Myometrial-derived CXCL12 promotes lipopolysaccharide induced preterm labour by regulating macrophage migration, polarization and function in mice.

Preterm birth is a major contributor to neonatal mortality and morbidity. Infection results in elevation of inflammation-related cytokines followed by infiltration of immune cells into gestational tissue. CXCL12 levels are elevated in preterm birth indicating it may have a role in preterm labour (PTL); however, the pathophysiological correlations between CXCL12/CXCR4 signalling and premature labour are poorly understood. In this study, PTL was induced using lipopolysaccharide (LPS) in a murine model. LPS induced CXCL12 RNA and protein levels significantly and specifically in myometrium compared with controls (3-fold and 3.5-fold respectively). Highest levels were found just before the start of labour. LPS also enhanced the infiltration of neutrophils, macrophages and T cells, and induced macrophage M1 polarization. In vitro studies showed that condition medium from LPS-treated primary smooth muscle cells (SMC) induced macrophage migration, M1 polarization and upregulated inflammation-related cytokines such as interleukin (IL)-1, IL-6 and tumor necrosis factor alpha (TNF-α). AMD3100 treatment in pregnant mice led to a significant decrease in the rate of PTL (70%), prolonged pregnancy duration and suppressed macrophage infiltration into gestation tissue by 2.5-fold. Further, in-vitro treatment of SMC by AMD3100 suppressed the macrophage migration, decreased polarization and downregulated IL-1, IL-6 and TNF-α expression. LPS treatment in pregnant mice induced PTL by increasing myometrial CXCL12, which recruits immune cells that in turn produce inflammation-related cytokines. These effects stimulated by LPS were completely reversed by AMD3100 through blocking of CXCL12/CXCR4 signalling. Thus, the CXCL12/CXCR4 axis presents an excellent target for preventing infection and inflammation-related PTL.

Adult bone marrow progenitors become decidual cells and contribute to embryo implantation and pregnancy.

Decidua is a transient uterine tissue shared by mammals with hemochorial placenta and is essential for pregnancy. The decidua is infiltrated by many immune cells promoting pregnancy. Adult bone marrow (BM)-derived cells (BMDCs) differentiate into rare populations of nonhematopoietic endometrial cells in the uterus. However, whether adult BMDCs become nonhematopoietic decidual cells and contribute functionally to pregnancy is unknown. Here, we show that pregnancy mobilizes mesenchymal stem cells (MSCs) to the circulation and that pregnancy induces considerable adult BMDCs recruitment to decidua, where some differentiate into nonhematopoietic prolactin-expressing decidual cells. To explore the functional importance of nonhematopoietic BMDCs to pregnancy, we used Homeobox a11 (Hoxa11)-deficient mice, having endometrial stromal-specific defects precluding decidualization and successful pregnancy. Hoxa11 expression in BM is restricted to nonhematopoietic cells. BM transplant (BMT) from wild-type (WT) to Hoxa11-/- mice results in stromal expansion, gland formation, and marked decidualization otherwise absent in Hoxa11-/- mice. Moreover, in Hoxa11+/- mice, which have increased pregnancy losses, BMT from WT donors leads to normalized uterine expression of numerous decidualization-related genes and rescue of pregnancy loss. Collectively, these findings reveal that adult BMDCs have a previously unrecognized nonhematopoietic physiologic contribution to decidual stroma, thereby playing important roles in decidualization and pregnancy.

Endometriosis promotes atherosclerosis in a murine model.

