1 Department of Medical Microbiology and Immunology, Aarhus University, The Bartholin Building, 8000 Aarhus C and 2 The Fertility Clinic & The Scientific Unit, Brædstrup and Horsens Hospitals, 8740 Brædstrup, Denmark
3 To whom Correspondence should be addressed. Email: agata{at}medmicro.au.dk
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Abstract |
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Key words: antibodies/ELISA/immunoblot/membrane proteins/Mycoplasma hominis
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Introduction |
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M. hominis is an opportunistic pathogen and a common inhabitant of the female lower genital tract and it is believed to be sexually transmitted (Elshibly et al., 1996). It has been isolated from cases of post-partum fever and sepsis. However, the most common infection is that of the genito-urinary tract. In women with bacterial vaginosis (BV), M. hominis is frequently found (at least in 2/3 of women with BV, compared with 10% of healthy women) (Rosenstein et al., 1996
). Isolation from cervix, endometrium and Fallopian tubes of women with salpingitis associated M. hominis with development of salpingitis and pelvic inflammatory disease (PID) (Mardh and Westrom, 1970
). By use of a microimmunofluorescence technique it was shown that antibodies to M. hominis were found three times more often in infertile women who had PID than in healthy controls (Moller et al., 1985
). It is therefore believed that M. hominis has the potential to cause infertility as a result of tubal diseases (Taylor-Robinson, 1996
). The exact mechanism of pathogenesis of this species, however, has not been revealed.
Serological tests are not easy to develop due to the significant heterogeneity and antigenic variation among different M. hominis isolates (Andersen et al., 1987). The study revealed that different isolates had only between 41 and 72% of their proteins in common. In later studies (Christiansen et al., 1990
; Olson et al., 1991
) it was specified that these differences were found in membrane proteins and less frequently in cytoplasmic proteins. The membrane proteins are believed to be most immunogenic and are referred to as surface antigens.
In this study we show that past infections with M. hominis can have a relation to human reproductive failure. The measurement of antibody response was performed with ELISA. We show that a mixture of membrane proteins from two different M. hominis isolates is preferred as antigen rather than membrane proteins from just one isolate, due to the significant heterogeneity among M. hominis isolates.
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Materials and methods |
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Patient serum sample collection
The study was a part of the project approved by the local Scientific Ethical Committee (ref. no: VF 20010161). Serum samples were obtained from 304 women undergoing IVF treatment during the years 19972000. For detailed description of patient material see Clausen et al. (2001). All women were examined by hysterosalpingography (HSG) and laparoscopy. The patient blood samples were taken when they began the first in vitro fertilization (IVF) treatment. The women were classified according to the cause of infertility into three diagnostic groups: (i) 132 women with tubal factor infertility (TFI), (ii) 64 in the male factor infertility group (MFI), and (iii) 108 in the unexplained infertility group (UFI).
As normal material we used 31 serum samples obtained from healthy female blood donors collected at Skejby Hospital, Aarhus, Denmark. However, no information of the tubal status of the blood donors was available.
SDSPAGE and immunoblotting
The pellets of each M. hominis isolate were suspended in SDS sample buffer (62.5 mM, 10% v/v glycerol, 2.3% v/w SDS, 5% v/v -mercaptoethanol, 0.05% w/v bromphenol blue), heated to 100 °C for 2 min and separated by SDSPAGE (7.5% SDS polyacrylamide gel with a 5% stacking gel). Proteins were electrotransferred to nitrocellulose membranes (Schleicher & Schull, Dassel, Germany). The molecular weight marker was cut out of the membrane and stained with Amido Schwartz. The membranes were blocked overnight with blocking buffer (20 mM Tris-base, 150 mM NaCl, 3% gelatine) at 4 °C. The immunostaining was performed as described previously (Birkelund and Andersen, 1988
).
Preparation of antigen and ELISA
Pellets obtained from harvesting 120 ml of culture were suspended each in 800 µl of TrisHCl (50 mM TrisCl pH 8.0, 0.15 M NaCl) and sonicated for 10 s by ultrasound using the Ultrasonic Processor Vibra Cell VCX 600 Watt (Sonics & Materials, Newtown, CT) set at amplitude 20%. After sonication the concentration of proteins was adjusted to 1 mg/ml and Triton X-114 (TX-114) was added up to the final concentration of 1%. The M. hominis protein solutions with Triton X-114 were kept for 1 h on ice. To remove insoluble proteins the solution was centrifuged for 15 min at 20 000 g and 4 °C. For separation of aqueous and detergent phases the procedure of Bordier (1981) was applied. The protein content of each phase was analyzed by SDSPAGE protein separation on 12.5% acrylamide gels and stained with Coomassie Blue.
