Meclizine improves endometrial repair and reduces simulated menstrual bleeding in mice with induced adenomyosis.

BACKGROUND: Adenomyosis is one of the structural causes of abnormal uterine bleeding, which often presents as heavy menstrual bleeding. Mostly because of the poor understanding of its pathophysiology, medical management of adenomyosis-induced heavy menstrual bleeding is still a challenge. We have previously reported that glycolysis is crucial to endometrial repair following menstruation and that suppressed glycolysis can cause heavy menstrual bleeding. OBJECTIVE: This study aimed to test the hypothesis that meclizine, a drug with an excellent safety profile, alleviates heavy menstrual bleeding in mice with induced adenomyosis using a simulated menstruation model. STUDY DESIGN: Adenomyosis was induced in 36 female C57BL/6 mice using endometrial-myometrial interface disruption. Three months after induction, the mice were randomly divided into the following 3 groups: low-dose meclizine, high-dose meclizine, and controls. Treatment with meclizine or vehicle started shortly before the simulated menstruation procedure and ended before progesterone withdrawal. The amount of blood loss was quantified and uterine tissue was harvested for histologic evaluation of the grade of endometrial repair. We performed immunohistochemistry analysis of 4 proteins critically involved in glycolysis: Glut1 (glucose transporter 1), Hk2 (hexokinase 2), Pfkfb3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3), and Pkm2 (pyruvate kinase M2). The extent of tissue fibrosis in both ectopic and eutopic endometria was evaluated using Masson trichrome staining. RESULTS: In mice with induced adenomyosis, meclizine accelerated endometrial repair in a dose-dependent manner and reduced the amount of menstrual bleeding. Meclizine administration raised endometrial immunoexpression of Hk2 and Pfkfb3 but not of Glut1 or Pkm2. The extent of endometrial fibrosis was reduced following the meclizine administration. Remarkably, these favorable changes were accompanied by the suppression of lesional progression, as evidenced by the dose-dependent reduction in the extent of fibrosis (a surrogate for lesional progression). CONCLUSION: These encouraging results, taken together, suggest that glycolysis may be a promising therapeutic target and that meclizine may hold therapeutic potential as a nonhormonal treatment for adenomyosis-induced heavy menstrual bleeding without exacerbating the disease.

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

RESEARCH QUESTION: What role, if any, does histone deacetylase 3 (HDAC3) play in adenomyosis-associated heavy menstrual bleeding (HMB)? DESIGN: 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. RESULTS: 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. CONCLUSIONS: Lesional fibrosis results in reduced endometrial HDAC3 expression and subsequent disruption in NF-κB signalling and inflammation, leading to adenomyosis-associated HMB.

Corroborating evidence for aberrant expression of histone deacetylase 8 in endometriosis.

Purpose: The aim of this study was to evaluate the dynamic change in staining of Class I HDACs and Hdac6 in lesions harvested serially from different time points in mice with induced endometriosis. In addition, the effect of Hdac8 activation as well as Hdac8 and Hdac6 inhibition on lesional progression and fibrogenesis was evaluated. Methods: Immunohistochemistry analysis of Class I HDACs and Hdac6 in serially harvested lesion samples in mouse. Hdac8 activation, as well as Hdac6/8 inhibition, was evaluated in mice with induced endometriosis. Results: We found a progressive increase in lesional staining of Hdac1, Hdac8, and Hdac6 and gradual decrease in Hdac2 staining and consistently reduced staining of Hdac3 during the course of lesional progression. The stromal Hdac8 staining correlated most prominently with all markers of lesional fibrosis. Hdac8 activation significantly accelerated the progression and fibrogenesis of endometriotic lesions. In contrast, specific inhibition of Hdac8 or Hdac6, especially of Hdac8, significantly hindered lesional progression and fibrogenesis. Conclusions: Hdac8 is progressively and aberrantly overexpressed as endometriotic lesions progress. This, along with the documented HDAC1 upregulation in endometriosis and the overwhelming evidence for the therapeutic potentials of HDACIs, calls for further and in-depth investigation of epigenetic aberrations of endometriosis in general and of HDACs in particular.