BACKGROUND: Epidemiologic studies have demonstrated an association between endometriosis and the subsequent development of cardiovascular disease. The direct effect of endometriosis on the progression of atherosclerotic, if any, has not been previously characterized. Endometriosis leads to systemic inflammation that could have consequences for cardiovascular health. Here, we reported the effects of endometriosis on the development of atherosclerosis in a murine model. OBJECTIVE: This study aimed to determine the contribution of endometriosis in promoting cardiovascular disease in a murine model of endometriosis. STUDY DESIGN: Endometriosis was induced in 18 apolipoprotein E-null mice, the standard murine model used to study atherosclerosis. Mice of the same strain were used as controls (n=18) and underwent sham surgery without inducing endometriosis. The formation of endometriotic lesions was confirmed after 25 weeks of induction. Atherosclerotic lesions were subjected to hematoxylin and eosin staining followed by measurement of the aortic root luminal area and wall thickness. The whole aorta was isolated, and Oil Red O staining was performed to quantify the lipid deposits or plaque formation; moreover, biochemical assays were carried out in serum to determine the levels of lipids and inflammatory-related cytokines. RESULTS: Apolipoprotein E mice with endometriosis exhibited increased aortic atherosclerosis compared with controls as measured using Oil Red O staining (7.9% vs 3.1%, respectively; P=.0004). Mice with endometriosis showed a significant 50% decrease in the aortic luminal area compared with sham mice (0.85 mm2 vs 1.46 mm2; P=.03) and a significant increase in aortic root wall thickness (0.22 mm vs 0.15 mm; P=.04). There was no difference in the lipoprotein profile (P<.05) between mice with endometriosis and sham mice. The serum levels of inflammatory cytokines interleukin 1 alpha, interleukin 6, interferon gamma, and vascular endothelial growth factor were significantly (P<.05)increased in the endometriosis mice. CONCLUSION: Our study used a murine model to determine the effect of endometriosis on atherosclerosis. Inflammation-related cytokines interleukin 1 alpha, interleukin 6, interferon gamma, and vascular endothelial growth factor (angiogenic factor) released by endometriotic lesions may contribute to the increased cardiovascular risks in women with endometriosis. To reduce the risk of cardiovascular disease, early identification and treatment of endometriosis are essential. Future treatments targeting inflammatory cytokines may help reduce the long-term risk of cardiovascular disease in women with endometriosis.

Tofacitinib alters STAT3 signaling and leads to endometriosis lesion regression.

Endometriosis is a widespread gynecologic condition affecting up to 15% of women of reproductive age. The Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathway is upregulated in endometriosis and is a therapeutic target. Here we sought to determine the effect of Tofacitinib, a JAK inhibitor in widespread clinical use, on JAK/STAT signaling in endometriosis and lesion growth. Endometriosis was surgically induced in C57BL/6 mice using homologous uterine horn transplantation. Lesions were allowed to form over 4 weeks followed by Tofacitinib (10 mg/kg) or vehicle administered by oral gavage over 4 weeks. Tofacitinib treatment in vivo led to endometriosis lesion regression and reduced adhesion burden compared to vehicle treatment. In vitro studies on Ishikawa cells showed that Tofacitinib reduced hypoxia-inducible factor 1α and vascular endothelial growth factor mRNA levels at 12 and 24 h. Western blot analysis showed that Tofacitinib effectively reduced STAT3 phosphorylation in Ishikawa cells and human primary stromal and epithelial cells from eutopic endometrium of patients with and without endometriosis. This study suggests that the inhibition of JAK/STAT signaling using Tofacitinib may be a viable method for the treatment of endometriosis.

Endometriosis stromal cells induce bone marrow mesenchymal stem cell differentiation and PD-1 expression through paracrine signaling.

Endometriosis is an estrogen-dependent, inflammatory gynecological disorder characterized by the growth of endometrial cells in lesions outside the uterus. Bone marrow-derived cells (BMDCs) engraft lesions and increase lesion size. Do endometriosis cells regulate differentiation of engrafted BMDCs in the pathogenesis and growth of endometriosis? Here, we report endometriosis derived stromal cells promote the differentiation of BMDCs to stromal, epithelial and leukocyte cell fates through paracrine signaling. In-vitro studies demonstrated that both mRNA and protein levels of vimentin, cytokeratin and PD-1 were significantly increased in BMDCs cocultured with stromal cells from endometriosis (ENDO) patients compared to stromal cells from normal endometrium (CNTL). Increased expression of PD-1 has been reported in malignancy where it promotes T cell quiescence and immune tolerance. Increased PD-1 was also confirmed in-vivo where we showed that PD-1 expression was induced in BMDCs engrafted into endometriotic lesions in a murine model of endometriosis. AMD3100, an antagonist for CXCR4 receptor inhibited PD-1 expression in BMDCs suggesting that PD-1 induction requires CXCL12. These results suggest that endometriosis stimulated BMDC differentiation through paracrine signaling and increased T cell PD-1 expression. Increased PD-1 expression may be one mechanism by which endometriosis avoids immune surveillance.

