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1.
Development ; 147(17)2020 09 08.
Artículo en Inglés | MEDLINE | ID: mdl-32747437

RESUMEN

The placenta releases large quantities of extracellular vesicles (EVs) that likely facilitate communication between the embryo/fetus and the mother. We isolated EVs from second trimester human cytotrophoblasts (CTBs) by differential ultracentrifugation and characterized them using transmission electron microscopy, immunoblotting and mass spectrometry. The 100,000  g pellet was enriched for vesicles with a cup-like morphology typical of exosomes. They expressed markers specific to this vesicle type, CD9 and HRS, and the trophoblast proteins placental alkaline phosphatase and HLA-G. Global profiling by mass spectrometry showed that placental EVs were enriched for proteins that function in transport and viral processes. A cytokine array revealed that the CTB 100,000  g pellet contained a significant amount of tumor necrosis factor α (TNFα). CTB EVs increased decidual stromal cell (dESF) transcription and secretion of NF-κB targets, including IL8, as measured by qRT-PCR and cytokine array. A soluble form of the TNFα receptor inhibited the ability of CTB 100,000  g EVs to increase dESF secretion of IL8. Overall, the data suggest that CTB EVs enhance decidual cell release of inflammatory cytokines, which we theorize is an important component of successful pregnancy.


Asunto(s)
Decidua/inmunología , Vesículas Extracelulares/inmunología , Interleucina-8/inmunología , Trofoblastos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Femenino , Antígenos HLA-G/inmunología , Humanos , Células K562 , FN-kappa B/inmunología , Embarazo , Tetraspanina 29/inmunología
2.
PLoS Genet ; 16(6): e1008601, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32555663

RESUMEN

Programmed cellular responses to cycling ovarian-derived steroid hormones are central to normal endometrial function. Abnormalities therein, as in the estrogen-dependent, progesterone-"resistant" disorder, endometriosis, predispose to infertility and poor pregnancy outcomes. The endometrial stromal fibroblast (eSF) is a master regulator of pregnancy success. However, the complex hormone-epigenome-transcriptome interplay in eSF by each individual steroid hormone, estradiol (E2) and/or progesterone (P4), under physiologic and pathophysiologic conditions, is poorly understood and was investigated herein. Genome-wide analysis in normal, early and late stage eutopic eSF revealed: i) In contrast to P4, E2 extensively affected the eSF DNA methylome and transcriptome. Importantly, E2 resulted in a more open versus closed chromatin, confirmed by histone modification analysis. Combined E2 with P4 affected a totally different landscape than E2 or P4 alone. ii) P4 responses were aberrant in early and late stage endometriosis, and mapping differentially methylated CpG sites with progesterone receptor targets from the literature revealed different but not decreased P4-targets, leading to question the P4-"resistant" phenotype in endometriosis. Interestingly, an aberrant E2-response was noted in eSF from endometriosis women; iii) Steroid hormones affected specific genomic contexts and locations, significantly enriching enhancers and intergenic regions and minimally involving proximal promoters and CpG islands, regardless of hormone type and eSF disease state. iv) In eSF from women with endometriosis, aberrant hormone-induced methylation signatures were mainly due to existing DNA methylation marks prior to hormone treatments and involved known endometriosis genes and pathways. v) Distinct DNA methylation and transcriptomic signatures revealed early and late stage endometriosis comprise unique disease subtypes. Taken together, the data herein, for the first time, provide significant insight into the hormone-epigenome-transcriptome interplay of each steroid hormone in normal eSF, and aberrant E2 response, distinct disease subtypes, and pre-existing epigenetic aberrancies in the setting of endometriosis, provide mechanistic insights into how endometriosis affects endometrial function/dysfunction.


