Ovarian endometrioma vascularization in women with pelvic pain

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1 ENDOMETRIOSIS Ovarian endometrioma vascularization in women with pelvic pain Juan Luis Alcázar, M.D., and Manuel García-Manero, M.D. Department of Obstetrics and Gynecology, Clínica Universitaria de Navarra, School of Medicine, University of Navarra, Pamplona, Spain Objective: To assess whether a correlation exists between angiogenesis in ovarian endometrioma with the presence of pelvic pain. Design: Prospective study. Setting: Tertiary-care university hospital. Patient(s): Sixty-five patients (mean age, 33.3 years; range, years) were diagnosed as having suspected cystic ovarian endometriosis, and were scheduled for surgery. Patients were classified into two groups according to clinical complaints: group A, asymptomatic patients or patients presenting mild dysmenorrhea; and group B, severe dysmenorrhea and/or chronic pelvic pain and/or dyspareunia. Intervention(s): Transvaginal power-doppler ultrasonography and immunohistochemical staining for CD-34 in histological specimens. Main Outcome Measure(s): The amount of blood flow, lowest pulsatility and resistance indexes, and microvessel density (MVD). Result(s): Five patients were excluded after surgery because no ovarian endometriosis was found in histological analysis. Thirty women were included in each group. Ovarian endometriomas were more frequently vascularized in group B (87%) than in group A (60%). The lowest pulsatility and resistance indexes were significantly lower, and MVD was significantly higher, in group B compared with group A. There was a correlation between the degree of vascularization detected by power-doppler ultrasound and MVD. Conclusion(s): We conclude that vascularization of ovarian endometriomas evaluated by transvaginal color Doppler and MVD is higher in patients who present with pelvic pain than in asymptomatic patients. This could be an indicator of the activity of endometriosis. (Fertil Steril 2007;87: by American Society for Reproductive Medicine.) Key Words: Ovarian endometriosis, pelvic pain, vascularization, Doppler ultrasound Endometriosis is a common disorder, affecting women of reproductive age, that causes pelvic pain and contributes to infertility in approximately 15% of infertile women. The main sites of the disease are the pelvic peritoneum, ovaries, and rectovaginal septum. Extragenital disease is also frequent. When endometriotic implant foci become walled off by adhesions or are embedded inside the tissue, endometrioma results. However, it is not precisely known why ovarian endometriomas develop only in some patients. The precise mechanism of pelvic pain associated with endometriosis is not completely understood; probably multiple factors are involved. Although it still remains unclear, it is thought that fresh peritoneal implants may cause functional pain such as dysmenorrhea, whereas deep-infiltrating Received July 10, 2006; revised November 15, 2006; accepted November 17, Reprint requests: Juan Luis Alcázar, M.D., Department of Obstetrics and Gynecology, Avenida Pio XII 36, Pamplona, Spain (FAX: ; jlalcazar@unav.es). endometriosis and ovarian endometriomas are responsible for organic-type pain such as dyspareunia and chronic pelvic pain (1, 2). Angiogenesis seems to be one of the processes involved in the pathogenesis of endometriosis (3), and was specifically associated with disease activity (4) and pain (5). Ultrasonography is commonly used as a diagnostic imaging tool in women with suspected endometriosis. Its efficiency was demonstrated in the diagnosis of ovarian endometrioma (6). Transvaginal color and power-doppler allows for the assessment of ovarian endometriomal vascularity (6, 7). We speculated that ovarian endometriomas in patients who present with pelvic pain would be more vascularized than those in asymptomatic women. The results of our previous study supported this hypothesis (8). However, this study was a retrospective analysis, and lacked a correlation with immunohistochemical or morphological vascular anal /07/$32.00 Fertility and Sterility Vol. 87, No. 6, June 2007 doi: /j.fertnstert Copyright 2007 American Society for Reproductive Medicine, Published by Elsevier Inc. 1271

