Objective
The objective of the study was to characterize endometrial inflammation associated with common genital tract pathogens.
Study Design
The design of the study was the immunohistochemical characterization of the endometrial leukocyte subpopulations from 37 controls and 45 women infected with Chlamydia trachomatis , Neisseria gonorrhoeae , or Trichomonas vaginalis .
Results
Compared with uninfected women, endocervical infection with C trachomatis , N gonorrhoeae , or T vaginalis was associated with significant increases in endometrial T cells, B cells, plasma cells, and polymorphonuclear leukocytes. Even more substantial increases in T cell, B cell, and plasma cell numbers were detected among women infected endocervically and endometrially with C trachomatis .
Conclusion
Because lower genital tract C trachomatis , N gonorrhoeae , or T vaginalis infections were associated with comparable increases in the same endometrial leukocyte subpopulations, our results suggest the underappreciated involvement of T vaginalis in upper genital tract inflammatory processes. The more robust inflammatory infiltrate associated with C trachomatis endometrial ascension may offer insight into host inflammatory responses associated with pelvic inflammatory disease development.
Endometritis is a histopathological diagnosis formed by the identification of abnormal endometrial inflammatory cell infiltrates and disturbances in normal endometrial growth. The leukocyte subpopulations most firmly established with diagnosis of histological endometritis are polymorphonuclear leukocytes (PMNs) and plasma cells. Although they are not necessarily indicative of disease duration, inflammatory cell infiltrates are also used to stratify endometritis into acute and chronic forms of disease.
Acute endometritis is identified by the presence of PMNs in the absence of menstrual endometrium, whereas chronic endometritis relies on plasma cell identification in the absence of neutrophilic infiltration. Although offending pathogens are not always found, migration of Chlamydia trachomatis or Neisseria gonorrhoeae from the lower to upper genital tract is the most common cause of chronic (also called plasma cell) endometritis.
Because the diagnosis of chronic endometritis has been associated with the development of pelvic inflammatory disease (PID), chronic endometritis may represent an intermediary step between lower genital tract infection and upper genital tract damage. Accurate identification of histological endometritis, however, is problematic because many leukocyte subpopulations are also present in normal endometrial tissue. Adding to this diagnostic complexity, the frequency of some endometrial leukocyte subpopulations varies with stages of the menstrual cycle.
In proliferative and early secretory phases, the predominant leukocyte subpopulations are macrophages, natural killer (NK) cells, and T cells (accounting for approximately 10% of endometrial stromal cells), whereas in the late secretory stage, these leukocyte subpopulations account for 20% of the stromal cells. This increase is primarily the result of an increase in NK cell numbers because T cell numbers remain relatively constant throughout the menstrual cycle. Unlike NK and T cells, plasma cells are infrequently detected in normal endometrial tissue, whereas B cells are only sporadically identified (comprising less than 2% of all endometrial leukocytes).
Although prior immunohistochemistry-based studies comparing leukocyte subpopulations among nonpregnant women with normal endometrium and women with chronic endometritis found no differences in the number of endometrial macrophages and NK cells, large increases in endometrial B cells, and more modest increases in endometrial T cells, have been associated with diagnosis of chronic endometritis. Less well described, however, is the endometrial inflammatory infiltrate associated with the specific genital tract pathogens causing chronic endometritis.
In the present study, we performed immunohistochemical analyses of endometrial tissue using markers specific for NK cells, T cells, B cells, plasma cells, and macrophages to characterize endometrial inflammatory responses associated with genital tract infection with C trachomatis , N gonorrhoeae , and a putative PID pathogen, Trichomonas vaginalis .
Materials and Methods
We selected 82 paraffin-embedded endometrial specimens from a previous investigation of the risk factors associated with PID. Both the parent and the current investigations were approved by the University of Pittsburgh’s Institutional Review Board.
In the parent study, endocervical swab specimens had been collected for culture-based detection of N gonorrhoeae and T vaginalis and polymerase chain reaction (PCR)–based detection of C trachomatis . Transcervical endometrial biopsy specimens had also been obtained, and a tissue portion had undergone identical diagnostic testing for C trachomatis , N gonorrhoeae , and T vaginalis , whereas another portion had been paraffin embedded.
