RESUMEN
BACKGROUND: Premature ovarian insufficiency (POI) is a condition characterized by a substantial decline or loss of ovarian function in women before the age of 40. However, the pathogenesis of POI remains to be further elucidated, and specific targeted drugs which could delay or reverse ovarian reserve decline are urgently needed. Abnormal DNA damage repair (DDR) and cell senescence in granulosa cells are pathogenic mechanisms of POI. Ubiquitin-specific protease 14 (USP14) is a key enzyme that regulates the deubiquitylation of DDR-related proteins, but whether USP14 participates in the pathogenesis of POI remains unclear. METHODS: We measured USP14 mRNA expression in granulosa cells from biochemical POI (bPOI) patients. In KGN cells, we used IU1 and siRNA-USP14 to specifically inhibit USP14 and constructed a cell line stably overexpressing USP14 to examine its effects on DDR function and cellular senescence in granulosa cells. Next, we explored the therapeutic potential of IU1 in POI mouse models induced by D-galactose. RESULTS: USP14 expression in the granulosa cells of bPOI patients was significantly upregulated. In KGN cells, IU1 treatment and siUSP14 transfection decreased etoposide-induced DNA damage levels, promoted DDR function, and inhibited cell senescence. USP14 overexpression increased DNA damage, impaired DDR function, and promoted cell senescence. Moreover, IU1 treatment and siUSP14 transfection increased nonhomologous end joining (NHEJ), upregulated RNF168, Ku70, and DDB1, and increased ubiquitinated DDB1 levels in KGN cells. Conversely, USP14 overexpression had the opposite effects. Intraperitoneal IU1 injection alleviated etoposide-induced DNA damage in granulosa cells, ameliorated the D-galactose-induced POI phenotype, promoted DDR, and inhibited cell senescence in ovarian granulosa cells in vivo. CONCLUSIONS: Upregulated USP14 in ovarian granulosa cells may play a role in POI pathogenesis, and targeting USP14 may be a potential POI treatment strategy. Our study provides new insights into the pathogenesis of POI and a novel POI treatment strategy.
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Senescencia Celular , Daño del ADN , Reparación del ADN , Células de la Granulosa , Insuficiencia Ovárica Primaria , Ubiquitina Tiolesterasa , Femenino , Insuficiencia Ovárica Primaria/patología , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/genética , Células de la Granulosa/metabolismo , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/patología , Senescencia Celular/efectos de los fármacos , Animales , Humanos , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Reparación del ADN/efectos de los fármacos , Ratones , Adulto , Ratones Endogámicos C57BL , Línea CelularRESUMEN
Premature ovarian insufficiency (POI) is a cause of infertility and endocrine dysfunction in women, defined by loss of normal, predictable ovarian activity before the age of 40 years. POI is clinically characterized by amenorrhoea (primary or secondary) with raised circulating levels of follicle-stimulating hormone. This condition can occur due to medical interventions such as ovarian surgery or cytotoxic cancer therapy, metabolic and lysosomal storage diseases, infections, chromosomal anomalies and autoimmune diseases. At least 1 in 100 women is affected by POI, including 1 in 1,000 before the age of 30 years. Substantial evidence suggests a genetic basis to POI. However, the cause of idiopathic POI remains unknown in most patients, indicating that gene variants associated with this condition remain to be discovered. Over the past 10 years, tremendous progress has been made in our knowledge of genes involved in POI. Genetic approaches in diagnosis are important as they enable patients with familial POI to be identified, with the opportunity for oocyte preservation. Moreover, genetic approaches could provide a better understanding of disease mechanisms, which will ultimately aid the development of improved treatments.
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Insuficiencia Ovárica Primaria , Humanos , Insuficiencia Ovárica Primaria/fisiopatología , Insuficiencia Ovárica Primaria/etiología , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/diagnóstico , Femenino , Hormona Folículo Estimulante/sangre , AdultoRESUMEN
Ovarian insufficiency is one of the common reproductive disorders affecting women with limited therapeutic aids. Mesenchymal stem cells have been investigated in such disorders before yet, the exact mechanism of MSCs in ovarian regeneration regarding their epigenetic regulation remains elusive. The current study is to investigate the role of the bone marrow-derived mesenchymal stem cells (BM-MSCs) lncRNA (Neat-1 and Hotair1) and miRNA (mir-21-5p, mir-144-5p, and mir-664-5p) in mitigating ovarian granulosa cell apoptosis as well as searching BM-MSCs in altering the expression of ovarian and hypothalamic IGF-1 - kisspeptin system in connection to HPG axis in a cyclophosphamide-induced ovarian failure rat model. Sixty mature female Sprague Dawley rats were divided into 3 equal groups; control group, premature ovarian insufficiency (POI) group, and POI + BM-MSCs. POI female rat model was established with cyclophosphamide. The result revealed that BM-MSCs and their conditioned media displayed a significant expression level of Neat-1, Hotair-1, mir-21-5p, mir-144-5p, and mir-664-5p. Moreover, BM-MSCs transplantation in POI rats improves; the ovarian and hypothalamic IGF-1 - kisspeptin, HPG axis, ovarian granulosa cell apoptosis, steroidogenesis, angiogenesis, energy balance, and oxidative stress. BM-MSCs expressed higher levels of antiapoptotic lncRNAs and microRNAs that mitigate ovarian insufficiency.
