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Abstract Objective Abnormalities in the eutopic endometrium of women with endometriosis may be related to disease-associated infertility. Although previous RNA-sequencing analysis did not show differential expression in endometrial transcripts of endometriosis patients, other molecular alterations could impact protein synthesis and endometrial receptivity. Our aim was to screen for functional mutations in the transcripts of eutopic endometria of infertile women with endometriosis and controls during the implantation window. Methods Data from RNA-Sequencing of endometrial biopsies collected during the implantation window from 17 patients (6 infertile women with endometriosis, 6 infertile controls, 5 fertile controls) were analyzed for variant discovery and identification of functional mutations. A targeted study of the alterations found was performed to understand the data into disease's context. Results None of the variants identified was common to other samples within the same group, and no mutation was repeated among patients with endometriosis, infertile and fertile controls. In the endometriosis group, nine predicted deleterious mutations were identified, but only one was previously associated to a clinical condition with no endometrial impact. When crossing the mutated genes with the descriptors endometriosis and/or endometrium, the gene CMKLR1 was associated either with inflammatory response in endometriosis or with endometrial processes for pregnancy establishment. Conclusion Despite no pattern of mutation having been found, we ponder the small sample size and the analysis on RNA-sequencing data. Considering the purpose of the study of screening and the importance of the CMKLR1 gene on endometrial
Resumo Objetivo Anormalidades no endométrio eutópico de mulheres com endometriose podem estar relacionadas à infertilidade associada à doença. Embora a análise prévia de sequenciamento de RNA não tenha evidenciado expressão diferencial em transcritos endometriais de pacientes com endometriose, outras alterações moleculares poderiam afetar a síntese de proteínas e a receptividade endometrial. Nosso objetivo foi rastrear mutações funcionais em transcritos de endométrios eutópicos de mulheres inférteis com endometriose e de controles durante a janela de implantação. Métodos Os dados do sequenciamento de RNA de biópsias endometriais coletados durante a janela de implantação de 17 pacientes (6 mulheres inférteis com endometriose, 6 controles inférteis, 5 controles férteis) foram analisados para a descoberta de variantes e a identificação de mutações funcionais. Um estudo direcionado das alterações encontradas foi realizado para compreender os dados no contexto da doença. Resultados Nenhuma das variantes identificadas foi comuma outras amostras dentro do mesmo grupo, assim como nenhuma mutação se repetiu entre pacientes com endometriose, controles inférteis e férteis. No grupo de endometriose, foram identificadas nove mutações deletérias preditas, mas apenas uma foi previamente associada a uma condição clínica sem impacto endometrial. Ao cruzar os genes mutados com os descritores endometriose e/ou endométrio, o gene CMKLR1 foi associado a resposta inflamatória na endometriose e a processos endometriais para estabelecimento da gravidez. Conclusão Apesar de nenhum padrão de mutação ter sido encontrado, ponderamos o pequeno tamanho da amostra e a análise dos dados de sequenciamento de RNA. Considerando o objetivo do estudo de triagem e a importância do gene CMKLR1 na modulação endometrial, este poderia ser um gene candidato para estudos adicionais que avaliem mutações no endométrio eutópico de pacientes com endometriose.
Subject(s)
Humans , Female , Pregnancy , Embryo Implantation , Sequence Analysis, RNA , Endometriosis/complications , Endometriosis/genetics , Endometrium/metabolism , Infertility, Female/etiology , Mutation , Computer Simulation , Case-Control Studies , Prospective Studies , Receptors, Chemokine/genetics , Infertility, Female/metabolismABSTRACT