2 yses as well as appropriate control over some possible confounding factors. In the present study, we aimed to confirm or rule out this hypothesis in a prospective study that correlated transvaginal power-doppler findings, immunohistochemical analysis of microvessel density (MVD), and complaints of pain in a series of selected women suspected of having ovarian cystic endometriosis. MATERIALS AND METHODS Patients A series of 65 women diagnosed as having ovarian cystic endometriosis by clinical history and B-mode ultrasonography between February 2003 February 2005 were included in this prospective study. After a detailed anamnesis was performed, patients were classified into two groups according to complaints of pelvic pain. Group A included asymptomatic patients or patients who presented with mild or moderate dysmenorrhea, but without either dyspareunia or chronic pelvic pain. Group B included patients who presented with severe dysmenorrhea (no response to a conventional type or dosage of analgesic treatment, and/or requiring bed rest) and/or dyspareunia (limiting pain during sexual intercourse) and/or chronic pelvic pain (pelvic pain throughout the menstrual cycle for 6 months). To establish the degree of pain, we used a visual analogue scale (VAS) (9). All patients underwent physical examination prior to ultrasound examination. Patients with a past history of confirmed pelvic endometriosis, pelvic surgery, pelvic inflammatory disease, or treatment with any kind of hormonal therapy (oral contraceptives, LH-releasing hormone (RH) analogues, clomiphen, or gonadotropins) in the previous 3 months were excluded. All patients provided written informed consent after the nature of the study was fully explained. Institutional review board approval (from the Clínica Universitaria de Navarra, Pamplona, Spain) was obtained before the study began. Ultrasound Examination All patients were evaluated by transvaginal power-doppler ultrasonography (TVPD) before surgery, with the use of a Voluson 730 (GE Healthcare, Zipf, Austria) with a MHz endovaginal probe and color, power, and pulsed Doppler during the follicular phase of the menstrual cycle. With the use of B-mode ultrasonography, an ovarian endometrioma was suspected in the presence of a typical pattern of a round-shaped homogeneous hypoechoic mass of low-levels echoes (6, 7). Endometriomal volume was calculated according to the prolate ellipsoid formula (height length width ), and expressed in milliliters. All scans were completed by means of TVPD to evaluate the vascularity of the endometrioma. Briefly, after morphological evaluation was performed, a power-doppler gate was activated to identify vessels within the mass. According to the criteria of Chui et al. (10), endometriomal vascularization was classified as absent (no color signals detected in the cyst wall), scanty ( 25% of the cyst wall with color signals), moderate (25% 50% of the cyst wall with color signals), or high ( 50% of cyst wall with color signals) (Fig. 1). Then, pulsed Doppler was used to interrogate each color signal identified, and a flow-velocity waveform (FVW) was obtained. Only arterial FVWs were processed. The pulsatility index (PI) and resistance index (RI) were electronically computed. When multiple signals were obtained, the lowest PI and RI were used for analytical purposes. Sample volume was set at a mm width. The high-pass filter was set at 50 Hz. The spatial peak temporal average intensity was 80 mw/cm 2 in B-mode, power-doppler, and pulsed Doppler modes. The pulse-repetition frequency was set at khz. One examiner (J.L.A.), who was blinded to patient complaints in order to avoid biased assessments of vascularity, performed all examinations. Surgery All patients underwent laparoscopic surgery for cyst removal within 10 days of ultrasound examination, to confirm the presence of endometriosis histologically. In all cases, the presence and type of pelvic adherences, the American Fertility Society (AFS) score, and the AFS stage were recorded (11). Pelvic adhesions were lysed, and peritoneal implants were treated by electrocoagulation. A definitive diagnosis of ovarian endometriosis was rendered in the presence of ectopic endometrium with both glandular and stromal components. Surgeons were blinded to patients complaints. Patients were evaluated 4 6 months after surgery. In group B, complaints of pain were reassessed with the use of the VAS. MVD Immunohistochemical Staining and Counting Tissue samples were fixed in a 10% formalin solution for 24 hours. They were then dehydrated in graded ethanol, cleaned in xylene, and embedded in paraffin. Sections were cut into 3 4- m slices, deparaffined in xylene, and rehydrated through a series of ethanol solutions, with a final rinse in water. After being dewaxed in xylol and rehydrated in graded alcohol, sections were washed with Tris-buffered saline (TBS, Tris-HCl 0.05 M, NaCl 0.5 M, ph 7.36), and treated with 3% hydrogen peroxide for 30 minutes at room temperature to block endogenous peroxidase. Immunostaining was performed with the use of primary antibodies against CD-34 (monoclonal antibody class II, 1272 Alcázar. Endometriomal vascularization and pelvic pain Vol. 87, No. 6, June 2007

