Dan C. Martin, M.D.*t Vivek K. Khare, M.D.:j: Brigitte E. Miller, M.D.*

FERTILITY AND STERILITY Copyright 1995 American Society for Reproductive Medicine Vol. 63, No.1, January 1995 Printed on acid-free paper in U. S. A. Association of Chlamydia trachomatis immunoglobulin gamma titers with dystrophic peritoneal calcification, psammoma bodies, adhesions, and hydrosalpinges Dan C. Martin, M.D.*t Vivek K. Khare, M.D.:j: Brigitte E. Miller, M.D.* Baptist Memorial Hospital, University of Tennessee, Memphis, Tennessee Objective: To correlate Chlamydia trachomatis immunoglobulin gamma (lgg) titers with psammoma bodies, dystrophic peritoneal calcification, degree of calcification, adhesions, and hydrosalpinges. Design: This is a prospective single-blinded histologic analysis of tissue and retrospective analysis of historical laboratory and clinical variables. Setting: Tertiary hospital and private practice patient charts. Patients: Sixty consecutive patients with C. trachomatis lgg titers reported on the coding sheets of a previous study for endometriosis. Main Outcome Measures: The histologic slides were reviewed in a blinded fashion for calcification. Previously used data sheets were reviewed for C. trachomatis lgg titers. Historical data, adhesion scores, hystrosalpingogram findings, and laparoscopic findings were obtained from charts. Results: Dystrophic calcification, psammoma bodies, moderate-to-severe dystrophic calcification and hydrosalpinges were associated with positive C. trachomatis lgg titers. Conclusion: This study suggests relationship of C. trachomatis with dystrophic calcification, psammoma bodies, adhesions, and hydrosalpinges. This relationship suggests that C. trachomatis lgg titers can be used as a marker to help determine those infertility patients who might best benefit from hysterosalpingogram or laparoscopy and in clinical studies of endometriosis, infertility, pain, or ovarian cancer. However, there is no current data to suggest a need for therapy on the basis of a positive C. trachomatis lgg titer or of dystrophic peritoneal calcification. Fertil Steril 1995;63:39-44 Key Words: Chlamydia trachomatis, C. trachomatis, infertility, dystrophic calcifications, psammoma bodies, lgg titers, endometriosis, adhesions, peritoneal adhesions, pelvic pain, ovarian cancer. Dystrophic calcification refers to the deposition of calcium salts in tissue. It is noted generally in infarcts or regions of fibrous scarring and is believed to represent crystallization of intracellular Received January 25, 1994; revised and accepted July 15, 1994. * Department of Obstetrics and Gynecology. t Reprint requests: Dan C. Martin, M.D., 910 Madison Avenue, Suite 805, Memphis, Tennessee 38103 (FAX: 901-525-0253). :j: Department of Pathology. Division of Gynecologic Oncology. calcium. Psammoma bodies are discrete and unique forms of dystrophic calcification that represent cross-sections of calcified papillary formations. Histologically, these lesions are concentric and laminated. Psammoma bodies have been described in a variety of benign and malignant pathologic states. These include endosalpinglosis, inflammatory lesions, endometriosis, ovarian cystadenofibroma, serous papillary tumors of the ovary, meningiomas, papillary carcinoma of the thyroid, tuberculosis and neoplastic papillary lesions of the kidney, Vol. 63, No. 1, January 1995 Martin et al. Calcification and chlamydia titers 39

breast, lung, pituitary gland, and endometrium (1-8). Previous gynecologic publications have spanned conclusions ranging from a need to exclude primary epithelial neoplasia whenever psammoma bodies are found (1, 2) to a need to distinguish benign from malignant features in papillary structures containing psammoma bodies (3). Psammoma bodies are nonspecific. The diagnosis of malignancy is based on malignant features such as epithelial component atypicality and desmoplastic response. Caution has been suggested to avoid a diagnosis of disseminated ovarian cancer when focal ovarian cancer is associated with disseminated psammoma bodies (4). In spite of these warnings, the finding of psammoma bodies on Papanicolaou smear, in the absence