RESUMEN Objetivos Analizar las características clínicas, bioquímicas, estudios complementarios, hallazgos moleculares y la prevalencia de glándula eutópica en neonatos con HC pertenecientes al Programa Provincial de Pesquisa Neonatal de Córdoba, Argentina, entre 1996 y 2015. Analizar la evolución de los pacientes que reunieron criterios para una reevaluación. Pacientes y métodos Se analizaron retrospectivamente las historias clínicas de 237 pacientes detectados por pesquisa neonatal en la provincia de Córdoba, Argentina, entre 1996-2015 con una incidencia promedio de 1/2146 pesquisados. Presentaron glándula eutópica 81 pacientes (34%) F35/M46; se excluyeron 10 con síndromes genéticos asociados. Se analizaron los niveles de: TSH, T4T, T4L, T3, TPOAb / TGAb y Tiroglobulina (ECLIA -ROCHE) (VR: >15 días: 6-83 ng/ ml; <15 días: 29-173 ng/ml), ecografía y centellografía de cuello con Tc-99m. El valor de corte de TSH sérica adoptado para la confirmación diagnóstica fue de ≥10 mUI/ml. Se realizaron estudios de biología molecular en casos seleccionados. Se reevaluaron niños mayores de 3 años, sin bocio, con valores normales de Tiroglobulina y sin requerimiento de incrementos en la dosis de LT4. Resultados: La prevalencia de HC y Tiroides Eutópica se mantuvo constante. El 50% de los pacientes (36/71) mostraron hiperplasia glandular tiroidea. El 84% (n: 60 de 71) presentó niveles de TSH sérica ≥20 uUI/ml (20-1186) y el 75% (n: 53 de 71) >40 uUI/ml (40-1186). TGAb and TPOAb fueron positivos en un niño. La determinación de TG fue normal en el 29% (21/71) de los casos, elevada en el 56% (39/71) y baja en el 14% (10/71). Los estudios de biología molecular resultaron diagnósticos en 26 pacientes de 18 familias, demostrándose mutaciones en los genes de: TPO: 9 pacientes, TG: 12 pacientes, NIS: 2 pacientes, DUOX2: 2 pacientes y TRβ: 1 paciente. Se encontraron 11 nuevas mutaciones: tres en TPO, cinco en TG, dos en NIS y una en DUOX2. Se informaron anomalías congénitas en el 11% (8/71) de los pacientes. Se reevaluó el 11% (8/71) de los niños, resultando: HC transitorio n: 5, permanente n: 2 y una niña con Síndrome de Resistencia a las Hormonas Tiroideas. La prevalencia de lactantes con HC y glándula eutópica se mantuvo constante a lo largo de 19 años del Programa. Conclusiones Nuestros estudios demuestran que la prevalencia de Hipotiroidismo Congénito con glándula eutópica se mantuvo estable en los períodos analizados. Este grupo de pacientes se caracterizó predominantemente por presentar HC de carácter permanente acompañado por fenotipos de moderada a severa intensidad. En el futuro deberá profundizarse el conocimiento respecto a la influencia de factores medioambientales, como posibles agentes de riesgo asociados a la génesis de Hipotiroidismo Congénito.
abstract Objectives To describe clinical, biochemical characteristics and complementary studies to diagnosis, molecular findings and the prevalence of eutopic gland in newborn with CH detected through our neonatal screening program in Córdoba, Argentina, between 1996 and 2015. To analyze the evolution of the patients who met criteria for re-evaluation. Patients and methods We retrospectively analysed medical records of 237 patients with CH detected by neonatal screening in Córdoba, Argentina, from 1996 to 2015 with an average incidence of 1/2146 researched. 81 patients (34%) F35/M46 had eutopic thyroid gland; 10 patients with associated genetic syndromes were excluded. TT4, FT4, T3, TSH, TPOAb, TGAb and Thyroglobulin (VR: >15 days: 6-83 ng/ml; <15 days: 29-173 ng/ml) (ECLIA ROCHE), thyroid ultrasonography and 99Tc scan were assessed. The serum TSH cutoff value adopted for diagnostic confirmation was ≥10 mIU/ml. Molecular biology studies were performed in selected cases. Those who had no goiter, with normal thyroglobulin, and had not required increases in L-T4 dose underwent re-evaluation after the age of 3 years. Results The prevalence of HC and thyroid Eutopic remained constant. 50% of the patients (36/71) showed glandular hyperplasia. In 84% (60/71) presented serum TSH levels ≥20 uUI/ml (20-1186) and in 75% (n: 53 of 71) levels >40 uUI/ml (40-1186). TGAb and TPOAb were positive only in one baby. TG levels were: normal in 29% (21/71) of the cases, elevated in 56% (39/71) and low in 14% (10/71). Gene mutations were found in 26 patients from 18 families: TPO: 9 patients, TG: 12 patients, NIS: 2 patients, DUOX2: 2 patients y TRβ: 1 patient. Eleven new mutations were found: three in TPO, five in TG, two in NIS and one in DUOX2. Congenital anomalies were reported in 11% (8/71) patients. The 11% (8/71) of children were re-evaluated resulting in: 5 Transient CH, 2 Permanent CH and 1 with Resistance to Thyroid Hormones. The prevalence of infants with CH and eutopic gland remained constant along 19 years of the Program. Conclusions Our studies show that the prevalence of congenital hypothyroidism with eutopic gland remained stable in the periods analyzed. This group of patients was predominantly characterized by permanent CH accompanied by moderate to severe phenotypes. In the future, knowledge about the influence of environmental factors, as possible risk agents associated with the genesis of Congenital Hypothyroidism, should be deepened.
