Extent Of Lesional Fibrosis Research Articles

Reduced endometrial expression of histone deacetylase 3 in women with adenomyosis who complained of heavy menstrual bleeding

What role, if any, does histone deacetylase 3 (HDAC3) play in adenomyosis-associated heavy menstrual bleeding (HMB)? Seventy-two women with adenomyosis-associated HMB were recruited. Of these, 37 women reported moderate/heavy bleeding (MHB) and the remaining 35 women reported excessive bleeding (EXB). The stiffness of adenomyotic lesions and neighbouring endometrial-myometrial interface (EMI) was measured by transvaginal elastosonography, and full-thickness uterine tissue columns were processed for Masson trichrome staining and immunohistochemistry analyses. The protein expression levels of HDAC3 in endometrial cells cultured on substrates of different stiffnesses, and the protein concentrations of nuclear factor-κB (NF-κB) p65 subunit with HDAC3 suppression were evaluated. Mouse experiments were performed to assess the effect of adenomyosis on Hdac3 expression, endometrial repair and bleeding, and to evaluate the effect of HDAC3 inhibition on endometrial repair. Compared with controls, the endometrial staining of HDAC3 was significantly lower in women with adenomyosis-associated HMB, concomitant with a greater extent of fibrosis. The stiffness of lesions and neighbouring EMI was significantly higher in the EXB group compared with the MHB group, as was the extent of fibrosis in lesions, their neighboring EMI and endometrium. Expression of HDAC3 was reduced significantly when endometrial epithelial cells were cultured in stiff substrates. Suppression of HDAC3 abrogated the activation and signalling of NF-κB. Mice with induced adenomyosis exhibited reduced Hdac3 staining and elevated fibrosis in endometrium, concomitant with disrupted endometrial repair and more bleeding. Hdac3 inhibition resulted in botched inflammation and increased bleeding. Lesional fibrosis results in reduced endometrial HDAC3 expression and subsequent disruption in NF-κB signalling and inflammation, leading to adenomyosis-associated HMB.

In Search of an Imaging Classification of Adenomyosis: A Role for Elastography?

Adenomyosis is a complex and poorly understood gynecological disease. It used to be diagnosed exclusively by histology after hysterectomy; today its diagnosis is carried out increasingly by imaging techniques, including transvaginal ultrasound (TVUS) and magnetic resonance imaging (MRI). However, the lack of a consensus on a classification system hampers relating imaging findings with disease severity or with the histopathological features of the disease, making it difficult to properly inform patients and clinicians regarding prognosis and appropriate management, as well as to compare different studies. Capitalizing on our grasp of key features of lesional natural history, here we propose adding elastographic findings into a new imaging classification of adenomyosis, incorporating affected area, pattern, the stiffest value of adenomyotic lesions as well as the neighboring tissues, and other pathologies. We argue that the tissue stiffness as measured by elastography, which has a wider dynamic detection range, quantitates a fundamental biologic property that directs cell function and fate in tissues, and correlates with the extent of lesional fibrosis, a proxy for lesional "age" known to correlate with vascularity and hormonal receptor activity. With this new addition, we believe that the resulting classification system could better inform patients and clinicians regarding prognosis and the most appropriate treatment modality, thus filling a void.

Identification of lesional attributes of dysmenorrhea severity and the serum antimüllerian hormone levels in women with ovarian endometriomas

To investigate whether lesional immunostaining of putative biomarkers of recurrence and the extent of lesional and cortical fibroses are correlated with the severity of dysmenorrhea and serum antimüllerian hormone (AMH) levels in women with ovarian endometriomas (OEs). Retrospective cohort study. Academic hospital. A total of 313 women with histologically confirmed OEs were recruited. Their demographic and clinical information and data on their preoperative AMH levels were collected. Additionally, samples of their lesional tissues and ovarian cortex tissues adjacent to the OE lesions were procured for histologic and immunohistochemistry analyses. None. All OE tissue samples were stained for phosphorylated nuclear factor κB p65 subunit, progesterone receptor isoform B, Slit2, and α-smooth muscle actin. In addition, the extent of lesional and cortical fibroses was quantitated by Masson trichrome staining. We evaluated the relationship between the lesion size; laterality; extent of lesional and cortical fibroses, along with the putative markers of recurrence; and severity of dysmenorrhea and preoperative serum AMH levels in patients with OE. We found that the extent of lesional fibrosis was positively correlated with the severity of dysmenorrhea but had no impact on the AMH levels. On the other hand, the extent of cortical fibrosis, along with age, was negatively correlated with the AMH levels. The correlation between lesional fibrosis and the severity of dysmenorrhea and between cortical fibrosis and the AMH levels would call an early intervention once OE is diagnosed or suspected to prevent further pain and diminished ovarian reserve.