CXCR4 or CXCR7 antagonists treat endometriosis by reducing bone marrow cell trafficking.

Adult stem cells have a major role in endometrial physiology, including remodelling and repair. However, they also have a critical role in the development and progression of endometriosis. Bone marrow-derived stem cells engraft eutopic endometrium and endometriotic lesions, differentiating to both stromal and epithelial cell fates. Using a mouse bone marrow transplantation model, we show that bone marrow-derived cells engrafting endometriosis express CXCR4 and CXCR7. Targeting either receptor by the administration of small molecule receptor antagonists AMD3100 or CCX771, respectively, reduced BM-derived stem cell recruitment into endometriosis implants. Endometriosis lesion size was decreased compared to vehicle controls after treatment with each antagonist in both an early growth and established lesion treatment model. Endometriosis lesion size was not effected when the local effects of CXCL12 were abrogated using uterine-specific CXCL12 null mice, suggesting an effect primarily on bone marrow cell migration rather than a direct endometrial effect. Antagonist treatment also decreased hallmarks of endometriosis physiopathology such as pro-inflammatory cytokine production and vascularization. CXCR4 and CXCR7 antagonists are potential novel, non-hormonal therapies for endometriosis.

Accurate diagnosis of endometriosis using serum microRNAs.

BACKGROUND: Endometriosis, a chronic disease that afflicts millions of women worldwide, has traditionally been diagnosed by laparoscopic surgery. This diagnostic barrier delays identification and treatment by years, resulting in prolonged pain and disease progression. Development of a noninvasive diagnostic test could significantly improve timely disease detection. We tested the feasibility of serum microRNAs as diagnostic biomarkers of endometriosis in women with gynecologic disease symptoms. OBJECTIVE: The objective of the study was to validate the use of a microRNA panel as a noninvasive diagnostic method for detecting endometriosis. STUDY DESIGN: This was a prospective study evaluating subjects with a clinical indication for gynecological surgery in an academic medical center. Serum samples were collected prior to surgery from 100 subjects. Women were selected based on the presence of symptoms, and laparoscopy was performed to determine the presence or absence of endometriosis. The control group was categorized based on absence of visual disease at the time of surgery. Circulating miRNAs, miR-125b-5p, miR-150-5p, miR-342-3p, miR-451a, miR-3613-5p, and let-7b, were measured in serum by quantitative real-time polymerase chain reaction in a blinded fashion without knowledge of disease status. Receiver-operating characteristic analysis was performed on individual microRNAs as well as combinations of microRNAs. An algorithm combining the expression values of these microRNAs, built using machine learning with a random forest classifier, was generated to predict the presence or absence of endometriosis on operative findings. This algorithm was then tested in an independent data set of 48 previously identified subjects not included in the training set (24 endometriosis and 24 controls) to validate its diagnostic performance. RESULTS: The mean age of women in the study population was 34.1 and 36.9 years for the endometriosis and control groups, respectively. Control group subjects displayed varying pathologies, with leiomyoma occurring the most often (n = 39). Subjects with endometriosis had significantly higher expression levels of 4 serum microRNAs: miR-125b-5p, miR-150-5p, miR-342-3p, and miR-451a. Two serum microRNAs showed significantly lower levels in the endometriosis group: miR-3613-5p and let-7b. Individual microRNAs had receiver-operating characteristic areas under the curve ranging from 0.68 to 0.92. A classifier combining these microRNAs yielded an area under the curve of 0.94 when validated in the independent set of subjects not included in the training set. Analysis of the expression levels of each microRNA based on revised American Society of Reproductive Medicine staging revealed that all microRNAs could distinguish stage I/II from control and stage III/IV from control but that the difference between stage I/II and stage III/IV was not significant. Subgroup analysis revealed that neither phase of the menstrual cycle or use of hormonal medication had a significant impact on the expression levels in the microRNAs used in our algorithm. CONCLUSION: This is the first report showing that microRNA biomarkers can reliably differentiate between endometriosis and other gynecological pathologies with an area under the curve >0.9 across 2 independent studies. We validated the performance of an algorithm based on previously identified microRNA biomarkers, demonstrating their potential to detect endometriosis in a clinical setting, allowing earlier identification and treatment. The ability to diagnose endometriosis noninvasively could reduce the time to diagnosis, surgical risk, years of discomfort, disease progression, associated comorbidities, and health care costs.