Asunto(s)
Metilación de ADN , Endometriosis/genética , Epigénesis Genética , Estradiol/metabolismo , Progesterona/metabolismo , Transcriptoma , Adulto , Cromatina/genética , Cromatina/metabolismo , Islas de CpG , Endometriosis/metabolismo , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Estradiol/farmacología , Femenino , Humanos , Progesterona/farmacología
3.
Reprod Biomed Online ; 44(3): 495-503, 2022 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-35031240

RESUMEN

RESEARCH QUESTION: Do women with laparoscopically confirmed endometriosis have higher plasma concentrations of circulating cell-free DNA (cirDNA) than those without endometriosis? DESIGN: Prospective study of women aged 18-45 years undergoing benign gynaecological laparoscopy at two tertiary hospitals. Venous blood was collected immediately before surgery, and women were allocated to the endometriosis or control groups based on surgical findings. Total plasma cirDNA and cirDNA integrity were measured by quantitative polymerase chain reaction (qPCR) targeting short (115 bases) and long (247 bases) ALU segments. Endometrial-derived cirDNA was measured by qPCR of bisulfite-treated cirDNA using primers selective for a FAM101A sequence uniquely unmethylated in endometrial tissue. Five cirDNA parameters were compared between the control and endometriosis cohorts: total cirDNA concentration, long-stranded cirDNA concentration, integrity ratio, endometrial cirDNA concentration and endometrial cirDNA proportion. RESULTS: Twenty-eight endometriosis and 15 control samples were included. Women with and without endometriosis had cirDNA concentrations of 2.24 ± 0.89 ng/ml and 2.56 ± 0.92 ng/ml, respectively. Analysis by phenotype of endometriosis revealed a significantly higher endometrial cirDNA concentration in women with superficial disease (n = 10) compared with deep endometriosis (n = 18) (mean difference 0.14 ng/ml; 95% CI 0.15 to 0.26; P = 0.025), but not with controls. CONCLUSIONS: No significant differences were found in any of the cirDNA parameters between women with and without endometriosis. The low statistical power and heterogenous pelvic pathology in the control group render it difficult to determine whether the negative results reflect a true lack of increase in cirDNA in endometriosis.


Asunto(s)
Ácidos Nucleicos Libres de Células , Endometriosis , Adolescente , Adulto , Endometriosis/genética , Endometrio , Femenino , Humanos , Persona de Mediana Edad , Estudios Prospectivos , Adulto Joven
4.
Hum Reprod ; 35(3): 617-640, 2020 03 27.
Artículo en Inglés | MEDLINE | ID: mdl-32219408

RESUMEN

STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)? SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization. WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis. STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization. PARTICIPANTS/MATERIALS, SETTING, METHODS: eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs. MAIN RESULTS AND THE ROLE OF CHANCE: SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization. LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro analyses. WIDER IMPLICATIONS OF THE FINDINGS: Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.


Asunto(s)
Decidua , Fibroblastos/citología , Interleucina-11/fisiología , Semen , Estudios Transversales , Decidua/fisiología , Endometriosis , Endometrio/citología , Femenino , Humanos , Interleucina-11/genética , Síndrome del Ovario Poliquístico
5.
Int J Mol Sci ; 21(7)2020 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-32283828

RESUMEN

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use.


Asunto(s)
Endometrio/efectos de los fármacos , Endometrio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Progesterona/farmacología , Progestinas/farmacología , Testosterona/farmacología , Supervivencia Celular/efectos de los fármacos , Estrógenos/metabolismo , Estrógenos/farmacología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Progesterona/química , Progestinas/química , Testosterona/química
6.
J Assist Reprod Genet ; 35(5): 757-770, 2018 May.
Artículo en Inglés | MEDLINE | ID: mdl-29404863

