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BACKGROUND: Endometriosis is a multifaceted gynecological condition that poses diagnostic challenges and affects a significant number of women worldwide, leading to pain, infertility, and a reduction in patient quality of life (QoL). Traditional diagnostic methods, such as the revised American Society for Reproductive Medicine (r-ASRM) classification, have limitations, particularly in preoperative settings. The Numerical Multi-Scoring System of Endometriosis (NMS-E) has been proposed to address these shortcomings by providing a comprehensive preoperative diagnostic tool that integrates findings from pelvic examinations and transvaginal ultrasonography. METHODS: This retrospective study aims to validate the effectiveness of the NMS-E in predicting surgical outcomes and correlating with the severity of endometriosis. Data from 111 patients at Nippon Medical School Hospital were analyzed to determine the correlation between NMS-E scores, including E-score-a severity indicator-traditional scoring systems, surgical duration, blood loss, and clinical symptoms. This study also examined the need to refine parameters for deep endometriosis within the NMS-E to enhance its predictive accuracy for disease severity. RESULTS: The mean age of the patient cohort was 35.1 years, with the majority experiencing symptoms such as dysmenorrhea, dyspareunia, and chronic pelvic pain. A statistically significant positive correlation was observed between the NMS-E's E-score and the severity of endometriosis, particularly in predicting surgical duration (Spearman correlation coefficient: 0.724, p < 0.01) and blood loss (coefficient: 0.400, p < 0.01). The NMS-E E-score also correlated strongly with the r-ASRM scores (coefficient: 0.758, p < 0.01), exhibiting a slightly more excellent predictive value for surgical duration than the r-ASRM scores alone. Refinements in the methodology for scoring endometriotic nodules in uterine conditions improved the predictive accuracy for surgical duration (coefficient: 0.752, p < 0.01). CONCLUSIONS: Our findings suggest that the NMS-E represents a valuable preoperative diagnostic tool for endometriosis, effectively correlating with the disease's severity and surgical outcomes. Incorporating the NMS-E into clinical practice could significantly enhance the management of endometriosis by addressing current diagnostic limitations and guiding surgical planning.
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[This corrects the article DOI: 10.3389/fimmu.2023.1251784.].
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Preterm birth (PTB) is the leading cause of neonatal mortality, and reducing the PTB rate is one of the most critical issues in perinatal medicine. Cervical insufficiency (CI), a major cause of PTB, is characterised by premature cervical ripening in the second trimester, followed by recurrent pregnancy loss. Although multiple clinical trials have suggested that progesterone inhibits cervical ripening, no studies have focused on progesterone-induced molecular signalling in CI. Here, we established a primary culture system for human uterine cervical fibroblasts using a sample of patients with refractory innate CI who underwent transabdominal cervical cerclage and patients with low Bishop scores who underwent elective caesarean section as controls. RNA sequencing showed that the progesterone response observed in the control group was impaired in the CI group. This was consistent with the finding that progesterone receptor expression was markedly downregulated in CI. Furthermore, the inhibitory effect of progesterone on lipopolysaccharide-induced inflammatory stimuli was also impaired in CI. These results suggest that abnormal cervical ripening in CI is caused by the downregulation of progesterone signalling at the receptor level, and provide a novel insight into the molecular mechanism of PTB.
