Oestrogen receptors and hypoxia inducible factor 1 alpha expression in abdominal wall endometriosis

Histological confirmation of endometrioma is based on detecting at least two of the three following features: endometrial stromal, endometrial like glands, and hemosiderin pigment ( Dwivedi et al., 2002 ). Immunostaining intensity for ERs and HIF-1α were characterized in the epithelial and stromal compartments of each specimen. Representative images are shown in Figure 1 and Figure 2 , and staining intensities are described in Table 2 and Table 3 . Generally, ERα is the dominant receptor in normal uteri. Positive ERα staining was detected in the nuclei of both endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). In CoEM, a significant decrease in ERα expression level was noted in the secretory phase endometrium (p=0.002 in EECs and p<0.0001 in ESCs) compared to in the proliferative phase. In AWEM, ERα distribution did not differ from the CoEM. Similarly, the positive rate for ERα in both EECs and ESCs were higher in the proliferative phase compared with those of the secretory phase during the menstrual cycle (p=0.0137 in EECs and p=0.0267 in ESCs). However, ERα expression levels in EECs and ESCs were lower in AWEM compared with the CoEM of the corresponding cycle (proliferative phase: p<0.0001 in EECs and p=0.0002 in ESCs; secretory phase: p=0.0052 in EECs and p=0.0302 in ESCs). Similarly to ERα, the expression pattern of ERβ was also found in the nuclei, and in both CoEM and AWEM, the expression level of ERβ was always higher in the proliferative phase than in the secretory phase (CoEM: p=0.0435 in EECs and p=0.0001 in ESCs; AWEM: p=0.0017 in EECs and p<0.0001 in ESCs). Unlike ERα, expression of ERβ in AWEM was significantly higher than that in CoEM in both EECs and ESCs in the corresponding menstrual cycle (proliferative phase: p=0.0003 in EECs and p<0.0001 in ESCs; secretory phase: p=0.0154 in EECsand p=0.0025). A low level of GPER expression was detected in CoEM, mainly in the cytoplasm of EECs. Expression of GPER, one of the estrogen receptors regulated by ovarian hormone, increased in the proliferative phase while decreased in secretory phase in both CoEM and AWEM (CoEM: p=0.0023 in EECs and p=0.0003in ESCs; AWEM: p=0.0012 in EECs and p=0.0047 in ESCs). Surprisingly, we observed a completely different GPER expression pattern in AWEM shown in two aspects, the first of which is its expression site. In AWEM, GPER accumulated in the nuclei of EECs and ESCs, although the cytoplasm was still expressed. The second is the expression level: expression of GPER was significantly higher in AWEM than that in CoEM (proliferative phase: p<0.0001 in EECs and p=0.421 in ESCs; secretory phase: p=0.0207 in EECs and p= 0.0262). Staining of endometrium with HIF-1α antibody showed distinctive expression patterns in CoEM and AWEM. HIF-1α was predominantly detected in the nuclei of both the stromal and epithelial compartments of endometrium. In CoEM, expression of HIF-1α was significantly higher during the secretory phase than that during the proliferative phase (p=0.0070 in EECs and p =0.0239 in ESCs). In addition, in AWEM, there was no difference in HIF-1α expression during the menstrual cycle (p=0.8971 in EECs and p=0.9481 in ESCs), which suggests that hormonal changes do not significantly affect HIF-1α expression in abdominal endometriotic cysts. Expression of HIF-1α in AWEM was also significantly lower than in CoEM than in AWEM (proliferative phase: p=0.0036 in EECs and p=0.0156 in ESCs; secretory phase: p=0.0085 in EECs and p=0.0002 in ESCs).

