Fallopian tubes are compared in Table 1. The mean age of women with blocked Fallopian tubes was two years higher as compared to those with patent tubes (P < 0.05). Women with blocked tubes were more likely to be nulliparous (P < 0.01), smokers (P < 0.01) and to have had a history of PID (P < 0.025). Serologic evidence of past chlamydial infection, as evidenced by the presence of anti-chlamydial IgG (P < 0.01), IgA (P < 0.01) and anti-CHSP (P < 0.01), was also more prevalent in the group with occluded Fallopian tubes than in the controls (Table 1). Only those patients seropositive for anti-C. trachomatis IgG or IgA were positive for anti-CHSP-60 antibodies.
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MBL-54 genotype distribution and Fallopian tube status
The MBL genotype and allele distribution in women with patent or occluded Fallopian tubes is compared in Table 2. The women with blocked tubes had a decreased incidence of wild-type allele A homozygosity and an increased incidence of allele A/allele B heterozygosity and variant allele B homozygosity compared to the controls (P < 0.01). Carriage of the variant allele B was 29.4% in women with occluded tubes as opposed to 8.7% in the women with patent tubes (P < 0.01). While the genotype distribution of the women with patent tubes was in Hardy–Weinberg equilibrium (P > 0.05), the genotype distribution among the women with occluded tubes was not (P = 0.03).
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MBL-54 genotype distribution, Fallopian tube status and chlamydial serology
Associations between Fallopian tube status, MBL genotype distribution and chlamydial serology is summarized in Table 3. Since the associations between MBL genotype and tubal status were not influenced by the type of anti-chlamydial antibody (IgG, IgA or CHSP-60), an overall evaluation of chlamydial serology was used. Patients who had any of the three types of circulating anti-chlamydial antibodies tested were categorized as positives, while those without immunization as negatives.
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Among the 97 women with occluded Fallopian tubes, 54 (56%) were positive in at least one of the chlamydial antibody tests. This was greater than the 36.5% (38/104) seropositivity rate detected in women with patent tubes (P < 0.01) (Table 3). Furthermore, in women positive for anti-chlamydial serology, MBL allele A homozygosity was associated with open Fallopian tubes (P = 0.01) while MBL allele A/allele B heterozygosity (P < 0.05) and homozygosity for the variant allele B were associated with blocked tubes (P < 0.05). In addition, carriage of the variant B allele was strongly correlated with tubal occlusion (P < 0.001).
In C. trachomatis seronegative women, allele A homozygosity was again associated with open tubes (P < 0.025) while the frequency of heterozygotes was similar in the two subgroups. Carriage of the variant allele B, however, remained associated with blocked tubes (P = 0.01).
In the whole study group, the frequency of homozygosity for the variant B allele was highest among seropositive women with blocked Fallopian tubes (20.4%). Conversely, homozygosity for the wild-type allele A was most frequent in women seronegative for C. trachomatis and with patents tubes (89.4%).
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Discussion |
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In the present study, carriage of the variant allele B of MBL-2 was strongly associated with occlusion of the Fallopian tubes in consecutive native Hungarian women undergoing laparoscopy. In addition, homozygosity for the wild-type allele A was protective against tubal damage. Analysis of patients according to their prior exposure to genital chlamydial infection revealed that it was the subset of patients with blocked tubes and also positive for antibodies to C. trachomatis who had the highest prevalence of allele B carriage. These results suggest that MBL participates in the defense against C. trachomatis and that codon 54 allele B carriage is an increased susceptibility factor for Fallopian tube damage in infected women. We hypothesize that a defect in the first line of the innate host defense in patients with the variant codon 54 B allele might contribute to the persistence of C. trachomatis in the female upper genital tract. Persistence of this microorganism has been associated with a higher risk for tissue scarring and sometimes occlusion of the Fallopian tubes (Cohen and Brunham, 1999
).