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Cerclaje Cervical , Nacimiento Prematuro , Humanos , Embarazo , Recién Nacido , Femenino , Progesterona/farmacología , Progesterona/uso terapéutico , Nacimiento Prematuro/tratamiento farmacológico , Cesárea , Cerclaje Cervical/métodos , Cuello del ÚteroRESUMEN
Objectives: The objective of this study was to assess the potential risk factors for abscess development in patients with endometrioma who present with an acute abdomen. Materials and Methods: We retrospectively reviewed the records of 51 patients who underwent emergency surgery for acute abdomen involving an endometrioma at our hospital between April 2011 and August 2021. The patients were divided into an infected group (n = 22) and a control group (n = 29). We analyzed patient characteristics; imaging findings; clinical data, including bacterial cultures; and perioperative outcomes to assess for differences between groups. Results: Patients in the infected group were significantly older than those in the control group (P = 0.03). They were more likely to have a history of endometriosis surgery (P = 0.04) and more likely to have undergone transvaginal manipulation within 3 months of presentation (P = 0.01). Body temperature on the day of admission was significantly higher in the infected group (P = 0.007), as were C-reactive protein levels on the day of admission and before surgery (P < 0.001; P = 0.018) and the white blood cell count on the day of admission (P = 0.016). Preoperative imaging showed significant thickening of the tumor wall (P < 0.001) and an enhanced contrast effect (P < 0.001) in the infected group. Conclusion: We identified several factors that suggest abscess in patients with an acute abdomen who have a complication of pathologically confirmed endometriosis. A recent vaginal procedure is a particular risk factor for abscess development in patients with endometriomas.
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BACKGROUND: Accurate diagnosis of retroflexed uterus in daily practice is essential because this condition is related to pelvic pain and deep endometriosis. Uterine flexion can be measured by transvaginal ultrasonography (TVUS), a cost-effective primary test, but the accuracy required for diagnosing retroflexed uterus is unclear. This study assessed the accuracy of TVUS for diagnosis of retroflexed uterus in patients with endometriosis and compared it with that of magnetic resonance imaging (MRI) -the gold standard for measuring the uterine axis. METHODS: The study included 123 patients who underwent endometriosis surgery in our department between 2012 and 2017. Uterine flexion angles were measured by retrospectively examining TVUS and MRI images, and the correlation was analyzed. Analysis of anteverted and retroverted uterine subgroups identified aspects of diagnosing uterine flexion with TVUS. RESULTS: Uterine flexion angles on TVUS were strongly positively correlated (r = 0.86) with MRI results. Additionally, TVUS yielded no false-positive diagnoses and 28 false-negative diagnoses of retroflexion. All false-negative diagnoses occurred in patients with anteverted retroflexed uteruses. CONCLUSIONS: TVUS was generally accurate for measuring uterine flexion angle, as indicated by its strong correlation with MRI. Misdiagnosis of anteverted retroflexed uterus was a limitation of using TVUS for retroflexion diagnosis.
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Endometriosis , Retroversión Uterina , Femenino , Humanos , Endometriosis/diagnóstico , Endometriosis/patología , Endometriosis/cirugía , Ultrasonografía/métodos , Estudios Retrospectivos , Sensibilidad y Especificidad , Imagen por Resonancia Magnética/métodosRESUMEN
On the basis of postoperative histopathological findings, a 29-year-old nulliparous woman was diagnosed as having ovotesticular disorder of sex development (DSD). She had undergone unilateral gonadectomy at age 6 years and vulvoplasty and vaginoplasty at age 8 years. Her karyotype was 46, XX. She had dyspareunia because of a narrow vagina, but her uterus and left gonad were normal. Spontaneous ovulation was confirmed, but sexual intercourse was impossible because of dyspareunia, despite vaginal self-dilatation with a vaginal dilator. Artificial insemination was initiated; however, five cycles failed to yield a viable pregnancy. We decided to perform in vitro fertilization (IVF), which resulted in conception. During IVF we administered intravenous anesthesia before oocyte collection to reduce her distress due to insufficient lumen expansion after vaginoplasty. The patient delivered a healthy male infant weighing 2,558 g at 37 weeks of gestation via cesarean section, which was performed because of gestational hypertension. This is the eighth report of a viable neonate born from a patient with ovotesticular DSD after gonadectomy and the first such pregnancy achieved by IVF. Therefore, IVF may be an effective option for infertile patients with ovotesticular DSD. Additionally, to prevent dyspareunia, self-management of the plastic vagina is important during the peri- and postoperative periods of early vaginoplasty.