Increased circulating miR-370-3p regulates steroidogenic factor 1 in endometriosis.

Endometriosis is a gynecologic disease common among reproductive-aged women caused by the growth of endometrial tissue outside the uterus. Altered expression of numerous genes and microRNAs has been reported in endometriosis. Steroidogenic factor 1 (SF-1), an essential transcriptional regulator of multiple genes involved in estrogen biosynthesis, is aberrantly increased and plays an important role in the pathogenesis of endometriosis. Here, we show the expression of SF-1 in endometriosis is regulated by miR-370-3p. Sera and tissue were collected from 20 women surgically diagnosed with endometriosis and 26 women without endometriosis. We found that miR-370-3p levels were decreased in the serum of patients with endometriosis while SF-1 mRNA levels were inversely upregulated in endometriotic lesions compared with respective controls. Transfection of primary endometriotic cells with miR-370-3p mimic or inhibitor resulted in the altered expression of SF-1 and SF-1 downstream target genes steroidogenic acute regulatory protein (StAR) and CYP19A1. Overexpression of miR-370-3p inhibited cell proliferation and induced apoptosis in endometriotic cells. This study reveals that miR-370-3p functions as a negative regulator of SF-1 and cell proliferation in endometriotic cells. We suggest a novel therapeutic strategy for controlling SF-1 in endometriosis.

Bone-marrow-derived endothelial progenitor cells contribute to vasculogenesis of pregnant mouse uterus†.

Angiogenesis is essential for cyclic endometrial growth, implantation, and pregnancy maintenance. Vasculogenesis, the formation of new blood vessels by bone marrow (BM)-derived endothelial progenitor cells (EPCs), has been shown to contribute to endometrial vasculature. However, it is unknown whether vasculogenesis occurs in neovascularization of the decidua during pregnancy. To investigate the contribution of BM-derived EPCs to vascularization of the pregnant uterus, we induced non-gonadotoxic submyeloablation by 5-fluorouracil administration to wild-type FVB/N female mice recipients followed by BM transplantation from transgenic mice expressing green fluorescent protein (GFP) under regulation of Tie2 endothelial-specific promoter. Following 1 month, Tie2-GFP BM-transplanted mice were bred and sacrificed at various gestational days (ED6.5, ED10.5, ED13.5, ED18.5, and postpartum). Bone-marrow-transplanted non-pregnant and saline-injected pregnant mice served as controls (n = 5-6/group). Implantation sites were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence. While no GFP-positive EPCs were found in non-pregnant or early pregnant uteri of BM-transplanted mice, GFP-positive EPCs were first detected in pregnant uterus on ED10.5 (0.12%) and increased as the pregnancy progressed (1.14% on ED13.5), peaking on ED18.5 (1.42%) followed by decrease in the postpartum (0.9%). The percentage of endothelial cells that were BM-derived out of the total endothelial cell population in the implantation sites (GFP+CD31+/CD31+) were 9.3%, 15.8%, and 6.1% on ED13.5, ED18.5, and postpartum, respectively. Immunohistochemistry demonstrated that EPCs incorporated into decidual vasculature, and immunofluorescence showed that GFP-positive EPCs colocalized with CD31 in vascular endothelium of uterine implantation sites, confirming their endothelial lineage. Our findings indicate that BM-derived EPCs contribute to vasculogenesis of the pregnant mouse decidua.

Bone marrow-derived cells or C-X-C motif chemokine 12 (CXCL12) treatment improve thin endometrium in a mouse model.