Coating of 96-well microtitre ELISA trays (Maxisorb, Nunc, Roskilde, Denmark) was done with 60 µl of antigen per well. Plates were incubated overnight at 4 °C. To block the antigen, the plates were incubated for 1 h with 75 µl of 15% FCS (fetal calf serum in PBS). Serum samples were diluted 1:50 in antibody buffer (medac, Hamburg, Germany), tested in duplicate wells and incubated for 1 h at 37 °C. Fifty microlitres of a mixture of goat anti-human IgG (Jackson Immunoresearch Laboratories, Inc., West Grove, USA) horseradish peroxidase labelled (dilution 1:40 000) and unlabelled (0.26 µg/ml) was used as a secondary antibody. Plates were developed with 50 µl tetramethyl benzidine (medac, Hamburg, Germany) for 30 min at 37 °C and the reaction was stopped with 100 µl of 1 N HCl. ELISA trays were washed between each incubation with PBS +0.05% Tween-20. Then the plates were read by Sunrise-reader (Tecan, Salzburg, Austria) at a 450 nm wavelength with a reference length of 620 nm.
Statistical analysis
SPSS-software was used for analysis of data. Receiver-operating characteristics (ROC) curves were used to analyse correlation between tubal factor infertility and seropositivity to M. hominis. Multiple logistic regression analysis was used to analyse the connection between seropositivity to M. hominis and a list of variables such as: indicators for each of three patient groups, patients' age, age squared and seropositivity to M. genitalium and C. trachomatis. The statistical model was reduced by backward selection, where insignificant variables are removed one after another.
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Results |
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Triton X-114 phase separation of proteins
The partition of proteins of the M. hominis isolates 132 and 4195 was performed in the detergent Triton X-114, which is used to solubilize membrane proteins from whole cells. The partition resulted in three different protein phases: the insoluble protein phase, which was collected and removed; the aqueous phase containing hydrophilic, mostly cytoplasmic proteins; and the detergent phase containing hydrophobic mostly integral membrane proteins with an amphophilic nature, which are believed to be most immunogenic and species specific. After each separation, detergent, aqueous and total protein phases were compared by SDSPAGE electrophoresis. The protein profiles of isolates 132 and 4195 were similar but small differences were noticed (Figure 2). When comparing the detergent and aqueous phase of the same isolate, elimination of many cytoplasmic proteins can be seen. Only the detergent phase was used as antigen in ELISA.
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Results of the ELISA test
Sera from 304 infertile women and from 31 healthy female blood donors were analyzed by the ELISA, using the mixture of detergent phases from two M. hominis isolates as antigen for coating ELISA plates. The OD450 values for patients belonging to each of the groups were analyzed and are shown in Figure 8. In all groups the largest number patients were in the negative range from 0 to 0.19. In the TFI group (Figure 7A) a generally greater frequency of the samples with the high OD450 values was seen, especially in the range between 1.4 and 2.19. A distribution most similar to the TFI group was observed in the UFI group (Figure 7B), where several high OD450 values were present. The least number of high OD450 values was seen in the MFI and blood donor groups (Figure 7C and D).
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Among 31 healthy women blood donors used as a negative control we found three (9.7%) patients that were positive in the ELISA.
Comparison of results from ELISA and immunoblotting
The results obtained from ELISA and immunoblotting were compared (Table II). In the ELISA, 97 patients were positive to M. hominis, while 84 patients were positive in immunoblotting. Sixty-nine (22.6%) patients were positive by both tests, 15 (4.9%) were positive in immunoblotting but not ELISA test, and 28 (9.2%) were positive only in ELISA.
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When analysing women from the TFI group for presence of antibodies against M. hominis, C. trachomatis and M. genitalium, 23 patients were positive only to M. hominis (Table III), 14 were positive for both M. genitalium and M. hominis, and 28 had antibodies to M. hominis and C. trachomatis. There were 10 patients that had antibodies to all three genital bacteria. In summary, out of 132, 106 (80%) women were seropositive to at least one of the three genital agents: M. hominis, M. genitalium or C. trachomatis (Table III).
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For the multiple logistic regression analysis we used binominal data (0, negative; 1, positive) transformed from the measurement data by introducing the cut-off OD450 value. In our statistical model we tested for a reduction where the aetiology was composed of only two groups: women with normal tubes: 172, and women with TFI: 132. The reduction was accepted (P=0.8177). We found significant correlation between tubal factor infertility and seropositivity to M. hominis (P=0.0015, OR = 2.21, 95%CI = 1.353.61). TFI patients had a 2.13-fold higher risk of having antibodies against M. hominis compared with patients with normal tubes, when odds ratios were interpreted as a relative risk.
We did not find any significant correlation between presence of antibodies against M. genitalium, C. trachomatis and seropositivity to M. hominis, when statistically tested (P>0.05).
Since M. genitalium and C. trachomatis are both correlated with development of TFI we analyzed whether M. hominis could be an independent predictor of TFI, by defining patients from the TFI group as a dependent variable and seropositivity to other species as independent variables in our statistical model. The presence of significant correlation to M. hominis with P=0.006, OD = 2.1, 95%CI = 1.2533.706, suggested that M. hominis can cause TFI independently of C. trachomatis and M. genitalium. Both M. genitalium and C. trachomatis were also significantly correlated to TFI as described by Clausen et al. (2001).
