F&S Science

Relugolix reduces leiomyoma extracellular matrix production via the transforming growth factor-beta pathway

To determine if the oral gonadotropin-releasing hormone antagonist relugolix affects leiomyoma extracellular matrix production through the transforming growth factor-beta (TGF-β) pathway. Laboratory study. None. Exposure of human leiomyoma cells to TGF-β and/or relugolix. Production of TGF-β, pSMAD2/3, SMAD2/3, collagen 1A1 (COL1A1), fibronectin (FN1), and versican (VCAN) in treated and untreated leiomyoma cells. Transforming growth factor-beta 3 production decreased at 24 hours with relugolix 10 nM (0.80 ± 0.09-fold) and 100 nM (0.86 ± 0.06-fold) and at 48 hours with relugolix 1 nM (0.86 ± 0.05-fold) and 100 nM (0.86 ± 0.06-fold). pSMAD2/3 production decreased at 24 hours with relugolix 1 nM (0.71 ± 0.01-fold), 10 nM (0.68 ± 0.01-fold), and 100 nM (0.41 ± 0.10-fold). Compared with relugolix treatment alone at the same concentration, combination treatment at 24 hours resulted in significantly increased COL1A1, FN1, and VCAN production with relugolix 1 nM, 10 nM, and 100 nM. At 48 hours, combination treatment resulted in significantly increased COL1A1, FN1, and VCAN production with relugolix 10 nM and 100 nM. Relugolix regulated leiomyoma size by decreasing COL1A1, FN1, and VCAN production. This effect is at least partly through the TGF-β pathway.

Transvaginal shear wave elastography for measuring uterine myometrial and leiomyoma stiffness: a protocol pilot study

Recent studies suggest that the stiffness of uterine leiomyomas may be related to their growth and behavior. Shear wave elastography (SWE), a quantitative method of measuring tissue stiffness, has been increasingly studied for use in gynecologic conditions. However, no protocols have been proposed for its use in this clinical setting. This study aimed to establish a standardized protocol for transvaginal SWE to measure uterine myometrial and leiomyoma stiffness and assess the reproducibility and reliability of SWE measurements. We also assessed myometrial vs. leiomyoma stiffness, compared myometrial stiffness in participants with and without leiomyomas, and assessed menstrual phase effects on stiffness values. Transvaginal SWE ultrasound measurements of myometrial and leiomyoma stiffness were obtained. All transvaginal SWE examinations were performed by an individual sonographer. Independent raters calculated stiffness measurements. Myometrial and leiomyoma stiffness were compared across menstrual phases. Twenty-seven premenopausal women, 16 with leiomyomas and 11 without, were enrolled. Seventeen participants completed examinations during multiple menstrual phases. Tissue type (myometrium/leiomyoma), leiomyoma status (presence/absence of leiomyomas), and menstrual phase (follicular/luteal). Interrater and test-retest reliability of SWE measurements. Myometrial and leiomyoma shear wave values. We successfully designed a streamlined protocol for obtaining SWE measurements in participants with and without leiomyomas. Fifty-one myometrial and 38 leiomyoma stiffness values measured by 2 independent raters achieved excellent interrater reliability: the intraclass correlation coefficients between the 2 raters were 0.990 and 0.994, respectively. Fifty myometrial and 42 leiomyoma stiffness measurements calculated by a single rater at 2 time points at least 90 days apart achieved excellent test-retest reliability: intraclass correlation coefficients of 0.992 and 0.995. The median stiffness values for leiomyomas were significantly higher than those for the surrounding myometrium (48.1 [interquartile range, 39.7-59.5] vs. 31.6 [interquartile range, 22.9-46.9] kPa). There was no significant change in myometrial or leiomyoma median stiffness values between the menstrual phases. Transvaginal ultrasound SWE showed excellent reproducibility and reliability in measuring myometrial and leiomyoma stiffness. Leiomyoma stiffness was significantly higher than that for the surrounding myometrium. Together, these findings support SWE's potential clinical utility in leiomyoma management.