Successful implantation and pregnancy is dependent on sufficient endometrial growth during each reproductive cycle. Here, we report the therapeutic effect of either bone marrow-derived cells (BMDCs) or the stem cell chemo-attractant C-X-C motif chemokine 12 (CXCL12) on endometrial receptivity in a murine ethanol induced thin endometrium model. Endometrial epithelial area was significantly increased in mice treated with BMDCs, CXCL12, or by co-treatment with both compared with PBS-treated controls. Ki-67 and CD31 immunoreactivity was significantly higher in mice treated with either BMDCs, CXCL12, or both. The mRNA expression levels of endometrial receptivity markers leukemia inhibitory factor, interleukin-1ß, and integrin beta-3 were increased in mice treated with either BMDCs, CXCL12, or both. The mRNA levels of matrix metalloproteinase-2 and -9 were significantly decreased by BMDCs but not by CXCL12. Pregnancy rates and litter size were increased after either treatment. Both BMDCs and CXCL12 displayed a comparable efficacy on endometrial regeneration in mice with thin endometrium. Our findings indicate the potential therapeutic effects of BMDCs and CXCL12 on infertility related to thin endometrium. Bone marrow-derived cells and CXCL12 displayed a comparable efficacy on endometrial regeneration in mice with thin endometrium.

Hematogenous Dissemination of Mesenchymal Stem Cells from Endometriosis.

Endometriosis is ectopic growth of endometrial tissue traditionally thought to arise through retrograde menstruation. We aimed to determine if cells derived from endometriosis could enter vascular circulation and lead to hematogenous dissemination. Experimental endometriosis was established by transplanting endometrial tissue from DsRed+ mice into the peritoneal cavity of DsRed- mice. Using flow cytometry, we identified DsRed+ cells in blood of animals with endometriosis. The circulating donor cells expressed CXCR4 and mesenchymal stem cell (MSC) biomarkers, but not hematopoietic stem cell markers. Nearly all the circulating endometrial stem cells originated from endometriosis rather than from the uterus. Cells expressing DsRed, CXCR4, and MSCs markers were identified in the peritoneal wall and surrounding vessels of recipient mice, contributing to both endometriosis and angiogenesis. Cells originating in endometriosis lesions migrated and implanted in lung tissue and displayed makers of differentiation, indicating retained multipotency. In vitro these cells demonstrated multipotency and were able to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Endometriosis lesions also expressed high levels of CXCL12, the CXCR4 receptor ligand. Serum CXCL12 levels were greater than in sham control mice. In humans with endometriosis, serum CXCL12 levels were significantly higher than controls, suggesting that the CXCL12/CXCR4 axis is operational in women with spontaneous endometriosis as well. Stem cells, rather than differentiated cells from endometriosis, enter the circulation in response to CXCL12. We identify an endometriosis-derived stem cell population, a potential mechanism of dissemination of this disease and a potential target for treatment of endometriosis. Stem Cells 2018;36:881-890.

Adipocyte alterations in endometriosis: reduced numbers of stem cells and microRNA induced alterations in adipocyte metabolic gene expression.

BACKGROUND: Endometriosis is an estrogen dependent, inflammatory disorder occurring in 5-10% of reproductive-aged women. Women with endometriosis have a lower body mass index (BMI) and decreased body fat compared to those without the disease, yet few studies have focused on the metabolic abnormalities in adipose tissue in women with endometriosis. Previously, we identified microRNAs that are differentially expressed in endometriosis and altered in the serum of women with the disease. Here we explore the effect of endometriosis on fat tissue and identified a role for endometriosis-related microRNAs in fat metabolism and a reduction in adipocyte stem cell number. METHODS: Primary adipocyte cells cultured from 20 patients with and without endometriosis were transfected with mimics and inhibitors of microRNAs 342-3p or Let 7b-5p to model the status of these microRNAs in endometriosis. RNA was extracted for gene expression analysis by qRT-PCR. PCNA expression was used as a marker of adipocyte proliferation. Endometriosis was induced experimentally in 9-week old female C57BL/6 mice and after 10 months fat tissue was harvested from both the subcutaneous (inguinal) and visceral (mesenteric) tissue. Adipose-derived mesenchymal stem cells in fat tissue were characterized in both endometriosis and non-endometriosis mice by FACS analysis. RESULTS: Gene expression analysis showed that endometriosis altered the expression of Cebpa, Cebpb, Ppar-γ, leptin, adiponectin, IL-6, and HSL, which are involved in driving brown adipocyte differentiation, appetite, insulin sensitivity and fat metabolism. Each gene was regulated by an alteration in microRNA expression known to occur in endometriosis. Analysis of the stem cell content of adipose tissue in a mouse model of endometriosis demonstrated a reduced number of adipocyte stem cells. CONCLUSIONS: We demonstrate that microRNAs Let-7b and miR-342-3p affected metabolic gene expression significantly in adipocytes of women with endometriosis. Similarly, there is a reduction in the adipose stem cell population in a mouse model of endometriosis. Taken together these data suggest that endometriosis alters BMI in part through an effect on adipocytes and fat metabolism.