Tetramethylpyrazine Retards the Progression and Fibrogenesis of Endometriosis

The development of more efficacious, non-hormonal therapeutics for endometriosis is still an unmet medical need begging to be fulfilled. Growing evidence indicates that endometriotic lesions are wounds undergoing repeated tissue injury and repair, and, as such, platelets play an important role in lesional progression. Tetramethylpyrazine (TMP), a compound derived from a herb that has been used for thousands of years to combat “blood stasis” in traditional Chinese medicine, is a prescription drug in China for the treatment of cerebrovascular disorders. We tested the hypothesis that TMP can decelerate lesional progression through arresting epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), and fibrogenesis. We found in our in vitro experiments that TMP treatment suppresses platelet-induced EMT, FMT, cellular contractility, and collagen production in a concentration-dependent manner. We also showed that in a mouse model of endometriosis, treatment with TMP significantly reduced lesion weight and the extent of lesional fibrosis and improved hyperalgesia, mostly likely through the reduction of lesional aggregation of platelets and the lesional expression of markers of EMT, FMT, and fibrogenesis. In light of our results and in view of its excellent safety profiles, TMP appears to be a promising drug candidate for treating endometriosis.

How does the extent of fibrosis in adenomyosis lesions contribute to heavy menstrual bleeding?

PurposeTo investigate how the extent of fibrosis in adenomyosis lesions contributes to heavy menstrual bleeding (HMB).MethodsWe recruited 57 women with histologically confirmed adenomyosis, 29 of whom reported moderate/heavy bleeding (MHB) (menstrual blood loss (MBL) ≥20 but <100 mL) and the remaining 28, excessive MBL (EXB; ≥100 mL). Lesional stiffness was measured by transvaginal elastosonography. Full‐thickness uterine tissue columns containing the lesion and its neighboring endometrial‐myometrial interface (EMI) and endometrial tissues were evaluated for tissue fibrosis and immunohistochemical analysis of HIF‐1α, COX‐2, EP2, and EP4.ResultsThe lesional stiffness in the EXB group was significantly higher than that of MHB, and consistently, the extent of lesional fibrosis and the extent of tissue fibrosis in both EMI and eutopic endometrium were also significantly higher. In adenomyotic lesions and their neighboring EMI and eutopic endometrial tissues, the immunostaining of HIF‐1α, COX‐2, EP2, and EP4 was significantly reduced. The extent of fibrosis and the immunostaining levels of HIF‐1α, COX‐2, EP2, and EP4 were negatively correlated in all tissues.ConclusionsLesional fibrosis begets stiffening matrix, propagating fibrosis to neighboring EMI and eutopic endometrium, resulting in reduced PGE2 and HIF‐1α signaling, and thus likely reduced hypoxia necessary for endometrial repair, leading to HMB.

Changing prostaglandin E2 (PGE2) signaling during lesional progression and exacerbation of endometriosis by inhibition of PGE2 receptor EP2 and EP4.

PurposeWe investigated the change, if any, in prostaglandin E2 (PGE2) signaling in endometriotic lesions of different developmental stages in mouse. In addition, we evaluated the effect of treatment of mice with induced deep endometriosis (DE) with inhibitors of PGE2 receptor subtypes EP2 and EP4 and metformin.MethodsThree mouse experimentations were conducted. In Experiment 1, female Balb/C mice were induced with endometriosis or DE and were serially sacrificed after induction. Experiments 2 and 3 evaluated the effect of treatment with EP2 and EP4 inhibitors and metformin, respectively, in mice with induced DE. Immunohistochemistry analysis of COX‐2, EP2, and EP4, along with the extent of lesional fibrosis, was evaluated.ResultsThe immunostaining of COX‐2, EP2, and EP4 turned from activation to a stall as lesions progressed. Treatment with EP2/EP4 inhibitors in DE mice exacerbated endometriosis‐associated hyperalgesia and promoted fibrogenesis in lesions even though it suppressed the PGE2 signaling dose‐dependently. In contrast, treatment with metformin resulted in increased PGE2 signaling, concomitant with improved hyperalgesia, and retarded lesional fibrogenesis.ConclusionsThe PGE2 signaling diminishes as endometriotic lesions progress. Treatment with EP2/EP4 inhibitors in DE mice exacerbates endometriosis, but metformin appears to be promising seemingly through the induction of the PGE2 signaling.