Compressive force induces differential gene and protein expression in uterine fibroids

To study how compressive forces influence fibroid and myometrial cells. Our work aimed to identify proteins and signaling pathways that are altered in fibroids in response to compressive forces. Laboratory-based. Patient-matched fibroid and myometrial cells were isolated from five women undergoing hysterectomy or myomectomy for the treatment of uterine fibroids. Only samples from women who had not had hormonal modulation within 3 months of surgery were used for this study. An embedded spheroid model was developed to model the fibroid tissue and provide a cushion that would help with the distribution of compressive force. Weights, 0 or 6.4 mm Hg, were added on top of an agarose cushion. Spheroids were cultured for 7 days. Histological evaluation, RNA-sequencing (n = 5), and proteomics characterization (n = 3). Paired multi-test t-tests were performed for statistical analysis. Differentially expressed genes (DEGs) were considered clinically relevant if the same genes were also significantly differentially expressed in at least one of the four existing fibroid and myometrium RNA-sequencing datasets. A total of 61 clinically relevant DEGs were identified between cell types that were only differentially expressed when the spheroids were under compression. This included EPHB1 which encodes ephrin signaling receptor EphB1; it was upregulated log2 fold-change of 2.81 in fibroid cells (q = 5.35 × 10 Compressive forces must be considered to study some of the important differences between fibroids and myometrium, including ephrin signaling. Enrichment analysis of the proteins with different abundances suggests that compression may also be involved in fibroid tumor initiation.

Stearoyl–coenzyme A desaturase enhances cell survival in human uterine leiomyoma

Stearoyl-CoA desaturase (SCD1) is an enzyme that catalyzes the conversion of saturated delta-9 fatty acids to monounsaturated fatty acids. SCD1 is highly expressed in various cancers and facilitates cancer cell survival, tumor growth, and metastasis. This study aimed to assess SCD1 expression and function in uterine leiomyoma and matched myometrial tissue and evaluate the impact of SCD1 inhibition on leiomyoma cell viability and apoptosis. Gene set enrichment analysis was performed to determine whether lipid metabolism pathways are dysregulated in leiomyoma. To assess the function of SCD1, primary leiomyoma and myometrial cells, as well as a CRISPR-engineered leiomyoma-relevant MED12 mutant human uterine smooth muscle (UtSM) cell line, were treated with SCD1 small interfering RNA or a small molecule inhibitor of SCD1, CAY10566. Cell viability and apoptosis assays, real-time quantitative polymerase chain reaction, and immunoblot analyses were performed to evaluate cell function in response to treatment. Leiomyoma and myometrial tissues were obtained from premenopausal individuals designated female at birth (n = 30) undergoing myomectomy or hysterectomy. SCD1 inhibition by small interfering RNA and CAY10566 treatment. Messenger RNA (mRNA) and protein levels and cell viability and apoptosis. Gene set enrichment analysis revealed that the cholesterol homeostasis pathway was significantly different in leiomyoma vs. adjacent myometrial tissues. Among the genes in this pathway, SCD1 mRNA levels were found to be significantly higher in leiomyoma than in matched myometrium. SCD1 inhibition by small interfering RNA or CAY10566 decreased antiapoptotic BCL2 mRNA and protein levels and cell viability in primary leiomyoma but not myometrial cells. SCD1 protein levels were significantly higher in the mutant MED12 UtSM cell line than in the wild-type MED12 UtSM cell line. CAY10566 treatment specifically decreased cell viability and increased apoptosis in mutant MED12 UtSM cells, with increased protein levels of cleaved caspase 3, cleaved PARP, and DDIT3 in mutant MED12 UtSM but not in wild-type MED12 UtSM cells. SCD1, an enzyme involved in lipid homeostasis, may play an important role in promoting leiomyoma growth and represents a novel target for the treatment of leiomyoma.