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Discussion |
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When the whole cell lysate from single isolates was used as an antigen for ELISA, none of the M. hominis isolates could detect more than 87% of positive sera (Brown et al., 1987). Therefore, the use of antigen pools from different isolates was suggested for optimal antibody detection. These serological findings are in agreement with our observations. When antigens from two different isolates were compared separately by ELISA some of the patients gave a positive response to only one of the isolates (Figure 4). Thus, a mixture of the two isolates 132 and 4195 was used in our serological study. In a recent study, where lipid-associated proteins (LAMPs) were used as antigen (Lo et al., 2003
) only one isolate of M. hominis was suggested for ELISA, despite the existence of antigenic variation between different isolates. This is, however, in disagreement with our findings.
The isolates 132 and 4195 were selected based on the isolation origin (both isolated from vagina) genomic, and phylogenetic differences (Andersen et al., 1987; Ladefoged et al., 1996
; Boesen et al., 1998
). A split graph analysis of different housekeeping genes (Sogaard et al., 2002
) revealed similarities and differences between M. hominis isolates where isolate 132 was very similar to isolate DC63 in all analyses, but different from isolate 4195. Moreover, the analysis of Triton X-114 protein profiles by SDSPAGE showed differences between those two isolates (Figure 2). The reason of excluding from ELISA isolate 7488, which was used in the immunoblotting, was based on the similarities in immunoblotting profiles of isolates 7488 and 132, and also that no additional sera were ELISA positive when detergent phase proteins of 7488 were included. Type strain PG21 was not selected, since in many tests, including what was found by Sogaard et al. (2002)
, it reacted differently from other isolates.
Differences observed in the results of immunoblotting and ELISA may reflect differences in the type of antigen used. Failure of the immunoblotting to detect 28 patients positive by ELISA may be related to increased sensitivity in ELISA. The antigen used for immunoblotting, unlike ELISA antigen, consisted of both cytoplasmic and membrane antigens. A cross-reactivity problem with cytoplasmic proteins could be one of the reasons why 16 patients were found positive only by immunoblotting and not by ELISA. Differences between immunoblotting and ELISA results even when the same antigen was used were described by Baseman et al. (2004), who suggested that the reason may involve different protein conditions used in the tests (reducing conditions in SDSpolyacrylamide gel electrophoresis are used in immunoblotting in contrast to nondenaturating conditions in the ELISA).
Statistical analysis of data from 304 infertile patients was performed using two tests, ROC curves (measurement data) and multiple logistic regression analysis (binominal data). To change data from measurement to binominal, a cut-off OD450 value was introduced. The estimation of the cut-off OD450 value can be performed by testing serum samples from the negative patients, calculating the mean OD450 value, plus three standard deviations. Mycoplasma hominis infections are mostly asymptomatic (none of the patients had any symptoms of PID when they visited the infertility clinics) and thus it was difficult to justify which patients were truly negative. Therefore, the use of both ELISA and immunoblotting was beneficial in estimating the negative patients. Among healthy blood donors expected to be less often positive to M. hominis, three had high IgG antibody titre, which could be explained by the retrospective character of the immunological assay. Antibodies may remain in the serum even when the patient does not harbour the microorganism anymore. Both statistical methods showed correlation between presence of antibodies to M. hominis and tubal factor infertility (TFI).
The results of this study suggested that M. hominis is related to development of TFI, probably due to tubal infection and scarring. The earlier study (Mardh and Westrom, 1970) where M. hominis was isolated in pure culture from the Fallopian tubes in 12.9% of the patients with acute salpingitis (and who also had M. hominis in the cervix), supports our findings. The influence that M. hominis can have on the epithelial cells in Fallopian tubes was analyzed in an in vitro organ study, where a noticeable swelling occurred in the cilia of epithelial cells after an overnight incubation with M. hominis (Mardh et al., 1976
). A study by Taylor-Robinson and Carney (1974)
failed, however, to show damage or loss of ciliary activity in mycoplasma-infected Fallopian tube organ culture.
When the TFI patient group was tested as a dependent variable and seropositivity to different genital microorganisms as independent variables, all three bacteria were significantly correlated with tubal factor infertility.
There was no correlation between positivity to M. hominis and positivity to the other two genital microorganisms, M. genitalium and C. trachomatis, and 23 TFI patients were positive only for M. hominis. This indicates that M. hominis can be an independent factor of tubal factor infertility.
Interestingly, 80% of the TFI cases were associated to a past bacterial infection with one of the three genital agents: M. hominis, M. genitalium, C. trachomatis, since the patients were seropositive to at least one of the microbes. The majority of patients (75) were seropositive to C. trachomatis; however, 31 had antibodies to M. hominis and/or M. genitalium which may indicate that genital mycoplasmas are important tubal pathogens.
Ten TFI patients had antibodies to all three microorganisms. It is, however, not known whether the patients had infections with those bacteria at the same time (co-infections) or at different time periods. In the case of co-infection it is important to diagnose all infectious microbes because different antibiotics should be given for C. trachomatis, M. genitalium and M. hominis infections (Falk et al., 2003; Ngan et al., 2004
).
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Acknowledgements |
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References |
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Submitted on November 10, 2004; resubmitted on December 17, 2004; accepted on January 11, 2005.
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