Immunoreactivity of Plasminogen Activator Inhibitor 1 and Its Correlation with Dysmenorrhea and Lesional Fibrosis in Adenomyosis

Adenomyosis is associated with dysmenorrhea, infertility, and lesional fibrosis. The pathogenesis of adenomyosis is still unclear. Plasminogen activator inhibitor 1 (PAI-1) plays important roles in pathological activities like tumor metastasis and endometriosis. Our objective was to investigate the expression and localization of PAI-1 in eutopic and ectopic endometrium with adenomyosis and in endometrium without adenomyosis. We also sought to determine the relationship between PAI-1 immunoreactivity and the severity of dysmenorrhea and the extent of lesional fibrosis in adenomyosis. PAI-1 expression was significantly higher in the ectopic endometrium of patients with adenomyosis than in both the eutopic endometrium of patients with adenomyosis and the endometrium of controls. Ectopic PAI-1 expression correlated positively with dysmenorrhea visual analog scale (VAS) scores and the extent of lesional fibrosis in adenomyosis. High PAI-1 expression increased the likelihood of moderate to severe dysmenorrhea in adenomyosis. These results suggest that PAI-1 is involved in the adenomyosis-associated dysmenorrhea and lesional fibrosis, which provide a potential target in treating symptomatic adenomyosis.

Diagnosing Deep Endometriosis Using Transvaginal Elastosonography.

Transvaginal ultrasound (TVUS) and MRI are currently two mainstream imaging techniques used to diagnose deep endometriosis (DE) with comparable accuracy, but there is still ample room for improvement. As endometriotic lesions progress to fibrosis concomitant with the increase in tissue stiffness, transvaginal elastosonography (TVESG) is well-suited for diagnosing DE. To test the hypothesis that lesional stiffness as measured by TVESG correlates with the extent of lesional fibrosis, the markers of progression, hormonal receptor expression, and vascularity, we recruited 30 patients suspected to have DE who went through pelvic examination, TVUS and/or MRI, and TVESG and were ultimately diagnosed by histology. Their lesional tissue samples were subjected to immunohistochemistry analysis of markers for epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), estrogen and progesterone receptors (ERβ and PR), microvessel density (MVD), and vascularity, as well as quantification of lesional fibrosis. We found that pelvic examination, TVUS, and MRI detected 83.3%, 66.7%, and 83.3% of all DE cases, respectively, while TVESG detected them all. The lesions missed by pelvic exam, TVUS and MRI were significantly smaller than those detected but nonetheless had higher lesional stiffness. Lesional stiffness correlated closely and positively with the extent of lesional fibrosis, negatively with the markers of EMT, MVD, vascularity, and PR expression, but positively with the marker for FMT and ERβ. Thus, through the additional use of information on differential stiffness between DE lesions and their surrounding tissues, TVESG improves diagnostic accuracy, provides a ballpark estimate on the developmental stage of the lesions, and may help clinicians choose the best treatment modality.

Enriched Environment Decelerates the Development of Endometriosis in Mouse.

We tested the hypothesis that enriched environment (EE), consisting of enlarged space, and increased physical activity and social interactions, hinders the development of endometriosis through attenuated adrenergic signaling, enhanced autophagy, and reduced leptin levels. Two mouse experiments were performed. In Experiment 1, 40 female Balb/C mice were randomly divided into four equal-sized groups, the SE (standard environment), EE, p-EE (EE instituted after endometriosis induction), and the d-EE (SE housing but received uterine fragments from EE donors) groups. Housing intervention was initiated 3weeks before the induction of endometriosis and continued for 3weeks after induction. In Experiment 2, 20 female mice were randomly divided into SE and EE groups, and the plasma leptin levels were measured. We measured lesion weight and hotplate latency and performed Masson trichrome staining as well as immunohistochemistry analysis of β2 adrenergic receptor (ADRB2), dopamine receptor D2 (DRD2), vascular endothelial growth factor (VEGF), and microtubule-associated protein light chain 3 (LC3). We found that EE reduced the lesion weight by 40.8% as compared with SE mice, but the reduction in p-EE and d-EE mice did not reach statistical significance. EE significantly reduced staining levels of ADRB2 and VEGF as well as the extent of lesional fibrosis but increased staining levels of LC3 and DRD2 in lesions as compared with the SE group. EE mice had reduced plasma leptin levels as compared with SE mice. Thus, EE decelerates the development of endometriosis and fibrogenesis and improved generalized hyperalgesia, possibly through increased DRD2 expression but decreased expression of ADRB2 and VEGF as well as enhanced autophagy and reduced leptin level.

Evidence in Support for the Progressive Nature of Ovarian Endometriomas.

