Adjacent Myometrium Research Articles

665 The state of local immune interrelations in patients with adenomyosis

ObjectiveThe frequency of adenomyosis reaches 70%. The intrusion of the basal layer of the endometrium into the myometrium is possible in case of a violation of local immunity, which is formed against the background of causative agents of genital infection. The study of immune interrelations in the uterus in patients with adenomyosis will expand the understanding of the pathogenesis. Study design: 159 women with adenomyosis were examined. In the cervical secretion, a quantitative assessment of the levels of cytokines associated with the activity of innate immune defense IL-4,8,10,18, TNF, INF, the concentration of sIgA, IgA, M, G, the concentration of C3, C4 components of complement and lysozyme. In the endometrium, Chlamydia trachomatis, Ureaplasma spp., Mycomlasma genitalium, HSV1,2 / CMV, HPV were determined by PCR. Results: In the presence of genital pathogens in patients with adenomyosis, disorders of innate immunity were revealed, manifested in an increased content of sIgA, IgA, G (p <0.05), which may be a consequence of impaired permeability of the mucous membranes. An increase in the level of pro-inflammatory (IL-8, IL-18, TNF) and a decrease in the level of anti-inflammatory cytokines (IL-4, IL-10), INF (p <0.05) were revealed. The complement system is activated, as evidenced by an increase in the level of C3, C4 (p <0.05). An increase in proinflammatory cytokines at the local level aims to limit inflammatory processes in the endometrium and promoting the development of a T-cell immune response. Conclusions: In patients with adenomyosis, signs of immunopathology are observed, leading to a weakening of immune control, which creates conditions for the development of endometrial heterotopies. Formed against the background of bacterial and viral pathogens, immune disorders can contribute to the invasion of endometrial cells into the adjacent myometrium.

Uterine Necrosis After Uterine Artery Embolization (UAE) - Rare Entity with a Dilemma

ABSTRACT Uterine artery embolization (UAE), a minimally invasive procedure used for controlling and managing profuse bleeding in conditions like uterine fibroids, and uterine hemorrhage, in cases of intractable postpartum hemorrhage (PPH). A wide array of complications due to UAE includes pulmonary embolism, uterine ischemia, necrosis, sepsis, and death. A 27 years old married woman (parity 1+2) presented with complaints of a mass protruding of the vagina with dirty brown vaginal discharge. Following UAE was done for postpartum hemorrhage (PPH) she had developed fever and burning micturition on which ultrasound was done to show an anteverted, enlarged, bulky uterus, with multiple endogenic foci, with endometrial canal and adjacent myometrium suggestive of infective pathology. In this case report complications of UAE for the management of PPH were discussed, however, research is required to comprehend the causes of uterine necrosis following uterine artery embolization and its management. Keywords: Uterine Artery Embolization; Postpartum Hemorrhage; Uterine Necrosis; Pathology.

Tryptophan 2,3-Dioxygenase-2 in Uterine Leiomyoma: Dysregulation by MED12 Mutation Status

Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures.

Increased Expression of Fibroblast Activation Protein is Associated with Autophagy Dysregulation and Oxidative Stress in Obese Women with Uterine Fibroids.

Uterine fibroids (UF) represent an immense health burden throughout the world. Obesity is considered one of the risk factors for UF development; however, the underlying mechanisms remain largely unexplored. We investigated the effect of obesity on fibroblast activation and its association with inflammation, autophagy dysfunction, and oxidative stress in UF patients. Thirty-five pre-menopausal UF patients were included in this study and classified into non-obese group (BM1 ≤ 30 kg/m2, n = 15) and obese group (BMI > 30 kg/m2, n = 20). Tissue samples were collected from fibroids and adjacent normal myometrium. Our results showed increased expression of fibroblast activation protein (FAP) together with markers of autophagy, inflammation, and oxidative stress in UF patients, which were all more markedly upregulated in obese compared to non-obese patients. In addition, BMI was significantly positive correlated with FAP and autophagy markers. In conclusion, the results of the present study suggest that obesity-associated autophagy dysregulation together with increased FAP expression may increase the risk of UFs in obese women.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid: a therapeutic approach to treat human uterine leiomyoma