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Apoptosis , Ciclofosfamida , Factor I del Crecimiento Similar a la Insulina , Células Madre Mesenquimatosas , MicroARNs , Insuficiencia Ovárica Primaria , ARN Largo no Codificante , Ratas Sprague-Dawley , Animales , Femenino , MicroARNs/genética , MicroARNs/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ciclofosfamida/efectos adversos , Ratas , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/inducido químicamente , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/genética , Ovario/metabolismo , Células de la Médula Ósea/metabolismo , AngiogénesisRESUMEN
BACKGROUND: POI (premature ovarian insufficiency) refers to premature and rapid decline of ovarian reserve function in women before the age of 40, which can be manifested as menstrual disorders, endocrine abnormalities and low fertility. Bu-Shen-Ning-Xin decoction (BSNXD) has been found to have therapeutic effects on POI. Nevertheless, how it exerts therapeutic effects remains elusive. PURPOSE: This research aims to clarify the pharmacological mechanisms of BSNXD. METHODS: We applied Ultra Performance Liquid Chromatography (UPLC) to identify the main components of BSNXD.4-vinylcyclohexene diepoxide(VCD)was used to induce POI models. ELISA detected the serum level of hormones. H&E staining evaluated the morphology of ovarian tissues.CircRNA and mRNA expression profiles in the ovaries of both POI rats and those treated with BSNXD were detected. Then, dysregulated circRNAs and mRNAs that were potentially altered by BSNXD were screened. Network pharmacology analysis was performed to identify drug targets of BSNXD active ingredients. A circRNA-miRNA-mRNA network and an oxidative stress(OS)-related subnetwork were constructed. Expression of rno_circRNA_012284, rno_miR-760-3p, and HBEGF(Heparin-binding epidermal growth factor-like growth factor) was measured by RT-PCR and their binding were verified by dual-luciferase reporter assays. ROS was measured through DCFH-DA fluorescence probes. The HBEGF target was selected for molecular docking with key active ingredients.Surface plasmon resonance(SPR) was applied to verify the binding ability and affinity between components and HBEGF. RESULTS: UPLC analysis indicated that 6 chemical compounds including berberine, paeoniflorin, morroniside,gallic acid, loganin, baicalin were identified.Elevated FSH and LH levels, suppressed E2 and AMH levels in the serum, and inhibited follicles and corpus luteums in the ovarian tissues of VCD-induced rats were notably reversed by BSNXD.In total, 992 up- and 1135 down-regulated circRNAs, and 205 up- and 243 down-regulated mRNAs were found in POI rat ovaries following BSNXD administration. Furthermore, 198 drug targets of BSNXD were identified. An OS-related and BSNXD-targeted ceRNA subnetwork composed of rno_circRNA_012284/rno_miR-760-3p/HBEGF was established. rno_circRNA_012284 and HBEGF were up-regulated and rno_miR-760-3p was down-regulated in POI ovarian granulosa cells (OGCs) after BSNXD administration. rno_circRNA_012284 was a sponge of rno_miR-760-3p to elevate HBEGF expression. Moreover, rno_circRNA_012284 overexpression alleviated POI-induced excessive ROS generation in ovarian granulosa cells, while rno_circRNA_012284 inhibition exerted the opposite effect. Finally,molecular docking speculated active ingredients of each herb acted on HBEGF to reduce the OS. SPR tests showed that Berberine,Baicalein,Quercetin,Pachymic acid,Paeoniflorin exhibited satisfying affinity with HBEGF protein. CONCLUSION: This study demonstrates that BSNXD ameliorates POI partly by attenuating OS in ovarian granulosa cells via rno_circRNA_012284/rno_miR-760-3p/HBEGF axis, uncovering the pharmacological mechanisms of BSNXD in alleviating POI.
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Medicamentos Herbarios Chinos , MicroARNs , Estrés Oxidativo , Insuficiencia Ovárica Primaria , ARN Circular , Animales , Femenino , Ratas , Ciclohexenos , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/farmacología , MicroARNs/metabolismo , MicroARNs/genética , Ovario/efectos de los fármacos , Ovario/metabolismo , Estrés Oxidativo/efectos de los fármacos , Insuficiencia Ovárica Primaria/tratamiento farmacológico , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/genética , Ratas Sprague-Dawley , ARN Circular/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Compuestos de Vinilo/farmacologíaRESUMEN
The relationship between premature ovarian insufficiency (FXPOI) and premutation in the FMR1 gene is well established. In recent years, though, a potential relationship between the latter and a low ovarian reserve has been suggested. To explore it, we conducted a retrospective study in an IVF program at a university tertiary referral center in Barcelona (Spain). Data were obtained retrospectively from a total of 385 women referred for FMR1 gene testing at our institution from January 2018 to December 2021. We compared the prevalence of FMR1 gene premutation between 93 of them, younger than 35 years, with a diminished ovarian reserve (DOR), characterized by levels of anti-Mullerian hormone < 1.1 ng/mL and antral follicle count < 5; and 132 egg donors screened by protocol that served as the controls. We found a higher prevalence of FMR1 premutation in the DOR group (seven patients (7.69%)) than in the control group (one patient (1.32%)), Fisher-exact test p-value = 0.012). We concluded that compared with the general population represented by young egg donors, the prevalence of FMR1 gene premutation is higher in young patients with a diminished ovarian reserve. Although these findings warrant further prospective validation in a larger cohort of patients within DOR, they suggest that, in clinical practice, FMR1 premutation should be determined in infertile young patients with DOR in order to give them adequate genetic counselling.