Whether endometriosis is a progressive disease is a highly contentious issue. While progression is reported to be unlikely in asymptomatic deep endometriosis, progression in symptomatic deep endometriosis has recently been reported, especially in menstruating women. However, pathophysiological reasons for these differences are unclear. This study was designed to investigate whether ovarian endometrioma (OE) is progressive or not. Thirty adolescent patients, aged 15 to 19 years, and 32 adult patients, aged 35 to 39 years, all laparoscopically and histologically diagnosed with OE, were recruited into this study after informed consent. Their demographic and clinical information were collected. Their OE tissue samples were collected and subjected to immunohistochemical analysis for E-cadherin, α-smooth muscle actin (α-SMA), desmin, and adrenergic receptor β2 (ADRB2), as well as quantification of lesional fibrosis by Masson trichrome staining. OE lesions from the adolescent and adult patients are markedly different, with the latter exhibiting more extensive and thorough progression and more extensive fibrosis, suggesting that lesions in adults progressed to a more advanced stage. Adult lesions and higher staining level of α-SMA and ADRB2 are positively associated with the extent of lesional fibrosis, while the lesion size and the E-cadherin staining are negatively associated. Our data provide a more definitive piece of evidence suggesting that OE is a progressive disease, since the adult lesions have had a longer time to progress. In addition, the pace of progression depends on lesional age as well as the severity of endometriosis-associated dysmenorrhea, if any.

Scutellarin Suppresses Platelet Aggregation and Stalls Lesional Progression in Mouse With Induced Endometriosis.

Platelets play an important role in the development of endometriosis. Scutellarin is a flavonoid isolated from a medicinal herb traditionally used as a potent antiplatelet agent. In this study, we sought to evaluate its potential therapeutic effect, if any, in mice with induced endometriosis. Endometriosis was induced in 27 female Balb/c mice by intraperitoneal injection of uterine fragments. Two weeks after the induction, the 27 mice were randomly divided in equal sizes into 3 groups: untreated, which received only vehicle, and low-dose and high-dose groups, which received low- and high dose of scutellarin treatment. Hotplate test was administrated to all mice before endometriosis induction, and before and after the scutellarin treatment. Two weeks after the treatment, a blood sample was drawn before sacrifice and all lesions were harvested. The peripheral platelet activation rate and total lesion weight were assessed, and immunohistochemistry and histochemistry analyses were performed to evaluate the extent of proliferation, angiogenesis, fibroblast-to-myofibroblast transdifferentiation (FMT), and fibrosis in lesions. Compared with untreated mice, mice in both low-dose and high-dose groups had significantly reduced lesion weight and improved hyperalgesia. Scutellarin also reduced the peripheral-activated platelets rate and resulted in significantly reduced platelet aggregation, cellular proliferation, angiogenesis, the extent of FMT, and the extent of fibrosis in lesions. Thus, we conclude that scutellarin is efficacious in treating endometriosis in vivo by suppressing platelet aggregation, inhibiting proliferation, angiogenesis, and fibrogenesis, resulting in reduced lesion size and improved pain behavior. As such, scutellarin may be a potentially promising therapeutics for the treatment of endometriosis.

Plasma High Mobility Group Box 1 (HMGB1), Osteopontin (OPN), and Hyaluronic Acid (HA) as Admissible Biomarkers for Endometriosis

Identification of biomarkers for endometriosis is an unmet medical need that demands to be fulfilled. In this study, we first used a mouse model of endometriosis and evaluated the potential utility of select biomarkers based on serial observations. Since fibrosis is the end result of lesional development, we chose high mobility group box 1 (HMGB1), osteopontin (OPN), and hyaluronic acid (HA), all three of them have been well documented to be involved in endometriosis and fibrosis, as potential biomarkers. In addition, we performed immunohistochemistry analysis of HMGB1, OPN, and the receptors for HMGB1, such as toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB), proliferating cell nuclear antigen (PCNA), interleukin-33 (IL-33), and receptor for advanced glycation endproducts (RAGE)–a pattern recognition receptor, with HMGB1 being its important ligand. We then evaluated the same set of putative markers in 30 women with ovarian endometriomas and 20 without endometriosis, and reevaluated the 3 plasma markers 3 months after the surgical removal of all visible endometriotic lesions. In mouse, the lesional staining levels of OPN, RAGE, and IL-33 were all significantly higher than that of normal endometrium, and increased progressively as lesions progressed. In contrast to HMGB1, TLR4, p-p65 and PCNA staining levels were decreased progressively. In humans, lesional staining levels of OPN correlated positively, while that of HMGB1 correlated negatively with the extent of fibrosis. All three plasma markers correlated positively with the extent of lesional fibrosis. Through this integrated approach, we identified plasma HMGB1, OPN and HA as promising admissible biomarkers for endometriosis.