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Dispareunia , Trastornos Ovotesticulares del Desarrollo Sexual , Embarazo , Humanos , Masculino , Femenino , Trastornos Ovotesticulares del Desarrollo Sexual/diagnóstico , Cesárea , Coito , Fertilización In VitroRESUMEN
Macrophages are essential for the proper inflammatory and reparative processes that lead to regeneration of skeletal muscle after injury. Recent studies have demonstrated close links between the function of activated macrophages and their cellular metabolism. Sterol regulatory element-binding protein 1 (SREBP1) is a key regulator of lipid metabolism and has been shown to affect the activated states of macrophages. However, its role in tissue repair and regeneration is poorly understood. Here we show that systemic deletion of Srebf1, encoding SREBP1, or macrophage-specific deletion of Srebf1a, encoding SREBP1a, delays resolution of inflammation and impairs skeletal muscle regeneration after injury. Srebf1 deficiency impairs mitochondrial function in macrophages and suppresses the accumulation of macrophages at sites of muscle injury. Lipidomic analyses showed the reduction of major phospholipid species in Srebf1 -/- muscle myeloid cells. Moreover, diet supplementation with eicosapentaenoic acid restored the accumulation of macrophages and their mitochondrial gene expression and improved muscle regeneration. Collectively, our results demonstrate that SREBP1 in macrophages is essential for repair and regeneration of skeletal muscle after injury and suggest that SREBP1-mediated fatty acid metabolism and phospholipid remodeling are critical for proper macrophage function in tissue repair.
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Macrófagos , Músculo Esquelético , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Fosfolípidos , Regeneración , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Animales , RatonesRESUMEN
Mitochondrial tRNAs are indispensable for the intra-mitochondrial translation of genes related to respiratory subunits, and mutations in mitochondrial tRNA genes have been identified in various disease patients. However, the molecular mechanism underlying pathogenesis remains unclear due to the lack of animal models. Here, we established a mouse model, designated 'mito-mice tRNALeu(UUR)2748', that carries a pathogenic A2748G mutation in the tRNALeu(UUR) gene of mitochondrial DNA (mtDNA). The A2748G mutation is orthologous to the human A3302G mutation found in patients with mitochondrial diseases and diabetes. A2748G mtDNA was maternally inherited, equally distributed among tissues in individual mice, and its abundance did not change with age. At the molecular level, A2748G mutation is associated with aberrant processing of precursor mRNA containing tRNALeu(UUR) and mt-ND1, leading to a marked decrease in the steady-levels of ND1 protein and Complex I activity in tissues. Mito-mice tRNALeu(UUR)2748 with ≥50% A2748G mtDNA exhibited age-dependent metabolic defects including hyperglycemia, insulin insensitivity, and hepatic steatosis, resembling symptoms of patients carrying the A3302G mutation. This work demonstrates a valuable mouse model with an inheritable pathological A2748G mutation in mt-tRNALeu(UUR) that shows metabolic syndrome-like phenotypes at high heteroplasmy level. Furthermore, our findings provide molecular basis for understanding A3302G mutation-mediated mitochondrial disorders.
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Enfermedades Mitocondriales , ARN de Transferencia de Leucina , Humanos , Animales , Ratones , ARN de Transferencia de Leucina/metabolismo , Enfermedades Mitocondriales/genética , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Mutación , Procesamiento Postranscripcional del ARNRESUMEN
BACKGROUND: Carboplatin, the key drug used in treating gynaecological cancer, has an approximately 12-16% risk of hypersensitivity reactions. We aimed to investigate the efficacy and adverse effects of carboplatin desensitisation therapy for gynaecological cancer. METHODS: The desensitisation protocol was standardised as a four-step, 4-h, carboplatin administration in the hospital. A retrospective medical record review was conducted on 15 patients who underwent carboplatin desensitisation for gynaecological malignancies at our hospital. Patients' data were analysed to evaluate the treatment success rate, therapeutic effect of desensitisation, adverse events, and treatment. RESULTS: Of 91 carboplatin desensitisation cycles scheduled; the completion rate was 93.4% (85/91). Adverse events occurred in 23 of these 91 (25.3%). In four (4.4%) of the 23 cycles, hypersensitivity reactions could be treated only by discontinuing the infusion and slowing the administration, while in the remaining 19 (20.9%), medication was administered intravenously after discontinuing the infusion to manage hypersensitivity reactions. No treatment-related deaths occurred. Overall, 23 series of anti-cancer agent regimens, including carboplatin desensitisation, were administered to the 15 patients. The therapeutic response rate was 82.6% and the disease control rate was 95.7%. CONCLUSIONS: Carboplatin desensitisation was beneficial in patients with a history of carboplatin-induced hypersensitivity reactions.