RESUMEN

PURPOSE: The study aims to test the hypothesis that platelet-rich plasma (PRP) stimulates cellular processes involved in endometrial regeneration relevant to clinical management of poor endometrial growth or intrauterine scarring. METHODS: Human endometrial stromal fibroblasts (eSF), endometrial mesenchymal stem cells (eMSC), bone marrow-derived mesenchymal stem cells (BM-MSC), and Ishikawa endometrial adenocarcinoma cells (IC) were cultured with/without 5% activated (a) PRP, non-activated (na) PRP, aPPP (platelet-poor-plasma), and naPPP. Treatment effects were evaluated with cell proliferation (WST-1), wound healing, and chemotaxis Transwell migration assays. Mesenchymal-to-epithelial transition (MET) was evaluated by cytokeratin and vimentin expression. Differential gene expression of various markers was analyzed by multiplex Q-PCR. RESULTS: Activated PRP enhanced migration of all cell types, compared to naPRP, aPPP, naPPP, and vehicle controls, in a time-dependent manner (p < 0.05). The WST-1 assay showed increased stromal and mesenchymal cell proliferation by aPRP vs. naPRP, aPPP, and naPPP (p < 0.05), while IC proliferation was enhanced by aPRP and aPPP (p < 0.05). There was no evidence of MET. Expressions of MMP1, MMP3, MMP7, and MMP26 were increased by aPRP (p < 0.05) in eMSC and eSF. Transcripts for inflammation markers/chemokines were upregulated by aPRP vs. aPPP (p < 0.05) in eMSC and eSF. No difference in estrogen or progesterone receptor mRNAs was observed. CONCLUSIONS: This is the first study evaluating the effect of PRP on different human endometrial cells involved in tissue regeneration. These data provide an initial ex vivo proof of principle for autologous PRP to promote endometrial regeneration in clinical situations with compromised endometrial growth and scarring.


Asunto(s)
Endometrio/citología , Plasma Rico en Plaquetas , Anciano , Movimiento Celular , Proliferación Celular , Células Cultivadas , Endometrio/fisiología , Transición Epitelial-Mesenquimal , Femenino , Fibroblastos/fisiología , Regulación de la Expresión Génica , Humanos , Masculino , Células Madre Mesenquimatosas/fisiología , Regeneración
7.
Biol Reprod ; 97(4): 564-576, 2017 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-29025102

RESUMEN

Uterine fibroids are a common finding in infertility patients. Impaired implantation and decidualization have been proposed to contribute to compromised fertility. Data are limited on the endometrial transcriptome from subjects with uterine fibroids, as well as endometrial receptivity and decidualization potential of endometrial stromal fibroblasts (eSF) from women with fibroids. Our objective was to investigate the endometrial transcriptome of women with noncavity-distorting intramural fibroids and compare them to control subjects with no uterine pathology throughout the menstrual cycle. We also evaluated endometrial receptivity gene expression and basic endometrial functions such as decidualization, proliferation, and apoptosis in women with fibroid uterus. Results showed that large numbers of transcripts were significantly dysregulated throughout the menstrual cycle in fibroid subjects compared to controls. However, there were essentially no differences in the expression of receptivity markers at the tissue level, as well as decidualization markers in tissue and eSF in subjects with fibroids compared to controls. However, eSF from women with a fibroid uterus exhibited decreased proliferation potential and increased apoptosis upon decidualization. These data indicate preserved implantation and decidualization potential despite observed gene expression changes in endometrium from women with noncavity-distorting fibroids compared to controls. How this phenomenon and altered proliferation/apoptosis may contribute to impairment of endometrial function in subfertile patients warrants further investigation.


Asunto(s)
Endometrio/metabolismo , Regulación de la Expresión Génica/fisiología , Leiomioma/metabolismo , Adulto , Apoptosis , Proliferación Celular , Femenino , Humanos , Persona de Mediana Edad , Análisis por Matrices de Proteínas
8.
Biol Reprod ; 95(5): 93, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27535958

RESUMEN

Endometriosis is an estrogen-dependent, progesterone-resistant disorder largely derived from retrograde transplantation of menstrual tissue/cells into the pelvis, eliciting an inflammatory response, pelvic pain, and infertility. Eutopic endometrium (within the uterus), giving rise to pelvic disease, displays cycle-dependent transcriptomic, proteomic, and signaling abnormalities, and although its DNA methylation profiles dynamically change across the cycle in healthy women, studies in endometriosis are limited. Herein, we investigated the DNA methylome and associated gene expression in three phases of the cycle in eutopic endometrium of women with severe endometriosis versus controls, matched for ethnicity, medications, smoking, and no recent contraceptive steroid use. Genome-wide DNA methylation and gene expression were coassessed in each sample. Cycle phase was determined by histology, serum hormone levels, and unsupervised principal component and hierarchical cluster analyses of microarray data. Altered endometrial DNA methylation in endometriosis was most prominent in the midsecretory phase (peak progesterone), with disruption of the normal pattern of cycle-dependent DNA methylation changes, including a bias toward methylation of CpG islands, suggesting wide-range abnormalities of the chromatin remodeling machinery in endometriosis. DNA methylation changes were associated with altered gene expression relevant to endometrial function/dysfunction, including cell proliferation, inflammation/immune response, angiogenesis, and steroid hormone response. The data provide insight into epigenetic reprogramming and steroid hormone actions in endometrium contributing to the pathogenesis and pathophysiology of endometriosis.