Aberrant expression of histone deacetylase 8 in endometriosis and its potential as a therapeutic target.

Purpose: To screen Zn2+-dependent histone deacetylase (HDAC) 1-11 in endometriotic cells and then evaluated the HDACs identified from the screening in ovarian endometrioma (OE) and deep endometriotic (DE) lesions, and to evaluate the therapeutic potential of HDAC8 inhibition in mice. Methods: Quantification of gene and protein expression levels of HDAC1-11 in endometriotic cells stimulated by TGF-ß1, and immunohistochemistry analysis of Class I HDACs and HDAC6 in OE/DE lesion samples. The therapeutic potential of HDAC8 inhibition was evaluated by a mouse model of deep endometriosis. Results: The screening identified Class I HDACs and HDAC6 as targets of interest. Immunohistochemistry analysis found a significant elevation in HDAC8 immunostaining in both OE and DE lesions, which was corroborated by gene and protein expression quantification. For other Class I HDACs and HDAC6, their lesional expression was more subtle and nuanced. HDAC1 and HDAC6 staining was significantly elevated in DE lesions while HDAC2 and HDAC3 staining was reduced in DE lesions. Treatment of mice with induced deep endometriosis with an HDAC8 inhibitor resulted in significantly longer hotplate latency, a reduction of lesion weight by nearly two-thirds, and significantly reduced lesional fibrosis. Conclusions: These findings highlight the progression-dependent nature of specific HDAC aberrations in endometriosis, and demonstrate, for the first titme, the therapeutic potential of suppressing HDAC8.

Activation of α7 nicotinic acetylcholine receptor retards the development of endometriosis.

BACKGROUND: Women with endometriosis have been shown to have a reduced vagal tone as compared with controls and vagotomy promoted while vagus nerve stimulation (VNS) decelerated the progression of endometriosis in mice. Extensive research also has shown that the activation of the cholinergic anti-inflammatory pathway by VNS activates α7 nicotinic acetylcholine receptor (α7nAChR), potently reducing inflammation. Yet whether α7nAChR plays any role in endometriosis is unknown. We evaluated its expression in normal endometrium, ovarian and deep endometriotic lesions, and evaluated its role in the development of endometriosis. METHODS: Immunohistochemistry analyses of α7nAChR in endometriotic lesions as well as control endometrium, and quantification of tissue fibrosis by Masson trichrome staining were performed. Mouse experiments were conducted to evaluate the impact of α7nAChR activation or suppression on lesional progression and possible therapeutic effect. Finally, in vitro experiments were conducted to evaluate the effect of activation of α7nAChR on epithelial-mesenchymal transition (EMT), fibroblast-to-myofibroblast transdifferentiation (FMT), smooth muscle metaplasia (SMM) and fibrogenesis in an endometriotic epithelial cell line and primary endometriotic stromal cells derived from ovarian endometrioma tissue samples. RESULTS: Immunostaining of α7nAChR was significantly reduced in human endometriotic epithelial cells as compared with their counterpart in normal endometrium. Lesional α7nAChR staining levels correlated negatively with lesional fibrosis and the severity of dysmenorrhea. The α7nAChR agonist significantly impeded the development of endometriotic lesions in mouse models possibly through hindrance of EMT and FMT. It also demonstrated therapeutic effects in mice with induced deep endometriosis. Treatment of endometriotic epithelial and stromal cells with an α7nAChR agonist significantly abrogated platelet-induced EMT, FMT and SMM, and suppressed cellular contractility and collagen production. CONCLUSIONS: α7nAChR is suppressed in endometriotic lesions, and its activation by pharmacological means can impede EMT, FMT, SMM, and fibrogenesis of endometriotic lesions. As such, α7nAChR can be rightfully viewed as a potential target for therapeutic invention. TRIAL REGISTRATION: Not applicable.

Higher fibrotic content of endometriotic lesions is associated with diminished prostaglandin E2 signaling.

