Histologic study of peritoneal endometriosis in infertile women

FERTILITY AND STERILITY Copyright" 1990 The American Fertility Society Printed on acid-free paper in U.S.A. Histologic study of peritoneal endometriosis in infertile women Michelle Nisolle, M.D. Benedicte Paindaveine, M.D. Anne Bourdon, M.D. Martine Berliere, M.D. Franc,:oise Casanas-Roux, B.S. Jacques Donnez, M.D., Ph.D.* Infertility Research Unit, Department of Gynecology, University of Louvain, Brussels, Belgium The present study included 118 patients undergoing a laparoscopy for infertility. In 86 patients with laparoscopically diagnosed endometriosis (group I), biopsies were taken from areas of apparent endometriosis (n = 86) and from a visually normal peritoneum (n = 52). Histology reveals the presence of endometriosis in 93% of positive sites and in 13% of negative sites. In 32 patients without endometriosis at laparoscopy (group II), biopsies were taken from normal uterosacral ligaments (n = 32). Endometriosis was observed in 6% of cases. Despite the increased ability to detect pigmented and nonpigmented endometriotic lesion, histological study revealed the presence of endometriosis in normal peritoneum in 13% (group I) and 6% (group II) of cases. Fertil Steril53:984, 1990 The diagnosis of peritoneal endometriosis at the time of laparoscopy is often made by observation of puckered black or bluish typical lesions. These lesions are the easiest to see but the diagnosis of endometriosis can be easily overlooked when typical black or bluish pigments are not seen. Vasquez et al. 1 documented microscopic implants of endometriosis on the surface of peritoneum by scanning electron microscopy (SEM) in patients with unexplained infertility in whom no endometriosis was seen at the time of laparoscopy. Murphy et al. 2 described endometrial surface epithelium in specimens thought to represent mesothelium. Steingold et al. 3 demonstrated peritoneal endom.etriotic foci in areas without evidence of disease. Moreover, the concept of subtle appearance of endometriosis was described by Jansen and RusselV Redwine, 5 Stripling et al., 6 Martinet al., 7 and Nisolle et al. 8 This has resulted in an increased di- Received September 25, 1989; revised and accepted February 13,1990. *Reprint requests: Jacques Donnez, M.D., Ph.D., Cliniques Universitaires St. Luc, Avenue Hippocrate 10, B-1200 Brussels, Belgium. agnosis of endometriosis. The aim of this study is to evaluate histologically, biopsies of peritoneal endometriosis and of visually normal peritoneum taken from patients undergoing a laparoscopy for infertility. MATERIALS AND METHODS In 118 patients who were undergoing laparoscopy for infertility, peritoneal biopsies were taken from areas of the pelvic peritoneum bearing foci of endometriosis and/or from areas of visually normal peritoneum (uterosacral ligaments). Biopsies were taken with a biopsy punch forceps (26175 DH; Storz, Tuttlingen, West Germany) and were 3 to 5 mmlarge. The laparoscope was placed 4 to 5 em from the peritoneum to evaluate its surface. Thereafter, the laparoscope was placed close to the peritoneum to achieve some magnification. The gross appearance of established endometriosis is usually obvious. It appears as brownish, bluish, or purplish hemorrhagic areas often associated with stellate scarring. Dark pigmented lesions are thus the typical visual criteria for diagnosis of endometriosis, but visualization of pigment is not 984 Nisolle et al. Peritoneal endometriosis

Table 1 Peritoneal Endometriosis and Infertility Site of biopsy Sites (+)a Sites (-)b Histologically proved endometriosis Sites(+) Sites(-) Group! 86 52 80/86 (93)c 7/52 (13) a Site ( + ), macroscopically visible endometriotic lesion. b Site (-), macroscopically normal peritoneum. c Values in parentheses are percents. Group II 32 2/32 (6) necessary to diagnose endometriosis. Several distinctive appearances of nonpigmented endometriotic peritoneal lesions have been described. 4-9 These include (1) white opacification of the peritoneum, (2) glandular excrescences, (3) red flamelike lesions, (4) yellow-brown patches, (5) subovarian adhesions, and (6) circular peritoneal defects. Areas of petechial peritoneum or areas with peritoneal hypervascularization are also considered as first stages of endometriosis. 8 In the present study, peritoneum was considered as normal peritoneum if no lesion described before was seen. The first group consisted of 86 patients with laparoscopically diagnosed endometriosis (Table 1). Most patients with pigmented endometriosis also had areas of nonpigmented endometriosis, either contiguous with pigmented lesions, or present as isolated areas of peritoneal abnormality. Biopsy was taken from the typical (puckered black) endometriotic implants in all patients of group I. In this group, biopsy was also taken from a visually normal peritoneum (n = 52 patients). The second group consisted of 32 women without any suspected endometriotic lesion; biopsy was taken from normal peritoneum of uterosacral ligaments. All biopsy specimens were fixed in formaldehyde and embedded in paraffin. Three micrometer serial sections were stained with Gomori's Trichrome and