Subject(s)
Humans , Male , Female , Infant, Newborn , Thyroid Gland/physiopathology , Congenital Hypothyroidism/etiology , Congenital Hypothyroidism/physiopathology , Thyroid Hormones/genetics , Congenital Abnormalities/diagnosis , Neonatal Screening/methods , Hyperplasia/geneticsABSTRACT
Endometriosis is an estrogen-regulated chronic inflammatory disease, characterized by the presence and growth of endometrium-like tissue in extrauterine locations. Its prevalence is 6 to 10% of women in the general population, and 35 to 40% of women with pain and/or infertility. Endometriosis is manifested in different forms, of which peritoneal endometriosis, rectovaginal endometriosis and ovarian endometriosis are most common. Several investigations have been conducted to investigate the genetic basis of endometriosis. However, these studies have been unsuccessful in identifying robust genetic variants associated with endometriosis. On the contrary, the advent of whole genome cDNA microarray approach has allowed for the identification of genes that display modulation in their expression in an endometriotic tissue. Several biological pathways involved in the pathogenesis of endometriosis have been identified. This review article compiles the inferences drawn from high throughput investigations of endometriotic tissue.
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BACKGROUND: Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. RESULTS: Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERa) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. CONCLUSION: These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERa and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.
Subject(s)
Humans , Female , Adult , Aromatase/metabolism , Gene Expression/genetics , Endometriosis/metabolism , Endometrium/metabolism , Estradiol/metabolism , Biopsy , Immunoblotting , Statistics, Nonparametric , Endometriosis/physiopathology , Endometriosis/pathology , Endometrium/cytology , Epithelial Cells/metabolism , Primary Cell Culture , Menstrual Cycle/metabolismABSTRACT
Objective To evaluate the changes in eutopic endometrial vascularity of endometriosis using three-dimensional power Doppler ultrasound.Methods Forty-four women with endometriosis and 51 normal controls were studied by three-dimensional power Doppler ultrasound.Eutopic endometrial volume,vascularization index (VI),flow index (FI) and vascularization flow index (VFI) were calculated using VOCAL software.Results In proliferative phase,the mean VI was (5.499 ± 5.153)％ in patients with endometriosis and ( 1.135 ± 1.333) ％ in normal controls ( P ＜0.001 ),the mean FI was (28.832 ± 4.279)dB in patients with endometriosis and (25.544 ± 3.465)dB in normal controls ( P =0.006),the VFI was (1.769 ± 1.981 )dB in patients with endometriosis and (0.321 ± 0.397)dB in normal controls( P =0.002).In secretory phase,the mean VI was (8.693 ± 5.940) ％ in patients with endometriosis and ( 1.014±0.968)％ in normal controls ( P ＜0.001); the mean FI was (30.689 ± 3.632)dB in patients with endometriosis and (24.748 ± 3.811 )dB in normal controls( P ＜0.001 ),the VFI was (2.753 ± 2.044)dB in patients with endometriosis and (0.27 ± 0.293)dB in normal controls( P =0.001 ).Both in proliferative and secretory phase,no significant difference in endometrial volume between the two groups was observed ( P =0.108 and 0.068,respectively).Conclusions Three-dimensional power ultrasound provides a useful tool to investigate eutopic endometrial vascularization of endimetriosis.
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Objective:Nerve fibres distribution in the functional layer of endometrium of women with endometdosis was investigated.Methods:Histological sections of endometrial tissue were prepared from endometrialcurettings and hysterectomies performed on women with endometnosis(n=25)and without endometriosis(n=40).Immunohistochemistry was used to detect nerve fibres by highly specific polyclonal rabbit antibody PGP 9.5.The assessment of nerve fibre density was performed bv Image Pro Plus Discovery.Results:Nerve fibres were identified throughout the functional layers of the endometrium in all endometriosis patients,but not found in the functional layer of the endometrium in women without endometriosis(P<0.01).Conclusions:Nerve fibres detectad in the functional layer in all women with endometriosis may have important implications for understanding the generation of pain in these patients.
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Objective To investigate the expression of neurotrophic tyresine kinase receptor TrkB in the eutopic and ectopic endometium of women with endometriosis and evaluate the role of TrkB in the pathogenesis of endometriosis. Methods Seventeen women with endometriosis and 9 controls were studied. Expression of TrkB was investigated using RT-PCR, immunohistochemistry and Western blot, respectively. Results TrkB mRNA was expressed in eutopic endometrium in secretory phase and ectopic endometium of women with endometriosis, (23.51±0.51)% vs (35.29±0.67) % (P<0.05), but TrkB mRNA expression was not detected in control endometrium and eutopic endometrium in proliferative stage of women with endometriosis by RT-PCR. TrkB was expressed both on membrane (glycosylated receptor) and in cytoplasm (non-glycosylated receptor) of the glandular epithelial cells of ectopic endometium, and more often observed in cytoplasm of glandular epithelial cells of eutopic endometrium in secretory phase of women with endometriosis by IHC. In eutopic endometrium in secretory phase and ectopic endometium of women with endometriosis, the expression of TrkB protein was (0.12±0.02)% vs (0.37±0.01)% (P<0.05) by Western blot. Conclusions There is an overexpression of TrkB in eutopic endometrium in secretory phase and ectopic endometium of women with endometriosis. Full-length (glycosylated receptor) TrkB more often is overexpressed in ectopic endometium of women with endometriosis, and TrkB may act as a pathogenic role in formation of endometriosis.