FIGURE 1 Transvaginal power-doppler ultrasound depicting endometrioma vascularization. (A) Scanty vascularization. (B) Moderate vascularization. (C) High vascularization.

3 FIGURE 1 Transvaginal power-doppler ultrasound depicting endometrioma vascularization. (A) Scanty vascularization. (B) Moderate vascularization. (C) High vascularization. M7165 DAKO diluted to a 1:100 ratio [DAKO Inc., Golstrup, Denmark]) in a humid chamber overnight at 4 C, followed by the second antibody (biotynilated anti-mouse antibody) and peroxidase-labeled avidin (Universal DAKO LSAB2 system K0675; DAKO Inc.) for 30 minutes each. The chromogen 3=-diaminobenzidine tetrahydrochloride (DAB) was then used, which resulted in a brown color at the antigen site. Finally, sections were counterstained with Harris hematoxylin. Positive controls for the reaction consisted of paraffinembedded sections from renal carcinoma. Negative controls were created by substituting the primary antibody with a nonimmune serum. The CD-34 labeled vessels were counted for assessment of vascular surface density in all samples. Slices were observed at 400 magnification to identify the areas with the densest vascularity near the surface endometrial epithelium. The presence of microvessels was assessed in three random fields in each slice. In each field, MVD was estimated in the internal wall area (subepithelial) and in the external wall (subcapsular) (Fig. 2). The CD-34-labeled vessels were counted by one examiner (M.G.M.) blinded to the status of specimens. This same examiner performed a second count 1 week later to assess intraobserver variability. The average number of microvessels counted was used for analysis. Statistical Analysis According to data from our previous study, we estimated that a sample size of 32 women in each group was needed ( 0.05, 80%). The Kolmogorov-Smirnov test was used to compare the data distribution of continuous variables. Data are presented as mean with SD or median with interquartile range (IQR), according to the distribution. Student s t-test or the Mann-Whitney U test was used to compare continuous variables. The 2 test was used to compare cat- Fertility and Sterility 1273

FIGURE 2 Microvessel density (CD-34-labeled) in a case of endometrioma. TABLE 1 Demographic data, surgical findings, and power-doppler vascularization in asymptomatic and symptomatic women.