Sensory nerve-derived neuropeptides accelerate the development and fibrogenesis of endometriosis.

Do sensory nerves play any role in the development of endometriosis? Sensory nerves participate in all major steps (epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT) and smooth muscle metaplasia (SMM)) in the development and fibrogenesis of endometriotic lesions. Endometriotic lesions are known to be hyperinnervated due to neurogenesis resulting from neutrophins secreted by endometriotic lesions and possibly platelets. These neutrophins seem to preferentially favour production of sensory neurons at the expense of sympathetic neurons. Three independent, yet complementary, prospective, randomized mouse experimentations were conducted. A total of 143 female Balb/C mice and 24 female immunodeficient nude Balb/C mice were used. The mice were sacrificed 2 or 4 weeks after the induction of endometriosis. In Experiment 1, 21 mice were randomly divided into three groups of equal size for sympathetic denervation, sensory denervation and controls. Denervation was carried out chemically. In Experiment 2, 24 nude mice were randomly divided into three equal-sized groups: the BEFORE and AFTER groups that respectively received surgical denervation 3 days before or after the induction of endometriosis by subcutaneous grafting of human endometriotic tissues, and the Control group that received a sham surgery without denervation 3 days before induction. For Experiments 1 and 2, all mice were sacrificed two weeks after induction of endometriosis. In Experiment 3, substance P (SP) and aprepitant, a potent and selective neurokinin 1 receptor (NK1R) antagonist, were used to activate and inhibit the NK1R signalling pathway, respectively. A total of 32 mice were randomly divided into four groups of equal sizes: control (CTL), SP, Before-Induction and After-Induction. One day before the induction of endometriosis, mice in CTL, SP and Before-Induction groups were infused with sterile saline, SP and aprepitant, respectively, via Alzet osmotic pumps. Two weeks after the induction, the After-induction group was infused with aprepitant in similar fashion. All mice were sacrificed four weeks after the induction of endometriosis. In all three experiments, the bodyweight and hotplate latency were evaluated before induction and sacrifice. In addition, all lesions were excised, weighed and processed for quantification and immunohistochemistry analysis of markers for EMT, FMT and SMM, and the extent of fibrosis was evaluated by Masson trichrome staining. In Experiment 1, chemical denervation of sympathetic and sensory nerves reduced the lesion weight by 43.2% (±23.1%) and 68.7% (±20.3%), respectively, as compared with controls. In particular, sensory denervation led to significantly greater reduction in lesion weight than sympathetic denervation. Sensory denervation also resulted in significantly improved hyperalgesia as compared with controls. In contrast, sympathetic denervation yielded only transient improvement in hyperalgesia. Both sympathetic and sensory denervation resulted in lower immunoreactivity against markers of proliferation and fibrosis, especially sensory denervation.In Experiment 2, surgical denervation before or after induction of endometriosis also decelerated the development of endometriosis, as manifested by significantly reduced lesion weight and extent of lesional fibrosis, along with improved hyperalgesia.In Experiment 3, NK1R activation by SP infusion accelerated lesional development, as evidenced by significantly increased lesional weight, more thorough progression of EMT, FMT, SMM, exaggerated lesional fibrosis and deteriorated hyperalgesia. In contrast, NK1R antagonism decelerated lesional development and improved hyperalgesia. N/A. This study is limited by the use of histologic and immunohistochemistry analyses only and the lack of molecular data. Since sensory nerves are known to be important in wound healing and fibrogenesis, our findings also give more credence to the notion that endometriotic lesions are wounds undergoing repeated tissue injury and repair. As such, sensory nerves or the NK1R signalling pathway in particular may be potential targets for intervention. This research was supported by Grants 81471434 (SWG), 81530040 (SWG), 81771553 (SWG), 81671436 (XSL) and 81871144 (XSL) from the National Natural Science Foundation of China and an Excellence in Centres of Clinical Medicine grant (2017ZZ01016) from the Science and Technology Commission of Shanghai Municipality. None of the authors have anything to disclose.

The establishment of a mouse model of deep endometriosis.