examined, on a blind basis, with a Leitz Orthoplan microscope (Leitz, Wetzlar, West Germany). In all cases, the mitotic index was calculated as previously described 10 by counting mitotic figures (prometaphase, metaphase, anaphase, and telophase) for 2,000 epithelial cells per biopsy. This is the only method available for women because administration of colchicine or tritiated thymidine is not ethical. The epithelial height was measured with the help of an ocular micrometer. Fifty cells were selected in which the plane of section clearly passed through the cell nucleus parallel to the lon- gitudinal axis of the cell. Blind interpretation of histological results was done systematically. Results (epithelial height) were' expressed as the mean ± SD. The x 2 test and the median test were used for statistical analysis. RESULTS There are three basic microscopic elements seen with endometriosis: (1) endometrial epithelium, (2) stroma, and (3) hemorrhage. In our study, the microscopic criteria for endometriosis were the presence of both glandular epithelium and stroma. Microscopic Findings Data from group I are seen in Table 1. Biopsies taken from peritoneal endometriotic implant (n = 86), easily recognized by direct visualization of puckered black or bluish lesion, showed the presence of endometrial elements, glands and stroma, in 80 cases (93%). Among the six other biopsies, three showed the presence of stroma cells without epithelial elements, and one showed the presence of hemosiderin-laden macrophages. The last two biopsies were very small ( <2 mm) and neither glandular epithelium nor stroma were found. Biopsies taken from a visually normal peritoneum (n = 52) showed the presence of endometriotic tissue in 7 cases (13%). In group II, biopsies taken from uterosacralligaments (n = 32) showed the presence of endometriotic tissue in two cases (6%). This rate was less than one-half the rate (13%) observed in women with visual endometriosis, but the difference was not significant. Morphological Characteristics Morphological characteristics of peritoneal endometriosis (Table 2) were studied in the 89 biopsies with histologically proved endometriosis. Peritoneal endometriosis was histologically character- Table 2 Morphological Characteristics in Peritoneal Endometriosis Biopsies Typical glandular epithelium and stroma Active endometriosis Oviduct-like epithelium Epithelial height (JLm) Mitotic index(%) a Values in parentheses are percents. b Value is mean± SD. 89 (100)" 68 (76) 49 (55) 14.8± 3.2b 0.06 Nisolle et al. Peritoneal endometriosis 985

Figure 1 Puckered black lesion at laparoscopy confirmed the combination of endometrial glands and stroma. Note the presence of intraluminal debris. (Gomori's Trichrome X32). Figure 3 Red flame-like lesions and glandular excrescences are characterized by the presence of numerous endometrial glands. (Gomori's Trichrome X32). ized by both epithelium and stroma of the endometrial type. An endometriotic lesion was considered "active" 10 when typical glandular epithelium appeared proliferative with typical stroma and not flattened. Such a lesion was found in 76% of cases. Areas of oviduct-like epithelium with ciliated cells were demonstrated in 55% of peritoneal endometriotic foci. The epithelial height and the mitotic index were calculated in typical glandular epithelium. Their values were 14.8 ± 3.2 J.lm (mean± SD) and 0.06%, respectively. Puckered black lesions were a combination of glands, stroma, scar, and intraluminal debris (Fig. 1). Brown and black coloration appears to be a function of the amount of intraluminal hemosiderin and debris. Most areas of pigmented lesions are surrounded by areas of nonpigmented endometriosis. Histologically, white or yellow-brown lesions were predominantly fibromuscular scar with few scattered areas of gland and stroma (Fig. 2). White opacification of the peritoneum is due to the presence of rare retroperitoneal glandular structure surrounded by scanty stroma. This type of lesion could not be diagnosed by SEM because of the absence of endometriotic surface elements. Red flame-like lesions and glandular excrescences appeared similar to native endometrium (Fig. 3). Sometimes, endometriosis could be seen in small polypoid lesions (Fig. 4). Serial sections from areas of petechial peritoneum or areas with hypervascularization showed numerous blood vessels and endome trial glands (Fig. 5). The size of endometriotic lesions found in normal appearing peritoneum was 313 ± 185 J.lm (range: 88 to 720 J.lm). Figure 2 White opacification and yellow-brown lesions are characterized histologically by retroperitoneal glandular structure and scanty stroma surrounded by fibrotic tissue (Gomori's Trichrome X8). 986 Nisolle et al. Peritoneal endometriosis DISCUSSION During the last decade, numerous studies have described not only the typical and characteristic endometriotic lesions, but also the numerous subtle appearances of peritoneal endometriosis. 4-9 11 12 The typical macroscopical aspect of peritoneal endometriosis is a pigmented lesion (puckered black Figure 4 Polypoid lesion that is a characteristic endometriotic peritoneal lesion. (Gomori's Trichrome X32).

