Abdominal Wall Endometriosis: Myofibroblasts as a ...

Novel Insights from Clinical Practice Received: March 15, 2016 Accepted after revision: September 26, 2016 Published online: November 4, 2016

Gynecol Obstet Invest DOI: 10.1159/000452101

Abdominal Wall Endometriosis: Myofibroblasts as a Possible Evidence of Metaplasia: A Case Report Mohamed Gamal Ibrahim a Eleonore Delarue a Elene Abesadze a Matthias Haas b Jalid Sehouli a Vito Chiantera a Sylvia Mechsner a

Endometriosezentrum Charité, a Gynecology Clinic, and b Radiology Clinic, Charité Universitätsmedizin – Campus Benjamin Franklin, Berlin, Germany

Established Facts • Abdominal wall endometriosis (AWE) is predictable among patients with recurrent endometriosis surgeries. • Ultrasonography is the first investigation for AWE.

Novel Insights • Myofibroblasts are a possible evidence of metaplasia in AWE.

Abstract In this study, we report about a patient with extra-uterine endometriosis (EM) in the abdominal wall muscle with evident metaplasia based on the abundant alpha smooth muscle actin (ASMA)-expressing myofibroblasts. Laparotomy excision of the abdominal wall EM was done following ultrasonographic evidence of a hypodense swelling in the right

© 2016 S. Karger AG, Basel 0378–7346/16/0000–0000$39.50/0 E-Mail [email protected] www.karger.com/goi

rectus abdominis, which was confirmed by MRI. Immunohistochemistry staining for ASMA and collagen I was done, with the results confirming that endometriotic stromal cells expressed both. Anterior abdominal wall endometriosis was suspected because of the patient’s history of recurrent EM combined with the cyclic nature of symptoms. MRI is useful in determining the extent of the disease. In case of persisting symptoms even under hormonal treatment, surgical excision is mandatory. The expression of both ASMA and collagen I in and around EM lesions supports the notion of the metaplastic process in the course of disease development. © 2016 S. Karger AG, Basel

Sylvia Mechsner, MD Department of Gynaecology Charité University of Medicine DE–12203 Berlin (Germany) E-Mail sylvia.mechsner @ charite.de

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Key Words Endometriosis · Myofibroblasts · Rectus abdominis · Metaplasia

Introduction

Endometriosis (EM) is an enigmatic disease affecting 10–15% of women of reproductive age [1, 2]. The finding of endometrial glands and/or stroma outside the uterine cavity is the golden histopathological diagnostic standard [3]. Many theories have been formulated to clarify the origin of the disease, yet the pathogenesis is still puzzling. While a haematogenous dissemination of endometrial tissue to distant organs can explain EM on the liver [4] or in the urinary tract [5], a lymphatic dissemination seems more reasonable to explain EM in the regional lymph nodes [6]. However, the concept of retrograde menstruation and the implantation of the shed endometrium on the pelvic organs are still widely accepted [7]. The implanted ectopic endometrium might invade and implant deeply in the peritoneum. Certain studies suggest that it is the coelomic epithelium that might undergo metaplasia and transform into endometrial-like glands and stroma [8, 9]. Recently, attention of many researchers aroused to study the metaplasia in EM lesions, which could be supported by finding metaplastic myofibroblasts originating either from the endometriotic stromal cells or the surrounding mesenchymal cells. Myofibroblasts were previously seen in all different forms of EM, for example, peritoneal, ovarian and recto-vaginal EM [4, 10– 14]. Besides these enigmatic theories, development of endometriotic lesions due to translocation of uterine endometrial cells during surgeries like C-section is also possible. This study investigates a patient with extra-uterine EM in the abdominal wall muscle with evident metaplasia based on the abundant alpha smooth muscle actin (ASMA)-expressing myofibroblasts.