PURPOSE: While the prevailing view holds that the prostaglandin E2 (PGE2) signaling plays a vital role in endometriosis, PGE2 also is known to be anti-fibrotic. We investigated the immunostaining of COX-2, EP2, and EP4, along with fibrotic content in ovarian endometrioma (OE) and deep endometriosis (DE) lesions, and in OE lesions from adolescent and adult patients. In addition, we evaluated the effect of substrate stiffness on the expression of COX-2, EP2, and EP4 in endometrial stromal cells. METHODS: Immunohistochemistry analysis of COX-2, EP2, and EP4, along with the quantification of lesional fibrosis, was conducted for OE and DE lesion samples and also OE lesion samples from adolescent and adult patients. The effect of substrate rigidity on fibroblast-to-myofibroblast transdifferentiation (FMT) and the expression of COX-2, EP2, and EP4, with or without TGF-ß1 stimulation, were investigated. RESULTS: The immunostaining of COX-2, EP2, and EP4 was substantially reduced in endometriotic lesions as lesions became more fibrotic. Both TGF-ß1 stimulation and stiff substrates induced FMT and reduced the expression of COX-2, EP2, and EP4. CONCLUSIONS: Since fibrosis is a common feature of endometriosis, our results thus cast doubts on the use of therapeutics that suppresses the PGE2 signaling pathway, either by inhibiting COX-2 or EP2/EP4.

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

PURPOSE: We 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. METHODS: Three 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. RESULTS: The 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. CONCLUSIONS: The 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.

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

Purpose: To investigate how the extent of fibrosis in adenomyosis lesions contributes to heavy menstrual bleeding (HMB). Methods: We 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. Results: The 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. Conclusions: Lesional 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.

Possible involvement of neuropeptide and neurotransmitter receptors in Adenomyosis.

BACKGROUND: Accumulating data indicate that sensory nerve derived neuropeptides such as substance P and calcitonin gene related-protein (CGRP) can accelerate the progression of endometriosis via their respective receptors, so can agonists to their respective receptors receptor 1 (NK1R), receptor activity modifying protein 1 (RAMP-1) and calcitonin receptor-like receptor (CRLR). Adrenergic ß2 receptor (ADRB2) agonists also can facilitate lesional progression. In contrast, women with endometriosis appear to have depressed vagal activity, concordant with reduced expression of α7 nicotinic acetylcholine receptor (α7nAChR). The roles of these receptors in adenomyosis are completely unknown. METHODS: Adenomyotic tissue samples from 30 women with adenomyosis and control endometrial tissue samples from 24 women without adenomyosis were collected and subjected to immunohistochemistry analysis of RAMP1, CRLR, NK1R, ADRB2 and α7nAChR, along with their demographic and clinical information. The extent of tissue fibrosis was evaluated by Masson trichrome staining. RESULTS: We found that the staining levels of NK1R, CRLR, RAMP1 and ADRB2 were all significantly elevated in adenomyotic lesions as compared with control endometrium. In contrast, α7nAChR staining levels were significantly reduced. The severity of dysmenorrhea correlated positively with lesional ADRB2 staining levels. CONCLUSIONS: Our results suggest that SP, CGRP and noradrenaline may promote, while acetylcholine may stall, the progression of adenomyosis through their respective receptors on adenomyotic lesions. Additionally, through the activation of the hypothalamic-pituitary-adrenal (HPA)-sympatho-adrenal-medullary (SAM) axes and the lesional overexpression of ADRB2, adenomyosis-associated dysmenorrhea and adenomyotic lesions may be mutually promotional, forming a viscous feed-forward cycle.

Preoperative and perioperative intervention reduces the risk of recurrence of endometriosis in mice caused by either incomplete excision or spillage and dissemination.