Figure 5 Areas of petechial peritoneum or areas with hypervascularization are characterized by the presence of numerous blood vessels. Endometrial glands are rare (arrow). or bluish). Dark pigmented lesions, which are the typical visual criteria for diagnosis of endometriosis, are the late consequence of cyclic proliferation where tissue bleeding and discoloration by blood pigment have taken place. A few authors have hinted at the variety of subtle appearances possible with endometriosis. 4-9 11 12 Jansen and Russell 4 have described distinctive appearances of nonpigmented endometriotic peritoneal lesions. These include (1) white opacification ofthe peritoneum, (2) glandular excrescences, (3) red flame-like lesions, (4) yellow-brown patches, (5) subovarian adhesions, and (6) circular peritoneal defects. Endometriosis was confirmed in 81% of white opacified lesions, 81% of red flame-like lesions, 67% of glandular lesions, 50% of subovarian adhesions, 4 7% of yellow-brown patches, and 45% of circular peritoneal defects. Their study confirmed the biological continuum between nonpigmented and pigmented endometriotic lesions, and it was considered as an argument in favor of the serosal metaplasia theory over the implantation theory of Sampson in the cause of peritoneal endometriosis. 13 Areas of petechial peritoneum or areas with peritoneal hypervascularization are also considered as first stages of endometriosis. Endometriosis was confirmed in 50% of petechial peritoneum and peritoneal hypervascularization. 8 Endometriosis, histologically defined as the presence of endometrial elements (glands and stroma) outside the uterine cavity was confirmed by histological examination in 68% of cases by Chatman, 9 72% by Portuondo et al./ 3 83% by Vasquez et al.,l and 85% by Murphy et al. 2 Recently, Stripling et al. 6 confirmed endometriosis by histological examination in 97% of black lesions, and Martinet al. 7 confirmed endometriosis in 99% of cases. Our study, with a confirmation rate of >90%, confirms these findings of Stripling et al. 6 and Martin et al. 7 In our study, care was taken to obtain an elective biopsy of the endometriotic lesions and serial sections for histological examination. We confirm Stripling et al.'s 6 findings that the ability to detect endometriotic lesions increases with experience of both the surgeon and the pathologist. Indeed, in our study, when biopsies of typical lesions were taken by the most experienced laparoscopists, the confirmation rate was 100%. 12 Identification of endometriosis in biopsy specimens from areas of normal peritoneum in patients with known endometriosis was reported by Murphy et al. 2 By scanning electron microscopy, 25% of their specimens, which appeared normal by gross inspection, were found to contain evidence of endometriosis. In our study, by light microscopy, wereported a percentage of 13%. Moreover, histological study of biopsies from visually normal peritoneum in infertile women without any typical or "subtle" endometriotic lesions revealed the presence of endometriosis in 6% of cases. Unsuspected peritoneal endometriosis can thus be found in a visually normal peritoneum of infertile women with or without associated endometriosis. Although the rate (13%) in women with visual endometriosis was twice the rate observed in women without endometriosis, the difference was not significant. The size of endometriotic lesions in normal appearing peritoneum (313 ± 185 ~Lm) probably explains why the peritoneum had a normal aspect and why the lesion was not visible even though a meticulous inspection was made to identify small and nonhemorrhagic lesions. As recently demonstrated in infertile women, diagnosis of endometriosis at laparoscopy increased. 