Endometrial tissues were obtained in accordance with the guidelines of the Declaration of Helsinki after full and informed patient consent and with the approval by the local Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science. Biopsies of normal endometrium were collected from 40 tubal infertility patients (including tubal ligation, tubal blockage, hydrosalpinx and tubal adhesion, 20 in proliferation phase, 20 in secretary phase, age [mean ±SD], 27± 6 years) who had no detectable endometriosis by laparoscopy and no history of pelvic inflammatory disease, chronic pelvic pain, dysmenorrhea, or dyspareunia. Samples of ectopic endometrium from 20 AWE patients (8 in proliferation phase, 12 in secretary phase, age, 29±7 years) were taken from patients with histological confirmation. The characteristics of patients with AWE is seen in Figure 1 . All of the endometriotic cysts were more than 3 cm in diameter and contained a glandular epithelium surrounded by stromal tissue. All recruited women had regular menstrual cycles. No history of autoimmune disease, malignant disease, hormonal therapy, or immune therapy was admitted into the patients. Immunohistochemistry staining was performed on paraffin sections using rabbit monoclonal antibodies against ERα (1:50 dilution, Affinity, USA), ERβ (1:50 dilution, Affinity, USA), GPER (1:50 dilution, Abcam, USA), and HIF-1α (1:150 dilution, Affinity, USA). Sections were deparaffinized and rehydrated in graded ethanol, and antigen retrieval was performed in citrate (pH 6.0) for 3 min at full pressure using a pressure cooker. Sections were then treated with 3% hydrogen peroxide for 5 min to inhibit endogenous peroxidase activity. After blocking in solution (0.01 mol/L Tris, 0.3% Triton X-100, and 10% normal goat serum) for 30 min, sections were incubated with primary antibodies at 4°C overnight. After washing with PBS, sections were incubated with peroxidase-labelled anti-rabbit IgG for 30 min. Finally, all slides were incubated with DAB-Substrate (Beyotime, China) and counterstained in hematoxylin before they were dehydrated and mounted. For each endometrial uterine curetting sample from normal control endometrium, five nonoverlapping fields per biopsy were evaluated under the microscope. For AWE pateints, five nonoverlapping fields including endometrial stromal, endometrial like glands per biopsy were evaluated under the microscope. Two hundred total cells (one hundred epithelial and stromal cells) were counted in a region of interest. The stained slides were evaluated using a light microscope and through digitally scanned images by two independent pathologists who were blinded to the clinicopathological information associated with the samples. The scoring process was performed unaware of patient outcome. The remmele semiquantitative scoring system (immunoreactive score, IRS) was calculated using the positive rate (PR) and staining intensity (SI) of cells reacting to the antibodies. The PR was categorized as 0 (<10% positive cells), 1 (10–25% positive cells), 2 (26 –50% positive cells), 3 (50%−75% positive cells), or 4 (76 – 100% positive cells), and the SI was categorized as 0 (negative), 1 (weak), 2(moderate), or 3 (strong). The scoring pattern for staining was multiplied to give a total IHS and IHS ranged from 0 to 12 for each sample. Scores between 0 – 2 points were considered as negative (0); 3 – 5 points as weak staining (+); 6 – 8 points as intermediate (++); and 9 – 12 points as strong staining (+++). Statistical analysis was performed by GraphPad Prism 5. The comparison of quantitative differences in the expression of ERs and HIF-1α between CoEM and AWEM were made using the nonparametric Mann-Whitney test. Differences with statistical significance was set at a p value of <0.05.

Endometriosis is a complex disease characterized by multifactorial etiology and a variety of clinical manifestations. Most studies typically investigate only one or two types of pelvic endometriosis and generalize conclusions to all types of endometriosis, despite the fact that endometriotic lesions at various sites are strikingly different. AWE is a rare but unique form of endometriosis. Few studies on AWE were reported, and most of them are case reports focusing on the clinical presentation, diagnostic and therapeutic methods. The current therapeutic success is unsatisfactory because of limited insight into mechanisms of the disease. No studies have been reported about pathogenesis research on AWE so far, and this is the first study to profile the distribution and expression of ERs and HIF-1α in AWE. Endometriosis, including AWE, is known to be estrogen dependent. As we all know that aromatase is a key enzyme for estrogen biosynthesis and Estrogen action is mediated primarily via nuclear estrogen receptors (ERs). but hormone