Asunto(s)
Metilación de ADN , Endometriosis/metabolismo , Endometrio/metabolismo , Expresión Génica , Ciclo Menstrual/metabolismo , Adulto , Islas de CpG , Endometriosis/genética , Epigénesis Genética , Femenino , Regulación de la Expresión Génica , Humanos , Ciclo Menstrual/genética , Proteómica
9.
Biol Reprod ; 94(5): 118, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27075616

RESUMEN

Human endometrium undergoes cyclic regeneration involving stem/progenitor cells, but the role of resident endometrial mesenchymal stem cells (eMSC) as progenitors of endometrial stromal fibroblasts (eSF) has not been definitively demonstrated. In endometriosis, eSF display progesterone (P4) resistance with impaired decidualization in vivo and in vitro. To investigate eMSC as precursors of eSF and whether endometriosis P4 resistance is inherited from eMSC, we analyzed transcriptomes of eutopic endometrium eMSC and eSF isolated by fluorescence-activated cell sorting (FACS) from endometriosis (eMSCendo, eSFendo) and controls (eMSCcontrol, eSFcontrol) and their derived primary cultures. Differentially expressed lineage-associated genes (LG) of FACS-isolated eMSC and eSF were largely conserved in endometriosis. In culture, eSFcontrol maintained in vitro expression of a subset of eSF LG and decidualized in vitro with P4 The eMSCcontrol cultures differentiated in vitro to eSF lineage, down-regulating eMSC LG and up-regulating eSF LG, showing minimal transcriptome differences versus eSFcontrol cultures and decidualizing in vitro. Cultured eSFendo displayed less in vitro LG stability and did not decidualize in vitro. In vitro, eMSCendo differentiated to eSF lineage but showed more differentially expressed genes versus eSFendo cultures, and did not decidualize in vitro, demonstrating P4 resistance inherited from eMSCendo Compared to controls, cultures from tissue-derived eSFendo uniquely had a pro-inflammatory phenotype not present in eMSCendo differentiated to eSF in vitro, suggesting divergent niche effects for in vivo versus in vitro lineage differentiation. These findings substantiate eMSC as progenitors of eSF and reveal eSF in endometriosis as having P4 resistance inherited from eMSC and a pro-inflammatory phenotype acquired within the endometrial niche.


Asunto(s)
Endometriosis/patología , Endometrio/anomalías , Endometrio/patología , Fibroblastos/fisiología , Inflamación/genética , Células Madre Mesenquimatosas/fisiología , Nicho de Células Madre/genética , Enfermedades Uterinas/genética , Estudios de Casos y Controles , Diferenciación Celular/genética , Proliferación Celular/genética , Células Cultivadas , Endometriosis/genética , Endometriosis/inmunología , Endometriosis/metabolismo , Endometrio/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Células Madre Mesenquimatosas/metabolismo , Fenotipo , Transcriptoma/fisiología
10.
iScience ; 27(4): 109388, 2024 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-38510116

RESUMEN

Existing medical treatments for endometriosis-related pain are often ineffective, underscoring the need for new therapeutic strategies. In this study, we applied a computational drug repurposing pipeline to stratified and unstratified disease signatures based on endometrial gene expression data to identify potential therapeutics from existing drugs, based on expression reversal. Of 3,131 unique genes differentially expressed by at least one of six endometriosis signatures, only 308 (9.8%) were in common; however, 221 out of 299 drugs identified, (73.9%) were shared. We selected fenoprofen, an uncommonly prescribed NSAID that was the top therapeutic candidate for further investigation. When testing fenoprofen in an established rat model of endometriosis, fenoprofen successfully alleviated endometriosis-associated vaginal hyperalgesia, a surrogate marker for endometriosis-related pain. These findings validate fenoprofen as a therapeutic that could be utilized more frequently for endometriosis and suggest the utility of the aforementioned computational drug repurposing approach for endometriosis.

