Genome-wide identification of Chlamydia trachomatis antigens associated with tubal factor infertility

Abstract

Objective: To identify Chlamydia trachomatis antigens that can be used to differentially diagnose tubal factor infertility in comparison with previously reported heat shock protein 60.

Design: In vitro study.

Setting: Academic medical center.

Patient(s): Infertile women with and without tubal pathology diagnosed laparoscopically.

Intervention(s): None.

Main outcome measure(s): Antibody responses to C. trachomatis in infertile women with or without tubal pathologies using a C. trachomatis genome-wide proteome array.

Result(s): Comparison of the antibody profiles revealed 30 C. trachomatis antigens that were preferentially recognized in women with tubal factor infertility, with a detection sensitivity and specificity of 80.6% and 56.5%, respectively, 10 of which showed 100% specificity. A combination of CT443 and CT381 antigens yielded the highest detection sensitivity (67.7%) while maintaining 100% specificity.

Conclusion(s): These findings have demonstrated that antibodies to CT443 and CT381, when used in combination, have higher sensitivity and specificity in predicting tubal factor infertility than other indicators for tubal factor infertility, such as heat shock protein 60 antibodies (35.5%, 100%) or hysterosalpingogram (65%, 83%). Using a panel of C. trachomatis antigens to serologically diagnose tubal factor infertility can save the patients from undertaking expensive and invasive procedures for determining tubal pathology and choosing treatment plans.

Figure 1. Reactivity of 54 infertility sera with 933 GST-chlamydial fusion proteins representing 908 unique ORFs of C. trachomatis

Each of the human Antibodies (displayed along the x-axis on top of each panel) after 1/2000 dilution was reacted with each of the 933 GST fusion proteins (listed along the y-axis) immobilized onto the 96-well microplates. Each colored bar represents a positive reactivity between a given fusion protein and a patient serum with the tubal factor infertility (TFI) patient sera in green and infertile control (IFC) in red. The results are expressed as optical density (OD) readings obtained at the wavelength of 405 nm. Any reaction with an OD ≥ mean + 2 Standard deviations calculated from the same plate is defined positive. The positive OD values are expressed as binding intensity in increasing brightness of fluorescent color. The negative OD values are black. The 933 fusion proteins representing the C. trachomatis genome are first listed in order of the ORFs from CT001 to pCT08 (panel A). Both letter and number extensions were used to distinguish multiple fusion proteins and protein fragments that share the same ORF names. (B) The 933 fusion proteins were then reordered based on binding frequency by the TFI sera from high (top) to low (bottom). As indicated on the right of the panel, a total of 265 ORFs were recognized by one or more sera. (C) By visual inspection of the expanded view of the positive antigens, the fusion proteins preferentially recognized by either TFI (green) or IFC (red) patients were marked with brackets on the right of the panel. (D) A total of 30 antigens were significantly recognized by TFI patients.

Figure 2. Reactivity of 30 C. trachomatis antigens with 54 patient sera at 1:4000 dilution

The 30 antigens were reacted with the 54 human sera as described in table Table 2 note except that each serum was diluted 1:4000 (data not shown). (A) Ten of the 30 antigens, failing to react with any of the 23 IFC sera, were thus presented in the figure. Note that HSP60 (CT110) and OmcB (CT443) maintained a detection sensitivity of 35.5% and 58% respectively. (B) The reactivity of each of the 10 antigens was analyzed at individual antiserum level. Note that the combinations of CT443 with CT381 or HSP60 with CT376, CT381 & CT798 can have the highest sensitivity of 67.7% while maintaining 100% specificity. (C) The reactivity intensity between each antigen and the 21 positive sera (measured at individual antiserum level) was expressed as mean OD plus standard deviation. (D) Each of the 10 antigen were reacted with an antiserum sample pooled from the 21 sera at equal ratio without (D) or with absorption with C. trachomatis (CT)-infected HeLa lysate (E) or HeLa alone lysate (F). Note that absorption with CT-HeLa but not HeLa alone lysates removed the reactivity of each antigen with the pooled antiserum.