Engineered uterine primary myometrial cells with high-mobility group AT-hook 2 overexpression display a leiomyoma-like transcriptional and epigenomic phenotype

To determine if engineered high-mobility group AT-hook 2 (HMGA2) overexpressing uterine primary myometrial cells recapitulate the transcriptional and epigenomic features of HMGA2-subtype leiomyomas. Isolated primary, "normal" myometrial cells from three patients were engineered to overexpress HMGA2 to determine how HMGA2 establishes transcriptomic and epigenomic features of HMGA2-overexpressing leiomyoma. Academic research laboratory. Primary myometrial cells were isolated from normal myometrium obtained from three patients undergoing hysterectomy. Not applicable. Determined genome-wide transcriptomic and epigenomic features of engineered HMGA2-overexpressing uterine primary myometrial cells. Engineered HMGA2-V5-overexpressing primary myometrial cells approximated the HMGA2 expression level observed in HMGA2-overexpression subtype leiomyoma. High-mobility group AT-hook 2-V5 expression resulted in differential expression of 1,612 genes (false discovery rate [FDR] < 0.05) that were found to be enriched in pathways associated with leiomyoma formation, including extracellular matrix organization. Comparative gene expression analysis between HMGA2-V5 engineered primary cells and HMGA2-overexpression subtype leiomyoma revealed significant overlap of differentially expressed genes. Mechanistically, HMGA2-V5 overexpression resulted in 41,323 regions with differential H3K27ac deposition (FDR < 0.05) and 205,605 regions of altered chromatin accessibility (FDR < 0.05). Transcription factor binding site analysis implicated the AP-1 family of transcription factors. High-mobility group AT-hook 2 overexpression induces leiomyoma-like transcriptomic and epigenomic modulations in myometrial cells.

HMGA2 overexpression induces plasticity in myometrial cells and a transcriptomic profile more similar to that of uterine fibroids

To study the possible role for HMGA2 overexpression in differentiated myometrial cells and its potential to induce a stem cell-like or dedifferentiating phenotype and drive fibroid development. Myometrial cells were immortalized and transduced with an HMGA2 lentivirus to produce HMGA2hi cells. In vitro stem cell assays were conducted, and ribonucleic acid from HMGA2hi and control cells as well as fibroid-free myometrial and HMGA2 fibroid (HMGA2F) tissues were submitted for ribonucleic acid sequencing. University research laboratory. Women who underwent hysterectomy for symptomatic uterine fibroids or other gynecological conditions. Not applicable. In vitro stem cell-like properties from myometrial cell lines. Ribonucleic acid sequencing and collagen production of HMGA2-overexpressing primary leiomyoma tissue and cell lines. HMGA2hi cells had enhanced self-renewal capacity, decreased proliferation, and a greater ability to differentiate into other mesenchymal cell types. HMGA2hi cells exhibited a stem cell-like signature and shared transcriptomic similarities with HMGA2F. Moreover, dysregulated extracellular matrix pathways were observed in both HMGA2hi cells and HMGA2F. Our findings show that HMGA2 overexpression may drive myometrial cells to dedifferentiate into a more plastic phenotype and provide evidence for an alternative mechanism for fibroid etiology, suggesting that fibroids arise not only from a mutated stem cell but also from a mutated differentiated myometrial cell.

Coenzyme Q-10 reduced the aberrant production of extracellular matrix proteins in uterine leiomyomas through transforming growth factor beta 3

