Endometrial tissue in peritoneal fluid

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1 FERTILITY AND STERILITY Copyright c 1986 The American Fertility Society Printed in UBA. Endometrial tissue in peritoneal fluid Delphine Bartosik, M.D. *t Samuel L. Jacobs, M.D.* Lynda J. Kelly, C.T.:J: Hahnemann University, Philadelphia, Pennsylvania Peritoneal fluid (PF) was studied for the presence of endometrial tissue in a consecutive series of 67 women (with documented tubal patency) undergoing diagnostic laparoscopy, tubal lavage, and hysteroscopy. PF was completely aspirated from the cul-de-sac both before and after uterine irrigation. The PF was then analyzed for the presence of endometrial tissue. In native PF no significant difference in the incidence of endometrial tissue between patients with (19%) and without (11%) endometriosis (P = 0.6) was observed. Refluxed PF, obtained after uterine irrigation, showed a significantly higher incidence of endometrial tissue in women with endometriosis (76%) as compared to controls (42%) (P = 0.03). We propose two models to explain the development of endometriosis. These are not mutually exclusive, may be independent of each other, and may represent two distinct pathophysiologic disease processes. Fertil Steril46:796, 1986 It is classically accepted that the retrograde passage of endometrial tissue, whether spontaneous, secondary to outflow obstruction, or iatrogenic, may be a significant factor in the development of endometriosis. Based on observations of endometrial tissue in peritoneal fluid,1-4 this theory implies that retrograde passage of endometrial tissue occurs in all women. Thus far, only one report4 has been published in which the incidence of endometrial tissue in peritoneal fluid of patients with endometriosis, compared with those without the disease, has been examined: no differences were seen. Thus, the Sampson theory1 Received March 10, 1986; revised and accepted July 15, *Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology. treprint requests: Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Hahnemann University, Broad and Vine, Philadelphia, Pennsylvania :f:department of Pathology. does not explain why endometrial tissue implants only in certain patients. Ayers and Friedenstab 5 recently reported that women with endometriosis have hypotonia of the uterotubal junction. This suggests that retrograde passage of endometrial tissue is more likely to occur in patients with endometriosis. In a consecutive series of patients who underwent diagnostic laparoscopy, we obtained peritoneal fluid both before and after uterine irrigation and studied it for the presence of endometrial tissue. PATIENTS MATERIALS AND METHODS We studied 83 consecutive patients in whom combined tubal lavage (for tubal patency), hysteroscopy, and laparoscopy were performed during a 12-month period. Fifteen patients were excluded because of tubal obstruction. Among the 52 remaining patients with endometriosis, 25 had 796 Bartosik et al. Pathophysiology of endometriosis

2 infertility and 27 had pelvic pain. They were 16 to 42 years of age (mean, 28.8 ± 6.0 years). Among the 15 patients without endometriosis, 6 had infertility and 9 had pelvic pain. They were aged 18 to 44 years (mean, 29.6 ± 7.1 years). None of the patients were undergoing hormonal therapy at the time of diagnostic laparoscopy. LAPAROSCOPY AND CHROMOPERTUBATION Laparoscopy was performed with the use of a lo-mm Eder laparoscope (Eder Instrument Co., Chicago, IL). Tubal lavage was performed with a solution of 10 ml Indigo Carmine (Hynson, Westcott and Dunning; Division of Becton, Dickinson, and Co., Baltimore, MD) in 250 ml of 0.9% saline, which was injected through a Rubin cannula (Eder Instrument Co.) inserted into the endocervical canal. A second puncture probe was inserted in all patients to enable a careful search for the presence of endometriosis. Findings at the time of diagnostic laparoscopy were evaluated based on the presence of tubal patency, endometriosis, pelvic adhesions, and pelvic inflammatory disease. Diagrams were prepared, and the quantification of the degree of endometriosis was based on the standards proposed by The American Fertility Society.6 HYSTEROSCOPY Hysteroscopy was performed with either a 7-mm Eder hysteroscope (Eder Instrument Co.) or a no. 17 (French) cystoscope (American V. Mueller Co., Blenheim, NJ). A 32% dextran 70 solution (Hyskon, Pharmacia Laboratories, Piscataway, NJ) was used to distend the uterine cavity. ASPIRATION OF PERITONEAL FLUID