To evaluate the effect of inhibition of histone deacetylases (HDACs) by suberoylanilide hydroxamic acid (SAHA) treatment of human uterine leiomyoma primary (HULP) cells invitro on cell proliferation, cell cycle, extracellular matrix (ECM) formation, and transforming growth factor β3 (TGF-β3) signaling. Prospective study comparing uterine leiomyoma (UL) vs. adjacent myometrium (MM) tissue and cells with or without SAHA treatment. Hospital and university laboratories. Women with UL without any hormone treatment. Myomectomy or hysterectomy surgery in women for leiomyoma disease. HDAC activity was assessed by enzyme-linked immunosorbent assay, and gene expression was assessed by quantitative real-time polymerase chain reaction. Effects of SAHA on HULP cells were analyzed by CellTiter(Promega, Madison, Wisconsin), Western blot, and quantitative real-time polymerase chain reaction. The expression of HDAC genes (HDAC1, fold change [FC] = 1.65; HDAC3, FC = 2.08; HDAC6, FC = 2.42) and activity (0.56 vs. 0.10 optical density [OD]/h/mg) was significantly increased in UL vs. MM tissue. SAHA decreased HDAC activity in HULP cells but not in MM cells. Cell viability significantly decreased in HULP cells (81.68% at 5 μM SAHA, 73.46% at 10 μM SAHA), but not in MM cells. Proliferating cell nuclear antigen expression was significantly inhibited in SAHA-treated HULP cells (5 μM SAHA, FC = 0.556; 10 μM SAHA, FC = 0.622). Cell cycle markers, including C-MYC (5 μM SAHA, FC = 0.828) and CCND1 (5 μM SAHA, FC = 0.583; 10 μM SAHA, FC = 0.482), were significantly down-regulated after SAHA treatment. SAHA significantly inhibited ECM protein expression, including FIBRONECTIN (5 μM SAHA, FC = 0.815; 10 μM SAHA, FC = 0.673) and COLLAGEN I (5 μM SAHA, FC = 0.599; 10 μM SAHA, FC = 0.635), in HULP cells. TGFβ3 and MMP9 gene expression was also significantly down-regulated by 10 μM SAHA (TGFβ3, FC = 0.596; MMP9, FC = 0.677). SAHA treatment inhibits cell proliferation, cell cycle, ECM formation, and TGF-β3 signaling in HULP cells, suggesting that histone deacetylation may be useful for treatment of UL.

Prolactin is Expressed in Uterine Leiomyomas and Promotes Signaling and Fibrosis in Myometrial Cells.

Uterine leiomyomas are benign, estrogen-sensitive, fibrotic smooth muscle cell tumors occurring in the uterine myometrium. Leiomyomas are a considerable health burden, with a lifetime prevalence of 80% and limited treatment options. Estrogen and progesterone have positive effects on leiomyoma growth, but little is known about the roles of other hormones. One hormone of interest is prolactin, as it has been described to be present and functional in leiomyomas. The current study investigates prolactin production within leiomyomas and its effects on myometrial cells. RNA isolation and quantitative-PCR of human leiomyoma samples relative to matched adjacent myometrium confirms significant expression of prolactin and dopamine receptor D2, a known regulator of prolactin production and release in the pituitary, with no difference in prolactin receptor expression. Immunohistochemistry confirms increased prolactin in leiomyomas compared to adjacent myometrium and uteri from women without leiomyomas. These results suggest that leiomyomas contain cells that produce prolactin, which may then promote signaling in leiomyoma cells to regulate leiomyoma development/growth. Accordingly, we find that prolactin robustly activates STAT5 and MAPK signaling in rat and human myometrial cell lines. Furthermore, prolactin stimulates expression of myofibroblast markers in rat myometrial cells. Our findings suggest that local prolactin production in leiomyomas may stimulate trans-differentiation of myometrial cells to myofibroblasts, which in turn contributes to the fibrotic nature of leiomyomas.