11.
Commun Biol ; 6(1): 780, 2023 08 16.
Artículo en Inglés | MEDLINE | ID: mdl-37587191

RESUMEN

Endometriosis is a leading cause of pain and infertility affecting millions of women globally. Herein, we characterize variation in DNA methylation (DNAm) and its association with menstrual cycle phase, endometriosis, and genetic variants through analysis of genotype data and methylation in endometrial samples from 984 deeply-phenotyped participants. We estimate that 15.4% of the variation in endometriosis is captured by DNAm and identify significant differences in DNAm profiles associated with stage III/IV endometriosis, endometriosis sub-phenotypes and menstrual cycle phase, including opening of the window for embryo implantation. Menstrual cycle phase was a major source of DNAm variation suggesting cellular and hormonally-driven changes across the cycle can regulate genes and pathways responsible for endometrial physiology and function. DNAm quantitative trait locus (mQTL) analysis identified 118,185 independent cis-mQTLs including 51 associated with risk of endometriosis, highlighting candidate genes contributing to disease risk. Our work provides functional evidence for epigenetic targets contributing to endometriosis risk and pathogenesis. Data generated serve as a valuable resource for understanding tissue-specific effects of methylation on endometrial biology in health and disease.


Asunto(s)
Endometriosis , Femenino , Humanos , Endometriosis/genética , Metilación de ADN , Dolor , Implantación del Embrión
12.
Nat Genet ; 55(3): 423-436, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36914876

RESUMEN

Endometriosis is a common condition associated with debilitating pelvic pain and infertility. A genome-wide association study meta-analysis, including 60,674 cases and 701,926 controls of European and East Asian descent, identified 42 genome-wide significant loci comprising 49 distinct association signals. Effect sizes were largest for stage 3/4 disease, driven by ovarian endometriosis. Identified signals explained up to 5.01% of disease variance and regulated expression or methylation of genes in endometrium and blood, many of which were associated with pain perception/maintenance (SRP14/BMF, GDAP1, MLLT10, BSN and NGF). We observed significant genetic correlations between endometriosis and 11 pain conditions, including migraine, back and multisite chronic pain (MCP), as well as inflammatory conditions, including asthma and osteoarthritis. Multitrait genetic analyses identified substantial sharing of variants associated with endometriosis and MCP/migraine. Targeted investigations of genetically regulated mechanisms shared between endometriosis and other pain conditions are needed to aid the development of new treatments and facilitate early symptomatic intervention.


Asunto(s)
Endometriosis , Femenino , Humanos , Endometriosis/genética , Endometriosis/metabolismo , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Dolor , Comorbilidad
13.
Hum Fertil (Camb) ; 25(4): 662-669, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-33464141

RESUMEN

In patients with high serum E2 embryo transfer is often postponed, as high E2 levels adversely affect embryo transfer outcome. We aimed to determine if stratified serum oestradiol (E2) and progesterone (P4) levels differentially affect endometrial histology and endometrial oestrogen and progesterone receptor protein levels. Endometrial biopsies were collected from oocyte donors. Samples were divided based on peak serum E2 levels into three groups: (i) low-E2 (n = 33) E2≤2999pg/mL; (ii) mid-E2 (n = 40) E2 3000-4999 pg/mL; and (iii) high-E2 (n = 15) E2≥5000 pg/mL. Oestrogen receptor alpha (ERα) and progesterone receptors A and B (PR) protein levels in endometrial stroma (S), glandular (GE) and luminal (LE) epithelia were assessed by immunohistochemistry. Samples in high-E2 group demonstrated strongest association with accelerated endometrial maturation (2 (1-2); 2 (1-3); and 3 (2.8-3) median days of advancement of endometrial maturation respectively in low, mid, high-E2 groups, p = 0.046). There were significant differences in ERα and PR immunoexpression in S, GE and LE among the groups (p < 0.05). Higher E2 levels were associated with decreased ERα expression (p < 0.017) in GE and LE, and increased PR expression in S and GE (p < 0.011 and p < 0.0001, respectively). Higher serum E2 levels were associated with impaired endometrial steroid hormone receptor expression, higher serum P4 and more advancement of endometrial maturation.