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Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.
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Replicación del ADN , ADN Mitocondrial/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Metiltransferasas/metabolismo , Proteínas Mitocondriales/metabolismo , Factores de Transcripción/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Células HEK293 , Células HeLa , Humanos , Metiltransferasas/genética , Proteínas Mitocondriales/genética , Factores de Transcripción/genéticaRESUMEN
A 31-year-old nulliparous Japanese woman visited the clinic due to worsening dysmenorrhea. A cystic endometriotic lesion was found in the vesico-uterine pouch. Laparoscopic surgery was chosen due to the severe dysmenorrhea. Her first oocyte retrieval attempt was performed at in-vitro fertilization clinic before the planned surgery. However, she complained of abdominal pain on day 6 after the retrieval. We diagnosed her with peritonitis with an abscessed cystic endometriotic lesion in the vesico-uterine pouch. Conservative treatment was ineffective. Therefore, laparoscopic surgery was performed. The cysts in the vesico-uterine pouch were drained of pus. No adhesions or lesions of endometriosis in the uterus, bilateral adnexa, or pelvic peritoneum were found. Although cystic endometriotic lesions in the vesico-uterine pouch are rare, they can form abscesses after oocyte retrieval. The possibility of abscesses formation risk must be considered. Moreover, following the management of endometrioma, sufficient medication should be administered to prevent this formation.
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AIM: The objective of this study is to identify the risk factors for cancellation after dispatch of rapid response cars (RRC) for prehospital emergency care. METHODS: We retrospectively extracted data from all RRC cases dispatched from our hospital between April 2017 and March 2019. A total of 1,440 cases were included in our study and divided into either the "cancelled" group (n = 723) or the "treated" group (n = 717), based on the occurrence of cancellation. The variables obtained from the request calls for RRC included patient characteristics, distance from the hospital to the scene, and reasons for RRC request. The variables were compared between the two groups and logistic regression analysis was carried out to identify the risk factors for RRC cancellation. RESULTS: Multivariable analysis showed that distance from the hospital to the scene (odds ratio [OR] 1.25; 95% confidence interval (CI), 1.21-1.28), suspicion of cardiopulmonary arrest with no witness information (OR 7.61; 95% CI, 4.13-14.00), dyspnea (OR 2.22; 95% CI, 1.19-4.11), and suicide by hanging (OR 3.49; 95% CI, 1.37-8.89) were independent risk factors for cancellation. CONCLUSIONS: In our study, a greater distance from the hospital to the scene, suspicion of cardiopulmonary arrest with no witness information, dyspnea, and suicide by hanging were identified as independent risk factors for cancellation after dispatch of RRC. Evaluating the risk factors for cancellation at individual facilities could help hospitals adjust their dispatch criteria to allocate limited medical resources more effectively.
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BACKGROUND: The efficacy of steroid treatment for coronavirus disease (COVID-19) is unknown. CASE PRESENTATION: A 67-year-old man was transported to our hospital due to impaired consciousness and respiratory failure. After admission, tracheal aspirate of the patient was harvested, and it tested positive for severe acute respiratory syndrome coronavirus 2 nucleic acid. He required veno-venous extracorporeal membrane oxygenation to sustain his oxygenation. However, his respiratory failure did not improve for 20 days. On day 20 of admission, we started to use i.v. steroid therapy. On day 23, lung opacity on the chest X-ray cleared and the patient's oxygen saturation improved significantly. We successfully removed extracorporeal membrane oxygenation on day 27. CONCLUSION: Our case report encourages more future trials to evaluate the therapeutic use of i.v. steroid in severe COVID-19-induced acute respiratory distress syndrome.