of malignant features, has been read as compatible with serous cystadenocarcinoma. This reading persisted in one patient and resulted in four surgical procedures ending with a total abdominal hysterectomy with bilateral salpingo-oophorectomy (5). Dystrophic peritoneal calcification has been reported in peritoneal biopsies of women with positive Chlamydia trachomatis immunoglobulin gamma (IgG) titers (9). These were described as grain-like lesions. Some of these has been diagnosed incorrectly as endometriosis while others were coexistent with endometriosis. In addition, psammoma bodies also have been found in clear and white lesions. Similar white lesions biopsied at laparoscopy have been metastatic breast cancer (9). In a study of patients with pain and/or infertility, Ripps (10) has documented a prevalence of positive C. trachomatis IgG titers in 35% of patients with endometriosis and 68% of patients with no endometriosis. Coexistence of these two diseases and the similarity of appearances require precise histology to come to a correct diagnosis. The purpose of this study is to correlate the prevalence of dystrophic calcification, tubal disease (i.e., hydrosalpinx), and level of The American Fertility Society (AFS) adhesion score (11) with C. trachomatis lgg titers. Other areas of investigation included Fitz-Hugh-Curtis adhesions and cervical C. trachomatis direct fluorescent antibody or culture. MATERIALS AND METHODS All patients with recorded C. trachomatis lgg titers (n = 60) were identified retrospectively from the prospective coding sheets of 282 consecutive patientsin a previous study on the appearance of en- dometriosis and peritoneal lesions (12). Surgeries were performed from January of 1988 through September of 1989. Chlamydia trachomatis lgg titers in these patients had been performed in patients with history or exam compatible with peritoneal inflammation. There was no prospective protocol for the use of titers. Tissue was taken from all abnormal-appearing peritoneum and was classified by the appearance. This was based on a prospective protocol designed to study subtle appearances of endometriosis (12). There were no blind biopsies taken of normal appearing tissue. Tissue had been excised using a combination of C0 2 laser, unipolar electrosurgery, scissors, and biopsy forceps. The peritoneal biopsies of these patients were evaluated by a pathologist blinded to the clinical history and to the results of C. trachomatis lgg titers. The presence or absence of dystrophic calcification and psammoma bodies was recorded. When calcification was identified, it was quantified as follows: mild ( <6 foci per 10 high power fields [HPF]), moderate (from 6 to 40 foci per 10 HPF), and severe (>40 foci per 10 HPF). Chlamydia trachomatis lgg titers were performed using Pharmacia Virgo C. trachomatis IgG immunofluorescent antibody test kit (Schiapparelli Biosystems, Inc., Colombia, MD) for the detection of C. trachomatis lgg antibodies. This fluorescent antibody assay uses the indirect method of fluorescent antibody staining. This test is specific for C. trachomatis when the C. trachomatis lgg titer is :2:1:8. The charts also were reviewed for the following variables: presence of hydrosalpinx on hysterosalpingogram, presence of hydrosalpinx at laparoscopy, presence of Fitz-Hugh-Curtis adhesions at laparoscopy, history of previous abdominal surgery, history of pelvic inflammatory disease (PID) and history of gonorrhea. The AFS adhesion scoring was performed retrospectively using the operative notes and drawings from the patients' charts. Of the 60 patients, 28 ( 4 7%) had positive C. trachomatis lgg titers (:2:1:8) and 32 (53%) had negative C. trachomatis lgg titers ( <1:8). Forty-five patients (75%) had endometriosis. Seventeen patients (28%) had both endometriosis and positive C. trachomatis lgg titers. Forty-one patients (68%) had a history of previous abdominal surgery. Fifty-two (87%) had pain and infertility while eight patients (13%) were being evaluated for pain only. Cervical C. trachomatis cultures and cervical C. trachomatis direct fluorescent antibody were per- 40 Martin et al. Calcification and chlamydia titers Fertility and Sterility

