Endometrial Cancer Detection by DNA Methylation Analysis in Cervical Papanicolaou Brush Samples.

Background: Endometrial cancer (EC) is the leading gynecological cancer worldwide, yet current EC screening approaches are not satisfying. The purpose of this retrospective study was to evaluate the feasibility and capability of DNA methylation analysis in cervical Papanicolaou (Pap) brush samples for EC detection. Methods: We used quantitative methylation-sensitive PCR (qMS-PCR) to determine the methylation status of candidate genes in EC tissue samples, as well as cervical Pap brushes. The ability of RASSF1A and HIST1H4F to serve as diagnostic markers for EC was then examined in cervical Pap brush samples from women with endometrial lesions of varying degrees of severity. Results: Methylated RASSF1A and HIST1H4F were found in EC tissues. Further, methylation of the two genes was also observed in cervical Pap smear samples from EC patients. Methylation levels of RASSF1A and HIST1H4F increased as endometrial lesions progressed, and cervical Pap brush samples from women affected by EC exhibited significantly higher levels of methylated RASSF1A and HIST1H4F compared to noncancerous controls (P < .001). Receiver operating characteristic (ROC) curves and area under the curve (AUC) analyses revealed RASSF1A and HIST1H4F methylation with a combined AUC of 0.938 and 0.951 for EC/pre-EC detection in cervical Pap brush samples, respectively. Conclusion: These findings demonstrate that DNA methylation analysis in cervical Pap brush samples may be helpful for EC detection, broadening the scope of the commonly used cytological screening. Our proof-of-concept study provides new insights into the field of clinical EC diagnosis.

Single-Cell Atlas of Human Ovaries Reveals The Role Of The Pyroptotic Macrophage in Ovarian Aging.

Female fecundity declines in a nonlinear manner with age during the reproductive years, even as ovulatory cycles continue, which reduces female fertility, disrupts metabolic homeostasis, and eventually induces various chronic diseases. Despite this, the aging-related cellular and molecular changes in human ovaries that occur during these reproductive years have not been elucidated. Here, single-cell RNA sequencing (scRNA-seq) of human ovaries is performed from different childbearing ages and reveals that the activation of the pyroptosis pathway increased with age, mainly in macrophages. The enrichment of pyroptotic macrophages leads to a switch from a tissue-resident macrophage (TRM)-involve immunoregulatory microenvironment in young ovaries to a pyroptotic monocyte-derived macrophage (MDM)-involved proinflammatory microenvironment in middle-aged ovaries. This remolded ovarian immuno-microenvironment further promotes stromal cell senescence and accelerated reproductive decline. This hypothesis is validated in a series of cell and animal experiments using GSDMD-KO mice. In conclusion, the work expands the current understanding of the ovarian aging process by illustrating a pyroptotic macrophage-involved immune mechanism, which has important implications for the development of novel strategies to delay senescence and promote reproductive health.

METTL3-dependent m6A methylation facilitates uterine receptivity and female fertility via balancing estrogen and progesterone signaling.

Infertility is a worldwide reproductive health problem and there are still many unknown etiologies of infertility. In recent years, increasing evidence emerged and confirmed that epigenetic regulation played a leading role in reproduction. However, the function of m6A modification in infertility remains unknown. Here we report that METTL3-dependent m6A methylation plays an essential role in female fertility via balancing the estrogen and progesterone signaling. Analysis of GEO datasets reveal a significant downregulation of METTL3 expression in the uterus of infertile women with endometriosis or recurrent implantation failure. Conditional deletion of Mettl3 in female reproductive tract by using a Pgr-Cre driver results in infertility due to compromised uterine endometrium receptivity and decidualization. m6A-seq analysis of the uterus identifies the 3'UTR of several estrogen-responsive genes with METTL3-dependent m6A modification, like Elf3 and Celsr2, whose mRNAs become more stable upon Mettl3 depletion. However, the decreased expression levels of PR and its target genes, including Myc, in the endometrium of Mettl3 cKO mice indicate a deficiency in progesterone responsiveness. In vitro, Myc overexpression could partially compensate for uterine decidualization failure caused by Mettl3 deficiency. Collectively, this study reveals the role of METTL3-dependent m6A modification in female fertility and provides insight into the pathology of infertility and pregnancy management.

Cervical HPV infection in Guangzhou, China: an epidemiological study of 198,111 women from 2015 to 2021.