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Fertilización In Vitro , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Reserva Ovárica , Insuficiencia Ovárica Primaria , Humanos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Femenino , Reserva Ovárica/genética , Adulto , Insuficiencia Ovárica Primaria/genética , Estudios Retrospectivos , Mutación , Prevalencia , España/epidemiologíaRESUMEN
Age at menopause (AOM) has a substantial impact on fertility and disease risk. While many loci with variants that associate with AOM have been identified through genome-wide association studies (GWAS) under an additive model, other genetic models are rarely considered1. Here through GWAS meta-analysis under the recessive model of 174,329 postmenopausal women from Iceland, Denmark, the United Kingdom (UK; UK Biobank) and Norway, we study low-frequency variants with a large effect on AOM. We discovered that women homozygous for the stop-gain variant rs117316434 (A) in CCDC201 (p.(Arg162Ter), minor allele frequency ~1%) reached menopause 9 years earlier than other women (P = 1.3 × 10-15). The genotype is present in one in 10,000 northern European women and leads to primary ovarian insufficiency in close to half of them. Consequently, homozygotes have fewer children, and the age at last childbirth is 5 years earlier (P = 3.8 × 10-5). The CCDC201 gene was only found in humans in 2022 and is highly expressed in oocytes. Homozygosity for CCDC201 loss-of-function has a substantial impact on female reproductive health, and homozygotes would benefit from reproductive counseling and treatment for symptoms of early menopause.
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Estudio de Asociación del Genoma Completo , Homocigoto , Insuficiencia Ovárica Primaria , Humanos , Femenino , Insuficiencia Ovárica Primaria/genética , Polimorfismo de Nucleótido Simple , Persona de Mediana Edad , Menopausia/genética , Reino Unido , Frecuencia de los Genes , Islandia , Dinamarca , Predisposición Genética a la EnfermedadRESUMEN
Premature Ovarian Insufficiency (POI) is a highly heterogeneous condition characterized by ovarian dysfunction in women occurring before the age of 40, representing a significant cause of female infertility. It manifests through primary or secondary amenorrhea. While more than half of POI cases are idiopathic, genetic factors play a pivotal role in all instances with known causes, contributing to approximately 20-25% of cases. This article comprehensively reviews the genetic factors associated with POI, delineating the primary candidate genes. The discussion delves into the intricate relationship between these genes and ovarian development, elucidating the functional consequences of diverse mutations to underscore the fundamental impact of genetic effects on POI. The identified genetic factors, encompassing gene mutations and chromosomal abnormalities, are systematically classified based on whether the resulting POI is syndromic or non-syndromic. Furthermore, this paper explores the genetic interplay between mitochondrial genes, such as Required for Meiotic Nuclear Division 1 homolog Gene (RMND1), Mitochondrial Ribosomal Protein S22 Gene (MRPS22), Leucine-rich Pentapeptide Repeat Gene (LRPPRC), and non-coding RNAs, including both microRNAs and Long non-coding RNAs, with POI. The insights provided serve to consolidate and enhance our understanding of the etiology of POI, contributing to establishing a theoretical foundation for diagnosing and treating POI patients, as well as for exploring the mechanisms underlying the disease.
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Insuficiencia Ovárica Primaria , Insuficiencia Ovárica Primaria/genética , Humanos , Femenino , Mutación , Predisposición Genética a la EnfermedadRESUMEN
BACKGROUND: Accumulating studies have highlighted the significant role of circulating metabolomics in the etiology of reproductive system disorders. However, the causal effects between genetically determined metabolites (GDMs) and reproductive diseases, including primary ovarian insufficiency (POI), polycystic ovary syndrome (PCOS), and abnormal spermatozoa (AS), still await thorough clarification. METHODS: With the currently most comprehensive genome-wide association studies (GWAS) data of metabolomics, systematic two-sample Mendelian randomization (MR) analyses were conducted to disclose causal associations between 1,091 blood metabolites and 309 metabolite ratios with reproductive disorders. The inverse-variance weighted (IVW) method served as the primary analysis approach, and multiple effective MR methods were employed as complementary analyses including MR-Egger, weighted median, constrained maximum likelihood (cML-MA), contamination mixture method, robust adjusted profile score (MR-RAPS), and debiased inverse-variance weighted method. Heterogeneity and pleiotropy were assessed via MR-Egger intercept and Cochran's Q statistical analysis. Outliers were detected by Radial MR and MR-PRESSO methods. External replication and metabolic pathway analysis were also conducted. RESULTS: Potential causal associations of 63 GDMs with POI were unearthed, and five metabolites with strong causal links to POI were emphasized. Two metabolic pathways related to the pathogenesis of POI were pinpointed. Suggestive causal effects of 70 GDMs on PCOS were detected, among which 7 metabolites stood out for strong causality with elevated PCOS risk. Four metabolic pathways associated with PCOS mechanisms were recognized. For AS, 64 GDMs as potential predictive biomarkers were identified, particularly highlighting two metabolites for their strong causal connections with AS. Three pathways underneath the AS mechanism were identified. Multiple assessments were conducted to further confirm the reliability and robustness of our causal inferences. CONCLUSION: By extensively assessing the causal implications of circulating GDMs on reproductive system disorders, our study underscores the intricate and pivotal role of metabolomics in reproductive ill-health, laying a theoretical foundation for clinical strategies from metabolic insights.
