Original Article
Deep Invasive Endometriosis Lesions of the Rectosigmoid May Be Related to Alterations in Cell Kinetics
Reproductive Sciences 1-7 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719115574341 rs.sagepub.com
Marco Antonio Bassi, MD, PhD1, Victor Arias, MD, PhD2, Nicolau D’Amico Filho, MD3, Ba´rbara Yasmin Gueuvoghlanian-Silva, PhD4, Mauricio Simoes Abrao, MD, PhD1, and Sergio Podgaec, MD, PhD1,4
Abstract Objectives: The aim of this study was to analyze cell kinetics through expression and apoptosis of topoisomerase 2-a (TOP2A), p53, and c-erb2 in rectosigmoid endometriotic lesions and in healthy endometrial tissue and to establish correlations between such findings and clinical data in patients with rectosigmoid endometriosis. Methods: Sixty patients with rectosigmoid endometriosis and 20 control women without endometriosis were included. Immunohistochemical assays were used to measure expression of TOP2A, p53, and c-erB-2. Apoptosis was quantified by directly counting the apoptotic bodies. Findings: The number of lesions was positively correlated with expression of TOP2A in the lesion. There was also significant correlation between the lesions’ size and number and cell turnover index. Apoptosis index (AI) was the same for endometriosis lesions and eutopic endometrium. Expression of TOP2A was significantly lower in the endometriosis group compared to the controls. Conclusions: Changes in cell proliferation but not in the AI in rectosigmoid endometriosis are indicative of an imbalance in cell kinetics that may lead to the development of the disease. Keywords apoptosis, bowel endometriosis, cell proliferation, deep infiltrating endometriosis, topoisomerase 2-a
Introduction Endometriosis is a heterogeneous disease characterized by the growth of endometrial tissue outside the uterine cavity and affects up to 15% of women in reproductive age.1 Although the etiology of endometriosis remains unknown, etiopathogenic theories include retrograde dissemination of eutopic tissue,2,3 coelomic metaplasia,3,4 lymphatic and hematogeneous dissemination,3 angiogenesis alterations,5 inhibition of matrix metalloproteinases,6 and immunological7 and genetic factors.8,9 Superficial peritoneal foci, ovarian endometriomas, and deep infiltrative lesions (those penetrating a depth of more than 5 mm) are considered 3 different entities.10 Approximately 5% to 10% of cases with endometriosis involve lesion in the rectosigmoid.11 Cell kinetics reflects the homeostatic balance in healthy tissues and can be understood as the balance between cell proliferation and cell death. Whether programmed cell death— known as apoptosis—is occurring can be assessed in different tissues by identifying the presence of apoptotic bodies with 1 or more of several characteristics, including small and condensed cytoplasmic volume and chromatin margination.12 Cell
proliferation can be estimated by the expression of topoisomerase 2-a (TOP2A), an enzyme involved in DNA transcription.13 The cell kinetics of endometriotic lesions may also be regulated by oncogene proteins such as p53 and c-erbB-2, which have been found in eutopic and ectopic endometrium of women with endometriosis.14 Apoptosis is an essential process in endometrium for the cycling turnover of this tissue. Usually, every month senescent or dysfunctional endometrium cells undergo apoptosis, and
1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Sao Paulo, Sa˜o Paulo, Brazil 2 Department of Pathology, Perola Byington Hospital, Sa˜o Paulo, Brazil 3 Department of Obstetrics and Gynecology, Samaritano Hospital, Sa˜o Paulo, Brazil 4 Jewish Teaching and Research Institute, Albert Einstein Hospital, Sa˜o Paulo, Brazil
Corresponding Author: Sergio Podgaec, Avenida Albert Einstein 627, Bloco A1, sala 113, Morumbi, Sa˜o Paulo, Sa˜o Paulo 05652-900, Brazil. Email: [email protected]
Downloaded from rsx.sagepub.com at Swinburne Univ of Technology on December 16, 2015
2
Reproductive Sciences
open the way for the proliferation of new cells that will reconstitute the endometrium layer.15 Nonphysiological changes in cell kinetics generally are due alterations in genes involved in cell cycle regulation. These alterations tend to result in accumulation of cells in the tissue, either as consequence of an increase in the number of cells in proliferation or from reduction in the number of cells entering apoptosis.16,17 Several studies have found significant reduction in apoptosis in glandular18-20 and stromal18,19 components of endometriotic tissue. Jones et al18 reported the absence of apoptosis in stromal cells of ectopic endometrial tissue. Other researchers have suggested that there is a loss of the cyclic variability in glandular cells of endometriotic lesions, which is a characteristic of the apoptosis that occurs in eutopic endometrial tissue.17,19 Ilad et al21 suggested that apoptosis alterations in stromal cells might play a crucial role in the development of endometriosis. Indeed, there is an imbalance of apoptotic factors in endometriosis, with higher expression of antiapoptotic and lower expression of proapoptotic factors by endometrial cells.22 The present study measures cell proliferation and apoptosis in both glandular and stromal compartments of endometriosis lesions, in order to assess how cell kinetics determine endometriotic infiltration of tissues distant from the uterus and correlate with clinical characteristics of patients with rectosigmoid endometriosis.