formed on 58 of these patients by multiple physicians at more than one laboratory and by more than one technique. This was determined by the geographical location of the patient when the test was performed. Statistical analyses for associations of C. trachomatis lgg titer, dystrophic calcification, psammomatous calcification, nonpsammomatous calcification, pelvic adhesions, Fitz-Hugh-Curtis adhesions, hydrosalpinges, and history ofpid were performed. Chi-square analysis was used when the expected number in each cell by the null hypothesis was five or greater (hydrosalpinges). Fisher's Exact twotailed analysis was used when the expected number in each cell by the null hypothesis was less than five (dystrophic calcification, psammoma bodies, and degree of calcification). Regression analysis was used to correlate C. trachomatis lgg titers with AFS adhesion scores. Chi-square analysis and Fisher's Exact two-tailed analysis was performed with SAS run on a VAX cluster main frame computer. Analysis of AFS adhesion scores and C. trachomatis IgG titers was performed using t-tests for independent samples and multiple-regression analysis using SPSS Release 4.0 for VAX/VMS (SPSS, Inc., Chicago, IL) on a main frame VAX 8800 (Digital Equipment Co., Maynard, MA). RESULTS Of the 60 patients' slides, 10 demonstrated dystrophic calcification and 50 did not. Two types of dystrophic calcifications were noted: psammoma bodies and nonpsammomatous calcification (Table 1). Of the 10 who had dystrophic calcification, eight had positive C. trachomatis lgg titers (two-tailed Fisher's Exact, P = 0.035). All eight of these patients had psammomatous calcification (two-tailed Fisher's exact, P = 0.001). Seven of these patients had a moderate-to-severe degree of psammomatous Table 1 biopsies* Summary of the data from evaluation of peritoneal Foci of dystrophic calcification* No. of patients None Mild Moderate Severe IgG (+) 28 20 1 4 3 lgg (-) 32 30 2 0 0 * Mild, <6 foci of dystrophic calcification per 10 HPF; moderate, from 6 to 40 foci of dystrophic calcification per 10 HPF; and severe, >40 foci of dystrophic calcification per 10 HPF. calcification (two-tailed Fisher's Exact, P = 0.003) and one patient had a mild degree of psammomatous calcification in their peritoneal biopsies. Four of these eight also had endometriosis. The other two patients had a mild degree of nonpsammomatous dystrophic calcification in their biopsies. These two patients had endometriosis, histories of previous surgery, and negative C. trachomatis lgg titers. The means for the AFS adhesion scores were 11.0 ± 16.9 and 22.2 ± 21.0 (t = -2.21, P = 0.031). Regression analysis of adhesion scores and the level of positive titer yielded a slope of 1.45, which was not statistically significant for a population of this size. Hysterosalpingogram revealed hydrosalpinges in 11 patients, which were confirmed at the time of laparoscopy. A twelfth patient had bilateral nonvisualization of the tubes at hysterosalpingogram and was noted to have hydrosalpinges at laparoscopy. One additional patient had hydrosalpinges diagnosed on hysterosalpingogram but, at laparoscopy, had normal appearing tubes that dilated with Methylene blue before prompt spill from delicate appearing fimbria. Of the 12 hydrosalpinx, 10 were in patients with positive C. trachomatis lgg titers and 2 were in patients with negative C. trachomatis lgg titers (x 2 = 6.37, P = 0.012). There were three patients with Fitz-Hugh-Curtis adhesions. All (100%) of these had positive C. trachomatis lgg titers. This is not statistically significant for a population of this size. No patients in this study had a prior history specific for C. trachomatis or gonorrhea. Of the eight patients with a previous history of PID, five had positive C. trachomatis lgg titers and three did not. Of the five with positive C. trachomatis lgg titers, four had hydrosalginges while one of three with negative C. trachomatis lgg titers had a unilateral hydrosalpinx. The other seven hydrosalpinges were in patients with no history of PID. These findings were not statistically significant for a population of