7 However, our data confirm that the operating surgeon did not make the diagnosis in at least 6% of patients, despite the significant increase in the diagnosis and documentation of endometriosis. When the incidence of the morphological characteristics was compared with the incidence observed in ovarian endometriosis, 8 no difference was found (active endometriosis: 76% versus 84% 8 ; oviduct-like epithelium 55% versus 62%). 8 The epithelial height was similar (14.8 versus 16.0 ~Lm), 8 but the mitotic index was significantly lower in peritoneal endometriotic epithelium (0.06%) than in ovarian endometriosis (0.39% ). The significantly different mitotic activity suggests that pathogeneses of peritoneal and ovarian endometriosis are not identical. This also could explain why Nisolle et al. Peritoneal endometriosis 987

ovarian endometriosis responds less well than peritoneal endometriosis to hormonal therapy. 8 14-16 In conclusion, endometriosis has multiple appearances: typical and numerous subtle lesions. Nevertheless, despite the increased ability to detect subtle lesions, histology proved the presence of endometriosis in normal peritoneum from infertile women with endometriosis in 13% of cases and from infertile women without endometriosis in 6% of cases. REFERENCES 1. Vasquez G, Cornillie F, Brosens IA: Peritoneal endometriosis: scanning electron microscopy and histology of minimal pelvic endometriotic lesions. Fertil Steril42:696, 1984 2. Murphy AA, Green WR, Bobbie D, de la Cruz ZC, Rock JA: Unsuspected endometriosis documented by scanning electron microscopy in visually normal peritoneum. Fertil Steril 46:522, 1986 3. Steingold KA, Cedars M, Lu JKH, Randle D, Judd HL, Meldrum DR: Treatment of endometriosis with a long-acting gonadotropin-releasing hormone agonist. Obstet Gynecol 69:403, 1987 4. Jansen RPS, Russell P: Nonpigmented endometriosis: clinical laparoscopic and pathologic definition. Am J Obstet Gynecol155:1154, 1986 5. Redwine DB: The distribution of endometriosis in the pelvis by age groups and fertility. Fertil Steril47:173, 1987 6. Stripling MC, Martin DC, Chatman DL, Vander Zwaag R, Poston WM: Subtle appearance of pelvic endometriosis. Fertil Steril49:427, 1988 7. Martin DC, Hubert GD, Vander Zwaag R, El-Zeky F: Laparoscopic appearances of peritoneal endometriosis. Fertil Steril51:63, 1989 8. Nisolle M, Paindaveine B, Bourdon A, Casanas-Roux F, Donnez J: Peritoneal endometriosis: typical aspect and subtle appearance. In Laser Operative Laparoscopy and Hysteroscopy, Edited by J Donnez. Leuven, Nauwelaerts Printing, 1989, p 25 9. Chatman DL: Pelvic peritoneal defects and endometriosis; Allen-Masters syndrome revisited. Fertil Steril 36:751, 1981 10. Nisolle-Pochet M, Casanas-Roux F, Donnez J: Histologic study of ovarian endometriosis after hormonal therapy. Fertil Steril 49:423, 1988 11. Goldstein DP, de Cholnoky C, Emans SJ, Leventhal JM: Laparoscopy in the diagnosis and management of pelvic pain in adolescents. J Reprod Med 24:251, 1980 12. Nisolle M, Donnez J: Unpublished data 13. Portuondo JA, Herran C, Echanojauregui AD, Riego AG: Peritoneal flushing and biopsy in laparoscopically diagnosed endometriosis. Fertil Steril 38:538, 1982 14. Buttram VC Jr, Reiter CR, WardS: Treatment of endometriosis with Danazol: report of a 6 year prospective study. Fertil Steril43:353, 1985 15. Donnez J, Lemaire-Rubbers M, Karaman Y, Nisolle-Pochet M, Casanas-Roux F: Combined (hormonal and microsurgical) therapy in infertile women with endometriosis. Fertil Steril 48:239, 1987 16. Donnez J, Nisolle-Pochet M, Clerckx-Braun F, Sandow J, Casanas-Roux F: Administration of nasal Buserelin as compared with subcutaneous Buserelin implant for endometriosis. Fertil Steril 52:27, 1989 988 Nisolle et al. Peritoneal endometriosis