Aspiration of peritoneal fluid to allow complete visualization of the cul-de-sac and aspiration of the cul-de-sac fluid after hysteroscopy are part of our standard medical practice. Peritoneal fluid was aspirated completely before and again after completion of the uterine irrigation (Le., before and after lavage) via a small cannula in the second puncture site, through which a no. 8 (French) red rubber catheter (Leon Stotter Co, Bridgeton, NJ) was inserted. SPECIMEN PREPARATION Each specimen was divided into two equal parts: one half of the specimen was submitted for cytologic analysis and the other half was mixed to make a 50% ethanol solution in preparation for cell block. Two milliliters of a 2% melted cabowax fixative was added to the undiluted specimen, which was then added to 98 ml of 50% ethanol. Carnoy's solution (100 ml acetic acid plus 300 ml of chloroform in 650 ml of 95% ethanol) was then added to lyse red blood cells. The specimen was centrifuged at 1500 rpm for 5 to 10 minutes, the supernatant was decanted, and a small amount of sediment was spread on glass sides. The slides were fixed and stained according to the Papanicolaou method. The peritoneal fluid originally diluted with ethanol was also centrifuged and the supernatant decanted. The sediment obtained was processed further by adding 40 drops of plasma and 10 drops of reconstituted topical thrombin. This was then placed in biopsy cassette from which cell blocks were processed in the usual manner. EVALUATION OF SAMPLES Both preparations were examined carefully for the presence of endometrial tissue. If insufficient peritoneal fluid was obtained, only a smear was prepared and evaluated for the presence of endometrial tissue. In addition, the samples were studied in a blind manner with respect to the diagnosis, and all slides were reviewed by an experienced cytopathologist. RESULTS CELLULAR MORPHOLOGIC FEATURES During the course of this study we found that cell blocks are much better than cytologic preparations for preserving the intactness of glandular structures. The major cell population observed was reactive mesothelial cells (Fig. 1). The presence of ciliated columnar cells was also noted in 10% of the specimens. Endometrial tissue was observed in gland-like formation, in actual glands, or in glands with stroma. Gland-like structures, probably of endometrial origin, are illustrated in Figure 2. An intact endometrial gland is illustrated in Figure 3. Endometrial stroma, suggesting predecidual change, is illustrated in Figure 4. For the purposes of tabulating data, only those specimens clearly demonstrating endometrial tissue were considered positive. Statistical analyses Bartosik et al. Pathophysiology of endometriosis 797

3 , Figure 1 Reactive mesothelial cells, The major cell population present in peritoneal fluid (x 40). Figure 3 An intact endometrial gland in peritoneal fluid (x 40). were performed with the use of nonparametric methods. ENDOMETRIAL TISSUE IN PERITONEAL FLUID ENDOMETRIAL TISSUE IN PERITONEAL FLUID Fifty-three patients with patent tubes were studied. We attempted to remove all peritoneal fluid before uterine irrigation. The peritoneal fluid aspirated after completion of uterine irrigation was evaluated for the presence or absence of endometrial tissue (Table 1). There were 41 patients with endometriosis (AFS score 6.8 ± 7.0), of whom 31 had endometrial cells in the peritoneal fluid (76%). Twelve patients did not have endometriosis; of these, only 5 had endometrial cells (42%). Differences in the occurrence of endometrial cells in peritoneal fluid observed between patients with and without endometriosis were statistically significant (P = 0.03). ASPIRATED BEFORE UTERINE IRRIGATION A total of 41 patients with patent tubes had sufficient peritoneal fluid aspirated before uterine irrigation for subsequent analysis (Table 1). There were 32 patients with endometriosis (AFS score = 7.5 ± 7.7; mean ± standard deviation), of whom 6 had endometrial cells in the peritoneal fluid (19%). There were nine patients without endometriosis, only one of whom had endometrial cells in the peritoneal fluid (11%). Differences in the incidence of endometrial cells in the peritoneal fluid of patients with and without endometriosis were not statistically significant (P = 0.6). Figure 2 Gland-like structures in peritoneal fluid (x 40). 798 Bartosik et ai. Pathophysiology of endometriosis ASPIRATED AFTER UTERINE IRRIGATION Figure 4 Endometrial stroma, showing predecidual changes, found in peritoneal fluid (x 40).