this size. Of the 58 patients who had cervical chlamydia culture or direct fluorescent antibody (28 with positive C. trachomatis lgg titers and 30 with negative C. trachomatis lgg titers), none (O%) had positive culture or direct fluorescent antibody. One patient in this study with a positive C. trachomatis lgg titer of 1:64 had a subsequent diagnosis of a low malignant potential tumor of the ovary and a coexistent multifocal lymphoma. This was found 42 months after the surgery of this study. At the time of her original surgery, the patient had Vol. 63, No. 1, January 1995 Martin et al. Calcification and chlamydia titers 41

infertility, psammoma bodies, bilateral hydrosalpinges, and right salpingitis isthmica nodosa. DISCUSSION This study documents a statistically significant correlation of positive C. trachomatis IgG titers with dystrophic calcification, psammoma bodies, moderate-to-severe dystrophic calcification, adhesions, and hydrosalpinges. The association with these diseases suggests a role for the use of C. trachomatis IgG titers in clinical studies of pain and infertility. The association of increased AFS adhesion scores and hydrosalpinges with positive C. trachomatis IgG titers is compatible with the inflammatory nature of this process and previous publications (13-16). With a larger study population, a statistically significant association of positive titers with Fitz-Hugh-Curtis adhesions or of titer levels and adhesion scores may be documented. Chlamydia trachomatis has been found in about one-half of the patients with proven PID. Those and other nongonococcal infections are associated with a more adverse reproductive outcome than gonococcal infections. The low historical recognition of PID and lack of correlation with positive C. trachomatis IgG titers in our study population is in agreement with previously published studies. Furthermore, none of these patients had a current positive or historical positive C. trachomatis direct fluorescent antibody or culture. Of note, 7 of the 12 women with documented tubal occlusion had no history ofpid. Moreover, one of these also had a negative C. trachomatis IgG titer. Cates (17) has concluded that efforts to identify women with upper genital tract Chlamydia infections may be made more effective with the inclusion of atypical symptoms or biochemical markers. Although C. trachomatis IgG titers may be useful in this effort, history and other markers also are needed. Evidence of a previously unrecognized C. trachomatis infection suggests that patients in this population are at risk of having had other unrecognized infectious diseases. Up to 68% of patients with tubal infertility have positive titers to both C. trachomatis and gonorrhea (18), which suggests that these patients are at high risk for having unrecognized diseases other than C. trachomatis, which may be the biologic cause of the findings in this paper. Our study correlates the histologic finding of dystrophic calcification with positive C. trachomatis IgG titers. All patients with positive C. trachomatis IgG titers and dystrophic calcification had psammoma bodies. The dystrophic calcification in the two patients with negative C. trachomatis IgG titers were not psammomatous in nature. It is possible this represents focal necrosis of the peritoneum and subsequent precipitation of calcium salts because both of these patients had previous histories of endometriosis and past surgery. Of ongoing clinical importance is the correct diagnosis of peritoneal abnormalities. Patients continue to be referred to one of the authors (D.C.M.) with a surgical diagnosis of endometriosis but with histology demonstrating dystrophic calcification. Endometriosis and psammoma bodies can occur separately and be confused for the other (12) or can coexist as they did in four patients in this study. Accurate histologic or cytologic diagnosis is needed for appropriate clinical care (9, 19), for research protocols and for studies in staging of adhesions or endometriosis. As a final concern, endosalpingiosis associated with psammoma bodies and with inflammatory tubal pathology, borderline malignancy of the ovaries, and with