Asunto(s)
Estradiol , Receptor alfa de Estrógeno , Femenino , Humanos , Receptor alfa de Estrógeno/metabolismo , Endometrio/metabolismo , Receptores de Progesterona/metabolismo , Progesterona , Oocitos/metabolismo
14.
Science ; 376(6594): eabl4896, 2022 05 13.
Artículo en Inglés | MEDLINE | ID: mdl-35549404

RESUMEN

Molecular characterization of cell types using single-cell transcriptome sequencing is revolutionizing cell biology and enabling new insights into the physiology of human organs. We created a human reference atlas comprising nearly 500,000 cells from 24 different tissues and organs, many from the same donor. This atlas enabled molecular characterization of more than 400 cell types, their distribution across tissues, and tissue-specific variation in gene expression. Using multiple tissues from a single donor enabled identification of the clonal distribution of T cells between tissues, identification of the tissue-specific mutation rate in B cells, and analysis of the cell cycle state and proliferative potential of shared cell types across tissues. Cell type-specific RNA splicing was discovered and analyzed across tissues within an individual.


Asunto(s)
Atlas como Asunto , Células , Especificidad de Órganos , Empalme del ARN , Análisis de la Célula Individual , Transcriptoma , Linfocitos B/metabolismo , Células/metabolismo , Humanos , Especificidad de Órganos/genética , Linfocitos T/metabolismo
15.
Front Immunol ; 12: 788315, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35069565

RESUMEN

The uterine lining (endometrium) exhibits a pro-inflammatory phenotype in women with endometriosis, resulting in pain, infertility, and poor pregnancy outcomes. The full complement of cell types contributing to this phenotype has yet to be identified, as most studies have focused on bulk tissue or select cell populations. Herein, through integrating whole-tissue deconvolution and single-cell RNAseq, we comprehensively characterized immune and nonimmune cell types in the endometrium of women with or without disease and their dynamic changes across the menstrual cycle. We designed metrics to evaluate specificity of deconvolution signatures that resulted in single-cell identification of 13 novel signatures for immune cell subtypes in healthy endometrium. Guided by statistical metrics, we identified contributions of endometrial epithelial, endothelial, plasmacytoid dendritic cells, classical dendritic cells, monocytes, macrophages, and granulocytes to the endometrial pro-inflammatory phenotype, underscoring roles for nonimmune as well as immune cells to the dysfunctionality of this tissue.


Asunto(s)
Endometriosis , Endometrio , RNA-Seq , Análisis de la Célula Individual , Endometriosis/genética , Endometriosis/inmunología , Endometriosis/patología , Endometrio/inmunología , Endometrio/patología , Femenino , Humanos
16.
Sci Rep ; 10(1): 6888, 2020 04 23.
Artículo en Inglés | MEDLINE | ID: mdl-32327684

RESUMEN

The contraceptive effectiveness of intrauterine devices (IUDs) has been attributed in part to a foreign body reaction in the endometrium. We performed this study to better understand mechanisms of action of contraceptives of by studying their effects on endometrial and cervical transcriptomes. We collected endometrial and cervical biopsies from women using the levonorgestrel-releasing intrauterine system (LNG-IUS, n = 11), copper intrauterine device (cu-IUD, n = 13) or levonorgestrel-containing combined oral contraceptives (COC, n = 12), and from women not using contraceptives (control group, n = 11). Transcriptional profiling was performed with Affymetrix arrays, Principal Component Analysis and the bioconductor package limma. In endometrial samples from cu-IUD users, there were no genes with statistically significant differential expression compared to controls. In LNG-IUS users, 2509 genes were differentially expressed and mapped predominantly onto immune and inflammatory pathways. The cervical samples showed no statistically significant differential gene expression compared to controls. Hormonal and copper IUDs have significantly different effects on the endometrial transcriptome, with the LNG-IUS transcriptome showing pronounced inflammation and immune activation compared to controls whereas the cu-IUD transcriptome was indistinguishable from luteal phase endometrium. These findings argue against a foreign body reaction as a common mechanism of action of IUDs.