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Estudio de Asociación del Genoma Completo , Análisis de la Aleatorización Mendeliana , Metaboloma , Síndrome del Ovario Poliquístico , Insuficiencia Ovárica Primaria , Humanos , Síndrome del Ovario Poliquístico/genética , Síndrome del Ovario Poliquístico/sangre , Síndrome del Ovario Poliquístico/metabolismo , Femenino , Masculino , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/sangre , Insuficiencia Ovárica Primaria/metabolismo , Metabolómica/métodos , Espermatozoides/metabolismoRESUMEN
This case report elucidates a scenario involving two sibling sisters born out of consanguineous marriage-one initially presenting with lower respiratory infection, concurrently exhibiting short stature and primary amenorrhoea. Investigation into the primary amenorrhoea unveiled hypergonadotropic hypogonadism, confirmed by the absence of ovaries and a hypoplastic uterus on pelvic MRI. Genetic analysis via whole exome sequencing identified a homozygous variant NM_001282717.2: c.808C>T in the MCM8 gene, located on exon 8 of chromosome 20, inherited in an autosomal recessive manner. The scarcity of primary ovarian insufficiency cases linked to MCM8 highlights the necessity of thoroughly investigating the genetic and clinical consequences of such variants.
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Proteínas de Mantenimiento de Minicromosoma , Mutación , Insuficiencia Ovárica Primaria , Hermanos , Útero , Humanos , Femenino , Insuficiencia Ovárica Primaria/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Útero/anomalías , Consanguinidad , Imagen por Resonancia Magnética , Secuenciación del Exoma , Amenorrea/genética , Amenorrea/etiología , Anomalías Urogenitales/genética , Anomalías Urogenitales/diagnóstico por imagenRESUMEN
Premature ovarian insufficiency (POI) is one of the important causes of female infertility. Yet the aetiology for POI is still elusive. FBXW7 (F-box with 7 tandem WD) is one of the important components of the Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase. FBXW7 can regulate cell growth, survival and pluripotency through mediating ubiquitylation and degradation of target proteins via triggering the ubiquitin-proteasome system, and is associated with tumorigenesis, haematopoiesis and testis development. However, evidence establishing the function of FBXW7 in ovary is still lacking. Here, we showed that FBXW7 protein level was significantly decreased in the ovaries of the cisplatin-induced POI mouse model. We further showed that mice with oocyte-specific deletion of Fbxw7 demonstrated POI, characterized with folliculogenic defects, early depletion of follicle reserve, disordered hormonal secretion, ovarian dysfunction and female infertility. Impaired oocyte-GCs communication, manifested as down-regulation of connexin 37, may contribute to follicular development failure in the Fbxw7-mutant mice. Furthermore, single-cell RNA sequencing and in situ hybridization results indicated an accumulation of Clu and Ccl2 transcripts, which may alter follicle microenvironment deleterious to oocyte development and accelerate POI. Our results establish the important role of Fbxw7 in folliculogenesis and ovarian function, and might provide valuable information for understanding POI and female infertility.
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Proteína 7 que Contiene Repeticiones F-Box-WD , Oocitos , Folículo Ovárico , Insuficiencia Ovárica Primaria , Animales , Femenino , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/patología , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Oocitos/metabolismo , Ratones , Folículo Ovárico/metabolismo , Folículo Ovárico/crecimiento & desarrollo , Folículo Ovárico/patología , Modelos Animales de Enfermedad , Eliminación de Gen , Ratones Noqueados , Infertilidad Femenina/genética , Infertilidad Femenina/metabolismo , Infertilidad Femenina/patología , Cisplatino/efectos adversosRESUMEN
Premature ovarian failure (POF) is characterized by a significant decline in the ovarian follicle pool and oocyte reserve, alongside an increase in the number of low-quality oocytes and apoptosis of granulosa cells (GCs). Exosome-derived miRNA plays a regulatory role in crucial cellular activities and contributes to the onset and progression of POF. In this study, we successfully established a rabbit model of POF and conducted in vitro and in vivo experiments that confirmed DiI-labeled Pla-Exos (exosomes derived from plasma) could enter the follicle through blood circulation, with GCs capable of uptaking these exosomes. Our RNA-seq analysis revealed elevated expression of miR-10a-5p in Pla-Exos from POF rabbits. Moreover, our findings demonstrate that exosomal miR-10a-5p suppresses GCs proliferation and induces apoptosis via the mitochondrial pathway. Additionally, exosomal miR-10a-5p inhibits the TrkB/Akt/mTOR signaling pathway by downregulating BDNF expression, thereby modulating the expression levels of proteins and genes associated with the cell cycle, follicle development, and GCs senescence. In conclusion, our study highlights the role of Pla-Exos miR-10a-5p in promoting rabbit POF through the TrkB/Akt/mTOR signaling pathway by targeting BDNF. These findings provide new insights into potential therapeutic targets for POF, offering valuable references for addressing concerns related to female reproductive function.