For the current study, we selected paraffin-embedded endometrial samples from 37 women who had no evidence of C trachomatis , N gonorrhoeae , or T vaginalis infection. Among the other selected samples, 27, 7, and 11, respectively, had been collected from women with endocervical C trachomatis , N gonorrhoeae , or T vaginalis infection. None of the cases selected were from women coinfected with any of 2 or 3 of these genital tract pathogens, whereas 10 of the 27 women with endocervical C trachomatis infection had also been diagnosed with endometrial C trachomatis infection.
Endometrial specimens were excluded from further analysis if hematoxylin and eosin (H&E)–stained slides demonstrated menstrual cycle stage consistent with late secretory or menses because increased interstitial hemorrhage, tissue necrosis, and neutrophilic infiltration make interpretation of endometrial inflammatory changes more problematic. H&E-stained slides were also used to grade PMN infiltrate in 10 high-power (×400) fields of endometrial surface epithelium using a 0-2 semiquantitative scale: grade 0, no PMNs detected; grade 1, mean number of 1-4 PMNs per field; grade 2, mean number 5 or more PMNs per field.
The monoclonal antibodies used in this investigation (and leukocyte subpopulations they identify) were CD3 (T cells), CD8 (CD8 + T cells), CD20 (B cells), CD56 (NK cells), CD68 (macrophages), CD138 (plasma cells), and the class II major histocompatibility complex (MHC) molecule ( Table ). MHC class II molecule staining was performed without antigen retrieval, determination of CD8, CD56, and CD138 expression utilized microwave antigen retrieval in citrate buffer and indirect immunolabeling, and CD3, CD20, and CD68 immunostaining was completed using Ventana prediluted antibodies (Ventana, Tucson, AZ).
| Antibody | Cluster of differentiation | Cellular reactivity | Source |
|---|---|---|---|
| 2GV6 | CD3 | CD3 + lymphocytes | Ventana |
| C8/144B | CD8 | CD8 + lymphocytes | Dako |
| L26 | CD20 | B lymphocytes | Ventana |
| 123C3.D5 | CD56 | NK cells | Neomarkers |
| KP-1 | CD68 | Macrophages | Ventana |
| B-B4 | CD138 | Plasma cells | AbD Serotec |
| CR3/43 | HLA-DP, DQ, DR | MHC II | Dako |
Positive control slides were included with each stain to confirm antibody activity, whereas absence of reactivity among negative controls confirmed the adequacy of blocking procedures and the specificity of positive results. Two surgical pathologists blinded to all infectious disease diagnoses independently assessed each slide.
Positive cells for each marker were graded using a 0-4 semiquantitative scale: grade 0 = no positive cells; grade 1, single scattered positive cells; grade 2, loosely scattered cells or occasional small aggregates; grade 3, densely scattered cells; and grade 4, dense collections of positive cells throughout entire specimen. Because the vast majority of CD3 + T cells in the endometrium are either CD8 or CD4 positive, immunohistochemical identification of CD3 + – and CD8 + -positive cells also allowed determination of endometrial CD4 + T cell numbers.
Statistical analyses were completed using GraphPad Prism 5 software (San Diego, CA). Calculation of medians and percentile distributions were used to determine semiquantitative differences in each of the measured leukocyte subpopulations between the following: (1) women with no genital tract infection or women diagnosed with lower genital tract C trachomatis , N gonorrhoeae , or T vaginalis infection and (2) women with no genital tract infection, women diagnosed with endocervical C trachomatis infection, or women diagnosed with endocervical and endometrial C trachomatis infection. Comparisons of scores between groups were performed using a Kruskal-Wallis test on ranks and Dunn’s multiple comparison post hoc test ( P < .05 was considered statistically significant).
Results
We first compared PMN numbers and the number of CD3, CD8, CD20, CD56, CD68, CD138, and MHC class II–positive cells in the endometria of women with no genital tract infection with the number of these cells seen in the endometrial tissue of women diagnosed with C trachomatis or N gonorrhoeae endocervical infection, the 2 most firmly established infectious causes of upper genital tract inflammation.
Similar to the results from prior comparisons of endometrial biopsies from healthy women and women diagnosed with chronic endometritis, we found no significant differences in the numbers of endometrial macrophages and NK cells among uninfected women and women with endocervical C trachomatis or N gonorrhoeae infection ( Figure 1 ).
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