Asunto(s)
Endometrio/metabolismo , Dispositivos Intrauterinos de Cobre , Dispositivos Intrauterinos Medicados , Transcriptoma/genética , Adulto , Biopsia , Anticonceptivos Femeninos/farmacología , Endometrio/patología , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Levonorgestrel/farmacología , Análisis de Componente Principal , Transducción de Señal/efectos de los fármacos , Transcriptoma/efectos de los fármacos
17.
Hum Reprod Update ; 25(5): 564-591, 2019 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-31424502

RESUMEN

BACKGROUND: Endometriosis, a common oestrogen-dependent inflammatory disorder in women of reproductive age, is characterized by endometrial-like tissue outside its normal location in the uterus, which causes pelvic scarring, pain and infertility. While its pathogenesis is poorly understood, the immune system (systemically and locally in endometrium, pelvic endometriotic lesions and peritoneal fluid) is believed to play a central role in its aetiology, pathophysiology and associated morbidities of pain, infertility and poor pregnancy outcomes. However, immune cell populations within the endometrium of women with the disease have had incomplete phenotyping, thereby limiting insight into their roles in this disorder. OBJECTIVE AND RATIONALE: The objective herein was to determine reproducible and consistent findings regarding specific immune cell populations and their abundance, steroid hormone responsiveness, functionality, activation states, and markers, locally and systemically in women with and without endometriosis. SEARCH METHODS: A comprehensive English language PubMed, Medline and Google Scholar search was conducted with key search terms that included endometriosis, inflammation, human eutopic/ectopic endometrium, immune cells, immune population, immune system, macrophages, dendritic cells (DC), natural killer cells, mast cells, eosinophils, neutrophils, B cells and T cells. OUTCOMES: In women with endometriosis compared to those without endometriosis, some endometrial immune cells display similar cycle-phase variation, whereas macrophages (Mø), immature DC and regulatory T cells behave differently. A pro-inflammatory Mø1 phenotype versus anti-inflammatory Mø2 phenotype predominates and natural killer cells display abnormal activity in endometrium of women with the disease. Conflicting data largely derive from small studies, variably defined hormonal milieu and different experimental approaches and technologies. WIDER IMPLICATIONS: Phenotyping immune cell subtypes is essential to determine the role of the endometrial immune niche in pregnancy and endometrial homeostasis normally and in women with poor reproductive history and can facilitate development of innovative diagnostics and therapeutics for associated symptoms and compromised reproductive outcomes.


Asunto(s)
Endometriosis/patología , Endometrio/inmunología , Endometrio/fisiología , Células Dendríticas/inmunología , Estrógenos/metabolismo , Femenino , Humanos , Inflamación/inmunología , Inflamación/patología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Embarazo , Linfocitos T Reguladores/inmunología
18.
Fertil Steril ; 112(6): 1118-1128, 2019 12.
Artículo en Inglés | MEDLINE | ID: mdl-31843088