Case Report

Materials and Methods To search for myofibroblasts as an evidence for tissue metaplasia, paraffin slides were immunohistochemistry stained for ASMA – as a marker for both myofibroblasts and smooth muscle cells, but not for fibroblasts [8, 9] – in addition, collagen I was also used (normally synthesised by fibroblasts and myofibroblasts) [15]. The paraffin block of the affected rectus abdominis muscle was 4 μm sliced. Deparaffinisation in xylene, followed by rehydration in a series of decreasing ethanol concentration, was performed as previously described [11]. Antigen retrieval was done by treating the slides in the corresponding buffer solution (citrate, pH 6 and target 9, pH 9) for ASMA and collagen I in the streamer (Multi Gourmet, type 3216, Braun, Germany) for 20 min, respectively. Then they were cooled down in Tris buffer for another 20 min. Subsequently, the slides were blocked with hydrogen peroxide 3% for 10 min, which was followed by blocking them in 10% fetal calf serum for another 30 min. This was done to minimise the background staining. Later, the slides were incubated in the primary antibody (smooth muscle actin, clone 1A4, 1: 100, mouse monoclonal IgG, Dako and collagen I ab 34710, rabbit polyclonal IgG, 1: 100, abcam) for 60 min. This was followed by incubating them in a suitable secondary antibody (rabbit anti-mouse IgG (H+L) and mouse anti-rabbit IgG (H+L) Biotin-SP-conjugated AffiniPure, 1: 400, Jackson ImmunoResearch) for 40 min at room temperature (RT) to mark the target protein. Streptavidin-AP was incubated for 40 min at RT, followed by 20 min incubation with 2-solution DAB kit (Invitrogen) to get the final colour. Finally, they were counterstained with Mayer’s hemalum solution (Merck K GaA, Germany) for 35 s followed by covering them with Eukitt quick-hardening mounting medium (Sigma-Aldrich) and cover slips. A light microscope (Carl Zeiss Axiophot Microscope, Göttingen, Germany) with ×100, ×200, and ×400 power of magnification was used for evaluation. The images were edited using a Photoscape 3.7 program (Mooi Tech).


Ibrahim/Delarue/Abesadze/Haas/Sehouli/ Chiantera/Mechsner


A 31-year-old multiparous patient presented at the outpatient clinic complaining of chronic persistent pain in the anterior abdominal wall. The pain was associated with cyclical localised swelling in her abdomen related to her regular menstrual cycle. The patient also suffered from a catamenial pleural pain, which is resistant to oral analgesics and local infiltration anaesthetics. Over a period of 5 years, the patient had undergone multiple laparoscopic surgeries for excision of recurrent EM. Two years earlier, the patient observed localised abdominal swelling together with chronic persistent lower abdominal pain, following her last laparoscopy. She suffered from severe disabling dysmenorrhea, cyclic migraines with nausea and vomiting. Her quality of life was great-

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ly compromised. The patient’s last delivery, which was a C-section, was performed in 2008. An abdominal examination revealed soft swelling in the right lower abdominal wall, and a superficial ultrasonography showed a cystic hypoechogenic swelling in the right lower rectus abdominis muscle, measuring 23 × 17 × 41 mm. The MRI showed a traversing intra-muscular T1-weighted high signal intensity lesion in the lower right rectus abdominis muscle (fig. 1). MRI imaging was performed with a clinical 1.5-T system (Magnetom Avanto; Siemens Medical Solutions, Germany) and a torso phased-array 12-element coil. The imaging protocol included axial and coronal fat-saturated T1-weighted images with a 5 mm slice thickness acquired after intravenous administration of a low-molecular weight Gadoliniumbased contrast agent (Gadothek, Medithek, Germany). A laparoscopy and a coagulation of the uterine serosa were performed, the latter being studded with endometrial shreds. A midline laparotomy with an excision of the involved segment of the right rectus abdominis muscle was also done. A vicryl mesh was inserted to replace the muscular defect (fig. 2). The post-operative period was a success, and the patient was discharged. Histopathologically, this confirmed the diagnosis of EM.

Right

Right

Fig. 1. Coronal (left) and axial T1 (middle) weighted fat-saturated, contrast-enhanced MR shows a hyperintense

Color version available online

mass (arrow) in the lower portion of the right rectus abdominis muscle. Axial T1-weighted MR (right), shows a hyperintense signal of the mass corresponding to blood products within the lesion.

a

b

d

Fig. 2. Intraoperative management of the rectus abdominis EM. a A midline laparotomy followed by laparoscopy revealed an indurated swelling (black circle), traversing the lower right segment of the rectus abdominis, with brownish discoloruation of the surface. A chocolate-like fluid is poured out, while manipulating it. b The affected segment (black arrow) was excised, but was attached either to the

vesico-uterine ligament or to the utero-vesical pouch peritoneum, traction of the urinary bladder (white arrow). The segment was excised and the bladder integrity was tested. c A vicryl mesh was inserted to replace the muscular defect in the right rectus abdominis muscle. d The excised segment of the right rectus abdominis muscle showed chocolate fluid-filled multiloculated lesion (white arrows).

AWE: Myofibroblasts as a Possible Evidence of Metaplasia


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Color version available online

a

b

c

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dometrial glands are ASMA stained, indicating the abundance of myofibroblasts. ×200. b While the epithelial cells are not stained,

Results

The endometriotic stromal cells around the endometriotic glands showed a strong ASMA cytoplasmic expression. The surrounding mesenchymal cells showed a moderate collagen I cytoplasmic staining (fig. 3).