4 FIGURE 2 Microvessel density (CD-34-labeled) in a case of endometrioma. TABLE 1 Demographic data, surgical findings, and power-doppler vascularization in asymptomatic and symptomatic women. Group A Group B P value egorical variables. P.05 was considered statistically significant. All statistical analyses were performed with the statistical package SPSS 11.0 for Windows (SPSS, Inc., Chicago, IL). Age a 35.4 (7.5) 31.3 (5.1).015 Pregnancies a 0.6 (0.9) 0.3 (0.5).091 Abortions a 0.1 (0.2) 0.1 (0.2).321 Body mass 22.2 (2.2) 22.6 (3.2).665 index a Adhesions.270 None 9 (30%) 14 (47%) Smooth 5 (17%) 6 (20%) Dense 16 (53%) 13 (33%) AFS stage.313 II 6 (20%) 4 (13%) III 19 (63%) 16 (53%) IV 5 (17%) 10 (33%) AFS score 34.6 (28.9) 44.5 (30.1).199 Vascularization Absent 12 (40%) 4 (13%) Scanty 17 (57%) 0 Moderate 1 (3%) 19 (63%) High 0 7 (24%).0001 a Data expressed as mean, with SD in parentheses. RESULTS Five of 65 women evaluated were excluded because their definitive histological diagnosis was other than ovarian endometriosis (hemorrhagic cyst 3, dermoid cyst 2). Therefore, 60 patients were ultimately included: 30 in group A, and 30 in group B. The equal number in each group is coincidental. The patients mean age was 33.3 years (SD, 6.7; range, years). However, women in group B were significantly younger than in group A (Table 1). In group A, 25 (83%) women were asymptomatic, and five (17%) had mild dysmenorrhea. In group B, 21 (70%) had severe dysmenorrhea, five (17%) had severe dysmenorrhea and chronic pelvic pain, and four (13%) had moderate dysmenorrhea and chronic pelvic pain. The VAS was significantly higher in group B (mean, 7.6; SD, 1.5) than in group A (mean, 0.4; SD, 0.9) (P.001). The sonographic appearance of endometriomas was similar in both groups. In group A, 27 (90%) had the typical appearance, and three (10%) had a heterogeneous hypoechoic pattern. In group A, 28 (93%) had the typical appearance, and two (6.7%) had a heterogeneous hypoechoic pattern (P.24). The median volumes of endometriomas in group A (23.1 ml; IQR, 72.8; range, ml) and group B (33.2 ml; IQR, 79.8; range, ml) were comparable (P.71). Blood flow was found in 44 (73.3%) cases. Moderately or highly vascularized endometriomas were significantly more frequent in group B (Table 1). The mean PI and RI were significantly lower in group B (Table 2). The mean MVD count was significantly higher in group B (Table 2). The mean MVD was significantly higher in moderately highly vascularized endometriomas (93.2; SD, 40.1), compared with avascular or scantily vascularized endometriomas (55.7; SD, 29.4) (P.001) (Fig. 3). Regarding surgical findings, no statistical differences were found between the two groups (Table 1). The mean size of TABLE 2 Pulsed Doppler indexes and MVD in asymptomatic and symptomatic women. Group A Group B P value Lowest RI a 0.60 (0.15) 0.49 (0.4).012 Lowest PI a 1.14 (0.7) 0.81 (0.29).03 MVD b 59.8 (28.5) 85.3 (44.3).01 a Data expressed as mean (SD). b Data expressed as median (IQR) Alcázar. Endometriomal vascularization and pelvic pain Vol. 87, No. 6, June 2007

FIGURE 3 Mean MDV and 95% confidence intervals in absent-or-scantily and moderately-or-highly vascularized endometriomas. endometriomas was 4.7 cm (SD, 2.1; range, 1 10 cm) in group A, and 5.

5 FIGURE 3 Mean MDV and 95% confidence intervals in absent-or-scantily and moderately-or-highly vascularized endometriomas. endometriomas was 4.7 cm (SD, 2.1; range, 1 10 cm) in group A, and 5.1 cm (SD, 2.1; range, cm) in group B (P.579). The mean VAS after surgery (2.3; SD, 2.6) in group B was significantly lower than before surgery (7.6; SD, 1.5) (P.0001). Eighty percent (24 out of 30) of these women reported significant improvement in their clinical complaints. DISCUSSION Pelvic pain is one of the most frequent complaints reported by women suffering from pelvic endometriosis, and more specifically in patients presenting with ovarian endometriosis (2). The pathophysiology of pelvic pain associated with endometriosis is not completely understood. Several factors, such as inflammation with release of prostaglandins, recurrent microbleeding, mass effect, adhesions, neuronal involvement, and psychological factors, may be involved. Inflammation and recurrent microbleeding were related to dysmenorrhea and chronic pelvic pain in women with deeply infiltrating endometriosis (12). Mass effect and neuronal involvement were also related to pelvic pain in deeply infiltrating endometriosis (13). Adhesions were described as playing an independent role in the genesis of dysmenorrhea and ovarian cystic endometriosis related-pain (14). However, some authors did not find such an association (15 17). Our data also confirm that the presence and type of pelvic adherences, AFS stage, and AFS score seem unrelated to symptoms of pain. Recently, angiogenesis was claimed as one of the mechanisms involved (18). In fact, angiogenic factors were shown to be increased in ovarian endometriomas (19), and vascular surface density was also shown to be increased (20). Therefore, a correlation between ovarian endometriomal vascularization and symptoms of pain could be possible. Most studies that assessed angiogenesis in endometriosis used morphometric or immunohistochemical staining techniques in endometriotic tissue (5, 19), or analyzed vascular activity by measuring serum or peritoneal fluid concentrations of angiogenic factors (21, 22). Transvaginal color and power Doppler allow noninvasive in vivo assessment of ovarian endometriomal vascularity (6, 7). In our previous study, we found that ovarian endometriomas in patients who presented with pelvic pain were more vascularized than in asymptomatic women (8). However, Fertility and Sterility 1275