RESUMEN

OBJECTIVE: To phenotype transcriptomically M1 macrophages (Mϕ1) and M2 macrophages (Mϕ2) in the endometrium of women with endometriosis. DESIGN: Prospective experimental study. SETTING: University research laboratory. PATIENT(S): Six women with endometriosis and five controls without disease, in the secretory phase of the menstrual cycle. INTERVENTION(S): Mϕ1, Mϕ2, uterine natural killer, and T regulatory cells were isolated from human endometrium using a uniquely designed cell-specific fluorescence activating cell sorting panel. Transcriptome profiles were assessed by RNA high sequencing, bioinformatics, and biological pathway analyses. MAIN OUTCOMES MEASURE(S): Differential gene expression between Mϕ1 and Mϕ2 in women with and without endometriosis and in Mϕ1 versus Mϕ2 in each group was determined and involved different biologic and signaling pathways. RESULT(S): Flow cytometry analysis showed no significant differences in total numbers of leukocytes between control and endometriosis groups, although Mϕ1 were higher in the endometriosis group versus controls. Statistical transcriptomic analysis was performed only in Mϕ1 and Mϕ2 populations due to larger sample sizes. Bioinformatic analyses revealed that in women with endometriosis, endometrial Mϕ1 are more proinflammatory than controls and that Mϕ2 paradoxically have a proinflammatory phenotype. CONCLUSION(S): As Mϕ are phenotypically plastic and their polarization state depends on their microenvironment, the altered endometrial environment in women with endometriosis may promote endometrial Mϕ2 polarization and an Mϕ1 proinflammatory phenotype. Moreover, aberrant phenotypes of Mϕ may contribute to abnormal gene expression of the eutopic endometrium and a proinflammatory environment in women with endometriosis relevant to the pathophysiology of the disease and compromised reproductive outcomes.


Asunto(s)
Plasticidad de la Célula , Endometriosis/inmunología , Endometrio/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Transcriptoma , Adulto , Estudios de Casos y Controles , Plasticidad de la Célula/genética , Separación Celular/métodos , Microambiente Celular , Endometriosis/genética , Endometriosis/microbiología , Endometriosis/patología , Endometrio/metabolismo , Endometrio/microbiología , Endometrio/patología , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica , Humanos , Activación de Macrófagos/genética , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/patología , Persona de Mediana Edad , Fenotipo , Estudios Prospectivos , RNA-Seq , Transducción de Señal , Adulto Joven
20.
Mol Endocrinol ; 28(7): 1118-35, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24877562

RESUMEN

Human endometrium undergoes major gene expression changes, resulting in altered cellular functions in response to cyclic variations in circulating estradiol and progesterone, largely mediated by transcription factors and nuclear receptors. In addition to classic modulators, epigenetic mechanisms regulate gene expression during development in response to environmental factors and in some diseases and have roles in steroid hormone action. Herein, we tested the hypothesis that DNA methylation plays a role in gene expression regulation in human endometrium in different hormonal milieux. High throughput, genome-wide DNA methylation profiling of endometrial samples in proliferative, early secretory, and midsecretory phases revealed dynamic DNA methylation patterns with segregation of proliferative from secretory phase samples by unsupervised cluster analysis of differentially methylated genes. Changes involved different frequencies of gain and loss of methylation within or outside CpG islands. Comparison of changes in transcriptomes and corresponding DNA methylomes from the same samples revealed association of DNA methylation and gene expression in a number of loci, some important in endometrial biology. Human endometrial stromal fibroblasts treated in vitro with estradiol and progesterone exhibited DNA methylation changes in several genes observed in proliferative and secretory phase tissues, respectively. Taken together, the data support the observation that epigenetic mechanisms are involved in gene expression regulation in human endometrium in different hormonal milieux, adding endometrium to a small number of normal adult tissues exhibiting dynamic DNA methylation. The data also raise the possibility that the interplay between steroid hormone and methylome dynamics regulates normal endometrial functions and, if abnormal, may result in endometrial dysfunction and associated disorders.


Asunto(s)
Metilación de ADN , ADN/metabolismo , Endometrio/metabolismo , Epigénesis Genética/genética , Regulación de la Expresión Génica/genética , Adulto , Proliferación Celular , Análisis por Conglomerados , Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Islas de CpG/genética , Receptor con Dominio Discoidina 1 , Endometrio/citología , Estradiol/sangre , Estradiol/farmacología , Femenino , Fibroblastos/citología , Perfilación de la Expresión Génica , Humanos , Metaloproteinasas de la Matriz Secretadas/genética , Proteínas de la Membrana/genética , Ciclo Menstrual/genética , Persona de Mediana Edad , Mucina-1/genética , Progesterona/sangre , Progesterona/farmacología , Proteínas Tirosina Quinasas Receptoras/genética , Células del Estroma/citología , Transcriptoma/genética
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