To evaluate the impact of coenzyme Q-10 (CoQ-10) on the dysregulated synthesis of extracellular matrix proteins mediated by transforming growth factor beta 3 (TGF-β3) in uterine leiomyomas. Laboratory study. University. None. Treatment of immortalized uterine myometrial and leiomyoma cells to TGF-β3 and CoQ-10. The protein concentrations of collagen 1A1 (COL1A1), collagen 3A1 (COL3A1), collagen 11A1 (COL11A1), and fibronectin (FN1) were assessed through western blot analysis after treatment of immortalized uterine myometrial and leiomyoma cells with both transforming growth factor beta (TGF-β) 3 and concentrations of CoQ-10 at 10, 50, and 100 μM concurrently for 24 hours. Immortalized uterine leiomyoma and myometrial cells exposed to TGF-β3 for 24 hours demonstrated a significant up-regulation of COL1A1, COL3A1, COL11A1, and FN1 compared with untreated cells. In leiomyoma cells, concurrent treatment with CoQ-10 over the same timeframe revealed a dose-dependent decrease in these protein concentrations compared with those in cells treated with TGF-β3 alone. At the highest concentration of 100 μM of CoQ-10, significant decreases in the amounts of COL1A1 (0.59 ± 0.10-fold), COL3A1 (0.46 ± 0.09-fold), COL11A1 (0.53 ± 0.09-fold), and FN1 (0.56 ± 0.09-fold) were observed. Similarly, myometrial cells exposed to both TGF-β3 and CoQ-10 demonstrated a dose-responsive decline in the amount of extracellular matrix protein compared with cells exposed to TGF-β3 alone. Significant reductions in the amounts of COL1A1 (0.75 ± 0.03-fold), COL3A1 (0.48 ± 0.06-fold), COL11A1 (0.38 ± 0.06), and FN1 (0.69 ± 0.04-fold) were appreciated at 100-μM CoQ-10. Coenzyme Q-10 mitigated the aberrant production of key biomarkers of the extracellular matrix mediated by TGF-β3 in uterine leiomyomas. Our findings highlight a promising nonhormonal compound that can counteract the fibroproliferative process inherent to leiomyomas.

Simvastatin induces degradation of the extracellular matrix in human leiomyomata: novel in vitro, in vivo, and patient level evidence of matrix metalloproteinase involvement

To assess the effect of simvastatin on uterine leiomyoma growth and extracellular matrix (ECM) deposition. Laboratory analysis of human leiomyoma cell culture, xenograft in a mouse model, and patient tissue from a clinical trial. Academic research center. Tissue culture from human leiomyoma tissue and surgical leiomyoma tissue sections from a placebo-controlled randomized clinical trial. Simvastatin treatment. Serum concentrations, xenograft volumes, and protein expression. Mice xenografted with 3-dimensional human leiomyoma cultures were divided as follows: 7 untreated controls; 12 treated with activated simvastatin at 10 mg/kg body weight; and 15 at 20 mg/kg body weight. Simvastatin was detected in the serum of mice injected at the highest dose. Xenograft volumes were significantly smaller (mean 53% smaller at the highest concentration). There was dissolution of compact ECM, decreased ECM formation, and lower collagen protein expression in xenografts. Membrane type 1 matrix metalloproteinase was increased in vitro and in vivo. Matrix metalloproteinase 2 and low-density lipoprotein receptor-related protein 1 were increased in vitro. Simvastatin exhibited antitumoral activity with ECM degradation and decreased leiomyoma tumor volume in vivo. Activation of the matrix metalloproteinase 2, membrane type 1 matrix metalloproteinase, and low-density lipoprotein receptor-related protein 1 pathway may explain these findings.

Uterine fibroid cell cytoskeletal organization is affected by altered G protein-coupled estrogen receptor-1 and phosphatidylinositol 3-kinase signaling

To determine whether cyclic strain affects fibroid cell cytoskeletal organization, proliferation, and collagen synthesis differently than myometrial cells. A basic science study using primary cultures of patient-matched myometrial and fibroid cells. Academic laboratory. Premenopausal women undergoing myomectomy or hysterectomy for the treatment of symptomatic uterine fibroids. Application of uniaxial strain patterns mimicking periovulation, menses, or dysmenorrhea using the Flexcell tension system or static control. Secondarily, inhibition of G protein-coupled estrogen receptor-1 and phosphatidylinositol 3-kinase. Cell alignment, cell number, and collagen content. Menses-strained cells demonstrated the most variation in cell alignment, cell proliferation, and procollagen content between myometrial and fibroid cells. Procollagen content decreased in myometrial cells with increasing strain amplitude and decreasing frequency. G protein-coupled estrogen receptor-1 inhibition decreases cellular alignment in the presence of strain. Mechanotransduction affecting cytoskeletal arrangement through the G protein-coupled estrogen receptor-1-phosphatidylinositol 3-kinase pathway is altered in fibroid cells. These results highlight the importance of incorporating mechanical stimulation into the in vitro study of fibroid pathology.