Is it possible to establish a mouse model of deep endometriosis (DE)? A mouse DE model that is macroscopically and microscopically similar to nodular lesions in humans can be constructed in as short as 3 weeks by intraperitoneal injection of uterine fragments along with the infusion of substance P (SP) and/or calcitonin gene-related peptide (CGRP). Although a baboon DE model was reported 5 years ago, its prohibitive cost and demand for facilities and expertise associated with the use of non-human primates put its use out of reach for most laboratories. A total of 48 female Balb/C mice were used for this study. Among them, 16 were randomly selected as donors that contributed uterine fragments, and the remaining 32 were recipient mice. The mice with induced endometriosis were followed up for 3-4 weeks. One day before the induction of endometriosis by intraperitoneal injection of uterine fragments, osmotic pumps were inserted into equal groups of recipient mice to infuse either sterile saline, SP, CGRP, or both SP and CGRP. The hotplate test was administrated to all mice at the baseline and before and after induction of endometriosis. Four (3 for the SP+CGRP group) weeks after induction, all mice were sacrificed. Their endometriotic lesions were excised, weighed and processed for histopathologic examination, and histochemistry, immunohistochemistry and immunofluorescence analyses of markers of proliferation, angiogenesis, epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM), mesothelial-mesenchymal transition (MMT) and endothelial-mesenchymal transition (EndoMT) were done. The extent of lesional fibrosis was evaluated by Masson trichrome staining. To further evaluate surrounding organ/tissue invasion, the peritoneal areas adhesive to the lesions were excised for immunohistochemical analysis. Endometriotic lesions in mice treated with SP and/or CGRP satisfied all requirements for DE, i.e. presence of endometrial epithelial and stromal cells, abundance of fibromuscular content, and encapsulation in surrounding tissues or organs. The lesion weight in the CGRP, SP and SP+CGRP groups was 1.62, 2.14 and 2.18-fold, respectively, heavier than that of control group. Concomitantly, the SP, CGRP and SP+CGRP groups had significantly shorter hotplate latency than that of control group. Lesions in mice treated with SP and/or CGRP, especially with SP+CGRP, exhibited characteristics consistent with EMT, FMT, SMM and extensive fibrosis, along with signs of MMT and EndoMT. Lesional invasion into surrounding tissues/organs was found to be 25.0, 75.0 and 87.5% in mice treated with CGRP, SP and SP+CGRP, but none in control mice. N/A. This study is limited by the use of histologic and immunohistochemistry analyses only and lacks molecular data. The establishment of a mouse DE model supports the idea that endometriotic lesions are wounds undergoing repeated tissue injury and repair and underscores the importance of microenvironments in shaping the lesions' destiny. In addition, signs consistent with MMT and EndoMT indicate that there may be more culpable factors that still remain unidentified and should be pursued in the future. Moreover, the close correlation between the extent of lesional fibrosis and markers of EMT, MMT, EndoMT, FMT and SMM as shown here should facilitate our understanding of the molecular mechanisms underlying the DE pathophysiology. Since this DE model is based on a biologically plausible and evidence-backed theory, it should shed much needed insight into the molecular mechanisms underlying the pathophysiology of DE. This research was supported by Grants 81471434 (S.W.G.), 81530040 (S.W.G.), 81771553 (S.W.G.), 81671436 (X.S.L.) and 81871144 (X.S.L.) from the National Natural Science Foundation of China. None of the authors has any conflict of interest to disclose.

The M2a macrophage subset may be critically involved in the fibrogenesis of endometriosis in mice.

Recent research has shown that endometriotic lesions are essentially wounds that undergo repeated tissue injury and repair, which results in epithelial-mesenchymal transition, fibroblast-to-myofibroblast transdifferentiation, smooth muscle metaplasia and ultimately fibrosis. Macrophages are a key regulator of tissue repair and fibrogenesis. But do macrophages also play a role in fibrogenesis of endometriosis, and, if yes, which subset of macrophages? To elucidate the role of macrophages in fibrogenesis of endometriosis, we conducted three experiments in mice. In experiment 1, endometriotic tissue samples from female Balb/C mice with induced endometriosis were serially harvested to evaluate the role of macrophages in fibrogenesis. In experiments 2 and 3, female transgenic mice (C57BL/6J background) expressing the human diphtheria toxin receptor under the control of the CD11b promoter had macrophage depletion by diphtheria toxin injection after induction of endometriosis. Additionally, in experiment 3, adoptive transfer of different subsets of macrophage was carried out after macrophage depletion. Lesional infiltration of M2 macrophages increased progressively as lesions progressed undisturbed, concomitant with progressive epithelial-mesenchymal transition, fibroblast-to-myofibroblast transdifferentiation and fibrosis. Macrophage depletion after induction of endometriosis significantly reduced lesional infiltration of total macrophages, significantly reduced lesional infiltration of M2 macrophages and significantly reduced lesional fibrotic content and lesion weight (P < 0.05). Finally, adoptive transfer of M2a, but not M1 or M2c macrophages, systemically after macrophage depletion significantly increased the extent of fibrosis in lesions (P = 1.6 × 10-10). The identification of a particular macrophage subset in fibrogenesis of endometriosis should further help to shed new light on the pathophysiology of endometriosis.