INHIBITION OF BRD PROTEINS SUPPRESSES THE PHENOTYPE OF UTERINE FIBROIDS VIA REGULATION OF N6-METHYLADENOSINE REGULATORS

Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uterine fibroids (UFs), the most common pelvic tumors in women of reproductive age, is unknown. The present study aimed to determine the expression pattern of BRD proteins in UFs and matching myometrium and assess the effect of BRD inhibitors on UF phenotype and epitranscriptomic changes. Human UFs (n=22) and adjacent myometrium tissues (MyoF, n=7) were collected at the time of hysterectomy at the University of Chicago. Western blot (WB) was performed to determine the levels of BRD proteins in UFs and myometrium. Fourteen BRD inhibitors from the Epigenetic Chemical Probe Library provided by the University of Illinois at Chicago were used to determine the cell proliferation by MTT assay. Two BRD inhibitors (I-BRD9 and TP-472) were further used to explore their mechanistic actions. The student’s t-test was used to determine the significant difference. Our studies demonstrated that among 22 UFs analyzed, 96% (21/22, p<0.01), 55% (12/22), 82% (18/22, p<0.01) exhibited the upregulation of BRD2, BRD3, and BRD9 respectively, suggesting the aberrant BRD protein expression may contribute to the pathogenesis of UFs. To examine the biological impact of BRD inhibition on the UFs, 14 BRD inhibitors were screened to identify specific and potent BRD inhibitors in UF cells. 43% (6/14) of these BRD inhibitors selectively inhibited UF, but not myometrial cell proliferation. Furthermore, targeting BRD proteins with BRD inhibitors (I-BRD9 and TP-472) decreased the protein levels of extracellular matrix proteins (Type I Collagen, fibronectin), PCNA (cell proliferation marker), and Bcl-2 (anti-apoptotic marker) in a dose- and time-dependent manner in UF cells. Notably, inhibition of BRD proteins with I-BRD9 and TP-472 downregulated the expression of METTL3, YTHDC2, and YTHDF2, which are the key regulators for N6-methyladenosine (m6A) modification, indicating the tight link between BRD proteins and epitranscriptomic landscape. These results demonstrate for the first time that BRD proteins are aberrantly expressed in UFs. BRD inhibitors suppress the UF phenotype while concomitantly altering the expression of the central m6A regulators.

5-aza-2\u2032-deoxycitidine inhibits cell proliferation, extracellular matrix formation and Wnt/\u03b2-catenin pathway in human uterine leiomyomas

BackgroundUterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2′-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option.MethodsProspective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR.ResultsDNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza-CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 μM (85.3%). Treatment with 10 μM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/β-catenin pathway final targets, including WISP1 protein expression (10 μM, FC = 0.699), c-MYC gene expression (2 μM, FC = 0.745 and 10 μM, FC = 0.728), and MMP7 gene expression (5 μM, FC = 0.520 and 10 μM, FC = 0.577).Conclusions5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/β-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma.

Babu and Magon uterine closure technique during cesarean section: A randomized double-blind trial.