therapy on AWE is not as effective as other types of endometriosis( Bektaş et al., 2010 ). Therefore, it is necessary to carry out estrogen related research in AWE. Using IHC technique, we successfully demonstrated cycle-regulated expression of ERs in AWEM, with maximal expression in proliferative phase, which indicates that AWEM is regulated by the ovary hormone. However, the expression of ERs in AWEM was not parallel to that in CoEM. ERα expression in AWEM is slightly decreased than that in the corresponding period of CoEM, while ERβ and GPER increased significantly, especially for GPER. GPER expression in the cytoplasm was significantly higher in AWEM compared to CoEM. Furthermore, an even distribution of GPER in the nuclei was found in AWEM. Results from above suggested that GPER was involved both in the non-genomic estrogen response (rapid) and in genomic (transcriptional) regulation in AWE. Previous studies in ovarian endometriosis showed that deficient methylation of the ERβ promoter results in pathological overexpression of ERβ in endometriotic stromal cells ( Xue et al., 2007 ), and high levels of ERβ suppresses ERα promoter activity and mRNA and protein expression in endometriotic cells. This seems to be consistent with the change of ERα and ERβ expression in AWEM ( Trukhacheva et al., 2009 ). However, research also demonstrated that estrogen, acting through ERα but not ERβ or GPER, stimulates GPER expression ( Plante et al., 2012 ), which is inconsistent to our findings. So far, the underlying mechanism leading to abnormal expression of ERs and the functions of abnormal ERs in AWE remain unclear. Whether the nuclear GPER was co-expressed with canonical estrogen receptors (ERα and ERβ) or ERs have synergic, antagonistic or independent nature need more investigation. HIF-1α is a master transcription factor that mediates hypoxic effects such as angiogenesis, metastasis, cell survival, and proliferation ( Semenza, 2012b ). In previous studies, hypoxia appears to play an important role in the pathogenesis of endometriosis, and elevated levels of HIF-1α induces leptin production, COX-2 expression, VEGF expression, enhances autophagy and so on, indicating that hypoxia promotes the development of endometriosis through various mechanisms ( Lee and Tsai, 2017 ). In our present study, we found that HIF-1α expression was decreased in AWEM and there was no difference between proliferative phase and secretary phase. Which was consistent with the expression of HIF-1α in deep infiltrating endometriosis, but significant difference within ovarian endometriosis ( Filippi et al., 2016 ). What caused the difference? Indeed, the AWEM we collected were rich in blood supply. A mouse model shown that hypoxia is an early phenomenon in the establishment of endometriosis-like lesions, while diminishes once new blood vessels start to invade into the tissue ( Becker et al., 2008 ), which suggests that hypoxia does not have an active role in the maintenance of AWE. The HIF-1α expression in our study suggests that the pathogenesis of AWE may be different from other types of endometriosis and some unique mechanisms including gene expression and regulation may be involved in the development of AWE. The relationship between estrogen and hypoxia are widely studied in endocrine-related cancer, such as breast cancer, thyroid cancer, prostate cancer and so on. However, very few studies were conducted in endometriosis: in our previous study, we found that estrogen stabilizes HIF-1α through GPER in eutopic endometrium of endometriosis ( Zhang et al., 2017 ) and Meng-Hsing Wu found that increased ERβ expression in ESCs may be regulated by HIF-1α ( Wu et al., 2012 ). The interaction of ERs and HIF-1α is still controversial. Our findings about ERs and HIF-1α in AWE may provide the possible candidate of pathophysiologic mediation in endometriosis. However, there were some limits in our present study. Due to the rarity of AWE, the sample size analyzed was small, and we did not collected eutopic endometrium of AWE patients in this study. In addition, the experimental design was simple, with only the protein expression and distribution of ERs and HIF-1α in CoEM and AWEM being analyzed. Further investigation with larger numbers is required, and molecular analysis would be expected. In conclusion, our results suggest that aberrant expression of ERs and HIF-1α in both glandular epithelial and stromal cells may be essential for the development and growth of AWE. The striking difference in GPER expression and distribution in AWE indicates GPER as a potential diagnosis and therapeutic target. These new findings shed lights on future investigations of delineating the etiology of AWE and further studies are required to delineate the precise roles of ERs and HIF-1α isoforms in endometriosis.