Early studies have shown that, besides C. trachomatis, other microorganisms including gram-positive and gram-negative bacteria of the lower genital tract, genital Mycoplasmatales and aerobic bacteria might also play an important role in the etiology of female upper genital tract infection and its late consequences (Paavonen and Lehtinen, 1996). Although less pronounced than in the antibody-positive women with damaged tubes, women with blocked Fallopian tubes who were negative for chlamydial antibodies also had an increased carriage rate of MBL allele B as compared to antibody-negative women with patent tubes. This supports that an upper genital tract infection caused by bacteria other than C. trachomatis can also induce Fallopian tube blockage, and implies that these microorganisms are also susceptible to MBL-mediated regulation. We suggest that in these cases, some microorganisms of the ascended mixed aerobic–anaerobic flora in patients with acute PID which contain sugar groups on their surface might evade first-line host defense elicited by decreased production of MBL, and, consequently, lead to prolonged infection/inflammation and a higher risk of tubal occlusion.
In both subgroups (i.e. women with patent Fallopian tubes positive for chlamydial serology (subgroup 1) and women with patent Fallopian tubes negative for chlamydial serology (subgroup 2)), the MBL genotype distribution of the women with patent Fallopian tubes was in Hardy–Weinberg equilibrium, emphasizing that these subjects were representative of the overall population. Deviation of the genotype distribution of the women with blocked tubes from Hardy–Weinberg equilibrium strongly supports the association between carriage of this genetic variant and risk for this specific pathology.
Our study did not focus on the possible genotype variations in other members of the pattern recognition receptors of the host organism, including toll-like receptors and their co-receptor cluster of differentiation, etc. In a recent study, compound SNP's in the pattern recognition system have been associated with an increased frequency of tubal pathology following C. trachomatis infection (Den Hartog et al., 2006). Similarly, an association between cellular immune response to CHSP-60, human leukocyte antigen class II alleles and interleukin-10 (IL-10) promoter genotypes has been demonstrated with C. trachomatis-associated tubal factor infertility (Kinnunen et al., 2002). In a subsequent study, Wang et al. (2005) provided further evidence on the involvement of IL-10 genotype in the susceptibility of high-risk adolescents to recurrent genital chlamydial infection. We can not exclude that some of our C. trachomatis seronegative patients with tubal occlusion had a variant genotype with these components of the host immune system. The responses to a microbial infection vary considerably among different individuals. We have suggested previously that identification and recognition of the variables influencing susceptibility to specific sequelae is critical for development of focused treatments with improved efficacy for individual women (Witkin et al., 2000; Ledger and Witkin, 2006).
Carriage of the MBL codon 54 B variant has been reported in 22–28% of European and North American populations (Turner, 2003), which is in accordance with the rate found in healthy women in our study. Similarly, homozygous carriage of the wild-type allele in healthy Hungarian male and female controls to patients with coronary artery disease (Rugonfalvi-Kiss et al., 2002) or in Hungarian asthmatic children seropositive for C. pneumoniae (Nagy et al., 2003) were in the same range as seen in our patients.
Our data suggest that women homozygous for the variant B allele, and with a documented C. trachomatis infection, are at increased risk for tubal occlusion. Therefore, the identification (by PCR testing) of women with current lower genital tract infections who need treatment is important, as is a risk assessment for tubal pathology by serologic and immunologic testing. It is interesting to speculate that women newly infected with C. trachomatis (e.g. acute cervicitis) might benefit from testing for the MBL polymorphism and that those positive for allele B be given a more prolonged course of antibiotic treatment to compensate for their relative impairment of an innate anti-chlamydial defense mechanism. In addition, they could be counseled to seek prompt medical evaluation including C. trachomatis test for early signs and symptoms of genital tract infection.
Further, immunogenetic studies evaluating the role of genetic variations in immunologically important host defense genes as determinants of the susceptibility, course and consequences of genital chlamydial infections are needed to provide a more thorough insight into the pathomechanisms leading to definitive tubal damage. Future strategies might consider not only single but also compound variations. This kind of research might contribute to a more individualized approach in the management of these infections.
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