Transvaginal Elastosonography as an Imaging Technique for Diagnosing Adenomyosis.

To test the hypothesis that the lesional stiffness as measured by transvaginal elastosonography (TVESG) correlates with the extent of fibrosis in adenomyotic (AM) lesions, and thus TVESG can be used to diagnose AM, we conducted 2 studies. The first evaluated the relationship, if any, between lesional stiffness and lesional histology in 35 women with histologically confirmed AM in comparison with tissue stiffness in 11 control myometrial (CM) and 8 uterine fibroids (UFs) tissue samples. The second validated the relationship between lesional stiffness and the severity of dysmenorrhea and the amount of menses in AM patients by recruiting 112 patients diagnosed with AM, 67 with UF, and 130 controls. Transvaginal ultrasound and TVESG were both performed. We found that the stiffness of AM lesions was significantly higher than that of UF, which, in turn, was significantly higher than that of CM. Lesional stiffness correlated positively with uterine size and the extent of lesional fibrosis but negatively with E-cadherin and progesterone receptor expression levels. Lesional stiffness also correlated with the severity of dysmenorrhea as well as the amount of menses. Thus, TVESG can improve the diagnostic accuracy for AM, especially in differential diagnosis of AM from UF. The correlation between lesional stiffness and the extent of fibrosis and hormonal receptor expression and the severity of symptomology strongly suggests that TVESG not only can provide an instant assessment of the developmental stage of AM lesions but also may be used to guide the choice of the best treatment modality for the patient.

Corroborating evidence for platelet-induced epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis.

Do platelets play any role in the development of adenomyosis? As in endometriosis, adenomyotic lesions show significantly increased platelet aggregation, increased expression of transforming growth factor (TGF)-β1, phosphorylated Smad3, markers of epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT), and smooth muscle metaplasia (SMM), in conjunction with increased fibrosis as compared with normal endometrium. Both EMT and FMT are known to play vital roles in fibrogenesis in general and in endometriosis in particular. EMT has been implicated in the development of adenomyosis. SMM is universally seen in endometriosis and also in adenomyosis, and is correlated positively with the extent of fibrosis. However, there has been no published study on the role of platelets in fibrogenesis in adenomyosis, even though adenomyotic lesions undergo repeated cycles of tissue injury and repair, which suggests the involvement of platelets and their possible roles in fibrogenesis. Cross-sectional studies of ectopic endometrial and control endometrial tissue samples from three sets of women with and without adenomyosis (n= 34 and 20, 12 and 10, and 8 and 8, respectively) were carried out from 2014 to 2015. Immunohistochemistry analysis of ectopic endometrial tissues from women with (n= 34) and without (n= 20) adenomyosis with respect to biomarkers of EMT, FMT and highly differentiated smooth muscle cells as well as TGF-β1, phosphorylated Smad3, markers of proliferation, angiogenesis and extracellular matrix (ECM) deposits. Masson trichrome staining, Van Gieson staining and Pico-Sirius staining were performed to evaluate and quantify the extent of fibrosis in lesions. Progesterone receptor isoform B (PR-B) staining also was performed. In addition, CD42b-positive platelets in ectopic (n= 12) and control (n= 10) endometrium were counted by confocal microscopy and compared. The protein expression levels of TGF-β1 and phosphorylated Smad3 in both ectopic (n= 8) and control (n= 8) endometrium were measured by western blot analysis. Immunofluorescent staining of both platelets and hepatocyte growth factor (HGF) was also performed for adenomyotic tissue samples (n= 10). Adenomyotic lesions had a significantly higher extent of platelet aggregation and increased staining for TGF-β1 and phosphorylated Smad3 (both P-values <0.001 versus control). In addition, E-cadherin staining was decreased while vimentin staining in adenomyotic epithelial cells was increased, along with increased staining of proliferating cell nuclear antigen, vascular endothelial growth factor and CD31 (all P-values <0.001), markers of proliferation and angiogenesis. Staining for α-SMA, a marker for myofibroblast, desmin, smooth muscle myosin heavy chain and oxytocin receptor was significantly increased in adenomyotic lesions versus control, concomitant with increased staining of collagen I and lysyl oxidase (all P-values <0.001). Histochemistry analysis indicates that the extent of fibrosis is high in adenomyotic lesions (P < 0.001), and the extent appeared to correlate negatively with the microvessel density (P < 0.05). PR-B staining was significantly decreased in adenomyotic lesion as compared with control endometrium (P < 0.001). Platelets and HGF were co-localized mostly in the stromal component of adenomyotic lesions, near the glandular epithelium. The results are limited by the cross-sectional nature of the study and the use of histochemistry and immunohistochemistry analyses only, but nonetheless is a validation of our previous finding in mouse experiments. The data presented are consistent with the notion that platelet-induced activation of the TGF-β/Smad signaling pathway may be a driving force in EMT, FMT and SMM in the development of adenomyosis, leading to fibrosis. This study provides the first piece of evidence that adenomyotic lesions are wounds that undergo repeated injury and healing, and, as such, platelets play critical roles in the development of adenomyosis by promoting proliferation, angiogenesis, increasing ECM deposits, and SMM, resulting in fibrosis. Platelets may also be involved in uterine hyperactivity and myometrial hyperinnervation. Our results provide one explanation as to why adenomyosis is a challenge for medical treatment, and shed new light onto the pathophysiology of adenomyosis. Support for data collection and analysis was provided by grants from the National Science Foundation of China. None of the authors has anything to disclose.