RESEARCH QUESTION: Can preoperative or perioperative intervention reduce the risk of recurrence of endometriosis caused by either incomplete excision or spillage and dissemination? DESIGN: A mouse model of endometriosis recurrence caused by spillage and dissemination was first established using 24 female Balb/c mice. The spillage and dissemination model was used to test the efficacy of preoperative use of ketorolac, perioperative use of aprepitant and combined use of propranolol and andrographolide in a prospective, randomized mouse experiment involving 75 mice. The efficacy of these preoperative and perioperative interventions in a mouse recurrence model caused by incomplete excision was also tested using 72 mice. In all experiments, the baseline body weight and hotplate latency of all mice were measured and recorded before the induction of endometriosis, before the primary surgery and before sacrifice. In addition, all lesions were excised, weighed and processed for quantification and immunohistochemistry analysis of E-cadherin, α-SMA, VEGF, ADRB2 and putative markers of recurrence PR-B, p-p65, as well as Masson trichrome staining. RESULTS: All interventions substantially and significantly suppressed the outgrowth of endometriotic lesions and reduced the risk of recurrence caused by either spillage and dissemination or incomplete excision (P = 0.0007 to 0.042). These interventions also significantly attenuated the generalized hyperalgesia, inhibited the staining of α-SMA, p-p65, VEGF and ADRB2 but increased staining of E-cadherin and PR-B, resulting in reduced fibrosis. CONCLUSION: Given the excellent safety profiles of these drugs, these data strongly suggest that preoperative and perioperative intervention may potentially reduce the risk of endometriosis recurrence effectively.

Early maternal separation accelerates the progression of endometriosis in adult mice.

BACKGROUND: A large body of research highlights the importance of early-life environmental impact on the health outcome in adulthood. However, whether early-life adversity (ELA) has any impact on the development of endometriosis is completely unclear. In this study, we tested the hypothesis that ELA, as manifested by neonatal separation, can accelerate the progression of endometriosis in mouse through activation of the adrenergic receptor ß2 (ADRB2) signaling pathway, leading to increased angiogenesis and progression of endometriotic lesions. METHODS: Eight female Balb/C mice, in late pregnancy, were used used for this study, which later gave birth to 22 female newborn pubs. Eleven additional female Balb/C mice were also used as donors of uterine tissues. The 22 newborn pubs were randomly divided into 2 equal-sized groups, maternal separation (MS) and no separation (NS). Pubs in the MS group were separated from their dams for 3 h/day from postnatal day (PND) 1 to 21, while those in the NS control remained in the home cage with their dams. In adulthood (8-week old), 3 mice in each group were randomly selected to undergo a battery of behavior tests. The remaining 8 mice in each group were induced with endometriosis by intraperitoneal injection of uterine fragments from donor mice. Four weeks after the induction, all mice were sacrificed and their endometriotic lesions were excised for quantification and then prepared for immunohistochemistry analysis. RESULTS: We confirmed that MS during infancy resulted in anxiety and depression-like behaviors as previously reported. We also found that in MS mice the lesion weight was increased by over 2 folds and generalized hyperalgesia was also significantly increased as compared with NS mice. Immunostaining analysis demonstrated that MS accelerated the development of endometriosis likely through decreased dopamine receptor D2 (DRD2) expression and activation of the ADRB2/cAMP-response element binding protein (CREB) signaling pathway, leading to increased angiogenesis and progression of endometriotic lesions. CONCLUSIONS: Exposure of female mouse pups to ELA such as MS during their infancy period accelerates the progression of endometriosis, possibly through altered neuronal wiring and hyperactivity of the hypothalamic-pituitary-adrenal axis.

Adenomyosis in mice resulting from mechanically or thermally induced endometrial-myometrial interface disruption and its possible prevention.