We compared the effectiveness of the Babu and Magon uterine closure technique and unlocked double-layer uterine closure on the integrity and thickness of the uterine scar. A randomized double-blind trial was performed at Hazrat-e Rasoul -e-Akram Hospital, Tehran, Iran, from March 2018 to December 2019, in 72 pregnant women who were candidates for cesarean section for the first time. Women were randomly assigned to the Babu and Magon uterine closure technique (intervention group, n=34) or double-layer closure of the uterine incision (control group, n=38). The primary outcome of the study was the frequency of myometrial defects at the site of the scar (niche), and a large niche. Secondary outcomes, including the time taken for uterine closure and postpartum hemorrhage (early and late), were compared between groups. Adjacent myometrium thickness (AMT) between the two groups was not statistically significant. A niche was reported in 23.5% (8/34) and 50% (19/38) of women in the intervention and controls, respectively (p=0.02). A large niche was reported in 2.9% (1/34) and 23.7% (9/38) of women in the intervention and controls, respectively (p < 0.01). The duration of uterine closure was not statistically significant between the two groups. Hemoglobin levels did not differ significantly between groups during the first 24 h post-surgery. The results of the study showed that the technique of uterine closure is one of the main potential determinants of myometrial healing. The Babu and Magon uterine closure technique seems to lead to tissue alignment during suturing and consequently cause better myometrial healing, although this issue calls for well-founded longer studies of appropriate design.

Transcervical microwave ablation in type 2 uterine fibroids via a hysteroscopic approach: analysis of ablation profiles

Type 2 uterine fibroids are challenging to resect surgically as ≥ 50% volume of myoma lies within the myometrium. A hysteroscopic approach for ablating fibroids is minimally-invasive, but places a considerable burden on the operator to accurately place the ablation applicator within the target. We investigated the sensitivity of transcervical microwave ablation outcome with respect to position of the ablation applicator within 1 – 3 cm type 2 fibroids. Methods: A finite element computer model was developed to simulate 5.8 GHz microwave ablation of fibroids and validated with experiments in ex vivo tissue. The ablation outcome was evaluated with respect to applicator insertion angles (30°, 45°, 60°) , depth and offset from the fibroid center (±2 mm for 3 cm fibroid and ±1 mm for 1 cm fibroid) with 35 W and 15 W applied power for 3 cm and 1 cm fibroids, respectively. Power deposition was stopped when thermal dose of 40 cumulative equivalent minutes at 43 °C (CEM43) was accrued in adjacent myometrium. Results: Within the range of all evaluated insertion angles, depths and offsets, the ablation coverage was less sensitive to variation in angle as compared to depth and offset, and ranged from 34.9 – 83.6% for 3 cm fibroid in 140 – 400 s and 34.1 – 67.9% for 1 cm fibroid in 30 – 50 s of heating duration. Maximum achievable ablation coverage in both fibroid cases reach ∼ 90% if thermal dose is allowed to exceed 40 CEM43 in myometrium. Conclusion: The study demonstrates the technical feasibility of transcervical microwave ablation for fibroid treatment and the relationship between applicator position within the fibroid and fraction of fibroid that can be ablated while limiting thermal dose in adjacent myometrium.

Imaging, clinical, and pathologic findings of Sertoli-leydig cell tumors.

To explore the clinical features, imaging findings, and pathological manifestations of ovarian Sertoli-Leydig cell tumors (SLCTs). The clinical and pathological manifestations, tumor location, size, morphology, vascularity, computed tomography (CT) density, magnetic resonance imaging (MRI) signal intensity, and contrast enhancement patterns in five cases with SLCTs were retrospectively reviewed. SLCTs most commonly occurred in young women. Virilization was observed in three cases (60%). All five tumors were unilateral and oval or round, with a clear boundary. The solid part of the tumor was isoattenuated on the conventional CT scan, and showed isoattenuation or slight hypoattenuation relative to adjacent myometrium on T1 weighted imaging (T1WI) and T2 weighted imaging (T2WI). On contrast-enhanced images, three tumors showed marked enhancement. DICER1 hotspot mutations were commonly seen in SLCTs. A highly vascularized mass with low signal intensity (SI) of the solid part on T2WI and androgen overproduction symptoms may suggest an SLCT.