Transforming growth factor β1 signaling coincides with epithelial-mesenchymal transition and fibroblast-to-myofibroblast transdifferentiation in the development of adenomyosis in mice.

Do platelets have any role in the development of adenomyosis? Activated platelets coincide with the release of transforming growth factor (TGF)-β1 and induction of the TGF-β/Smad signaling pathway as well as evidence of epithelial-mesenchymal transition (EMT) and fibroblast-to-myofibroblast transdifferentiation (FMT) in a mouse model of adenomyosis, resulting ultimately in fibrosis, as in adenomyosis. Both EMT and FMT are known to play vital roles in fibrogenesis in general and in endometriosis in particular. EMT has been implicated in the development of adenomyosis, but this was based primarily on cross-sectional observation. It is unclear as to whether adenomyotic lesions and their microenvironment have the machinery to promote EMT and FMT, resulting ultimately in fibrosis. There has not been any published study on the role of platelets in the development of adenomyosis, even though adenomyotic lesions undergo repeated cycles of tissue injury and repair, which implicates the involvement of platelets and constitutes an environment conducive for fibrogenesis. Adenomyosis was induced in 28 female ICR mice by neonatal dosing of tamoxifen. Another 32 were neonatally dosed without tamoxifen. These mice were sacrificed serially and their tissue samples were subsequently evaluated. Female ICR mice with and without induced adenomyosis were sacrificed in batch at 5, 10, 15, 42 and 60 days of age. The depth of myometrial infiltration of endometrial tissues was assessed and immunohistochemistry analysis of biomarkers of EMT and FMT, as well as TGF-β1, phosphorylated Smad3 (p-Smad3) and markers of proliferation, angiogenesis and extracellular matrix (ECM) deposits was performed in ectopic (for adenomyotic mice) and eutopic (controls) endometrial tissue samples. Masson trichrome and Van Gieson stainings were performed to quantify the extent of fibrosis in lesions. Progesterone receptor isoform B (PR-B) staining also was performed. While TGF-β1 immunoreactivity was consistently low in control endometrium, its level was increased dramatically starting from Day 10, along with the extent of platelet aggregation. Staining for TGF-β1 and p-Smad3 increased progressively as adenomyosis progressed, along with markers for proliferation, angiogenesis and ECM deposits. Consistently, staining of vimentin (a marker for stromal or mesenchymal cells) was also increased while that of E-cadherin (a marker for epithelial cells) was reduced. PR-B staining also decreased progressively. Starting from Day 42, α-SMA staining, a marker for myofibroblasts, was elevated in lesions, while in control endometrium, it was negative. Concomitantly, the extent of fibrosis also was increased. This study is limited by the use of histochemistry and immunohistochemistry analyses only and the lack of intervention. Like their endometriotic counterpart, adenomyotic lesions and their microenvironment may contain all the necessary molecular machinery to promote fibrogenesis. Platelet-induced activation of the TGF-β/Smad signaling pathway may be a driving force in EMT and FMT in the development of adenomyosis, leading to fibrosis. This study provides the first piece of evidence that adenomyotic lesions are wounds that undergo repeated injury and healing, and as such, platelets play critical roles in the development of adenomyosis. It suggests the potential for the use of anti-platelet therapy in the treatment of adenomyosis, and also opens a new venue for developing novel biomarkers for diagnostic or prognostic purposes. Support for data collection and analysis was provided by grants from the National Science Foundation of China. None of the authors has anything to disclose.