Material and Method Patient Selection Between June 2008 and May 2011, a cross-sectional, prospective, case–control study was conducted by the Obstetrics and Gynecology Department of the University of Sa˜o Paulo School of Medicine. The study protocol was approved by the institutional review board (Ethical Approval #1328/06), and informed consent was obtained from all patients. The case cohort included patients with histologically confirmed deep infiltrative endometriosis of the rectosigmoid. The control group consisted of women without endometriosis, verified during laparoscopy for tubal ligation. Inclusion criteria for both groups were women between 25 and 45 years old, regular menstrual cycles (intervals between 25 and 32 days), no clinical signs of or laboratory test results consistent with ovarian failure, and no use of any hormonal treatment during the 3 months prior to surgery. During the study period, 100 patients with pelvic pain and ultrasound and/or magnetic resonance imaging findings23,24 consistent with deep infiltrating endometriosis lesions in the rectosigmoid met criteria for surgical treatment of the disease. The failure of clinical treatment to control the pain and the presence of rectosigmoid lesions exceeding 2 cm affecting the internal muscularis, submucosa, or mucosa layer of the bowel wall was considered an indication for surgery. All patients underwent laparoscopic rectosigmoid segmental resection. Of the 100 patients, 60 met inclusion criteria of the study and had 2
Table 1. Clinical Data of Patients With Rectosigmoid Endometriosis and of Control Group. Clinical Data Age, mean (SD) Menstrual cycle: follicular phase, n (%) Menstrual cycle: secretory phase, n (%) Dysmenorrhea, n (%) Deep dyspareunia, n (%) Chronic pelvic pain, n (%) Cyclic bowel symptoms, n (%)
Endometriosis (n ¼ 60) 33.8 (+5.9) 30 (50.0) 30 (50.0) 60 49 48 50
(100.0) (81.6) (80.0) (83.3)
Control (n ¼ 20)
P Value
32.9 (+4.3) .528 10 (50.0) 1.0 10 (50.0) 3 (15.0) 2 (10.0) 0 (0) 1 (5.0)
1.0
Deep Invasive Endometriosis Lesions of the Rectosigmoid May Be Related to Alterations in Cell Kinetics
Reproductive Sciences 1-7 ª The Author(s) 2015 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/1933719115574341 rs.sagepub.com
Marco Antonio Bassi, MD, PhD1, Victor Arias, MD, PhD2, Nicolau D’Amico Filho, MD3, Ba´rbara Yasmin Gueuvoghlanian-Silva, PhD4, Mauricio Simoes Abrao, MD, PhD1, and Sergio Podgaec, MD, PhD1,4
Abstract Objectives: The aim of this study was to analyze cell kinetics through expression and apoptosis of topoisomerase 2-a (TOP2A), p53, and c-erb2 in rectosigmoid endometriotic lesions and in healthy endometrial tissue and to establish correlations between such findings and clinical data in patients with rectosigmoid endometriosis. Methods: Sixty patients with rectosigmoid endometriosis and 20 control women without endometriosis were included. Immunohistochemical assays were used to measure expression of TOP2A, p53, and c-erB-2. Apoptosis was quantified by directly counting the apoptotic bodies. Findings: The number of lesions was positively correlated with expression of TOP2A in the lesion. There was also significant correlation between the lesions’ size and number and cell turnover index. Apoptosis index (AI) was the same for endometriosis lesions and eutopic endometrium. Expression of TOP2A was significantly lower in the endometriosis group compared to the controls. Conclusions: Changes in cell proliferation but not in the AI in rectosigmoid endometriosis are indicative of an imbalance in cell kinetics that may lead to the development of the disease. Keywords apoptosis, bowel endometriosis, cell proliferation, deep infiltrating endometriosis, topoisomerase 2-a
Introduction Endometriosis is a heterogeneous disease characterized by the growth of endometrial tissue outside the uterine cavity and affects up to 15% of women in reproductive age.1 Although the etiology of endometriosis remains unknown, etiopathogenic theories include retrograde dissemination of eutopic tissue,2,3 coelomic metaplasia,3,4 lymphatic and hematogeneous dissemination,3 angiogenesis alterations,5 inhibition of matrix metalloproteinases,6 and immunological7 and genetic factors.8,9 Superficial peritoneal foci, ovarian endometriomas, and deep infiltrative lesions (those penetrating a depth of more than 5 mm) are considered 3 different entities.10 Approximately 5% to 10% of cases with endometriosis involve lesion in the rectosigmoid.11 Cell kinetics reflects the homeostatic balance in healthy tissues and can be understood as the balance between cell proliferation and cell death. Whether programmed cell death— known as apoptosis—is occurring can be assessed in different tissues by identifying the presence of apoptotic bodies with 1 or more of several characteristics, including small and condensed cytoplasmic volume and chromatin margination.12 Cell