RESEARCH QUESTION: Do uterine procedures potentially disrupting the endometrial-myometrial interface (EMI) induce adenomyosis? DESIGN: Six prospective, randomized controlled experiments were conducted involving a total of 106 female BALB/c and 12 female C57BL/6 mice. The incidence of adenomyosis was evaluated in these two strains of mouse after mechanically induced EMI disruption (EMID), or thermally induced EMID using electrocoagulation of different intensities. Finally, the incidence was evaluated in mice that had received perioperative administration of aprepitant (an NK1R inhibitor), propranolol (a beta-blocker) or vehicle. Body weight, hot plate latency and grade of myometrial infiltration were evaluated. Histology, immunohistochemistry and histochemistry analyses were also performed. RESULTS: Mechanical injury to the EMI caused EMID. Adenomyosis developed in the majority of mice in the EMID groups 3 months after mechanically induced EMID but did not develop in the control group (83.3% in C57BL/6 mice, P = 0.015; 100% in BALB/c mice, P = 0.0002). With thermally induced EMID, adenomyosis was found in 30% of the EMID mice 10 weeks later, but the incidence increased to 66.7% if the extent of EMID damage was increased. In mice with perioperative administration of aprepitant or propranolol, the incidence of adenomyosis was reduced from 100% to 58.3% (both P = 0.00034). CONCLUSIONS: This study provides the first piece of experimental evidence that EMID resulting from iatrogenic uterine procedures can substantially increase the risk of developing adenomyosis, with the risk in proportion to the severity of disruption. More intriguingly, perioperative administration of an NK1R antagonist or beta-blocker significantly reduced the risk of developing adenomyosis.

Sodium tanshinone IIA sulfonate restrains fibrogenesis through induction of senescence in mice with induced deep endometriosis.

RESEARCH QUESTION: Does sodium tanshinone IIA sulfonate (STS) induce cellular senescence in endometriotic lesions and thus restrict lesional development and fibrogenesis in a recently established mouse model of deep endometriosis? DESIGN: Prospective randomized animal experiment in which deep endometriosis was induced in female Balb/C mice, which were then randomly divided into three groups (low-dose STS, high-dose STS and inert vehicle control) and received treatment for 2 weeks. All mice were then sacrificed and their lesions excised and harvested. Lesion weight was quantified and all lesion samples were subjected to histochemical analysis of the extent of lesional fibrosis by Masson trichrome staining, and of cellular senescence by senescence-associated ß-galactosidase (SA-ß-gal), along with immunohistochemistry analyses of p53, CCN1, activate Salvador 1 (Sav1), hyaluronan synthase 2 (HAS2), survivin, granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD163-positive M2 macrophages. Plasma P-selectin and hyaluronic acid levels were also quantified. Hotplate testing was also administered before the induction, then before and after treatment. RESULTS: STS treatment resulted in significantly reduced lesion weight, stalled lesional fibrogenesis and improved hyperalgesia, seemingly through the induction of cellular senescence by activating p53, Sav1 and CCN1 while suppressing HAS2, survivin and GM-CSF, resulting in increased apoptosis and reduced lesional infiltration of alternatively activated macrophages. In addition, STS treatment significantly reduced the plasma concentration of P-selectin and hyaluronic acid, possibly leading to reduced lesional platelet aggregation. CONCLUSIONS: STS appears to be a promising compound for treating endometriosis. The results suggest that senescence may restrict lesional progression and fibrogenesis, and targeting the senescence pathway may have desirable therapeutic potential.

Platelets induce endothelial-mesenchymal transition and subsequent fibrogenesis in endometriosis.