Persistent high-risk human papillomavirus (HPV) infection is the pivotal cause of cervical carcinogenesis. HPV types distribution varies greatly by region, and its long-term changes of prevalence remain to be fully characterized in China. Here, the largest population of 198,111 consecutive women who underwent routine cervical screening were investigated from 2015 to 2021 in Guangzhou, south China. The results showed that the overall HPV prevalence was 21.66% (42,911/198,111), and the annual prevalence increased significantly from 2015 to 2021 (p < 0.001). HPV52, 16, 58, CP8304, 51, 53, 39, and 68 were the most prevalent HPV types. The relative HPV-positive rate correlated positively with the progression of cervical intraepithelial neoplasia (p < 0.001); HPV16 was the predominant carcinogenic type, followed by HPV52 and HPV18. HPV infections were significantly age-specific, and 26.51% (11,375/42,911) of cases were caused by multiple HPV types. In addition, HPV infections typically cleared over a median time of 16 (interquartile range 9-31) months, and the clearance of HPV16 was significantly faster than that of other types (p < 0.001). These findings may serve as a guide for local governments to evaluate HPV vaccination and cervical cancer prevention strategies in south China.

mTORC1 signaling pathway integrates estrogen and growth factor to coordinate vaginal epithelial cells proliferation and differentiation.

The mouse vaginal epithelium cyclically exhibits cell proliferation and differentiation in response to estrogen. Estrogen acts as an activator of mTOR signaling but its role in vaginal epithelial homeostasis is unknown. We analyzed reproductive tract-specific Rptor or Rictor conditional knockout mice to reveal the role of mTOR signaling in estrogen-dependent vaginal epithelial cell proliferation and differentiation. Loss of Rptor but not Rictor in the vagina resulted in an aberrant proliferation of epithelial cells and failure of keratinized differentiation. As gene expression analysis indicated, several estrogen-mediated genes, including Pgr and Ereg (EGF-like growth factor) were not induced by estrogen in Rptor cKO mouse vagina. Moreover, supplementation of EREG could activate the proliferation and survival of vaginal epithelial cells through YAP1 in the absence of Rptor. Thus, mTORC1 signaling integrates estrogen and growth factor signaling to mediate vaginal epithelial cell proliferation and differentiation, providing new insights into vaginal atrophy treatment for post-menopausal women.

The effectiveness of urinary TIMP-2 and IGFBP-7 in predicting acute kidney injury in critically ill neonates.

BACKGROUD: Urinary tissue inhibitor of metalloproteinase-2 (TIMP-2), insulin-like growth factor binding protein-7 (IGFBP-7) and the combination of TIMP-2 and IGFBP-7 ([TIMP-2]•[IGFBP7]) are proposed to be predictive biomarkers for acute kidney injury (AKI). The intention of our study was to determine whether there is any significant predictive value of these biomarkers for the occurrence of AKI and severe AKI in critically ill neonates. METHODS: Urinary samples were serially collected in 237 neonates during neonatal intensive care unit (NICU) stay for measurements of TIMP-2 and IGFBP-7 in this prospective study. AKI diagnosis was based on KDIGO classification without urine output or serum creatinine >1.2 mg/dL. RESULTS: Twenty neonates developed AKI, including 11 with KDIGO stage 1, defined as mild AKI, and 9 with stages 2 and 3, defined as severe AKI. Urinary IGFBP-7 and [TIMP-2]•[IGFBP7] remained associated with AKI after adjustment for gestational age, gender and illness severity. Urinary [TIMP-2]•[IGFBP7] achieved an AUC of 0.71 (P = 0.034) and displayed a sensitivity of 88.9% and a specificity of 50.9% for discriminating severe AKI at the optimal cut-off value of 0.045. CONCLUSION: The combination of TIMP-2 and IGFBP-7 had independent discriminative value for severe AKI in critically ill neonates.

Urinary Nephrin as a Biomarker of Glomerular Maturation and Injury Is Associated with Acute Kidney Injury and Mortality in Critically Ill Neonates.

BACKGROUND: Nephrin is a key component of the slit diaphragm of the glomerular podocyte, and increased urinary nephrin level may reflect glomerular injury. OBJECTIVES: To determine whether urinary nephrin is a useful biomarker of glomerular maturation and injury and whether it is associated with acute kidney injury (AKI) and neonatal intensive care unit (NICU) mortality in critically ill neonates. METHODS: Urinary samples were serially collected in 234 neonates during NICU stay for measurements of nephrin, cystatin C (CysC), and albumin. AKI diagnosis was based on neonatal Kidney Disease: Improving Global Outcome (KDIGO) criteria. RESULTS: Of the neonates, 26 developed AKI and 24 died during NICU stay. The independent contributors to the initial urinary nephrin level obtained on the first 24 h admitted to NICU were gestational age (p = 0.004) and initial urinary CysC level (p < 0.001). Both initial (p = 0.037) and peak (p = 0.039) urinary nephrin were significantly associated with AKI, even after controlling for significant covariates, and had an area under the receiver-operating characteristic curve (AUC) of 0.71 and 0.70, respectively, for predicting AKI. At the optimal cutoff value of 0.375 µg/mg urinary creatinine, the initial urinary nephrin displayed sensitivity of 61.5% and specificity of 76.9% for predicting AKI. The AUCs for initial and peak urinary nephrin to predict NICU mortality were 0.81 and 0.83, respectively. CONCLUSIONS: Urinary nephrin, which may decrease with increasing glomerular maturity, is significantly associated with increased risk for AKI and NICU mortality even after adjustment for potential confounders. A higher level of urinary nephrin may be independently predictive of AKI and NICU mortality in critically ill neonates.