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Factor Neurotrófico Derivado del Encéfalo , Exosomas , Células de la Granulosa , MicroARNs , Insuficiencia Ovárica Primaria , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Serina-Treonina Quinasas TOR , Animales , Femenino , Exosomas/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Conejos , Células de la Granulosa/metabolismo , Receptor trkB/metabolismo , Receptor trkB/genética , Apoptosis/genética , Proliferación Celular , Humanos , Folículo Ovárico/metabolismoRESUMEN
BACKGROUND: The KITL-KIT interaction is known as an important initiator in oocyte activation through the downstream pathway of PI3K-AKT-FOXO3 signalling. Previous studies utilising germ cell-specific Kit mutant knockin and kinase domain knockout models with Vasa-Cre suggested the crucial role of KIT in oocyte activation at the primordial follicle stage. METHODS: We utilised mice with complete postnatal deletion of KIT expression in oocytes via Gdf9-iCre and conducted analyses on ovarian follicle development, specific markers, hormone assays, and fertility outcomes. FINDINGS: Our findings reveal contrasting phenotypes compared to previous mouse models with prenatal deletion of Kit. Specifically, postnatal deletion of Kit exhibit no defects in germ cell nest breakdown, follicle activation, and folliculogenesis during development. Remarkably, upon reaching full maturity, mice with postnatal deletion of Kit experience a complete loss of ovarian reserve, growing follicles, and ovarian function. Furthermore, mice display smaller ovarian size and weight, delayed folliculogenesis, and phenotypes indicative of primary ovarian insufficiency (POI), including elevated serum levels of FSH, reduced AMH, and absence of ovarian follicles, ultimately resulting in infertility. Additionally, the ovaries exhibit randomly distributed expression of granulosa and theca cell markers such as Inhibin α, ACVR2B, and LHR. Notably, there is the uncontrolled expression of p-SMAD3 and Ki67 throughout the ovarian sections, along with the widespread presence of luteinised stroma cells and cleaved Caspase-3-positive dying cells. INTERPRETATION: These genetic studies underscore the indispensable role of KIT in oocytes for maintaining the survival of ovarian follicles and ensuring the reproductive lifespan. FUNDING: This work was supported by National Institutes of Health grant R01HD096042 and startup funds from UNMC (S.Y.K.).
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Oocitos , Folículo Ovárico , Proteínas Proto-Oncogénicas c-kit , Animales , Femenino , Oocitos/metabolismo , Ratones , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Folículo Ovárico/metabolismo , Supervivencia Celular/genética , Reproducción , Ratones Noqueados , Biomarcadores , Factor 9 de Diferenciación de Crecimiento/metabolismo , Factor 9 de Diferenciación de Crecimiento/genética , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/genéticaRESUMEN
PURPOSE: To analyze the copy number variation (CNV) in the X-linked genes BCORL1, POF1B, and USP9X in idiopathic diminished ovarian reserve (DOR). METHODS: This case-control study included 47 women, 26 with DOR and 21 in the control group. Age, weight, height, BMI, and FSH level were evaluated, as well as antral follicle count (AFC), oocyte retrieval after controlled ovarian stimulation, and metaphase II (MII) oocytes. The CNVs of BCORL1, USP9X, and POF1B genes were measured by quantitative real time PCR (qPCR) using two reference genes, the HPRT1 (X-linked) and MFN2 (autosomal). Protein-protein interaction network and functional enrichment analysis were performed using the STRING database. RESULTS: The mean age was 36.52 ± 4.75 in DOR women and 35.38 ± 4.14 in control. Anthropometric measures did not differ between the DOR and control groups. DOR women presented higher FSH (p = 0.0025) and lower AFC (p < .0001), oocyte retrieval after COS (p = 0.0004), and MII oocytes (p < .0001) when compared to the control group. BCORL1 and POF1B did not differ in copy number between DOR and control. However, DOR women had more copies of USP9X than the control group (p = 0.028). CONCLUSION: The increase in the number of copies of the USP9X gene may lead to overexpression in idiopathic DOR and contribute to altered folliculogenesis and oocyte retrieval.