Discussion

Anterior abdominal wall endometriosis (AWE) is a very rare case, with an estimated prevalence of 0.01–1%. It can be clinically suspected when the typical cyclical abdominal pain occurs, though it is only evident in 50% of the cases. Non-cyclic pain should not be excluded in the diagnosis, as it might be the main manifestation in onethird of all patients. The clinical palpation of an abdominal swelling proves to be a reliable diagnostic sign, being previously evident in all cases of AWE, as in our case. The 4


the surrounding stromal cells expressed ASMA. ×400. c Collagen I expression is seen localised to the periphery of the lesion. ×100, d characterizing the mesenchymal cells in the periphery ×400.

prevalence of AWE, developing after caesarean section is higher, reaching 0.07–0.47% [16]. A differential diagnosis should always be considered: incisional hernia, desmoid tumour, suture granuloma, lipoma, and hematoma, primary or secondary malignancy. EM is histologically composed of 3 components: endometriotic glands, surrounding stromal cells, and smooth muscle-like cells [17, 18]. The origin of lesions can be discussed in 2 different ways: primary umbilical EM seems to have its origin from a metaplastic process of persisting coelom epithelial cells. However, in our case the origin of the endometriotic lesion seems to be the translocated endometrial tissue, during C-section. Translocated endometrial tissue undergoes metaplasia, constructing an endometriotic lesion. Interestingly, the endometriotic stromal cells of the abdominal wall showed a strong expression of ASMA, as a marker of myofibroblasts [8, 9]. This theory could point to a metaplastic process, which usually occurs during the developIbrahim/Delarue/Abesadze/Haas/Sehouli/ Chiantera/Mechsner


Fig. 3. ASMA and collagen immune expression in the ectopic endometrial tissue. a The stromal cells surrounding the ectopic en-

ment of EM [19, 20]. Myofibroblasts are specialised cells, usually recruited to the site of injured tissue and may originate from local fibroblasts. They produce collagen during the wound healing process. Furthermore, they acquire a contractile phenotype, which allows them to contract the granulation tissue, therefore, minimising its size [21]. They were previously seen in different forms of EM, for example, the peritoneal [11], rectovaginal, pulmonary [14], and hepatic EM [4]. However, others suggest a smooth muscle metaplasia in EM lesions. Immature smooth muscle cells are mainly localised in the centre of the lesions, where they express only ASMA. But on the periphery mature smooth muscle cells are seen expressing ASMA, caldesmon, and desmin, the latter as markers of differentiated smooth muscle cells [22]. Myofibroblasts are not only seen in EM lesions. An in vitro study showed that primary endometrial stromal cells express ASMA, after incubation with TGFβ1 (unpublished data) and have the potential to differentiate into myofibroblasts. Myofibroblasts can be a potential therapeutic target for EM, as the inhibition of ASMA expression in in vitro can reduce the contractility of myofibroblasts, and hence reduce the mechanical tension exerted on EM lesions. Reduced mechanical tension on the tissue may help reduce tissue micro-disruption, and subsequently reduce the possibility of implantation of new shed endometrium in the abdominal cavity [23]. Furthermore, myofibroblasts and smooth muscle-like cells in EM can cause EMassociated chronic pain. This can be a nidus for a new research to elucidate the role of myofibroblasts and smooth muscle cells [24].

Abdominal wall EM mostly occurs after obstetrical and gynaecological surgeries, especially in caesarean section scars; on the contrary, the disease has also been recorded in women without a previous history of surgery [25]. However, as discussed earlier, the origin of the endometriotic lesion is supposed to be the translocated endometrial tissue undergoing metaplasia. Therefore, surgical techniques used during C-section have to be sufficiently distinct to avoid all recurrence. This is essential to avoid contamination with decidualised endometrial cells, such as by cleaning the cavum after removal of the placenta. Furthermore, it is strongly recommended that the abdominal wound be kept clean from endometrial tissue at the end of the surgical procedure [26]. This can prevent the implantation of endometrium out of the uterine cavity.

Conclusion

AWE should be suspected when the patient has a history of recurrent EM and a cyclical nature of symptoms. MRI is very valuable in determining the extent of the disease. In case of persisting symptoms even under hormonal treatment, surgical excision is mandatory. The expression of both ASMA and collagen I in and around EM lesions supports a metaplastic process in the course of disease development and elucidates the role of these cells in EM. Disclosure Statement There are no conflicts of interest for any of the authors and coauthors that might relate to the research or its results.

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