Sphingosine 1-phosphate signaling in uterine fibroids: implication in activin A pro-fibrotic effect

To explore the link between sphingosine 1-phosphate (S1P) signaling and leiomyoma and the possible S1P cross-talk with the fibrotic effect of activin A. Case-control laboratory study. University institute and university hospital. Patients with uterine fibroids (n = 26). Tissue specimens of leiomyoma and normal myometrium were obtained from patients undergoing myomectomy or total hysterectomy. Expression of mRNA levels of the enzyme involved in S1P metabolism, S1P receptors, and S1P transporter Spns2 was evaluated in matched leiomyoma/myometrium specimens and cell populations. The effects of inhibition of S1P metabolism and signaling was evaluated on activin A-induced fibrotic action in leiomyoma cell lines. The expression of the enzymes responsible for S1P formation, sphingosine kinase (SK) 1 and 2, and S1P2, S1P3, and S1P5 receptors was significantly augmented in leiomyomas compared with adjacent myometrium. In leiomyoma cells, but not in myometrial cells, activin A increased mRNA expression levels of SK1, SK2, and S1P2. The profibrotic action of activin A was abolished when SK1/2 were inhibited or S1P2/3 were blocked. Finally, S1P augmented by itself mRNA levels of fibrotic markers (fibronectin, collagen 1A1) and activin A in leiomyomas but not in myometrial cells. This study shows that S1P signaling is dysregulated in uterine fibroids and involved in activin A-induced fibrosis, opening new perspectives for uterine fibroid treatment.

TGF-β1 role in uterine leiomyoma and endometrial polyp: an insight to drug-based treatment instead of surgical techniques

ObjectiveConsidering the high prevalence of leiomyoma and endometrial polyps, investigating the contributing factors and determining the pathophysiology of these lesions are essential. Target therapy is now an acceptable method for the treatment of some diseases. We aimed to determine the expression of transforming growth factor (TGF)-β1 in endometrial polyps and leiomyomas to discover a drug-based method to overcome surgical treatments.MethodsIn this cross-sectional study, 55 patients with leiomyoma and 55 patients with polyps were included. Prepared slides from leiomyoma and adjacent myometrium or polyp lesions and adjacent endometrium were obtained and investigated for TGF-β1. Then, data were collected and analyzed using SPSS version 22.ResultsThe mean age of participants was 40.6±5.8 years. Based on their reports, 88.2% (n=97) of patients in the study population had abnormal uterine bleeding with similar distributions among both groups. In contrast, 63.5% of the leiomyoma group did not express TGF-β1. However, in normal myometrium, 23.6% had the highest degree of TGF-β1 expression. Polyp tissue did not show staining for TGF-β1 in any patients. Additionally, 89.1% of non-polypoid endometrium did not express TGF-β1. Normal tissue had a significantly greater amount of TGF-β1 compared to leiomyoma and endometrial polyps.ConclusionTGF-β1 is expressed more prominently in normal myometrium with mostly high-intensity features compared to leiomyoma. Additionally, polyps showed no staining for TGF-β1, while normal endometrium showed a low-density staining pattern.

Abstract PO-001: Heterogeneous overexpression of HES6 promotes the growth of uterine leiomyomas