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Objective To evaluate endometrial biopsy and curettage in detecting small nerve fibers in eutopic endometrium for diagnosis of endometriosis.Methods Endometrial biopsies and curettings were taken in 65 women with menorrhalgia.Endometrial nerve fibers were immunohistochemically detected using the pan-neuronal marker PGP 9.5.All patients underwent laparoseopie approach.Results The specificity,sensitivity,positive and negative predictive value were 100% of endometrial biopsy and curettings for diagnosis of endometriosis.Condusions Careful endometrial biopsy combined with immunohistochemical staining for nerve fibers may be a reliable means of diagnosing or excluding endometriosis.
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OBJECTIVE: To examine expression of survivin and Bcl-2 in eutopic endometrium with endometriosis and without endometriosis by immunohistochemical stain. METHODS: Expression of survivin and Bcl-2 was immunohistochemically investigated in eutopic endometrium with endometriosis (n=30, study group I; laparoscopy-proved endometriosis, study group II; adenomyosis) or without endometriosis (n=34, control group). The score of immunohistochemical staining was evaluated semi-quantitative method modified by Sinicrope and Lu. Statistical analyses was carried out using one-way ANOVA with Turkey test. RESULTS: Survivin expression was significantly higher in nucleus of glandular epithelium of eutopic endometrium with endomtriosis compared to that without endometriosis (p<0.05). Increased expression of Bcl-2 was found in glandular epithelium of eutopic endometrium with endometriosis than without endometriosis, but there was no significantly difference. CONCLUSION: Our findings suggest that increased survivin expression may contribute to survival of endometriosis. Moreover, survivin could play an important role in pathogenesis of endometriosis.
Subject(s)
Female , Endometriosis , Endometrium , Epithelium , TurkeyABSTRACT
Objective DNA methylation in the pathogenesis of endometriosis is drawing more and more attention from the medical world.This study aimed to evaluate the methylation status of the Homeobox-A10 (HOXA10) gene promoter region and the expression of the HOXA10 protein in endometriosis,and to explore the relationship of endometriosis with the aberrant methylation and expression of HOXA10 and its clinical significance.Methods The methylation-specific PCR(MSP) and the immunohistochemical SP methods were employed to detect both the methylation status of the HOXA10 promoter and its expression in the eutopic and ectopic endometrial tissues from 36 endometriosis patients,with normal endometrial tissue samples from 12 healthy women as controls.Results Hypermethylation of the HOXA10 promoter region was present in 38.9% (14/36) of the ectopic endometrial tissue samples and 25.0% (9/36) of the eutopic endometrial tissue samples,all in endometriosis of stages Ⅲ and Ⅳ,but no hypermethylation was found in the endometrial tissues of the normal controls,with significant differences between the two groups (P
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Objective:To study the expression of integrin ?_5,?_3 in eutopic endometrium and ectopic endometrium of endometriosis. Methods:Reverse transcription polymerase chain reaction(RT-PCR) method was utilized to detect and quantitate integrin ?_5,?_3 mRNA expression levels in eutopic and ectopic endometrial tissues from 18 patients with endometriosis and the endometrial tissues from 18 non-endometriosis women.Serum concentration of estradiol(E_2),progesterone(P) and CA125 were measured by radioimmunoassay.AFS-r marking was used to describe the state of pelvic cavity during operation. Results:Various levels of positive expression of integrin subunits ?_5,?_3 were shown in both eutopic and ectopic endometrial tissues of endometriosis and the endometrium of non-endometriosis.The expressions of integrin ?_5 mRNA in eutopic endometrium,ectopic endometrium of endometriosis group and endometrium of control group were as 0.773?0.113,1.077?0.032,0.924?0.120,respectively.(Among) the groups,the differences were statistically significant(P0.05).Both CA125 level and AFS-r were positively correlated with integrin ?_5,?_3(P
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Objective:To investigate the expression of chemokine receptor in eupotic and ectopic endometrial tissues of women with endometriosis, and in endometrium of women without endometriosis.Methods:Normal endometrium, eutopic endometrium and endometriotic tissues were obtained from patients with endometriosis at laparoscopy. Total RNA was then extracted using the TRIzol reagent. The expression of chemokine receptors in these tissues were analyzed by way of semi-quantitative reverse transcriptase-polymerase chain reaction.Results:Compared to normal endometrium, the eutopic endometrium expressed significantly more CCR6, CCR8, CCR9 and CX3CR1(P