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Variaciones en el Número de Copia de ADN , Reserva Ovárica , Ubiquitina Tiolesterasa , Humanos , Femenino , Reserva Ovárica/genética , Adulto , Variaciones en el Número de Copia de ADN/genética , Ubiquitina Tiolesterasa/genética , Estudios de Casos y Controles , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/patología , Recuperación del Oocito , Proteínas Represoras/genética , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Oocitos/patologíaRESUMEN
Folliculogenesis is the process where follicles in the ovaries develop and eventually lead to ovulation. Any disruption to this process can cause premature ovarian failure. miR-326 is one of the microRNAs whose expression leads to Th17 production. Th17 activates the immune system to respond more vigorously, and by producing interlukins and cytokines causes inflammation and autoimmune disorders. Th17-induced inflammation and Th17/Treg imbalance can result in POF. This investigation took samples from 30 POF patients and 30 healthy people. The study utilized PCR to assess the expression levels of cytokines, specific transcription factor (ROR-γt), and miR-326. Additionally, ELISA was employed to analyze serum levels of IL-17, IL-21, IL-23. Furthermore, flow cytometry was utilized to determine the frequency of Th17. Compared to the control group, our results demonstrated a rise in the transcription factor RORɣt and a considerable rise in the frequency of Th17 cells in patients with POF. The level of inflammatory cytokines IL-17, IL-21, and IL-23 secreted in serum samples of patients with POF increased significantly compared to the control group. Results of investigating microRNA associated with Th17 cells also showed increased expression of miR-326 in females suffering from POF. The elevation of pro-inflammatory markers in women with POF contrary to the control group underscores the significant involvement of the immune system in pregnancy disorders pathogenesis. Consequently, immunological factors may serve as promising biomarkers for predicting POF likelihood in high-risk women in the future.
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MicroARNs , Insuficiencia Ovárica Primaria , Células Th17 , Humanos , Femenino , MicroARNs/sangre , MicroARNs/genética , MicroARNs/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Insuficiencia Ovárica Primaria/inmunología , Insuficiencia Ovárica Primaria/sangre , Insuficiencia Ovárica Primaria/genética , Adulto , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/sangre , Citocinas/sangre , Citocinas/metabolismo , Adulto Joven , Regulación de la Expresión Génica/inmunologíaRESUMEN
Objective: Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development. Premature ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles, constituting a significant cause of female infertility. In recent years, with the help of the rapid development in genetic sequencing technology, it has been demonstrated that genetic factors play a crucial role in the onset of POI. Among the population suffering from POI, genetic studies have revealed that genes involved in processes such as meiosis, DNA damage repair, and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified. FA complementation group M (FANCM) is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including FANCA-FANCW. Abnormalities in the FANCM genes are associated with female infertility and FANCM gene knockout mice also exhibit phenotypes similar to those of POI. During the genetic screening of POI patients, this study identified a suspicious variant in FANCM. This study aims to explore the pathogenic mechanisms of the FANCM genes of the FA pathway and their variants in the development of POI. We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals. Methods: One POI patient was included in the study. The inclusion criteria for POI patients were as follows: women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L (with a minimum interval of 4 weeks inbetween tests), alongside clinical symptoms of menstrual disorders, normal chromosomal karyotype analysis results, and exclusion of other known diseases that can lead to ovarian dysfunction. We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG). Subsequently, the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis. Plasmids containing wild-type and mutant FANCM genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human FANCM plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP FANCM-WT group, the EGFP FANCM-MUT group, and the EGFP group, respectively. To validate the production of truncated proteins, cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody. In order to investigate the impact on DNA damage repair, immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP FANCM-WT group and the EGFP FANCM-MUT group to examine whether the variant affected FANCM's ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in vitro in both the EGFP FANCM-WT group and the EGFP FANCM-MUT group, which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody. Results: In a POI patient from a consanguineous family, we identified a homozygous variant in the FANCM gene, c.1152-1155del:p.Leu386Valfs*10. The patient presented with primary infertility, experiencing irregular menstruation since menarche at the age of 16. Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone (AMH) level of 0.07 ng/mL. Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia. The patient's parents were a consanguineous couple, with the mother having regular menstrual cycles. The patient had two sisters, one of whom passed away due to osteosarcoma, while the other exhibited irregular menstruation, had been diagnosed with ovarian insufficiency, and remained childless. Bioinformatics analysis revealed a deletion of four nucleotides (c.1152-1155del) in the exon 6 of the patient's FANCM gene. This variant resulted in a frameshift at codon 386, introducing a premature stop codon at codon 396, which ultimately led to the production of a truncated protein consisting of 395 amino acids. In vitro experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization, with the protein being present only in the cytoplasm. Following treatment with mitomycin C, there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid (P<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant. Conclusions: The homozygous variant in the FANCM gene, c.1152-1155del:p.Leu386Valfs*10, results in the production of a truncated FANCM protein. This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex, preventing its entry into the nucleus and the subsequent recognition of DNA damage. Consequently, the localization of the FA core complex on chromatin is disrupted, impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs. By disrupting the rapid proliferation and meiotic division processes of primordial germ cells, the reserve of oocytes is depleted, thereby triggering premature ovarian insufficiency in females.