Systemic administration of bone marrow-derived cells leads to better uterine engraftment than use of uterine-derived cells or local injection.

Stem cells are recruited to the uterus where they differentiate into endometrial cells and have been suggested as potential therapy for uterine injury such as Asherman's syndrome. However, it is unknown whether local intrauterine injection may result in better stem cell engraftment of the uterus compared with systemic administration, and whether uterine-derived cells (UDCs) may confer an advantage over BM-derived cells (BMDCs). Mice underwent local injury to a single uterine horn. Green fluorescent protein (GFP)-expressing BMDCs, UDCs or saline (control) were injected either intravenously or locally (uterine lumen) into wild-type recipients. Two or 3 weeks post-transplant, uterine tissues were collected for fluorescence-activated cell sorting (FACS) and immunohistochemistry/immunofluorescence studies. Mice injected intravenously with BMDCs or UDCs had increased GFP+ cells recruitment to the non-injured or injured uterus compared to those injected locally. No significant differences were noted in GFP+ cell recruitment to the injured versus non-injured horn. In addition, systemic injection of BMDCs led to greater recruitment of GFP+ cells at 2 weeks and 3 weeks compared with UDCs. Immunohistochemical staining demonstrated that GFP+ cells were found in stroma but not in epithelium or blood vessels. Immunofluorescence analysis revealed that GFP+ cells were mostly CD45-negative, and negative for CD31 and cytokeratin, confirming their stromal identity. In conclusion, the systemic route of administration results in better recruitment of BMDCs or UDCs to the injured uterus than local injection. In addition, BMDCs recruitment to the uterus is greater than UDCs. These findings inform the development of stem cell-based therapies targeting the uterus.

microRNA Let-7b: A Novel treatment for endometriosis.

Endometriosis is an oestrogen-dependent, chronic inflammatory disease that affects 10% of reproductive-aged women. Current treatment options depend on female sex steroid hormone modulation; however, all have side effects and are not useful in women who want to conceive. microRNAs treatments have provided promising results for some chronic diseases and cancers. We have previously shown the microRNA Let-7b is repressed in endometriosis and that loss of Let-7 contributes to the pathophysiology of the disease. Here, we propose using microRNA Let-7b for the treatment of endometriosis in a murine model. Endometriosis was treated using microRNA Let-7b or a scrambled control microRNA. Let-7b treatment resulted in reduced endometriosis lesion size. Decreased gene expression was noted in several genes known to promote endometriosis growth including ER-α, ER-ß, Cyp19a, KRAS 4A, KRAS 4B and IL-6. These results indicate that microRNA Let-7b has a pleiotropic role in endometriosis pathophysiology affecting oestrogen signalling, inflammation and growth factor receptors. Local treatment of endometriosis with Let-7b is a promising therapy for endometriosis that simultaneously affects multiple pathways driving endometriosis without systemic hormonal side effects.

Endometriosis alters brain electrophysiology, gene expression and increases pain sensitization, anxiety, and depression in female mice.