Differences in menstrual cytokine profiles of women with and without symptomatic uterine fibroids

Uterine pathology and microbiome among patients with endometrial polyps and fibroids

To evaluate the uterine microbiome among women with endometrial polyps and submucosal fibroids and to compare results between endometrial sampling techniques. Patients with polyps or fibroids were prospectively recruited before hysteroscopy, whereas patients undergoing retrieval for planned oocyte cryopreservation were recruited prospectively as controls. Three specimen types obtained for each patient were the distal 5 mm of an embryo catheter passed to the uterine fundus (C), endometrial tissue from an endometrial biopsy (T), and formalin-fixed paraffin-embedded (FFPE) endometrial tissue from the same endometrial biopsy. 16S ribosomal RNA gene amplicon sequencing was performed to analyze the structure of the endometrial microbiome. Thirty-seven participants including 28 women with polyps and/or fibroids and 9 controls. None. Microbial taxonomic alpha and beta diversity; differential abundance of taxa. Across all sample types, participants with polyps had higher microbial alpha diversity than controls (4.3 vs. 5.1, q = 0.049), and microbial communities were significantly different (pairwise Permutational Multivariate Analysis of Variance (PERMANOVA) pseudo-F = 2.1, q = 0.003). These differences were observed when examining C specimens alone (5.4 vs. 6.4, q = 0.001; pairwise PERMANOVA pseudo-F = 2.5, q = 0.003), although they did not reach significance when examining either T or FFPE specimens alone. Participants with fibroids had similar alpha diversity yet significant differences in beta diversity compared with controls in analyses combining all specimens (pairwise PERMANOVA pseudo-F = 1.475, q = 0.030); however, these differences did not achieve significance when analyzing C, T, or FFPE specimens alone. When comparing C and T specimens vs. FFPE specimens overall, alpha diversity was significantly higher (q < 0.001 and q < 0.001, respectively) and there were significant differences in beta diversity (q < 0.003 and q < 0.003, respectively). Analyses of C specimens generated a larger number of significantly differentially abundant taxa compared with other sampling methods. Although not statistically significant, relative abundance of putative pathogens was higher in participants with polyps than controls regardless of sampling technique. Results of this exploratory study suggest that significant microbial differences exist among patients with endometrial polyps vs. healthy controls. However, results varied by sampling technique, highlighting a need to identify optimal sampling methods before validating findings in larger prospective cohort studies.

The immune landscape of uterine fibroids as determined by mass cytometry

To study the differences in immune cell profiles in uterine fibroids (Fibs) and matched myometrium (Myo). Observational study. Laboratory study. The study included tissue that was collected from 10 pairs of Fib and matched Myo from women, not on hormonal medications, undergoing hysterectomy and myomectomy. None. Differences in immune cell and cytokine composition between Fib and matched Myo. The mass cytometry analysis indicated that Fibs had a significantly higher number of natural killer (NK) cells, total macrophages, M2 macrophages, and conventional dendritic cells when compared with matched Myo from the same patient. In contrast, Fibs had significantly fewer CD3 and CD4 T cells when compared with Myo. The mass cytometry analysis results did not show any significant difference in the number of resting mast cells. Immunoflurorescent and immunohistochemical imaging confirmed the cytometry by time of flight results, showing a significantly higher number of NK cells, tryptase-positive mast cells indicative of mast cell activation, total macrophages, and M2 cells in Fibs and a significantly lower number of CD3 and CD4 T cells. The cytokine assay revealed significantly increased levels of human interferon α2, interleukin (IL)-1α, and platelet-derived growth factor AA and significantly lower levels of macrophage colony-stimulating factor and IL-1 receptor antagonist in Fib. Our results show significant differences in immune cell populations and cytokine levels between Fib and Myo. These differences could account for the increased inflammation in fib and a potential mechanism by which these tumors evade the immune system. These findings provide a foundation for further studies exploring the role of immune cells in Fib development.