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Chemical acetylation is postulated to occur in mitochondria. Mitochondrial transcription factor A (TFAM or mtTFA), a mitochondrial transcription initiation factor as well as the major mitochondrial nucleoid protein coating the entire mitochondrial genome, is proposed to be acetylated in animals and cultured cells. This study investigated the properties of human TFAM, in conjunction with the mechanism and effects of TFAM acetylation in vitro. Using highly purified recombinant human TFAM and 3 kb circular DNA as a downsized mtDNA model, we studied how the global TFAM-DNA interaction is affected/regulated by the quantitative TFAM-DNA relationship and TFAM acetylation. Results showed that the TFAM-DNA ratio strictly affects the TFAM property to unwind circular DNA in the presence of topoisomerase I. Mass spectrometry analysis showed that in vitro chemical acetylation of TFAM with acetyl-coenzyme A occurs preferentially on specific lysine residues, including those reported to be acetylated in exogenously expressed TFAM in cultured human cells, indicating that chemical acetylation plays a crucial role in TFAM acetylation in mitochondria. Intriguingly, the modification significantly decreased TFAM's DNA-unwinding ability, while its DNA-binding ability was largely unaffected. Altogether, we propose TFAM is chemically acetylated in vivo, which could change mitochondrial DNA topology, leading to copy number and gene expression modulation.
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Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Acetilación , ADN/genética , Humanos , Lisina/química , Modelos Moleculares , Conformación Proteica , Transcripción GenéticaRESUMEN
We report a rare case of an ovarian steroid cell tumor with a diagnosis prompted by heart failure symptoms. A 28-year-old Japanese nulligravida/nullipara with a chief complaint of respiratory discomfort during physical exertion and exhibiting heart failure symptoms was referred to our hospital. She also had signs of virilization, including secondary menorrhea since the age of 20, hirsutism and balding. Cushing's syndrome was suspected, and further examinations showed hypertestosteronemia and right ovarian tumor. Symptomatic treatment for heart failure with diuretics and antihypertensives was followed by abdominal right adnexectomy performed due to the androgen-producing ovarian tumor. The tumor was solid and larger than a fist, and confirmed as a steroid cell tumor through postoperative histopathology. Serum total testosterone levels normalized at day 3 postoperatively, and menstruation resumed 2 months later. Our case was diagnosed due to heart failure symptoms, and its treatment resulted in improvement in virilization signs.
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Insuficiencia Cardíaca , Neoplasias Ováricas , Tumores de los Cordones Sexuales y Estroma de las Gónadas , Adulto , Femenino , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/etiología , Humanos , Neoplasias Ováricas/complicaciones , Neoplasias Ováricas/diagnóstico , Esteroides , Virilismo/etiologíaRESUMEN
Human DNA polymerase γ (POLG) is a mitochondria-specific replicative DNA polymerase consisting of a single catalytic subunit, POLGα, and a dimeric accessory subunit, POLGß. To gain a deeper understanding of the role of POLGß, we knocked out this protein in cultured human cybrid cells and established numerous knockout clones. POLGß-knockout clones presented a clear phenotype of mitochondrial DNA loss, indicating that POLGß is necessary for mitochondrial DNA replication. Moreover, POLGß-knockout cells showed a severe decrease in POLGα levels and acute suppression of POLGß expression efficiently down-regulated POLGα levels. These results suggest that, in addition to its role as the processivity factor of POLG, POLGß acts as a POLGα stabilizer, an important role for POLGß in mitochondrial DNA maintenance.