Endometriosis is an estrogen-dependent inflammatory disorder among reproductive-aged women associated with pelvic pain, anxiety, and depression. Pain is characterized by central sensitization; however, it is not clear if endometriosis leads to increased pain perception or if women with the disease are more sensitive to pain, increasing the detection of endometriosis. Endometriosis was induced in mice and changes in behavior including pain perception, brain electrophysiology, and gene expression were characterized. Behavioral tests revealed that mice with endometriosis were more depressed, anxious and sensitive to pain compared to sham controls. Microarray analyses confirmed by qPCR identified differential gene expression in several regions of brain in mice with endometriosis. In these mice, genes such as Gpr88, Glra3 in insula, Chrnb4, Npas4 in the hippocampus, and Lcn2 in the amygdala were upregulated while Lct, Serpina3n (insula), and Nptx2 (amygdala) were downregulated. These genes are involved in anxiety, locomotion, and pain. Patch clamp recordings in the amygdala were altered in endometriosis mice demonstrating an effect of endometriosis on brain electrophysiology. Endometriosis induced pain sensitization, anxiety, and depression by modulating brain gene expression and electrophysiology; the effect of endometriosis on the brain may underlie pain sensitization and mood disorders reported in women with the disease.

Cross-sex testosterone therapy in ovariectomized mice: addition of low-dose estrogen preserves bone architecture.

Cross-sex hormone therapy (XHT) is widely used by transgender people to alter secondary sex characteristics to match their desired gender presentation. Here, we investigate the long-term effects of XHT on bone health using a murine model. Female mice underwent ovariectomy at either 6 or 10 wk and began weekly testosterone or vehicle injections. Dual-energy X-ray absorptiometry (DXA) was performed (20 wk) to measure bone mineral density (BMD), and microcomputed tomography was performed to compare femoral cortical and trabecular bone architecture. The 6-wk testosterone group had comparable BMD with controls by DXA but reduced bone volume fraction, trabecular number, and cortical area fraction and increased trabecular separation by microcomputed tomography. Ten-week ovariectomy/XHT maintained microarchitecture, suggesting that estrogen is critical for bone acquisition during adolescence and that late, but not early, estrogen loss can be sufficiently replaced by testosterone alone. Given these findings, we then compared effects of testosterone with effects of weekly estrogen or combined testosterone/low-dose estrogen treatment after a 6-wk ovariectomy. Estrogen treatment increased spine BMD and microarchitecture, including bone volume fraction, trabecular number, trabecular thickness, and connectivity density, and decreased trabecular separation. Combined testosterone-estrogen therapy caused similar increases in femur and spine BMD and improved architecture (increased bone volume fraction, trabecular number, trabecular thickness, and connectivity density) to estrogen therapy and were superior compared with mice treated with testosterone only. These results demonstrate estradiol is critical for bone acquisition and suggest a new cross-sex hormone therapy adding estrogens to testosterone treatments with potential future clinical implications for treating transgender youth or men with estrogen deficiency.

Low Body Mass Index in Endometriosis Is Promoted by Hepatic Metabolic Gene Dysregulation in Mice.

The gynecological disease endometriosis is characterized by the deposition and proliferation of endometrial cells outside the uterus and clinically is linked to low body mass index (BMI). Gene expression in the liver of these women has not been reported. We hypothesized that endometriosis may impact hepatic gene expression, promoting a low BMI. To determine the effect of endometriosis on liver gene expression, we induced endometriosis in female mice by suturing donor mouse endometrium into the peritoneal cavity and measuring the weight of these mice. Dual-energy X-ray absorptiometry (DEXA) scanning of these mice showed lower body weight and lower total body fat than controls. Microarray analysis identified 26 genes differentially regulated in the livers of mice with endometriosis. Six of 26 genes were involved in metabolism. Four of six genes were upregulated and were related to weight loss, whereas two genes were downregulated and linked to obesity. Expression levels of Cyp2r1, Fabp4, Mrc1, and Rock2 were increased, whereas Igfbp1 and Mmd2 expression levels were decreased. Lep and Pparg, key metabolic genes in the pathways of the six genes identified from the microarray, were also upregulated. This dysregulation was specific to metabolic pathways. Here we demonstrate that endometriosis causes reduced body weight and body fat and disrupts expression of liver genes. We suggest that altered metabolism mediated by the liver contributes to the clinically observed low BMI that is characteristic of women with endometriosis. These findings reveal the systemic and multiorgan nature of endometriosis.

Overexpression of ERBB4 JM-a CYT-1 and CYT-2 isoforms in transgenic mice reveals isoform-specific roles in mammary gland development and carcinogenesis.