RESEARCH QUESTION: Do endometriotic lesions undergo endothelial-mesenchymal transition (EndoMT)? DESIGN: Lesion samples from 30 patients with ovarian endometriomas and deep endometriosis, and control endometrial tissue samples from 30 women without endometriosis, were analysed. In-vitro experimentation using the human umbilical vein endothelial cell (HUVEC) line were conducted. Immunofluorescence staining and immunohistochemistry analysis using antibodies against endothelial cell and mesenchymal cell markers were conducted. The HUVEC cells were co-cultured with activated platelets or control medium with and without neutralization of TGF-ß1 PDGFR, or both. Their morphology, proliferation and expression levels of genes and proteins known to be involved in EndoMT were evaluated, along with their migratory and invasive propensity, contractility and collagen production capability. RESULTS: The proportion of CD31 and FSP-1 dual-positive cells in FSP-1+ fibroblasts was 74.7% (±5.4%) in ovarian endometrioma lesions, significantly higher than that in deep endometriosis lesions (26.8% ± 26.0%; P = 5.7 × 10-5), and was zero in normal endometrium. The extent of lesional fibrosis correlated positively with staining levels of the lesional mesenchymal markers FSP-1 and α-SMA (r = 0.91; P < 2.2 × 10-16, r = 0.81; P = 5.8 × 10-15, respectively). Human endothelial cells co-cultured with activated platelets acquire a morphology suggestive of EndoMT, concomitant with increased proliferation, loss of CD31 but marked increase in expression of mesenchymal markers. Morphological and gene and protein expression changes are accompanied by functional differentiation reflected by increased migratory and invasive capacity, contractility and collagen production. Neutralization of TGF-ß1 and PDGFR signalling abolished platelet-induced EndoMT in human endothelial cells. CONCLUSIONS: Multiple sources of myofibroblasts exist in endometriotic lesions, and implicates platelets, EndoMT, or both, as potential therapeutic targets for treating endometriosis.

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

STUDY QUESTION: Do sensory nerves play any role in the development of endometriosis? SUMMARY ANSWER: 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. WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study is limited by the use of histologic and immunohistochemistry analyses only and the lack of molecular data. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

STUDY QUESTION: Is it possible to establish a mouse model of deep endometriosis (DE)? SUMMARY ANSWER: 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). WHAT IS KNOWN ALREADY: 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. STUDY DESIGN, SIZE, DURATION: 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. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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. MAIN RESULTS AND THE ROLE OF CHANCE: 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. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study is limited by the use of histologic and immunohistochemistry analyses only and lacks molecular data. WIDER IMPLICATIONS OF THE FINDINGS: 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

Concurrent Learning Curves of 3-Dimensional and Robotic-Assisted Laparoscopic Radical Hysterectomy for Early-Stage Cervical Cancer Using 2-Dimensional Laparoscopic Radical Hysterectomy as a Benchmark: A Single Surgeon's Experience.

BACKGROUND For early-stage cervical cancers, radical hysterectomy (RH) with pelvic lymphadenectomy has been the standard care. This study compared the learning curves and intra-, peri-, and post-operative outcomes for 3-dimensional laparoscopic RH (3D-LRH) and robotic-assisted (RA)-LRH by a surgeon highly skilled in 2-dimensional (2D)-LRH for treatment of early-stage cervical cancer. MATERIAL AND METHODS Two hundred and thirty-nine patients with early-stage cervical cancer (FIGO stage: Ia2-IIa2) admitted to Shanghai Obstetrics and Gynecology Hospital, Fudan University were recruited into this prospective study: 54, 85, and 100 patients underwent 2D-, 3D-, and RA-LRH, respectively and were followed up. Patients' demographic, clinical, and operative information was retrieved and compared. CUSUM (cumulative summation) analysis using a benchmark derived from previously performed 2D-LRHs. RESULTS Both 3D- and RA-LRH had a steep learning curve. 3D-LRH was superior to 2D- and RA-LRH in terms of significantly shorter operating time. For all approaches, the operating time was associated with the uterus size of the patient and was not affected by other parameters. All approaches of LRH yielded comparable radicality and operative results other than operative time. CONCLUSIONS Both 3D- and RA-LRH approaches had similar radicality, and intra-operative and post-operative complication rates, however, 3D-LRH had the shortest operating time and lowest amount of blood loss. After reaching proficiency, RA-LRH had comparable operating time with that of 2D-LRH, and might be even shorter in cases where surgeon has acquired more experience. In countries where labor costs are low; 3D-LRH might be preferable to 2D- and RA-LRH for early-stage cervical cancer.

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

RESEARCH QUESTION: 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? DESIGN: 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. RESULTS: 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). CONCLUSIONS: The identification of a particular macrophage subset in fibrogenesis of endometriosis should further help to shed new light on the pathophysiology of endometriosis.

Platelets impair natural killer cell reactivity and function in endometriosis through multiple mechanisms.