6 this was a retrospective analysis, with biases inherent to this type of study design. In the present study, we prospectively evaluated this hypothesis. Our results confirm that ovarian endometriomal vascularization tends to be higher in symptomatic women who present with ovarian endometriomas. In this study, we used power Doppler instead of conventional color Doppler because of the higher sensitivity of the former, thus overcoming one possible bias of our previous study. To the best of our knowledge, no other study addressing the same question has been published. On the other hand, we also assessed angiogenesis by analyzing MVD in a specimen of ovarian endometrioma. Some authors found that MVD is increased in ovarian endometriomal tissue compared with controls (20). We found that MVD is even more increased depending on the degree of pain, confirming that pelvic pain is related to the degree of vascularization of these lesions. Another question concerns whether vascularization, as assessed by power Doppler, correlates with MVD in the histological specimen. Several studies showed that MVD correlates with Doppler findings in breast cancer (23), endometrial cancer (24), and ovarian cancer (25). In the present study, we demonstrated for the first time that this correlation also exists in the case of ovarian endometrioma. We found that MVD was significantly higher in moderately or highly vascularized endometriomas compared with those without vascularization or with scanty vascularization, as detected by Doppler ultrasound. Our findings are interesting from not only the pathophysiological but also the clinical point of view. Our study demonstrates that a noninvasive technique might be used to assess in vivo endometriomal vascularization that power- Doppler findings correlate with MVD in the histological specimen and that, in turn, both are related to the severity of pain. Therefore, a possible application of our findings in clinical practice might be the use of power Doppler to select patients for new therapies, such as antiangiogenic drugs (26) in endometriosis, or to assess the response to conventional therapy. REFERENCES 1. Koninckx PR, Meuleman C, Demeyere S, Lesaffre E, Cornillie FJ. Suggestive evidence that pelvic endometriosis is a progressive disease, whereas deeply infiltrating endometriosis is associated with pelvic pain. Fertil Steril 1991;55: Vercellini P. Endometriosis: what a pain it is? Semin Reprod Endocrinol 1997;15: Healy DL, Rogers PA, Hii L, Wingfield M. Angiogenesis: a new theory for endometriosis. Hum Reprod Update 1998;4: Cornillie FJ, Oosterlynck D, Lauweryns JM, Koninckx PR. Deeply infiltrating endometriosis: histology and clinical significance. Fertil Steril 1990;53: Nisolle M, Casanas-Roux F, Anaf V, Mine JM, Donnez J. Morphometric study of stromal vascularization in peritoneal endometriosis. Fertil Steril 1993;59: Alcázar JL, Laparte C, Jurado M, López-García G. The role of transvaginal ultrasonography combined with color velocity imaging and pulsed Doppler in the diagnosis of endometrioma. Fertil Steril 1997; 67: Guerriero S, Ajossa S, Mais V, Risalvato A, Lai MP, Melis GB. The diagnosis of endometriomas using colour Doppler energy imaging. Hum Reprod 1998;13: Alcázar JL. Transvaginal color Doppler in patients with ovarian endometriomas and pelvic pain. Hum Reprod 2001;16: Price