Abstract Introduction: Uterine leiomyomas are a common cause of infertility, pelvic pain and irregular vaginal bleeding. Robust evidence suggests that leiomyomas arise from a distinct population of myometrial-specific stem cells (mSC). However, the mechanisms responsible for driving the mal-differentiation of mSC and subsequent leiomyoma growth remain poorly understood. Here, we explore the role of HES6, a basic helix-loop-helix (bHLH) co-factor critical for promoting myogenic differentiation, in uterine leiomyomas. Methods: After obtaining IRB approval, real time PCR (qPCR) and Western blot were used to evaluate patterns of HES6 expression using RNA and protein prepared from matched specimens of myometrium, leiomyoma and uterine leiomyosarcoma (uLMS). Primary cultures were generated as previously described. HES6-specific siRNAs were used to target gene expression in vitro. Standardized assays were used to measure proliferation (CellTiter-Glo, Promega) and apoptosis (Caspase 3/7 assay). A commercially available antibody specific for HES6 was used to localize HES6 expression in formalin-fixed, paraffin-embedded (FFPE) tissue specimens by immunohistochemistry (IHC). Patterns of gene expression and splicing were profiled by Next Generation Sequencing. Student’s t-test was used to assess statistical significance. Results: We found that, on average, levels of HES6 transcript were &amp;gt;15-fold higher when specimens of leiomyomas were compared to adjacent normal myometrium (p&amp;lt;0.01). No relationship between expression of HES6 in myometrium or leiomyoma, menstrual phase (n=58), menopausal status (n=12) or clinical treatment of pre- or postmenopausal women with medroxyprogesterone (Pre: n=12; Post: n=6) was observed. When matched FFPE specimens were evaluated by IHC, we found that HES6 expression was selectively limited to organized subsets of cells within large leiomyomas as well as the blood vessels surrounding these tumors. Levels of HES6 were &amp;gt;4-fold lower in specimens of uLMS (n=6) than proliferative or secretory phase myometrium (p&amp;lt;0.01). In vitro, knockdown of HES6 expression significantly reduced rates of proliferation and enhanced rates of apoptosis across multiple primary cultures derived from matched specimens of myometrium and leiomyomas (n=10, p&amp;lt;0.05). Of the 123 gene products who expression was significantly altered (p&amp;lt;0.05) in response to targeting HES6 expression, levels of the mitochrondrial solute transporter SLC25A22 was most dramatically disrupted. Conclusions: Overexpression of HES6 is a reliable feature important for promoting the growth of benign but not malignant uterine smooth muscle tumors within metabolically defined subsets of uterine smooth muscle cells. We are currently working to determine how this highly novel pattern of intratumoral heterogeneity promotes leiomyoma growth and its hierarchical relationship to early events in the mal-differentiation of mSC. Citation Format: Joie Z. Guner, Venkata A. Jonnalagadda, Ninad A. Patel, Ramya P. Masand, Cecilia Valdes, Williams E. Gibbons, Matthew L. Anderson. Heterogeneous overexpression of HES6 promotes the growth of uterine leiomyomas [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-001.

Effectiveness of hysteroscopic resection of a uterine caesarean niche can be predicted: a prospective cohort study

This study aimed to develop and validate a model for the preoperative prediction of the effectiveness of hysteroscopic resection of a uterine cesarean niche in patients with postmenstrual spotting. The predictive model was developed in a primary prospective cohort consisting of 208 patients with niche treated by hysteroscopic resection. Multivariable logistic regression analysis was performed to develop the predictive model, which incorporated preoperative menstrual characteristics and magnetic resonance imaging (MRI) findings. Surgical efficacy was defined as a decrease in postmenstrual spotting duration of at least 3 days at the 3-month follow-up compared with baseline. The predictive model was presented with a nomogram, and the performance was assessed with respect to its calibration, discrimination, and clinical use. Internal validation was performed using tenfold cross-validation. The predictive factors in the final model were as follows: preoperative menstrual duration, thickness of the residual myometrium (TRM), length, TRM/thickness of the adjacent myometrium ratio, angle γ, area, and presence of a lateral branch of the niche. The model showed good performance in predicting the effectiveness of hysteroscopic niche resection. Incorporating the preoperative duration of the menstrual period and MRI findings of the niche into an easy-to-use nomogram facilitates the individualized prediction of the effectiveness of a hysteroscopic niche resection by 26 Fr resectoscope, but multicenter prospective studies are needed to validate it.