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Insuficiencia Ovárica Primaria , Femenino , Insuficiencia Ovárica Primaria/genética , Humanos , Mutación , Anemia de Fanconi/genética , Adulto , Infertilidad Femenina/genética , Infertilidad Femenina/etiología , ADN HelicasasRESUMEN
PURPOSE: Oocytes from women presenting primary ovarian insufficiency (POI) generate viable embryos at a lower rate than non-POI women, but the mechanisms responsible for the lower oocyte quality remain elusive. Due to the scarcity of human oocytes for research, animal models provide a promising way forward. We aimed at investigating the molecular events characterizing final maturation in POI oocytes in a well-defined POI-like bovine model. METHODS: Single-cell RNA-sequencing of bovine control and POI-like, GV, and MII oocytes (n = 5 per group) was performed. DEseq2 was used to identify differentially expressed genes. Further, a Gene set enrichment analysis and a transcriptomic meta-analysis between bovine and human oocytes were performed. RESULTS: In control cows, we found 2223 differentially expressed genes between the GV and MII stages. Specifically, the affected genes were related to RNA processing and transport, protein synthesis, organelle remodeling and reorganization, and metabolism. The meta-analysis with a set of young human oocytes at different maturation stages revealed 315 conserved genes through the GV-MII transition in cows and humans, mostly related to meiotic progression and cell cycle. Gene expression analysis between GV and MII of POI-like oocytes showed no differences in terms of differentially expressed genes, pointing towards a substantial failure to properly remodel the transcriptome in the POI model, and with the clustering analysis indicating that the cow's genetic background had a higher impact than the oocyte's maturation stage. CONCLUSION: Overall, we have identified and characterized a valuable animal model of POI, paving the way to identifying new molecular mechanisms involved in POI.
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Meiosis , Oocitos , Insuficiencia Ovárica Primaria , Bovinos , Femenino , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/patología , Animales , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Oocitos/patología , Meiosis/genética , Humanos , Transcriptoma/genética , Modelos Animales de Enfermedad , Oogénesis/genéticaRESUMEN
Premature ovarian failure (POF), which is often comorbid with dry eye disease (DED) is a key issue affecting female health. Here, we explored the mechanism underlying comorbid POF and DED to further elucidate disease mechanisms and improve treatment. Datasets related to POF (GSE39501) and DED (GSE44101) were identified from the Gene Expression Omnibus (GEO) database and subjected to weighted gene coexpression network (WGCNA) and differentially expressed genes (DEGs) analyses, respectively, with the intersection used to obtain 158 genes comorbid in POF and DED. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses of comorbid genes revealed that identified genes were primarily related to DNA replication and Cell cycle, respectively. Protein-Protein interaction (PPI) network analysis of comorbid genes obtained the 15 hub genes: CDC20, BIRC5, PLK1, TOP2A, MCM5, MCM6, MCM7, MCM2, CENPA, FOXM1, GINS1, TIPIN, MAD2L1, and CDCA3. To validate the analysis results, additional POF- and DED-related datasets (GSE48873 and GSE171043, respectively) were selected. miRNAs-lncRNAs-genes network and machine learning methods were used to further analysis comorbid genes. The DGIdb database identified valdecoxib, amorfrutin A, and kaempferitrin as potential drugs. Herein, the comorbid genes of POF and DED were identified from a bioinformatics perspective, providing a new strategy to explore the comorbidity mechanism, opening up a new direction for the diagnosis and treatment of comorbid POF and DED.
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Síndromes de Ojo Seco , Redes Reguladoras de Genes , Insuficiencia Ovárica Primaria , Mapas de Interacción de Proteínas , Humanos , Femenino , Síndromes de Ojo Seco/genética , Síndromes de Ojo Seco/diagnóstico , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/diagnóstico , Mapas de Interacción de Proteínas/genética , Biomarcadores , Perfilación de la Expresión Génica , Ontología de Genes , Bases de Datos Genéticas , Biología Computacional/métodosRESUMEN
INTRODUCTION: Premature ovarian insufficiency (POI) is one of the causes of female infertility. Unexplained POI is increasingly affecting women in their reproductive years. However, the etiology of POI is diverse and remains elusive. We and others have shown that brain-derived neurotrophic factor (BDNF) plays an important role in adult ovarian function. Here, we report on a novel role of BDNF in the Developmental Origins of POI. METHODS: Placental BDNF knockout mice were created using CRISPR/CAS9. Homozygous knockout (cKO(HO)) mice didn't survive, while heterozygous knockout (cKO(HE)) mice did. BDNF reduction in cKO(HE) mice was confirmed via immunohistochemistry and Western blots. Ovaries were collected from cKO(HE) mice at various ages, analyzing ovarian metrics, FSH expression, and litter sizes. In one-month-old mice, oocyte numbers were assessed using super-ovulation, and oocyte gene expression was analyzed with smart RNAseq. Ovaries of P7 mice were studied with SEM, and gene expression was confirmed with RT-qPCR. Alkaline phosphatase staining at E11.5 and immunofluorescence for cyclinD1 assessed germ cell number and cell proliferation. RESULTS: cKO(HE) mice had decreased ovarian function and litter size in adulthood. They were insensitive to ovulation induction drugs manifested by lower oocyte release after superovulation in one-month-old cKO(HE) mice. The transcriptome and SEM results indicate that mitochondria-mediated cell death or aging might occur in cKO(HE) ovaries. Decreased placental BDNF led to diminished primordial germ cell proliferation at E11.5 and ovarian reserve which may underlie POI in adulthood. CONCLUSION: The current results showed decreased placental BDNF diminished primordial germ cell proliferation in female fetuses during pregnancy and POI in adulthood. Our findings can provide insights into understanding the underlying mechanisms of POI.