DD, McGrath PA, Rafii A, Buckingham B. The validation of visual analogue scales as ratio scale measures for chronic and experimental pain. Pain 1983;17: Chui DK, Pugh ND, Walker SM, Gregory L, Shaw RW. Follicular vascularity: the predictive value of transvaginal power Doppler ultrasonography in an in-vitro fertilization programme: a preliminary study. Hum Reprod 1997;12: American Society for Reproductive Medicine classification of endometriosis. Fertil Steril 1997;67: Chapron C, Fauconnier A, Dubuisson JB, Barakat H, Vieira M, Breart G. Deep infiltrating endometriosis: relation between severity of dysmenorrhoea and extent of disease. Hum Reprod 2003;18: Anaf V, Simon P, El Nakadi I, Fayt I, Buxant F, Simonart T, et al. Relationship between endometriotic foci and nerves in rectovaginal endometriotic nodules. Hum Reprod 2000;15: Fauconnier A, Chapron C. Endometriosis and pelvic pain: epidemiological evidence of the relationships and implications. Hum Reprod Update 2005;11: Fedele L, Parazzini F, Bianchi S, Arcaini L, Candiani GB. Stage and localization of pelvic endometriosis and pain. Fertil Steril 1990;53: Gruppo Italiano per lo Studio dell Endometriosi. Relationship between stage, site and morphological characteristics of pelvic endometriosis and pain. Hum Reprod 2001;16: Perper MM, Nezhat F, Goldstein H, Nezhat CH, Nezhat C. Dysmenorrhea is related to the number of implants in endometriosis patients. Fertil Steril 1995;63: Groothuis PG, Nap AW, Winterhager E, Grummer R. Vascular development in endometriosis. Angiogenesis 2005;8: Fujimoto J, Sakaguchi H, Hirose R, Tamaya T. Expression of plateletderived endothelial cell growth factor (PD-ECGF) related to angiogenesis in ovarian endometrioma. J Clin Endocrinol Metab 1998;84: Inan S, Kuscu NK, Vatansever S, Ozbilgin K, Koyuncu F, Sayhan S. Increased vascular surface density in ovarian endometriosis. Gynecol Endocrinol 2003;17: Donnez J, Smoes P, Gillerot S, Casanas-Roux F, Nisolle M. Vascular endothelial growth factor (VEGF) in endometriosis. Hum Reprod 1998; 13: McLaren J, Prentice A, Charnock-Jones DS, Sharkey AM, Smith SR. Vascular endothelial growth factor (VEGF) concentrations are elevated in peritoneal fluid of women with endometriosis. Hum Reprod 1996; 11: Yang WT, Tse GM, Lam PK, Metreweli C, Chang J. Correlation between color power Doppler sonographic measurement of breast tumor vasculature and immunohistochemical analysis of microvessel density for the quantitation of angiogenesis. J Ultrasound Med 2002; 21: Lee CN, Cheng WF, Chen CA, Chu JS, Hsieh CY, Hsieh FJ. Angiogenesis of endometrial carcinomas assessed by measurement of intratumoral blood flow, microvessel density, and vascular endothelial growth factor levels. Obstet Gynecol 2000;96: Emoto M, Iwasaki H, Mimura K, Kawarabayashi T, Kikuchi M. Differences in the angiogenesis of benign and malignant ovarian tumors, demonstrated by analyses of color Doppler ultrasound, immunohistochemistry, and microvessel density. Cancer 1997;80: Nap AW, Griffioen AW, Dunselman GA, Bouma-Ter Steege JC, Thijssen VL, Evers JL, et al. Antiangiogenesis therapy for endometriosis. J Clin Endocrinol Metab 2004;89: Alcázar. Endometriomal vascularization and pelvic pain Vol. 87, No. 6, June 2007

Rectovaginal Septum Endometriosis: An Immunohistochemical Analysis of 62 Cases

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