Endometriosis is a challenging and complex gynecologic disorder, pathologically defined as the implantation of endometrial foci outside the uterine cavity and clinically associated with chronic pelvic pain, dysmenorrhea, and infertility. At present, the exact etiology of endometriosis has not been fully clarified, even though it was described as early as 1860 ( Giudice and Kao, 2004 ). One of the possible reasons is that endometriosis is a complex multifactorial disease, but most studies focused on pelvic endometriosis (ovary, peritoneum, and deep infiltrative endometriosis)( Tosti et al., 2015 ), ignoring extrapelvic endometriosis. Extrapelvic endometriosis can be found in almost all areas of the body, but most frequently in the abdominal wall ( Bektaş et al., 2010 ). Abdominal wall endometriosis (AWE) is defined as ectopic endometrium embedded in the subcutaneous fatty tissue and the abdominal wall muscle layers ( Savelli et al., 2012 ), which accounts for 0.03% - 3.5% of reported incidences and is mainly associated with surgical procedures such as cesarean delivery or hysterotomy ( Ding and Zhu, 2013 ). AWE is often delayed in diagnosis due to its rarity. Most studies of AWE are either case series or case report ( Khan et al., 2017 ); thus basic research on the mechanism of AWE is urgently needed. Iatrogenic direct implantation is the most accepted cause for AWE ( Ding and Zhu, 2013 ). The endometrial cells escape through an incision in the uterus during the surgical procedure and are implanted within the abdominal wound. However, this theory cannot explain why the incidence after cesarean section is only 0.03% −0.04% ( Ozel et al., 2012 ). There must be other factors contributing to the occurrence of AWE. According to the discoveries in recent years, two stimulating signal factors were found to be closely related to endometriosis: one is estrogen and the other is hypoxia environment ( Yang and Yang, 2017 ). Estrogen has been known to play a key role in endometriosis, and its influence on endometrium is complicated by the local expression and distribution of several types of estrogen receptors (ERs), including two isoforms of nuclear estrogen receptor (ERα and ERβ) and one membrane-associated receptor (G protein-coupled estrogen receptor, GPER, formerly known as GPR30 ( Huhtinen et al., 2012 ). ERα is the dominant receptor in the uterus, and it mediates various estrogen actions, including the stimulation of proliferation. In contrast, ERβ modulates the effects of ERα and antagonizes its proliferative effects ( Shang, 2006 ). The effects of ERα and ERβ in pelvic endometriosis have been studied intensively. Recently, the newly discovered GPER has prompted the reexamination of estrogen on endometriosis. However, none of the ERs have been studied in AWE to date. As AWE also responds to hormonal stimuli ( Zhu et al., 2017 ), an investigation into ERs expression patterns and effects in AWE is warranted. According to the implantation theory, the cast-off endometrial tissues immediately encounter hypoxic stress due to the lack of support from vascular network. Hypoxia-inducible factors-1α (HIF-1α) is the most characterized regulator of low oxygen levels via transcriptional regulation ( Semenza, 2012a ). Recent studies demonstrated that HIF-1α is crucially associated with endometriosis: HIF-1α induces expression of many important downstream genes to regulate the implantation, survival, and maintenance of endometriotic lesions ( Hsiao et al., 2015 ). However, the expression of HIF-1α differs during the stage of implantation and maintenance in the progression of endometriosis ( Van Den Berg et al., 2013 ), as well as in different types of endometriosis ( Filippi et al., 2016 ). The role of HIF-1α in endometriosis requires further investigation, and studies on the expression of HIF-1α in AWE will be a powerful supplement to clarify the relationship between hypoxia and endometriosis. Interestingly, studies have shown a close relationship between ERs and HIF-1α: ERα has been shown to play a crucial role in decreasing HIF-1α protein levels ( Miyauchi et al., 2013 ; Kim et al., 2014 ). Further studies demonstrated that ERα increased HIF-1α protein accumulation under E 2 stimulation ( Sudhagar et al., 2011 ). Cho J et al demonstrated that HIF-1α activates ERα under hypoxic conditions ( Cho et al., 2006 ). Additionally, Ryu K at el showed that HIF-1α represses the transcription of the ERα gene in human breast cancer cells ( Ryu et al., 2011 ). Even though most studies revealed that ERβ inhibited HIF-1α transactivation ( Park and Lee, 2014 ; Lim et al., 2011 ; Mak et al., 2010 ; Lim et al., 2009 ), it was also reported that ERβ increased HIF-1α expression ( Dey et al., 2015 ). As for GPER, some studies revealed that GPER was up-regulated by HIF-1α ( Recchia et al., 2011 ) while others showed that GPER mediated the activation of HIF-1α signaling ( De Francesco et al., 2014 ). Unfortunately, the findings above are rather controversial, and further studies on the relationship between ERs and HIF-1α are needed. The objective of the present study is to further elucidate the role of ERs and HIF-1α in AWE. We analyzed the expression of ERα, ERβ, GPER, and HIF-1α in AWEM of 20 patients and compared these levels with those in 40 CoEM during comparable phases of the menstrual cycle. Our results demonstrate that ERs and HIF-1α are important roles in the pathogenesis of AWE.