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ADN Polimerasa gamma/metabolismo , ADN Mitocondrial/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Mitocondrias/genética , ADN Polimerasa gamma/química , ADN Polimerasa gamma/genética , ADN Mitocondrial/química , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Estabilidad de Enzimas , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Células HeLa , Humanos , FenotipoRESUMEN
AIM: Acinetobacter baumannii is commonly associated with outbreaks and antibiotic-resistant nosocomial infection. This study aimed to determine the relationship between antibiotic resistance and genotypes of A. baumannii. METHODS: A study was undertaken in the critical care center (CCC) of Juntendo University Urayasu Hospital (Urayasu, Japan) between January 2012 and September 2015. Antimicrobial susceptibility tests were carried out according to the Clinical and Laboratory Standards Institute guidelines. All A. baumannii isolates were verified to carry carbapenemase genes and the ISA ba1 element using polymerase chain reaction. The genetic relationship of all A. baumannii isolates was determined by pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS: During the study period, 1634 patients were admitted to the CCC. Acinetobacter baumannii was detected in 43 patients (average age, 58 ± 19 years; 67.4% men). Six patients were determined to be extensively drug-resistant A. baumannii and 21 patients determined to be multidrug-resistant A. baumannii. Antimicrobial susceptibility linked genotypes of A. baumannii. Molecular characterization by pulsed-field gel electrophoresis and multilocus sequence typing showed that closely related clones of A. baumannii had spread in the CCC. CONCLUSION: Resistance to antimicrobial drugs was significantly associated with certain A. baumannii genotypic types and molecular types. Thus, we might be able to predict whether the genotype has spread in the CCC or not when the susceptibility is examined, facilitating the appropriate isolation of patients.
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AIM: This study aimed to assess whether hysteroscopic metroplasty using the incision method for septate uterus is a risk factor for adverse obstetric outcomes during pregnancy or delivery. METHODS: This retrospective, single-center cohort study of obstetric complications included 41 patients with recurrent pregnancy loss or unexplained infertility who underwent hysteroscopic metroplasty using the incision method for septate uterus. As controls, we recruited 1139 women who delivered at our hospital during the same period. The primary outcomes were mean weeks of delivery, mean birthweight, rate of cesarean section, rate of breech presentation, rate of post-partum hemorrhage, rate of preterm delivery, rate of placental abruption, rate of placenta previa, rate of placenta accreta and uterine rupture during pregnancy and delivery. RESULTS: The two groups did not differ in terms of age, mean weeks of delivery, mean birthweight, rate of post-partum hemorrhage, rate of preterm delivery, rate of placental abruption, rate of placenta previa or rate of placenta accreta. The rates of cesarean section and breech presentation were significantly higher in the study group than in the control group (56.1 vs 27.7%; P = 0.0002 and 19.5 vs 6.8%; P = 0.007, respectively). There were no cases of uterine rupture during pregnancy or delivery following hysteroscopic metroplasty. CONCLUSION: Hysteroscopic metroplasty using the incision method for septate uterus is not a risk factor for adverse obstetric outcomes. No severe complications, such as placenta abruption, placenta previa, placenta accreta, uterine rupture or heavy hemorrhage, were observed in the postoperative live birth group.
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Presentación de Nalgas/etiología , Histeroscopía/efectos adversos , Enfermedades Placentarias/etiología , Resultado del Embarazo , Rotura Uterina/etiología , Útero/cirugía , Adulto , Cesárea , Femenino , Humanos , Embarazo , Estudios Retrospectivos , Factores de RiesgoRESUMEN
Whilst 5-methylcytosine (5mC) is a major epigenetic mark in the nuclear DNA in mammals, whether or not mitochondrial DNA (mtDNA) receives 5mC modification remains controversial. Herein, we exhaustively analysed mouse mtDNA using three methods that are based upon different principles for detecting 5mC. Next-generation bisulfite sequencing did not give any significant signatures of methylation in mtDNAs of liver, brain and embryonic stem cells (ESCs). Also, treatment with methylated cytosine-sensitive endonuclease McrBC resulted in no substantial decrease of mtDNA band intensities in Southern hybridisation. Furthermore, mass spectrometric nucleoside analyses of highly purified liver mtDNA preparations did not detect 5-methyldeoxycytidine at the levels found in the nuclear DNA but at a range of only 0.3-0.5% of deoxycytidine. Taken together, we propose that 5mC is not present at any specific region(s) of mtDNA and that levels of the methylated cytosine are fairly low, provided the modification occurs. It is thus unlikely that 5mC plays a universal role in mtDNA gene expression or mitochondrial metabolism.