proliferation can be estimated by the expression of topoisomerase 2-a (TOP2A), an enzyme involved in DNA transcription.13 The cell kinetics of endometriotic lesions may also be regulated by oncogene proteins such as p53 and c-erbB-2, which have been found in eutopic and ectopic endometrium of women with endometriosis.14 Apoptosis is an essential process in endometrium for the cycling turnover of this tissue. Usually, every month senescent or dysfunctional endometrium cells undergo apoptosis, and
1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Sao Paulo, Sa˜o Paulo, Brazil 2 Department of Pathology, Perola Byington Hospital, Sa˜o Paulo, Brazil 3 Department of Obstetrics and Gynecology, Samaritano Hospital, Sa˜o Paulo, Brazil 4 Jewish Teaching and Research Institute, Albert Einstein Hospital, Sa˜o Paulo, Brazil
Corresponding Author: Sergio Podgaec, Avenida Albert Einstein 627, Bloco A1, sala 113, Morumbi, Sa˜o Paulo, Sa˜o Paulo 05652-900, Brazil. Email: [email protected]
Downloaded from rsx.sagepub.com at Swinburne Univ of Technology on December 16, 2015
2
Reproductive Sciences
open the way for the proliferation of new cells that will reconstitute the endometrium layer.15 Nonphysiological changes in cell kinetics generally are due alterations in genes involved in cell cycle regulation. These alterations tend to result in accumulation of cells in the tissue, either as consequence of an increase in the number of cells in proliferation or from reduction in the number of cells entering apoptosis.16,17 Several studies have found significant reduction in apoptosis in glandular18-20 and stromal18,19 components of endometriotic tissue. Jones et al18 reported the absence of apoptosis in stromal cells of ectopic endometrial tissue. Other researchers have suggested that there is a loss of the cyclic variability in glandular cells of endometriotic lesions, which is a characteristic of the apoptosis that occurs in eutopic endometrial tissue.17,19 Ilad et al21 suggested that apoptosis alterations in stromal cells might play a crucial role in the development of endometriosis. Indeed, there is an imbalance of apoptotic factors in endometriosis, with higher expression of antiapoptotic and lower expression of proapoptotic factors by endometrial cells.22 The present study measures cell proliferation and apoptosis in both glandular and stromal compartments of endometriosis lesions, in order to assess how cell kinetics determine endometriotic infiltration of tissues distant from the uterus and correlate with clinical characteristics of patients with rectosigmoid endometriosis.
Material and Method Patient Selection Between June 2008 and May 2011, a cross-sectional, prospective, case–control study was conducted by the Obstetrics and Gynecology Department of the University of Sa˜o Paulo School of Medicine. The study protocol was approved by the institutional review board (Ethical Approval #1328/06), and informed consent was obtained from all patients. The case cohort included patients with histologically confirmed deep infiltrative endometriosis of the rectosigmoid. The control group consisted of women without endometriosis, verified during laparoscopy for tubal ligation. Inclusion criteria for both groups were women between 25 and 45 years old, regular menstrual cycles (intervals between 25 and 32 days), no clinical signs of or laboratory test results consistent with ovarian failure, and no use of any hormonal treatment during the 3 months prior to surgery. During the study period, 100 patients with pelvic pain and ultrasound and/or magnetic resonance imaging findings23,24 consistent with deep infiltrating endometriosis lesions in the rectosigmoid met criteria for surgical treatment of the disease. The failure of clinical treatment to control the pain and the presence of rectosigmoid lesions exceeding 2 cm affecting the internal muscularis, submucosa, or mucosa layer of the bowel wall was considered an indication for surgery. All patients underwent laparoscopic rectosigmoid segmental resection. Of the 100 patients, 60 met inclusion criteria of the study and had 2
Table 1. Clinical Data of Patients With Rectosigmoid Endometriosis and of Control Group. Clinical Data Age, mean (SD) Menstrual cycle: follicular phase, n (%) Menstrual cycle: secretory phase, n (%) Dysmenorrhea, n (%) Deep dyspareunia, n (%) Chronic pelvic pain, n (%) Cyclic bowel symptoms, n (%)
Endometriosis (n ¼ 60) 33.8 (+5.9) 30 (50.0) 30 (50.0) 60 49 48 50
(100.0) (81.6) (80.0) (83.3)
Control (n ¼ 20)
P Value
32.9 (+4.3) .528 10 (50.0) 1.0 10 (50.0) 3 (15.0) 2 (10.0) 0 (0) 1 (5.0)
1.0