4 Table 1. Endometrial Tissue in Peritoneal Fluid Obtained Before and After Uterine Irrigation With endometriosis (no.) % Positive for tissue Without endometriosis (no.) % Positive for tissue Before irrigation 6/ EFFECT OF MENSTRUAL CYCLE STAGE ON THE After irrigation /12 42 INCIDENCE OF ENDOMETRIAL CELLS IN PERITONEAL FLUID AFTER UTERINE IRRIGATION Patients were grouped according to preovulatory (days 1 through 13), early postovulatory (days 14 through 20), and late postovulatory (days 21 through 28). Among these groups, patients with endometriosis were contrasted to those without endometriosis and the incidence of endometrial cells in peritoneal fluid was compared (Table 2). During the preovulatory and early luteal phases of the menstrual cycle, there were no significant differences in retrograde seeding among patients with and without endometriosis. Only those patients with endometriosis who were in the late luteal phase of the menstrual cycle showed a significantly higher rate of retrograde seeding of endometrial tissue. However, the number of patients without endometriosis was so small that there is a risk of a type II statistical error. 7 PAIRED SAMPLES OF PERITONEAL FLUID: DIFFERENCES AMONG PATIENTS Twenty-seven patients had sufficient peritoneal fluid obtained both before and after uterine irrigation to permit analysis for endometrial cells. For these 27 paired specimens, the presence or absence of endometrial cells was tabulated (Table 3) and the results were analyzed according to the Wilcoxon Signed Ranks test. In the six paired specimens of patients without endometriosis, there was no significant retro- Table 2. Endometrial Tissue in Peritoneal Fluid Obtained After Uterine Irrigation: Effects of Menstrual Cycle Stage Follicular phase Endometriosis (day 1-13) Present with tissue Absent with tissue n 8 2 % Luteal phase Day Day n % n % o o grade tubal seeding (P = 0.3). In the 21 paired specimens of patients with endometriosis, there was a highly significant incidence of retrotubal seeding of endometrial tissue (P = 0.01). DISCUSSION Although the classically accepted model for endometriosis suggests that retrograde seeding of endometrial tissue is the etiologic factor, our data challenge this hypothesis. First, in patients with patent tubes, we observed the presence of endometrial tissue in native peritoneal fluid equally often, regardless of whether or not the patient had endometriosis. Second, the incidence of iatrogenically refluxed endometrial cells in peritoneal fluid was significantly higher among patients with endometriosis, compared with those without the disease. Although red blood cells are observed in the peritoneal fluid of most women at the time of menstruation,8,9 the real issue concerns documentation of the presence of endometrial cells in this fluid. Koninckx and colleagues 4 observed that endometrial tissue in the peritoneal fluid was found just as often in patients with or without endometriosis. Portuondo and colleagues lo found endometrial cells in the peritoneal fluid of 25% of patients with endometriosis, but they did not study normal controls. Badawy and colleagues ll described an increased prevalence of endometrial tissue in the peritoneal fluid of endometriosis patients, compared with control. However, the control group consisted of patients undergoing laparoscopy for sterilization and for infertility, mainly tubal factors. Clearly, the analysis of their data did not exclude patients with tubal obstruction. Therefore, their experiment cannot be compared with ours. These cumulative observations that endometrial tissue in peritoneal fluid is seen as commonly in patients with and without endometrio- Table 3. Endometrial Tissue in Peritoneal Fluid Obtained Before and After Uterine Irrigation Endometrial tissue in Endometriosis peritoneal fluid Present Absent Before After n % n % No No No Yes Yes No Yes No Bartosik et al. Pathophysiology of endometriosis 799

