The monthly remodeling, shedding, and regeneration of the endometrium defining the human menstrual cycle is driven by gene expression changes in the underlying tissue hierarchy. Significant heterogeneity exists among cell types in the endometrium, such that multiple cell types vary dramatically in state through a monthly cycle and undergo various forms of differentiation at rapid rates. Histologic analysis and whole-tissue transcriptomic profiling have defined a specific molecular state as the optimal timing of the window of implantation (WOI) for in vitro fertilization transfer.This single-cell transcriptomic analysis aimed to characterize the transcriptomic transformation of human endometrium at single-cell resolution across the menstrual cycle, including at the WOI. Endometrial biopsies were collected from 19 healthy ovum donors between 4 and 27 days following menses, and single cells were captured and complementary DNA was generated using Fluidigm C1 medium chips. Six cell types were identified across the menstrual cycle: stromal fibroblast, endothelium, macrophage, lymphocyte, ciliated epithelium, and unciliated epithelium.Endometrial transformation was analyzed by within-cell type t-SNE using whole-transcriptome data from unciliated epithelia and stromal fibroblasts, the 2 major contributing cell types to endometrial transformation. This revealed 4 major, time-associated phases of both cell types. Among unciliated epithelia, single-cell gene dynamics were relatively continuous across phases 1 to 3 until an abrupt activation of genes consistently reported in whole-tissue transcriptomic data sets as overexpressed in the WOI marked entrance into phase 4. Among stromal fibroblasts, the WOI was characterized by widespread decidualization that became gradually upregulated through phase progression. Likewise, the WOI closed with more gradual transition dynamics in both cell types.The traditional definition of endometrial phases, consisting of the proliferative and secretory phases, correlated with the 4 phases identified here through single-cell analysis. Cell-cycling was elevated in phases 1 and 2 and ceased in later phases, suggesting the transition from proliferative to secretory occurred between phase 2 and 3. At the transcriptomic level, proliferative endometrium can be divided into 2 distinct phases with unique transcriptomic signatures.This study involved the systematic characterization of the human endometrium across the menstrual cycle through dynamic gene expression mapping. The results demonstrate that ciliated epithelium are a transcriptomically distinct endometrial cell type that are highly prevalent in the human endometrium and constantly changing in abundance across the cycle. This study likewise demonstrated an abrupt and strong transcriptomic activation in unciliated epithelia and a gradual activation in stromal fibroblasts to define the opening of the WOI, indicating a potential diagnostic target for more precise in vitro fertilization and embryo transfer.
Significance Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape of circulating RNA in human subjects. By focusing on tissue-specific genes, we were able to identify the relative contributions of these tissues to circulating RNA and monitor changes during tissue development and neurodegenerative disease states.
Noninvasive blood tests that provide information about fetal development and gestational age could potentially improve prenatal care. Ultrasound, the current gold standard, is not always affordable in low-resource settings and does not predict spontaneous preterm birth, a leading cause of infant death. In a pilot study of 31 healthy pregnant women, we found that measurement of nine cell-free RNA (cfRNA) transcripts in maternal blood predicted gestational age with comparable accuracy to ultrasound but at substantially lower cost. In a related study of 38 women (23 full-term and 15 preterm deliveries), all at elevated risk of delivering preterm, we identified seven cfRNA transcripts that accurately classified women who delivered preterm up to 2 months in advance of labor. These tests hold promise for prenatal care in both the developed and developing worlds, although they require validation in larger, blinded clinical trials.
BACKGROUND Detecting tumor-derived cell-free DNA (cfDNA) in the blood of brain tumor patients is challenging, presumably owing to the blood–brain barrier. Cerebral spinal fluid (CSF) may serve as an alternative “liquid biopsy” of brain tumors by enabling measurement of circulating DNA within CSF to characterize tumor-specific mutations. Many aspects about the characteristics and detectability of tumor mutations in CSF remain undetermined. METHODS We used digital PCR and targeted amplicon sequencing to quantify tumor mutations in the cfDNA of CSF and plasma collected from 7 patients with solid brain tumors. Also, we applied cancer panel sequencing to globally characterize the somatic mutation profile from the CSF of 1 patient with suspected leptomeningeal disease. RESULTS We detected tumor mutations in CSF samples from 6 of 7 patients with solid brain tumors. The concentration of the tumor mutant alleles varied widely between patients, from <5 to nearly 3000 copies/mL CSF. We identified 7 somatic mutations from the CSF of a patient with leptomeningeal disease by use of cancer panel sequencing, and the result was concordant with genetic testing on the primary tumor biopsy. CONCLUSIONS Tumor mutations were detectable in cfDNA from the CSF of patients with different primary and metastatic brain tumors. We designed 2 strategies to characterize tumor mutations in CSF for potential clinical diagnosis: the targeted detection of known driver mutations to monitor brain metastasis and the global characterization of genomic aberrations to direct personalized cancer care.
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