Vitamin D as an effective treatment in human uterine leiomyomas independent of mediator complex subunit 12 mutation

To study whether vitamin D (VitD) inhibits cell proliferation and Wnt/β-catenin and transforming growth factor-β (TGFβ) signaling pathways in uterine leiomyomas independent of mediator complex subunit 12 (MED12) mutation status. Prospective study comparing leiomyoma vs. myometrial tissues and human uterine leiomyoma primary (HULP) cells treated with or without VitD and analyzed by MED12 mutation status. Hospital and university laboratories. Women with uterine leiomyoma without any treatment (n = 37). Uterine leiomyoma and myometrium samples were collected from women undergoing surgery because of symptomatic leiomyoma pathology. Analysis of Wnt/β-catenin and TGFβ pathways and proliferation by quantitative real-time polymerase chain reaction in leiomyoma and myometrial tissue as well as in VitD-treated HULP cells analyzed by Sanger sequencing. Sequencing data showed that 46% of leiomyomas presented MED12 mutation, whereas no mutations were detected in adjacent myometrium. Expression of Wnt/β-catenin and TGFβ pathway genes was significantly increased in MED12-mutated leiomyomas compared to matched myometrium; no significant differences were found in wild-type (WT) leiomyomas. In HULP cells, VitD significantly decreased PCNA expression of both MED12-mutated and WT groups. VitD treatment decreased WNT4 and β-catenin expression in both groups compared to controls, with significance for WNT4 expression in MED12-mutated samples. Similarly, VitD significantly inhibited TGFβ3 expression in cells from both groups. MMP9 expression also decreased. Despite molecular differences between MED12-mutated and WT leiomyomas, VitD inhibited Wnt/β-catenin and TGFβ pathways in HULP cells, suggesting VitD as an effective treatment to reduce proliferation and extracellular matrix formation in different molecular subtypes of uterine leiomyomas.

Circular RNA expression profiling identifies novel biomarkers in uterine leiomyoma

BackgroundCircular RNAs (circRNAs) have recently been identified in the development and progression of multiple human diseases. However, the significance of circRNAs in uterine leiomyoma (ULM) remains to be elucidated. Here, we aim to explore the expression profile of circRNAs in ULM and the potential of cicRNAs to be used as biomarkers or therapeutic targets.MethodsGlobal circRNA expression Profiles for ULM was performed by microarray in ULM tissue and matched adjacent normal myometrium counterpart. Bioinformatics analysis, qRT-PCR validation, and receiver operating characteristic (ROC) diagnostic accuracy was applied for differentially expressed circRNAs. Cell proliferation and spheroid formation assay were performed to assess the functional role of candidate circRNA.Results579 up- and 625 down-regulated circRNAs were identified between ULMs and adjacent normal myometrium tissues. Bioinformatics analysis suggested that most differentially expressed circRNAs participate in pathways were related to pathological processes of ULM. The qRT-PCR validation results for 6 circRNAs (hsa_circ_0083920, hsa_circ_0056686, hsa_circ_0062558, hsa_circ_0020376, hsa_circ_0043597, hsa_circ_0026353, and circ_0017248) matched the microarray results. ROC analysis showed that hsa_circ_0083920, hsa_circ_0056686, hsa_circ_0062558, hsa_circ_0020376, and hsa_circ_0043597 could accurately distinguish the ULM samples from the myometrium samples. Additionally, hsa_circ_0056686 was validated to be upregulated in ULM and was associated with the leiomyoma size (P = 0.0446). Reduction of endogenous hsa_circ_0056686 expression significantly suppressed leiomyoma cell proliferation and spheroid formation capacity.ConclusionsThis study provides an integrated analysis of circRNAs in ULM, and gives new insight into the complex epigenetic mechanisms of ULM. Aberrantly expressed circRNAs may contribute to the pathogenesis of ULM and hsa_circ_0056686 might be a potential therapeutic target.

SAT-003 Prolactin Is Expressed in Uterine Leiomyomas and May Promote Their Growth