5 sis are consistent with the paradigm that most women have the potential to develop endometriosis but the condition develops only in women who have other predisposing factors. Thus there must be at least one additional mechanism contributing to the development of endometriosis. Dmowski and colleagues 12 demonstrated one such additional factor: the presence of impaired cellular immune factors in subjects who developed endometriosis. Although several groups have documented endometrial tissue in the peritoneal fluid after tubal lavage or hysteroscopy, 13, 14 these studies have not differentiated between patients with and without endometriosis. Our observations are the first to show that endometrial tissue is refluxed more often into the peritoneal cavities of women with endometriosis than in those of the control group. This suggests a deficient uterotubal control mechanism in patients destined to acquire endometriosis. In support of this theory, Ayers and Friedenstab 5 observed relative hypotonia of the uterotubal junction in women with endometriosis. Among such women with uterotubal hypotonia, any additional factor resulting in increased uterine tone could be expected to result in the development of earlier (or more severe) endometriosis. Such possibilities include patients with increased uterine tone associated with primary dysmenorrhea 15 or partial outflow obstruction. In addition, altered uterotubal migration of sperm and/or fertilized ova may contribute to the infertility observed in these patients with endometriosis. In summary, we propose two models to explain the development of endometriosis. In the first, because all women have the same chance of having endometrial cells in the peritoneal fluid, there must be a second mechanism that permits the disease to develop. One possible explanation is the altered cellular immune response that allows for implantation and growth of the ectopic endometrium. 12 In the second model, women are more likely to develop endometriosis if there is an additional factor, such as the increased uterine tone of primary dysmenorrhea or partial outflow obstruction, superimposed upon the uterotubal hypotonia. These two models for the development of endometriosis are not mutually exclusive, may be independent of each other, and may represent two distinct pathophysiologic disease processes. REFERENCES 1. Sampson JA: Peritoneal endometriosis due to the menstrual dissemination of endometrial tissue into the peritoneal cavity. Am J Obstet Gynecol 14:422, Watkins RE: Uterine retrodisplacements, retrograde menstruation and endometriosis. West J Surg Obstet Gynecol 46:480, Sampson JA: The development of the implantation theory for the origin of peritoneal endometriosis. Am J Obstet Gynecol 40:549, Koninckx PR, Ide P, Vandenbroucke W, Brosens IA: New aspects of the pathophysiology of endometriosis and associated infertility. J Reprod Med 24:257, Ayers JWT, Friedenstab AP: Utero-tubal hypotonia associated with pelvic endometriosis (Abstr). Presented at the Forty-First Annual Meeting of The American Fertility Society, Chicago Illinois, September 28 to October 2, Published by The American Fertility Society: Abstracts of the Scientific and Poster Sessions, Birmingham, 1985, p The American Fertility Society: Classification of endometriosis. Fertil Steril 32:633, Colton T: Statistics in Medicine, 1st Ed. Boston, Little Brown & Co., 1974, p Blumenkrantz MJ, Gallagher N, Bashore RA, Tenckhoff H: Retrograde menstruation in women undergoing chronic peritoneal dialysis. Obstet Gynecol 57:667, Halm~ J, Hammond MG, Hulka JF, Raj SG, Talbert LM: Retrograde menstruation in healthy women and in patients with endometriosis. Obstet Gynecol 64:151, Portuondo JA, Herran C, Echanojauregui AD, Riego AG: Peritoneal flushing and biopsy in laparoscopically diagnosed endometriosis. Fertil Steril 38:538, Badawy SZA, Cuenca V, Marshall L, Munchback R, Rinas AC, Coble DA: Cellular components in peritoneal fluid in infertile patients with and without endometriosis. Fertil Steril 42:704, Dmowski WP, Steele RW, Baker GF: Deficient cellular immunity in endometriosis. Am J Obstet Gynecol 141: 377, Beyth Y, Yaffe H, Levij ISh, Sadovsky E: Retrograde seeding of endometrium: sequela of tubal flushing. Fertil Steril 26:1094, Nagel TC, Kopher Ra, Tagatz GE, Okagaki T, Brooker DC: Tubal reflux of endometrial tissue during hysteroscopy. In Hysteroscopy: Principles and Practice, Edited by AM Siegler, HJ Lindemann. Philadelphia, JB Lippincott, 1984, p Ulmsten U, Andersson KE: Multichannel intrauterine pressure recording by means of microtransducers. Acta Obstet Gynecol 58:115, Bartosik et al. Pathophysiology of endometriosis