ovarian malignant cancer has been published (1, 2). Furthermore, infertility has been associated with ovarian cancer (20). Although the increase of cancer in infertility patients may be due to chronic ovulation and endocrine reasons, the possibility also exists that this may be related to chronic peritoneal irritation secondary to infection. The one patient of this study, with a low malignant potential tumor, had infertility and psammoma bodies. In addition to that patient, a second patient with a low grade ovarian malignancy subsequently has been diagnosed and treated. The second patient also had infertility, a positive C. trachomatis IgG titer, psammoma bodies, and endosalpingiosis. Current investigation into a possible association of C. trachomatis with ovarian tumor is in progress using C. trachomatis IgG titers in patients with a diagnosis of ovarian cancer and borderline malignant potential tumors. LIMITATIONS The limitations of the data of this paper are those inherent in a retrospective chart review of a tertiary care patient practice and the use of a data base designed for a previous study. The selective nature of this patient population is noted by the finding that all patients in this study had pain in addition to at least a positive C. trachomatis IgG titer, endometriosis, or adhesions. Chlamydia trachomatis titers 42 Martin et al. Calcification and chlamydia titers Fertility and Sterility

had been performed during routine clinical management and were not based on protocol. There were no serologic tests used for diseases such as gonorrhea, herpes, and mycoplasma, which can be coexistent with C. trachomatis. This is important because these diseases can be coexistent, and patient histories for C. trachomatis, gonorrhea, and other sexually transmitted diseases frequently are unreliable (17, 18). The data on AFS adhesion scores, history of C. trachomatis, history of gonorrhea, Fitz-Hugh-Curtis adhesions, and hysterosalpingograms was added retrospectively. The historical data was inadequate to quantitate the relative levels of pain and/or infertility. The population size is too small for adequate analysis of pregnancy rates. There were no random biopsies of normal appearing peritoneum. This study does not attempt to determine the prevalence of subclinical calcification. This study is inadequate to discuss advantages and disadvantages of antibiotic therapy, surgery, IVF, no therapy, or any therapy for patients with these findings. These results may be specific for patients with pain and infertility and not applicable to a general gynecologic population. The adhesion analysis of this study is limited by an inability to distinguish the adhesions of infectious pelvic inflammation from those of endometriosis or previous surgery. This is a problem of this study, the AFS adhesion classification (11), the revised AFS endometriosis classification (21), and the AFS instrument for pain associated with endometriosis (22). Lacking a clinically obvious marker for the origin of the adhesions, this problem will persist. CONCLUSIONS Chlamydia trachomatis IgG titers may be more useful as a marker for peritoneal and/or tubal abnormalities than a history of PID. Although positive, the C. trachomatis titers do not necessarily identify C. trachomatis as the cause of the peritoneal abnormalities. The impact of other possible coexistent diseases such as gonorrhea, herpes, or mycoplasma was not assessed. When peritoneal abnormalities are found, histology is needed for accurate diagnosis. Although clear or white lesions may be psammoma bodies, these are more commonly endometriosis and may be endosalpingiosis or cancer. At present, the finding of apparent psammoma bodies suggests the need for histology and examination for coexistent epithelial neoplasia but does not demonstrate an increased risk of neoplasia. Furthermore, in the absence of malignant cells, the finding of psammoma bodies is not an indication for extirpative surgical therapy. In addition, the lack of association of positive cultures or direct fluorescent antibodies fails to suggest a need for antibiotic therapy in these patients. The inclusion of