Uterine leiomyomas, commonly referred to as fibroids, are benign, estrogen sensitive smooth muscle tumors that occur in the myometrium of the uterine wall. Leiomyomas are common, as it is estimated that 60% of reproductive-aged women are affected, and 80% of women develop leiomyomas during their lifetime. In fact, uterine leiomyomas are the leading cause of abnormal menstrual bleeding or menstrual pain, as well as the number one reason for hysterectomy. Novel treatment options are necessary as current treatments are limited to anti-estrogen therapy or hysterectomy. Estrogen is known to have an effect on the etiology of leiomyomas, but little is known about the proliferative roles of other hormones in leiomyomas. One hormone of interest is prolactin (PRL) which is primarily secreted from the pituitary to regulate lactation, but has been linked to proliferation in breast cancer, perhaps via local prolactin production in breast tissue. With this background, we examined local PRL production and its effects on leiomyomas. RNA isolation and quantitative PCR of human leiomyoma samples (n=20) relative to adjacent myometrium in the same patients confirmed significant expression of both PRL (p= 0.0028) and dopamine receptor D2, a known regulator of PRL production in the pituitary (p<0.0001), with no difference in prolactin receptor expression. Using both immunohistochemistry and immunofluorescence of human leiomyomas samples, we find increased prolactin expression in leiomyomas when compared to adjacent myometrium or control uteri. These results suggest that leiomyomas contain cells producing PRL, which in turn may promote signaling in smooth muscle leiomyoma cells to regulate proliferation. Accordingly, we find that PRL robustly activates STAT5 and MAPK signaling in the rat leiomyoma cell line ELT3. Functional assays were also conducted to evaluate the ability of PRL to induce migration, invasion and proliferation of ELT3 cells. Together, our findings suggest that local prolactin production in leiomyomas may promote their growth, migration, invasion and proliferation. It is possible that this local production is mediated by the dopamine receptor D2. Thus, anti-PRL therapy or dopamine receptor D2 modulation may prove useful in treating this prevalent and often debilitating disease.

SWATH-MS Analysis of FFPE Tissues Identifies Stathmin as a Potential Marker of Endometrial Cancer in Patients Exposed to Tamoxifen

A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.

Successful conservative treatment for massive uterine bleeding with non-septic disseminated intravascular coagulation after termination of early pregnancy in a woman with huge adenomyosis: case report

BackgroundAdenomyosis is a benign gynecological condition in which endometrial tissue or endometrial-like tissue develops within the uterine myometrium. Few cases of disseminated intravascular coagulation has been reported in the patients with adenomyosis. Although hysterectomy is indicated for refractory massive uterine bleeding in the patients with advanced uterine adenomyosis, conservative treatment is often desired in women in the late reproductive age. Recently such cases are increasing due to the social trend of late marriage.Case presentationA 37-year-old woman with huge adenomyosis, gravida 2 para 0, was referred to our hospital to terminate her pregnancy. Acute, non-septic, disseminated intravascular coagulation (DIC) developed after early pregnancy was terminated in a woman with huge adenomyosis. Massive bleeding and DIC occurred 3 days after the dilatation and curettage. There was no evidence of infection as the cause of the DIC, because neither bacteria nor endotoxin could be detected in her blood, and antithrombin 3 (AT3), which would be expected to decrease in septic patients, was not decreased. Hemorrhage in the adenomyotic tissue after the termination presumably developed inflammation, with numerous microthrombi and necrosis in the adenomyotic tissue, which subsequently promoted coagulation and fibrinolysis, leading to the onset of massive uterine bleeding and DIC. Although severe hyperfibrinolysis is observed in peripheral blood, the fibrinolysis state in the uterine myometrium is considered to be even more severe. The newly formed clots for hemostasis under the uterine mucosa could be removed due to the excessive activation of fibrinolytic system happened in the adjacent myometrium, leading to the onset of massive uterine bleeding. Massive bleeding and DIC resolved quickly after the patient was treated with nafamostat mesilate, which is effective for both excessive coagulation and fibrinolysis.ConclusionsAdenomyosis could cause massive bleeding and DIC when pregnancy is terminated. Massive bleeding was considered to occur because the excessive fibrinolysis system inside adenomyosis affected the adjacent endometrium. Before considering hysterectomy to control refractory uterine bleeding, nafamostat mesilate should be considered as one option, thinking the pathophysiology of the massive bleeding due to uterine adenomyosis.