INTRODUCTION: Human Epidermal Growth Factor Receptor (ERBB4/HER4) belongs to the Epidermal Growth Factor receptor/ERBB family of receptor tyrosine kinases. While ERBB1, ERBB2 and ERBB3 are often overexpressed or activated in breast cancer, and are oncogenic, the role of ERBB4 in breast cancer is uncertain. Some studies suggest a tumor suppressor role of ERBB4, while other reports suggest an oncogenic potential. Alternative splicing of ERBB4 yields four major protein products, these spliced isoforms differ in the extracellular juxtamembrane domain (JM-a versus JM-b) and cytoplasmic domain (CYT-1 versus CYT-2). Two of these isoforms, JM-a CYT-1 and JM-a CYT-2, are expressed in the mammary gland. Failure to account for isoform-specific functions in previous studies may account for conflicting reports on the role of ERBB4 in breast cancer. METHODS: We have produced mouse mammary tumour virus (MMTV) -ERBB4 transgenic mice to evaluate potential developmental and carcinogenic changes associated with full length (FL) JM-a ERBB4 CYT-1 versus ERBB4 CYT-2. Mammary tissue was isolated from transgenic mice and sibling controls at various developmental stages for whole mount analysis, RNA extraction, and immunohistochemistry. To maintain maximal ERBB4 expression, transgenic mice were bred continuously for a year after which mammary glands were isolated and analyzed. RESULTS: Overexpressing FL CYT-1 isoform resulted in suppression of mammary ductal morphogenesis which was accompanied by decreased number of mammary terminal end buds (TEBs) and Ki-67 positive cells within TEBs, while FL CYT-2 isoform had no effect on ductal growth in pubescent mice. The suppressive ductal phenotype in CYT-1 mice disappeared after mid-pregnancy, and subsequent developmental stages showed no abnormality in mammary gland morphology or function in CYT-1 or CYT-2 transgenic mice. However, sustained expression of FL CYT-1 isoform resulted in formation of neoplastic mammary lesions, suggesting a potential oncogenic function for this isoform. CONCLUSIONS: Together, we present isoform-specific roles of ERBB4 during puberty and early pregnancy, and reveal a novel oncogenic property of CYT-1 ERBB4. The results may be exploited to develop better therapeutic strategies in breast cancer.

Bone marrow mesenchymal stem cell-derived exosomes shuttle microRNAs to endometrial stromal fibroblasts that promote tissue proliferation /regeneration/ and inhibit differentiation.

BACKGROUND: Human bone marrow-derived stem cells (hBMDSCs) are well characterized mediators of tissue repair and regeneration. An increasing body of evidence indicates that these cells exert their therapeutic effects largely through their paracrine actions rather than clonal expansion and differentiation. Here we studied the role of microRNAs (miRNAs) present in extracellular vesicles (EVs) from hBMDSCs in tissue regeneration and cell differentiation targeting endometrial stromal fibroblasts (eSF). METHODS: Extracellular vesicles (EVs) are isolated from hBMDSCs, characterized by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) techniques. Extracted total RNA from EVs was subjected to RNA seq analysis. Transfection and decidualization studies were carried out in endometrial stromal fibroblasts (eSF). Gene expression was analyzed by qRTPCR. Unpaired t-test with Welch's correction was used for data analysis between two groups. RESULTS: We identified several microRNAs (miRNAs) that were highly expressed, including miR-21-5p, miR-100-5p, miR-143-3p and let7. MiR-21 is associated with several signaling pathways involved in tissue regeneration, quiescence, cellular senescence, and fibrosis. Both miR-100-5p and miR-143-3p promoted cell proliferation. MiR-100-5p specifically promoted regenerative processes by upregulating TGF-ß3, VEGFA, MMP7, and HGF. MiR-100-5p blocked differentiation or decidualization as evidenced by morphologic changes and downregulation of decidualization mediators including HOXA10, IGFBP1, PRL, PR-B, and PR. CONCLUSION: EVs delivered to tissues by hBMDSCs contain specific miRNAs that prevent terminal differentiation and drive repair and regeneration. Delivery of microRNAs is a novel treatment paradigm with the potential to replace BMDSCs in cell-free regenerative therapies.