C. trachomatis IgG titers, serologic markers for other sources of pelvic inflammation, and observation for psammoma bodies appears to be prudent in research protocols in the study of endometriosis, infertility, pain, and ovarian cancer. Acknowledgements. We thank Ms. Marty Mauney for her aid in the preparation of this manuscript; the Baptist Memorial Hospital Department of Pathology staff for their aid in gathering the histologic slides and Roger Vander Zwaag, Ph.D, Baptist Memorial Hospital Department of Clinical Data Management, and Kristopher Arheart, Ed.D., Division of Biostatistics and Epidemiology, University of Tennessee, Memphis, for performing the statistical analyses. REFERENCES 1. Holmes MD, Levin HS, Ballard LA Jr. Endosalpingiosis. Cleveland Clinic Quarterly 1981;48:345-52. 2. Zinsser KR, Wheeler JE. Endosalpingiosis in the omentum. A study of autopsy and surgical material. Am J Surg Pathol 1982;6:109-17. 3. Kern WH. Benign papillary structures with psammoma bodies in culdocentesis fluid. Acta Cytol 1969;13:178-80. 4. Chen KTK. Psammoma bodies in pelvic washings [letter]. Acta Cytol1983;27:377-9. 5. Hallman KB, Nahhas WA, Connelly PJ. Endosalpingiosis as a source of psammoma bodies in a papanicolaou smear. J Reprod Med 1991;36:675-8. 6. Picoff RC, Meeker Cl. Psammoma bodies in the cervicovaginal smear in association with benign papillary structures of the ovary. Acta Cytol1970;14:45-7. 7. Valicenti JF, Priester SK. Psammoma bodies of benign endometrial origin in cervicovaginal cytology. Acta Cytol 1977;21:550-2. 8. Kanbour A, Poshi N. Psammoma bodies and detached ciliary tufts in a cervicovaginal smear associated with benign ovarian cystadenofibroma. Acta Cytol 1980;24:549-52. 9. Martin DC, Jansen R. Look-a-like lesions. In: Martin DC, editor. Atlas of endometriosis. London: Gower Medical Publishers, 1993:16.1-.6. 10. Ripps BA, Martin DC. Focal pelvic tenderness, pelvic pain and dysmenorrhea in endometriosis. J Reprod Med 1991;36:470-2. 11. The American Fertility Society. The American Fertility Society classification of adnexal adhesions, distal tubal occlusion, tubal occlusion secondary to tubal ligation, tubal pregnancies, Mullerian anomalies and intrauterine adhesions. Fertil Steril 1988;49:944-55. 12. Martin DC, Hubert GD, Vander Zwaag R, El-Zeky FA. Laparoscopic appearances of peritoneal endometriosis. Fertil Steril1989;51:63-7. Vol. 63, No.1, January 1995 Martin et al. Calcification and chlamydia titers 43

13. Sweet RL. Chlamydia! salpingitis and infertility. Fertil Steril 1982;38:530-3. 14. Quinn PA, Petrie M, Barkin M, Butany J, Derzko C, Gysler M, et a!. Prevalence of antibody to Chlamydia trachomatis in spontaneous abortion and infertility. Am J Obstet Gynecol1987;156:291-6. 15. Rowland GF, Forsey T, Moss TR, Steptoe PC, Hewitt J, Darougar S. Failure of in vitro fertilization and embryo replacement following infection with Chlamydia trachomatis. J In Vitro Fert Embryo Transf 1985;2:151-5. 16. Witkin SS, Ledger WJ. Antibodies to Chlamydia trachomatis in sera of women with recurrent spontaneous abortions. Am J Obstet Gynecol 1992;167:135-9. 17. Cates W J r, J oesoef R, Goldman MB. Atypical pelvic in flammatory disease: can we identify clinical predictors? Am J Obstet Gynecol1993;169:341-6. 18. Cates W Jr, Wasserheit JN. Genital chlamydia! infections: epidemiology and reproductive sequalae. Am J Obstet Gynecol1991;164:1771-81. 19. Sneige N, Fernandez T, Copeland LJ, Kalz RL. Mullerian inclusions in peritoneal washings, potential source of error in cytologic diagnosis. Acta Cytol 1986;30:271-6. 20. Whittemore AS, Harris R, Intyre J, the Collaborative Ovarian Cancer Group. Characteristics relating to ovarian cancer risk: collaborative analysis of twelve US case-control studies, II: invasive epithelial ovarian cancers in white women. Am J Epidemiol1992;3:1-15. 21. The American Fertility Society. Revised American Fertility Society classification of endometriosis: 1985. Fertil Steril 1985;43:351-2. 22. American Fertility Society. Management of endometriosis in the presence of pelvic pain. Fertil Steril1993;60(6):952-5. 44 Martin et al. Calcification and chlamydia titers Fertility and Sterility