Sphingosine 1-phosphate acts as proliferative and fibrotic cue in leiomyoma cells

To determine whether the bioactive sphingolipid sphingosine 1-phosphate (S1P) modulates cellular proliferation and synthesis of fibrotic proteins in leiomyoma differently than myometrial cells. A basic science study using human leiomyoma and myometrial cells. Not applicable. This is an in vitro study performed on cellular models. Leiomyoma and myometrial cells were treated with S1P, as well as with selective antagonists for S1P-specific G protein-coupled receptors and secondarily with inhibitors of extracellular signal-regulated kinase 1/2 (ERK1/2) and ezrin. The main outcome measures included cellular proliferation and fibrogenesis. Bromodeoxyuridine Cell Proliferation Assay was employed to measure deoxyribonucleic acid synthesis and proliferation, whereas western blot analysis was used to assess the expression of the fibrotic markers N-cadherin, α-smooth muscle actin, transgelin, and collagen type I alpha 1. Sphingosine 1-phosphate stimulates cellular proliferation of leiomyoma but not myometrial cells. The mitogenic effect elicited by S1P relies on the engagement of its specific receptor S1P These results, besides extending the knowledge on the molecular mechanism underlying uterine leiomyoma development and fibrosis, demonstrate the pathogenetic role of S1P in leiomyoma and support the rationale for targeting S1P signaling pathway as innovative potential treatment.

The combination of natural compounds Crila and epigallocatechin gallate showed enhanced antiproliferative effects on human uterine fibroid cells compared with single treatments

To investigate the combined effects of Crila and green tea extract, epigallocatechin gallate (EGCG), compared with single treatments, on human uterine fibroid cells. Human uterine leiomyoma (HuLM) cells were treated with different concentrations of Crila, alone or in combination with EGCG, and several experiments were employed. A laboratory study. N/A. Crila, EGCG. Cell proliferation assay, drug synergy using combination index, protein and gene expression analysis of proliferation marker proliferating cell nuclear antigen, and apoptosis marker BAX using western blotting and quantitative polymerase chain reaction, respectively. Results showed that tested Crila concentrations, when combined with 25 and 50 μM EGCG, exerted synergistic growth inhibitory effects on HuLM viability. This inhibitory effect on HuLM cell viability was because of decreased cell proliferation, as shown by a decrease in the proliferation marker proliferating cell nuclear antigen at messenger RNA and protein levels, rather than inducing apoptosis. Our study concludes that the utility of natural compounds may provide a safe and cost-effective alternative to currently used short-term hormonal therapies against uterine fibroids.

Angiotensin II drives proliferation and extracellular matrix deposition in human uterine fibroid cells in vitro

To investigate the effect of angiotensin II (Ang II) on proliferation and extracellular matrix (ECM) deposition in human uterine leiomyoma cells and normal myometrial cells. Experimental in vitro study using immortalized human leiomyoma (HuLM) cells, immortalized human uterine smooth muscle (UTSM) cells, and patient-derived primary fibroid and myometrial cells. Women with uterine fibroids who underwent hysterectomy. Administration of physiological and supraphysiological levels of Ang II (to mimic essential hypertension) to cultured HuLM, UTSM, primary fibroid, and myometrial cells to assess effects on cellular proliferation and ECM deposition. We evaluated HuLM, UTSM, primary fibroid, and UTSM cells for the presence of the Ang II type 1 receptor. Angiotensin II-induced proliferation and ECM deposition was assessed through MTS assay, Western blot analysis, immunofluorescence, and real-time polymerase chain reaction. Immortalized and primary leiomyoma and myometrial cells expressed Ang II type 1 receptor. Leiomyoma cells responded to Ang II with increased cellular proliferation measured by MTS assay, proliferating cell nuclear antigen protein levels, and Ki67 staining. The Ang II treatment of fibroid cells showed increased expression of Collagen 1A1, the predominant fibroid and myometrial collagen. Integrin β1, an upstream regulator of fibrosis, also showed an increase in protein and messenger ribonucleic acid expression in fibroid cells treated with Ang II. No difference in proliferation or ECM production was observed in myometrial cell controls. Angiotensin II promoted growth and matrix accumulation in fibroid cells, highlighting a potential link between fibroids and cardiovascular disease.