Study question: Do platelets have any role in the reduced cytotoxicity of natural killer (NK) cells in endometriosis? Summary answer: Platelets impair NK cell reactivity and function in endometriosis through multiple mechanisms. What is known already: Platelets play an important role in the development of endometriosis, and platelet-derived transforming growth factor-ß1 (TGF-ß1) suppresses the expression of NK Group 2, Member D (NKG2D) on NK cells, resulting in reduced cytotoxicity in women with endometriosis. Study design size, duration: Experiments on mice with induced endometriosis in which either platelets, NK cells or both were depleted and controls (none depleted). In vitro experiments with NK cells, platelets and, as target cells, endometriotic epithelial cell and endometrial stromal cell lines. Participants/materials setting methods: Immunohistochemistry analysis of ectopic endometrial tissues from mice with induced endometriosis receiving either platelet depletion (PD), NK cell depletion, or both or none. Immunofluorescence, flow cytometry and gene expression analysis for major histocompatibility complex class I (MHC-I) expression in target cells. Cytotoxicity and degranulation assays and the measurement of interferon (IFN)-γ secretion for the evaluation of NK cytotoxicity. Flow cytometry and gene expression for the expression of NK cell receptors. Main results and the role of chance: PD resulted in significantly reduced lesion weight in mice with induced endometriosis, but NK cell depletion as well as concomitant platelet and NK cell depletion increased the weight, suggesting that the anti-endometriosis effect of PD is mediated, at least in part, by increased NK cell cytotoxicity against endometriotic cells. Co-incubation of target cells with platelets resulted in rapid platelet coating as well as increased MHC-I expression in these cells, effectively providing a cloak of 'pseudo-self' to these cells to shield against NK cell lysis. It also reduced the expression of NKG2D ligands MICA and MICB and reduced the NK cell cytotoxicity. In addition, co-incubation of NK cells with platelets impaired the NK cell cytotoxicity as well. This impaired NK cell cytotoxicity was not due to the increased NK cell apoptosis, but, rather, through reduced NK cell degranulation and IFN-γ production, and reduced expression of activating receptors NKG2D and NKp46 and increased expression of inhibitory receptor KIR2DL1 in NK cells. Inhibition of TGF-ß1 signaling partially restored the aberrant expression of NKG2D, NKp46 and KIR2DL1, and partially restored the impaired NK cell cytotoxicity induced by activated platelets and their releasate. Large scale data: Not applicable. Limitations reasons for caution: This study is confined by the limitation of animal and in vitro experimentation and the lack of direct human data. Wider implications of the findings: Anti-platelet treatment holds promise in treating endometriosis. Study funding/competing interests: The National Natural Science Foundation of China (81471434 to S.W.G., 81270676 to S.W.G., 81370695 to X.S.L. and 81671436 to X.S.L). None of the authors has anything to disclose.

Social psychogenic stress promotes the development of endometriosis in mouse.

Exposure to chronic stress before and well after the induction of endometriosis is reported to increase lesion sizes in rats, but it is unclear whether stress, exposed shortly after the induction of endometriosis, would also promote the development of endometriosis, nor is it clear what the underlying possible molecular mechanism is. This study was undertaken to test the hypothesis that chronic stress can promote the development of endometriosis. A prospective randomized mouse experiment was conducted that subjected mice with induced endometriosis to predator stress. In addition, a cross-sectional immunohistochemistry study was performed in ectopic and eutopic endometrial tissue samples from age- and roughly menstrual phase-matched women with ovarian endometriomas. It was found that the chronic psychogenic stress induced epigenetic changes in the hippocampus in mouse independent of endometriosis. It was also found that chronic psychogenic stress induced epigenetic changes in the hippocampus of mice with endometriosis, and seemingly activated the adrenergic signalling in ectopic endometrium, resulting in increased angiogenesis and accelerated growth of endometriotic lesions. Thus, chronic psychogenic stress promotes endometriosis development, raising the possibility that the use of anti-depressants in cases of prolonged and intense stress might forestall the negative impact of stress on the development of endometriosis.