The clinical significance of FAM19A4 methylation in high-risk HPV-positive cervical samples for the detection of cervical (pre)cancer in Chinese women.

BACKGROUND: To explore the diagnostic value of FAM19A4 methylation in high-risk human papilloma virus (hrHPV)-positive cervical samples from Chinese women for estimating cervical cancer or its precancerous lesions. METHODS: Cervical samples from 215 women infected with high-risk HPV were collected by smear testing. We purposely chose 61 patients with cervical cancer, 57 with high-grade squamous intraepithelial lesions (HSIL), 31 with low-grade squamous intraepithelial lesions (LSIL), and 66 without cervical intraepithelial neoplasia (CIN) after histological confirmation. Taqman probe-based quantitative PCR (qPCR) was utilized to detect the methylation status of FAM19A4 in the cervical samples and further evaluate the use of this gene in the diagnosis of cervical cancer. RESULTS: (1) An increasing level of FAM19A4 methylation was detected with increasing progression of cervical lesions, with methylation rates of 10.61%(7/66), 35.48%(11/31), 56.14%(32/57) and 93.44%(57/61) in no CIN, LSIL, HSIL and cervical carcinoma samples respectively. (2) In all hrHPV-positive samples, the levels of FAM19A4 methylation in HPV16/18 groups were higher than that in 12 other hrHPV groups (P < 0.05), but there was no significant difference between two groups after grouping cervical lesions into cervical cancer, HSIL, LSIL and no CIN groups (P>0.05). (3)There were no significant differences of FAM19A4 methylation in different clinicopathological parameters of cervical cancer. (4) Though the sensitivity of FAM19A4 methylation test was inferior to that of cytology and FAM19A4 combining with HPV16/18 genotyping, but showed the best specificity with 81.44% both for detection HSIL alone and ≥ HSIL, with favorable youden index (YI) and area under curve (AUC). CONCLUSION: FAM19A4 is a specific biomarker of cancerous lesions of the cervix. FAM19A4 methylation analysis may serve as an auxiliary screening method for diagnosis of cervical (pre)cancer. However, in consideration of the limitations of this retrospective study, prospective population-based studies are necessary for further confirmation of the diagnostic value of FAM19A4 methylation for detection of cervical (pre)cancer in Chinese women.

Subclinical acute kidney injury is associated with adverse outcomes in critically ill neonates and children.

BACKGROUND: Research on acute kidney injury (AKI) has focused on identifying early biomarkers. However, whether AKI could be diagnosed in the absence of the classic signs of clinical AKI and whether the condition of subclinical AKI, identified by damage or functional biomarkers in the absence of oliguria or increased serum creatinine (sCr) levels, is clinically significant remains to be elucidated in critically ill children. The aims of the study were to investigate the associations between urinary cystatin C (uCysC) levels and AKI and mortality and to determine whether uCysC-positive subclinical AKI is associated with adverse outcomes in critically ill neonates and children. METHODS: In this prospective cohort study, uCysC levels were serially measured during the first week after intensive care unit (ICU) admission in a heterogeneous group of patients (n = 510) presenting to a tertiary neonatal and pediatric ICU. The diagnosis of neonatal AKI that developed during the first week after admission was based on neonatal KDIGO criteria or sCr >1.5 mg/dL, and pediatric AKI was based on Kidney Disease: Improving Global Outcomes (KDIGO) criteria. The term "uCysC(-)" or "uCysC(+)", indicating the absence or presence of tubular injury, was defined by the optimal peak uCysC cutoff value for predicting ICU mortality. RESULTS: The initial and peak uCysC levels were significantly associated with AKI and mortality, and had an area under the receiver operating characteristic curve of 0.76 and 0.81, respectively, for predicting mortality. At the optimal cutoff value of 1260 ng/mg uCr, the peak uCysC displayed sensitivity of 79.2% and specificity of 72.3% for predicting mortality. Among all patients, 130 (25.5%) developed uCysC(+)/AKI(-) status during the first week after admission. The adjusted odds ratio for patients with uCysC(+)/AKI(-) status in association with an increased risk of mortality compared with that for patients with uCysC(-)/AKI(-) was 9.34 (P < 0.001). Patients with uCysC(+)/AKI(-) spent 2.8 times as long in the ICU as those with uCysC(-)/AKI(-) (P < 0.001). CONCLUSIONS: Both initial and peak uCysC levels are associated with AKI and mortality and are independently predictive of mortality in critically ill neonates and children. Subclinical AKI may occur without detectable loss of kidney function, and uCysC-positive subclinical AKI is associated with worse clinical outcomes in this population.