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Factor Neurotrófico Derivado del Encéfalo , Ratones Noqueados , Placenta , Insuficiencia Ovárica Primaria , Animales , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/patología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Femenino , Ratones , Embarazo , Placenta/metabolismo , Ovario/metabolismo , Ovario/patología , Modelos Animales de Enfermedad , Oocitos/metabolismoRESUMEN
BACKGROUND: The therapeutic potential of exosomes from human umbilical cord mesenchymal stem cells (HUMSCs-Exo) for delivering specific circular RNAs (circRNAs) in treating premature ovarian failure (POF) is not well understood. This study aimed to explore the efficacy of HUMSCs-Exo in delivering hsa_circ_0002021 for POF treatment, focusing on its effects on granulosa cell (GC) senescence and ovarian function. METHODS: Bioinformatic analysis was conducted on circRNA profiles using the GSE97193 dataset from GEO, targeting granulosa cells from varied age groups. To simulate granulosa cell senescence, KGN cells were treated with cyclophosphamide (CTX). HUMSCs were transfected with pcDNA 3.1 vectors to overexpress hsa_circ_0002021, and the HUMSCs-Exo secreted were isolated. These exosomes were characterized by transmission electron microscopy (TEM) and Western blotting to confirm exosomal markers CD9 and CD63. Co-culture of these exosomes with CTX-treated KGN cells was performed to assess ß-galactosidase activity, oxidative stress markers, ROS levels, and apoptosis via flow cytometry. Interaction between hsa_circ_0002021, microRNA-125a-5p (miR-125a-5p), and cyclin-dependent kinase 6 (CDK6) was investigated using dual-luciferase assays and RNA immunoprecipitation (RIP). A POF mouse model was induced with CTX, treated with HUMSCs-Exo, and analyzed histologically and via immunofluorescence staining. Gene expression was quantified using RT-qPCR and Western blot. RESULTS: hsa_circ_0002021 was under expressed in both in vivo and in vitro POF models and was effectively delivered by HUMSCs-Exo to KGN cells, showing a capability to reduce GC senescence. Overexpression of hsa_circ_0002021 in HUMSCs-Exo significantly enhanced these anti-senescence effects. This circRNA acts as a competitive adsorbent of miR-125a-5p, regulating CDK6 expression, which is crucial in modulating cell cycle and apoptosis. Enhanced expression of hsa_circ_0002021 in HUMSCs-Exo ameliorated GC senescence in vitro and improved ovarian function in POF models by modulating oxidative stress and cellular senescence markers. CONCLUSION: This study confirms that hsa_circ_0002021, when delivered through HUMSCs-Exo, can significantly mitigate GC senescence and restore ovarian function in POF models. These findings provide new insights into the molecular mechanisms of POF and highlight the therapeutic potential of circRNA-enriched exosomes in treating ovarian aging and dysfunction.
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Exosomas , Células de la Granulosa , Células Madre Mesenquimatosas , Insuficiencia Ovárica Primaria , ARN Circular , Cordón Umbilical , Insuficiencia Ovárica Primaria/terapia , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/genética , Femenino , Exosomas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Humanos , Células de la Granulosa/metabolismo , ARN Circular/genética , ARN Circular/metabolismo , Animales , Cordón Umbilical/citología , Cordón Umbilical/metabolismo , Ratones , Senescencia Celular , Apoptosis , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
Estrogen and estrogen receptors (ERα and ERß) regulate a multitude of complicated physiological and pathological processes. Jan-Ake Gustafsson's group discovered ERß in 1996, this crucial finding gives us new insights into the understanding of estrogen signaling. ERß is highly expressed in the ovary and particularly exists in granulosa cells (GCs). ERß is a key transcription factor in the maintenance of ovarian granulosa cell growth, differentiation, and homeostasis, and the ovulation function of ovarian follicles and oocytes. Additionally, ERß can modulate the steroidogenic transcriptional program through phosphorylation and regulate both gonadotropin response and FOXL2 expression within the ovary. In this review, we focus on the role of ERß in regulating ovarian granulosa cell development and homeostasis, particularly its significance in ovarian cancer (OC), premature ovarian failure (POF), and polycystic ovary syndrome (PCOS). It also highlights the prospects of small molecule compounds targeting ERß, providing a new strategy for the treatment of ovarian-related diseases.